Intracellular Signaling

2 

Intracellular Signaling Aphrothiti J. Hanrahan, Gopa Iyer, and David B. Solit

S UMMARY

OF

K EY

P OI N T S

• Ligand binding and activation of cell surface and internal receptors trigger the activation and/or suppression of signaling cascades that regulate diverse cellular processes including cell growth, proliferation, survival, and invasion, among others. • Multiple nodes within these intracellular signaling networks are genetically and epigenetically altered in human cancers, leading to constitutive pathway activation or suppression. • Some cancers are dependent on genomic alterations in oncogenes or tumor suppressor genes for their

growth and survival, a phenomenon known as oncogene addiction. • Drugs that selectively target mutated proteins critical for the maintenance of the transformed phenotype have shown unprecedented clinical activity in genetically defined cancer subsets. • Precision medicine refers to the use of genetic and epigenetic information unique to an individual cancer patient to develop treatment regimens that target the driver oncogenes and tumor suppressors responsible for tumor progression. Potential challenges to the application of this approach include

The underlying basis of the cancer phenotype is deregulated cell growth, which stems from two main hallmarks of cancer: uncontrolled proliferation, and loss of programmed cell death (enhanced survival). In normal cells, these processes are tightly controlled through integration of signaling cascades that translate extracellular and intracellular cues into specific output responses. These signaling pathways are often initiated on binding of ligand to the extracellular domain of a receptor, followed by recruitment of adaptor proteins or kinases that activate an intracellular cascading network of protein and lipid intermediaries that ultimately produce a cellular response. In normal cells, the specificity, amplitude, and duration of signaling are tightly regulated, and these constraints are often abrogated in human cancers. Investigation of the signal transduction pathways that regulate normal cellular functions has revealed that key components of these networks are commonly altered in cancer cells by mutation, amplification and deletion, chromosomal translocation, overexpression, or epigenetic silencing. These alterations lead to activation or suppression of signaling cascades that underlie the various hallmarks of the cancer phenotype. This chapter reviews the major signal transduction cascades, with a focus on those that are frequently altered in human cancers. Individual sections highlight signaling intermediaries that have been validated as drug targets in patients with cancer. Table 2.1 summarizes actionable gene-level and mutation-level alterations in cancer and the drugs that are currently approved by the US Food and Drug Administration (FDA) for treatment, that are recommended standard of care biomarkers, or that have promising clinical or preclinical efficacy.1 24

the current inability to directly inhibit some oncogenic proteins (i.e., mutant KRAS), the development of drug resistance, technical hurdles posed by limited tissue availability for prospective molecular characterizing, and intratumoral and lesion-to-lesion genomic heterogeneity. • Routine genomic analysis of tumors or tumor-derived cell free DNA in plasma is now a component of standard care in an increasing number of cancer types, with the results used to guide treatment selection.

RECEPTOR TYROSINE KINASE SIGNALING The receptor tyrosine kinases (RTKs) comprise a family of transmembrane (TM) cell surface receptors that transduce extracellular signals internally to promote growth and survival and/ or to regulate other cellular phenotypes.2,3 Members of this protein family share a similar modular domain structure. Growth factors bind to the extracellular ligand-binding domain of RTKs and induce dimerization of two receptor monomers, juxtaposing the intracellular tyrosine kinase domains of each monomer.4 This results in transphosphorylation of tyrosine residues within the cytoplasmic domains of the RTK dimer. Following transphosphorylation, a variety of intracellular proteins are recruited to the activated RTK through Src homology 2 (SH2) domains that recognize the phosphotyrosine plus a specific amino acid sequence motif C-terminal to the tyrosine residues.5,6 Over 117 SH2 domains have been characterized, each with unique phosphotyrosine sequence specificities.7 Each domain is part of a larger adaptor protein involved in transducing extracellular signals to activate, or in some cases suppress, specific intracellular signaling cascades. Thus the complement of signaling pathways that a given RTK regulates is dictated by the profile of phosphorylated tyrosine residues plus flanking amino acids within their intracellular domains.8,9 However, more than one adaptor protein can often recognize individual context-dependent phosphotyrosine motifs within an RTK, underscoring how this system is designed to provide both specificity and diversity of intracellular signaling. Text continued on p. 29

Intracellular Signaling  •  CHAPTER 2 25

Table 2.1  Targeted Therapy for Disease-Specific Alterations of Actionable Oncogenes in Cancer Gene

Variant

Cancer Type

ABL1

BCR-ABL1 fusion

ALL

AKT1

E17K

ALK

Fusions Oncogenic mutations Fusions

ARAF ATM BRAF

L1196M, L1196Q R1275Q S214A S214C N2875K, R3008C, truncating mutations V600D, V600E, V600G, V600K, V600M, V600R

BRCA1

V600E, V600K Fusions K601E L597Q, L597R, L597S, L597V D594E, D594N, G466V, G469A, G469V, G596C KIAA1549-BRAF Fusion L597Q, L597V Oncogenic mutations

BRCA2

Oncogenic mutations

CDK4

Amplification

CDKN2A

Oncogenic mutations

EGFR

729_761del, 729_761indel, L858R

E709_T710delinsD, E709K, G719A, G719C, G719D, G719S, A763_Y764insFQEA, L747P, A750P, A763_Y764insFQEA, L833V, L861Q, L861R, S768I, EGFR-KDD T790M 762_823ins 762_823ins, G719A, L861R, S768I

Drug

Dasatinib Imatinib Dasatinib CML Imatinib Nilotinib Breast AZD5363 Ovarian AZD5363 All Tumors ARQ 751 NSCLC Alectinib Ceritinib Crizotinib NSCLC Brigatinib Soft tissue sarcoma Ceritinib Crizotinib NSCLC Brigatinib Embryonal tumor Crizotinib Histiocytosis Sorafenib NSCLC Sorafenib Prostate cancer Olaparib Melanoma Cobimetinib + vemurafenib Dabrafenib Dabrafenib + trametinib Vemurafenib Histiocytosis Vemurafenib NSCLC Dabrafenib Dabrafenib + trametinib Vemurafenib Colorectal Binimetinib + cetuximab + encorafenib Panitumumab + vemurafenib Colorectal Fluorouracil + radiation + trametinib Melanoma Trametinib Ovarian Paclitaxel + selumetinib Melanoma Trametinib Melanoma Trametinib Melanoma Trametinib Soft tissue sarcoma Sorafenib + temsirolimus Melanoma BGB659 Ovarian Niraparib Rucaparib Olaparib Ovarian Niraparib Rucaparib Olaparib Dedifferentiated liposarcoma Abemaciclib, palbociclib Well-differentiated Abemaciclib, palbociclib liposarcoma

Evidencea 1 1 1 1 1 3 3 4 1 1 1 1 2 2 3 4 3 3 4 1 1 1 1 2 2 2 2 3 3 4 1 3 3 3 4 4 4 1 1 2 1 1 2 2 2

NSCLC

Letrozole + palbociclib Palbociclib Afatinib Erlotinib Gefitinib Osimertinib Afatinib, erlotinib, gefitinib

4 4 1 1 1 4 1

NSCLC NSCLC NSCLC

Osimertinib EGF816 AP32788

1 4 4

Breast Esophagogastric NSCLC

Continued

26 Part I: Science and Clinical Oncology

Table 2.1  Targeted Therapy for Disease-Specific Alterations of Actionable Oncogenes in Cancer—cont’d Evidencea

Gene

Variant

Cancer Type

Drug

ERBB2

Amplification

Breast

All liquid tumors Leukemia GIST Thymic tumor

Ado-trastuzumab emtansine Lapatinib Lapatinib + trastuzumab Pertuzumab + trastuzumab Trastuzumab Trastuzumab Neratinib Neratinib Lapatinib AP32788 Cisplatin AZD9496, fulvestrant GDC-0810 GSK126 Tazemetostat AZD4547 Debio1347 Ponatinib Debio1347 JNJ-42756493 Debio1347 JNJ-42756493 BGJ398 Debio1347 Debio1347 JNJ-42756493 Debio1347 JNJ-42756493 Debio1347 JNJ-42756493 Debio1347 JNJ-42756493 Debio1347 JNJ-42756493 Debio1347 Sorafenib AG-120 BAY1436032 CB-839 AG-221 Ruxolitinib Sunitinib Sunitinib

1 1 1 1 1 1 3 3 3 4 3 3 4 4 4 3 3 4 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 4 3 3 4 4 3 3 1 2

GIST

Imatinib

1

GIST

Regorafenib

1

GIST

Dasatinib

2

GIST

Nilotinib

2

GIST Thymic tumor Melanoma

Sorafenib Sorafenib Imatinib

2 2 2

EZH2

Oncogenic mutations V659E E770_K831indel, E770_K831ins Oncogenic mutations Oncogenic mutations D538G, Y537S Oncogenic mutations

FGFR1

Amplification

FGFR2

BCR-FGFR1 fusion Fusions

ERCC2 ESR1

Esophagogastric Breast Breast NSCLC NSCLC Bladder Breast Breast Diffuse large B-cell lymphoma Lung squamous cell carcinoma Leukemia Adrenocortical carcinoma Bladder Cholangiocarcinoma Endometrial

FGFR3

Fusions

Adrenocortical carcinoma Bladder Glioma

G370C, G380R, K650E, K650M, K650N, K650Q, K650R, K650T, R248C, S249C, S371C, Y373C FLT3 IDH1

Y572_Y630ins R132C, R132G, R132H, R132Q, R132S

IDH2 JAK2 KIT

R140Q, R172G, R172K, R172M, R172S PCM1-JAK2 fusion 449_514mut, 550_592mut, A502_Y503dup, D579del, D820G, E554_K558del, H697Y, K550_W557del, K558delinsNP, L576P, P551_M552del, V555_L576del, V560D, V560del, V654A 449_514mut, 550_592mut, D419del, D579del, E554_I571del, E554_K558del, E554_V559del, F522C, I563_L576del, I653T, K550_W557del, K558N, K558_E562del, K558_V559del, K558delinsNP, K642E, L576P, M541L, M552_W557del, N564_Y578del, N822H, N822Y, P573_D579del, P577_ W582delinsPYD, P838L, Q556_K558del, T417_D419delinsI, T417_D419delinsRG, T574insTQLPYD, V530I, V555_L576del, V555_V559del, V559C, V559D, V559G, V559_V560del, V559del, V560D, V560G, V560del, V569_L576del, W557G, W557R, W557_K558del, Y553N, Y553_K558del, Y570H, Y578C 449_514mut, 550_592mut, D820G, D820Y, K550_W557del, K558delinsNP, N822K, V560D D816F, D816Y, D820G, D820Y, L576P, N822I, V559D, V560G, W557_K558del D816V, D820A, D820G, D820Y, K642E, L576P, V555_L576del, V559C, V559D, V654A, W557_K558del D820A, D820E, D820G, D820Y, K642E, N505I, P577_D579del, V559D, W557_K558del K642E, L576P, V559A

Bladder Breast AML AML All tumors

Intracellular Signaling  •  CHAPTER 2 27

Table 2.1  Targeted Therapy for Disease-Specific Alterations of Actionable Oncogenes in Cancer—cont’d Gene

Variant

Cancer Type

Drug

KRAS

Wild type

Colorectal

Cetuximab Panitumumab Regorafenib Cabozantinib + panitumumab Panitumumab + regorafenib Pembrolizumab alpelisib + binimetinib Cobimetinib + GDC-0994 Atezolizumab + cobimetinib Fluorouracil + radiation therapy + trametinib Abemaciclib, PD0325901 + palbociclib, palbociclib, ribociclib, ribociclib + trametinib Binimetinib + erlotinib Binimetinib, selumetinib, trametinib Docetaxel + trametinib Cobimetinib, selumetinib, trametinib Cobimetinib, selumetinib, trametinib Cobimetinib, selumetinib, trametinib Cobimetinib, selumetinib, trametinib DS-3032b RG7112 SAR405838 Crizotinib Cabozantinib Capmatinib Crizotinib Cabozantinib Crizotinib Cabozatinib Capmatinib Everolimus Everolimus, rapamycin, temsirolimus Trametinib Trametinib Binimetinib PLX3397 Atezolizumab + cobimetinib Binimetinib Binimetinib + ribociclib Radioiodine uptake therapy + selumetinib Fluorouracil + radiation therapy + trametinib Larotrectinib Entrectinib Entrectinib Larotrectinib Entrectinib Larotrectinib

Oncogenic mutations

All tumors Colorectal NSCLC

MAP2K1

Oncogenic mutations

Histiocytic disorder Low-grade serous ovarian Melanoma NSCLC

MDM2

Amplification

Liposarcoma

MET

963_D1010splice, 981_1028splice, X1006_splice, X1007_ splice, X1008_splice, X1009_splice, X1010_splice, X963_ splice Amplification

NSCLC

D1010H, D1010N, D1010Y

NSCLC RCC NSCLC

MTOR

E2014K C1483F, F1888L, L2230V, S2215F, T1977K

Bladder RCC (clear cell)

NF1

Oncogenic mutations

NRAS

Oncogenic mutations

Glioblastoma Melanoma Neurofibroma Neurofibroma Colorectal Melanoma Thyroid Colorectal

NTRK1

Fusions

NTRK2

Fusions

All tumors Salivary gland Salivary gland

NTRK3

Fusions

Salivary gland

Evidencea 1 1 1 4 4 4 4 4 4 4 4

4 4 4 3 3 3 3 3 3 4 2 3 3 2 2 2 3 3 3 4 4 4 4 4 3 3 3 3 4 3 3 3 3 3 3 Continued

28 Part I: Science and Clinical Oncology

Table 2.1  Targeted Therapy for Disease-Specific Alterations of Actionable Oncogenes in Cancer—cont’d Gene

Variant

PDGFRA

FIP1L1-PDGFRA fusion Fusions

PDGFRB

PIK3CA

Cancer Type

Leukemia Myelodysplasia Myeloproliferative Neoplasm 560_561insER, A633T, C450_K451insMIEWMI, C456_N468del, GIST C456_R481del, D568N, D842I, D842_H845del, D842_ M844del, D846Y, E311_K312del, G853D, H650Q, H845Y, H845_N848delinsP, I843del, N659K, N659R, N659S, N848K, P577S, Q579R, R748G, R841K, S566_E571delinsR, S584L, V469A, V536E, V544_L545insAVLVLLVIVIISLI, V561A, V561D, V561_I562insER, V658A, W559_R560del, Y375_K455del, Y555C, Y849C, Y849S D842V GIST Fusions Dermatofibrosarcoma Protuberans Myelodysplasia Myeloproliferative neoplasm Oncogenic mutations Breast

All tumors Endometrial Ovarian

PTCH1

Truncating mutations

Embryonal tumor Skin cancer, nonmelanoma

PTEN

Oncogenic mutations

All tumors

RAF1 RET

S257L Fusions

Endometrial Prostate Lung adenocarcinoma NSCLC

ROS1

Fusions D2033N

NSCLC

Drug

Evidencea

Imatinib Imatinib Imatinib Imatinib

1 1 1 2

Dasatinib Imatinib

2 1

Imatinib Imatinib Alpelisib Alpelisib + fulvestrant Buparlisib Buparlisib + fulvestrant Copanlisib Fulvestrant + taselisib GDC-0077 Serabelisib Taselisib Alpelisib + everolimus Alpelisib + letrozole, Alpelisib + letrozole + ribociclib Alpelisib + LJM716 + trastuzumab Alpelisib + olaparib, buparlisib + olaparib AZD5363 + fulvestrant AZD8835 + fulvestrant MLN0128 + serabelisib ARQ 751 AZD5363 + olaparib GDC-0077 Alpelisib + fulvestrant Buparlisib + fulvestrant Fulvestrant + taselisib Alpelisib + fulvestrant Buparlisib + fulvestrant Fulvestrant + taselisib Sonidegib Sonidegib Vismodegib ARQ 751 AZD5363 + olaparib AZD8186 Gedatolisib + palbociclib GSK2636771 LY3023414 Olaparib Enzalutamide + LY3023414 Sorafenib Cabozantinib Vandetanib Crizotinib Cabozantinib

1 1 3 3 3 3 3 3 3 3 3 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 3 3 3 4 4 4 4 4 4 4 4 4 2 3 1 3

Intracellular Signaling  •  CHAPTER 2 29

Table 2.1  Targeted Therapy for Disease-Specific Alterations of Actionable Oncogenes in Cancer—cont’d Gene

Variant

Cancer Type

Drug

TSC1

Oncogenic mutations

TSC2

Oncogenic mutations

CNS RCC CNS

Everolimus Everolimus Everolimus

Evidencea 2 2 2

a

Levels of evidence: • Level 1: FDA-recognized biomarker predictive of response to an FDA-approved drug in this indication • Level 2: Standard care biomarker predictive of response to an FDA-approved drug in this indication or another indication; including those recommended by NCCN, but not FDA recognized as standard of care • Level 3: Evidence of clinical activity in this indication, or another indication • Level 4: Preclinical or biologic evidence of activity ALL, Acute lymphoblastic leukemia; AML, acute myeloid leukemia; CML, chronic myelogenous leukemia; CNS, central nervous system; FDA, US Food and Drug Administration; GIST, gastrointestinal stromal tumor; NCCN, National Comprehensive Cancer Network; NSCLC, non-small cell lung cancer; RCC, renal cell carcinoma. Data modified from oncokb.org1; Chakravarty D, Gao J, Phillips S Kundra R, Zhang H, Wang J. OncoKB: a precision oncology knowledge base. JCO Precis Oncol. Published online May 16, 2017.

Recruitment of signaling intermediaries to the plasma membrane facilitates their interaction with membrane-bound proteins responsible for stimulating a diverse array of downstream pathways (Fig. 2.1). As an example, the lipid kinase phosphatidylinositol 3-kinase (PI3 kinase), described in more detail in a later section, recognizes and binds to a pattern of phosphorylated tyrosine residues present within multiple activated RTKs through the SH2 domain located in its p85 regulatory subunit. Binding of the p85 regulatory subunit in turn results in activation of its kinase activity. Approximately 20 classes of RTKs have been defined based on growth factor specificity. This section will focus on those RTK classes for which specific cancer therapies exist or are in development.

Epidermal Growth Factor Receptor Signaling Historically, the growth factors that stimulate RTKs were first discovered, followed by the structural and functional characterization of the RTKs themselves.10,11 Epidermal growth factor (EGF) was initially purified from mouse submaxillary glands in 1962 by Stanley Cohen and was found to stimulate premature eyelid opening and incisor eruption, phenotypes that suggested a role for EGF in the regulation of cellular proliferation.12 In 1978, the epidermal growth factor receptor (EGFR) was identified as the cell surface binding site for EGF.13 Over the next several years, tyrosine phosphorylation was identified in cells, followed by the discovery that the viral Src oncogene, which induces transformation of cells in vitro, is itself a tyrosine kinase, underscoring the potential importance of tyrosine phosphorylation for oncogenesis.14,15 Once the complete sequence of the EGFR protein was elucidated in the 1980s,16 the amino acid sequence of the receptor cytoplasmic domain was found to be similar to Src, suggesting that EGFR also possessed tyrosine phosphorylation activity. The connection between RTK activation and oncogenesis was further solidified when the amino acid sequence of EGFR was found to be homologous to the avian erythroblastosis virus erbB oncogene, which, when infected into chicken red blood cell precursors, is sufficient to induce erythroleukemia.17,18 The erbB oncogene encodes a TM protein that lacks the extracellular ligand binding domain of EGFR but possesses a cytoplasmic kinase domain that, when expressed in cells, can signal in a growth factor–independent manner. Subsequent studies have since identified within human cancers numerous alterations of EGFR and other RTKs that enhance proliferation without the need for growth factor stimulation. The EGFR class of RTKs comprises four receptor proteins encoded by four genes (in parentheses): EGFR (ERBB1), HER2/Neu (ERBB2), HER3 (ERBB3), and HER4 (ERBB4). EGFR binds to and is activated by a number of ligands, including EGF, transforming growth factor–α (TGF-α), HB-EGF, amphiregulin, betacellulin, epiregulin, and

epigen.19–21 Growth factor binding promotes either homodimerization or heterodimerization with other HER family members, followed by transphosphorylation.22 A ligand for HER2 has not yet been identified; instead, HER2 is activated through heterodimer formation with one of the other three ligand-bound receptors.23 Notably, HER2 is the preferred dimerization partner for EGFR, and EGFR-HER2 heterodimers are more stable than EGFR homodimers, remaining at the cell surface for a longer duration and undergoing endocytosis at a lower rate than EGFR homodimers.24,25 Furthermore, HER2 reduces the dissociation rate of EGF from EGFR, allowing for a more sustained period of EGF-induced signaling.19 The EGFR and HER2 components of the EGFR-HER2 heterodimer are also more likely to be recycled back to the cell surface than EGFR homodimers, which are more readily targeted for degradation.26 In addition, HER2-HER3 heterodimers possess the most potent mitogenic activity among the heterodimer and homodimer HER kinase combinations.27 In contrast to the other HER kinase family members, HER3 does not have intrinsic kinase activity and preferentially forms heterodimers with HER2.28 The ligands for HER3 and HER4 are the neuregulins, including heregulin. A number of tumor types frequently exhibit alterations within the EGFR family of RTKs.21 Sustained activation of these pathways can result in oncogene- or pathway-addicted tumors, and selective HER kinase inhibitors are now a component of the standard treatment of several malignancies. Alterations that affect RTK activity include mutations that result in constitutive activation of the tyrosine kinase; overexpression of the receptor, often due to gene amplification; and elevated levels of RTK ligands that stimulate signaling. EGFR mutations are found in 10% to 25% of non–small cell lung cancers (NSCLCs), with variation in the frequency of such alterations influenced by ethnicity and geographic location; in-frame microdeletions in exon 19 and point mutations in exon 21 (most commonly L858R or L861Q) or exon 18 (G719X) comprise over 80% of these alterations.29–31 In glioblastoma multiform (GBM), EGFR mutations, indels (including the EGFRvIII variant in which exons 2 to 7 of the extracellular domain are deleted, generating a ligand-independent, activated protein), amplification, splice variants, and rearrangements occur in 57% of tumors.32–34 However, because of the heterogeneity of GBM tumors, targeting EGFR is complicated; EGFR alteration is often concurrent with amplification or mutation of another RTK such as PDGFR, MET, or FGFR, or the presence of EGFRvIII on extrachromosomal DNA, or activation of IDH1.35–38 Overexpression of wild-type EGFR as a result of gene amplification has been observed in NSCLC and breast, gastric, colorectal, and head and neck cancers, and less commonly in other tumor types.39–41 Up to 30% of breast cancers display overexpression of HER2, which is an unfavorable prognostic factor, and therapy for these

30 Part I: Science and Clinical Oncology Growth Factor Ligand

oncogene tumor suppressor

Receptor Tyrosine Kinase

P Shc P Grb2

Ras-GDP Sos

NF1

Ras-GTP

Vermurafenib Dabrafenib

PLCε

Raf

P P

RalA/B

RalGDS

MEKK1/NF-κB

TIAM1

Trafficking/ proliferation p85

PIK3CAp110α

P P

Trametinib

MEK

Selumetinib Cobimetinib

CDC42/RAC P P

SCH772984 BVD-523

ERK

DEL-22379

DUSPs

AKT

NF- κB/actin

mTORC1 P p90RSK translation

NF- κB, Myt-1, GSK3, PP-1

P Fos/Jun/etc.

Proliferation/growth Negative feedback

Figure 2.1  •  Canonical Ras/MAPK signaling pathway. Ras proteins cycle between GDP-bound inactive and GTP-bound active states. Ras is often activated in response to ligand-specific binding to its cognate receptor. Ras can also be activated via intracellular cross talk. This schematic depicts the classic Ras/MAPK signal transduction cascade. Growth factor stimulation induces receptor tyrosine kinase (RTK) dimerization and autophosphorylation of tyrosine residues located within the intracellular domain of the receptor. These phosphorylated tyrosine residues serve as docking sites for scaffold proteins that facilitate activation of intracellular signaling cascades. For example, the adaptor protein Grb2, via its SH3 domain, recruits the Ras GEF (guanine-nucleotide exchange factor) SOS. Colocalization of SOS and Ras facilitates substitution of GTP for GDP and thus Ras activation. Active GTP-bound Ras binds and recruits the Raf (A-, B-, and C-Raf ) serine/threonine kinases to the plasma membrane and facilitates their activation. Active Raf in turn phosphorylates and activates MEK, which in turn phosphorylates and activates ERK. ERK phosphorylates substrates in the cytoplasm (p90RSK) and in the nucleus (Jun, Fos, Ets-2, Elk-1, CREB1, AP-1, ATF-2, among others), which regulate cell proliferation and survival. Ras interacts with more than 20 effector proteins, including the p110α subunit of PI3 kinase, RasGDS, PLCε, and TIAM1, which in sum control transcription, translation, vesicular trafficking, cell cycle progression, cytoskeletal changes, metabolic processes, immune inflammatory responses, and survival. Induction of Ras signaling also upregulates negative feedback elements that inhibit the pathway (e.g., DUSPs and SPROUTYs/SPREDs). Several proteins within the Ras signaling cascade are proto-oncogenes (green) and tumor suppressors (red) that are mutated, amplified, or deleted in many cancers. A number of selective inhibitors of Ras effectors have been tested as anticancer therapies. Examples include kinase inhibitors, which selectively target B-Raf and its downstream effectors MEK and ERK (see red boxes).

ERBB2-amplified breast cancers is now distinct from that of breast cancers with normal HER2 expression levels.21,42 ERBB2 amplification is also a driving event in gastric and to a lesser extent in bladder, endometrial, and cervical cancer.43–45 More recently, activating mutations and in-frame insertions/indels in ERBB2 were found to occur in 1% to 2% of all cancer patients, most commonly in patients with bladder cancer.44 Mutations in ERBB2 localize to either the extracellular domain, where they are presumed to promote dimer formation, or the kinase domain.46,47 In addition, activating mutations in ERBB3 have also been identified in bladder, colon, and gastric cancers.48,49 Numerous targeted agents have been developed that selectively inhibit EGFR-induced signaling (see Table 2.1 and Fig. 2.2).21,50 Cetuximab, a chimeric monoclonal antibody that binds to the extracellular domain of EGFR and competitively inhibits ligand binding, thereby preventing receptor activation, is approved for the treatment

of KRAS wild-type colorectal and head and neck cancers.51–56 Panitumumab, another human monoclonal anti-EGFR antibody, is also approved for KRAS wild-type metastatic colorectal cancer.56 The first-generation reversible EGFR tyrosine kinase inhibitors gefitinib and erlotinib, as well as the second-generation irreversible inhibitor afatinib, are FDA approved for the treatment of NSCLC, with greatest efficacy in patients with EGFR mutations or in-frame deletions.57–60 A second site mutation in EGFR (T790M) is a common mechanism of acquired resistance to first generation EGFR inhibitors. Osimertinib (AZD9291), a third-generation EGFR inhibitor, is highly active in patients with NSCLC in which resistance is mediated by the EGFR T790M mutation and is now FDA approved for this indication.61,62 The development of osimertinib and the fourth-generation EGFR inhibitor EAI04563 highlights how studies of acquired resistance can lead to the rational development of more effective kinase inhibitors.

Intracellular Signaling  •  CHAPTER 2 31

XL147 Buparlisib Alpelisib (α-specific) AZD8186 (β-specific) Copanlisib (α/δ-specific) Idelalisib (δ-specific)

AZD5363 Afureser­b

AKT

PIP2/3 P P

PI3Kp110α

PTEN PIP3

PDK1

P

mTORC2

SGK

Bad P NF-κB

GSK3β

P

P Cyclin D1

p85

IRS1

RasGTP CDC42/RAC NF-κB/ac­n

RHEB-GDP TSC1 TSC2

apoptosis

EGFR:Cetuximab EGFR/HER2:Lapa­nib, Trastuzumab

Receptor Tyrosine Kinase

PIP2

P IκB

Growth Factor Ligand

P P

P P

EGFR:gefi­nib, erlo­nib, osimer­nib EGFR/HER2:lapa­nib, afa­nib, nera­nib Other: ima­nib, alec­nib, larotrec­nib, etc

Dual PI3K-mTOR inhibitors: dactolisib, voxtalisib

RHEB-GTP

P

mTOR inhibitors: rapamycin, everolimus, temsirolimus, AZD8055, RapaLink

mTORC1

PRAS40 degrada­on

P 4EBP1

P p70S6K P

Protein transla­on

S6

P FOXO1/4

Arrest/Apoptosis oncogene tumor suppressor

prolifera­on and survival

Figure 2.2  •  PI3K/mTOR signaling pathway. The PI3 kinase family proteins are lipid kinases that transduce signals from receptor tyrosine kinases (RTK)

and G protein–coupled receptors to intracellular cascades that control proliferation, survival, and other cellular phenotypes. As an example, growth factor binding causes receptor dimerization and subsequent phosphorylation of tyrosine residues in the intracellular domain of the receptor. These tyrosine phosphorylation sites serve as docking sites for the p85 regulatory subunit of PI3 kinase and adaptor proteins such as IRS-1 in the case of signaling induced by the insulin/ IGF1 receptors. This results in allosteric activation of the catalytic subunit of PI3 kinase, which converts PIP2 to PIP3. PIP3 recruits PDK1 and AKT to the membrane via their pleckstrin homology (PH) domains. Colocalization of PDK1 and AKT results in phosphorylation (on threonine 308) and activation of AKT. Phosphorylation of AKT on Ser473 by mTORC2 is required for full activation of AKT. Activated AKT phosphorylates several effectors, including GSK3β, Bad, PRAS40, IκB, the FOXO1/4 transcription factors, and TSC2. AKT phosphorylation of TSC2, which is bound to TSC1, inhibits the GTPase function of this complex, thereby allowing activation of Rheb and subsequent activation of mTORC1. In turn, mTORC1 phosphorylates p70S6 kinase (p70S6K) and 4EBP1, an inhibitor of the eIF4E component of the cap-dependent translation initiation complex. p70S6K and mTOR also function to negatively regulate the pathway by initiating the phosphorylation and inhibition of IRS-1. PI3 kinases can also signal to other effectors such as Rac/CDC42 and the serum-glucocorticoid kinase (SGK) family to promote cellular survival, motility, and cytoskeletal rearrangement. Many components of the PI3 kinase signaling pathway are mutationally altered in cancer (oncogenes in green, tumor suppressors in red). A variety of compounds have been developed that selectively inhibit PI3 kinase signaling components. US Food and Drug Administration (FDA)–approved drugs and novel inhibitors in clinical testing that target RTKs, PI3 kinase, mTOR kinase, and AKT are highlighted in the red boxes.

ERBB2 amplification strongly correlates with HER2 protein overexpression, and the presence of either marker predicts for trastuzumab response in certain cancers.64 Trastuzumab, a humanized antibody that binds to the extracellular domain of HER2, has been FDA approved for the treatment of breast65–71 and esophagogastric72 cancers displaying HER2 overexpression. In patients with breast and gastric cancers, trastuzumab has modest activity when administered as singleagent therapy and is most commonly used in combination with chemotherapy.72–74 The combination of docetaxel, trastuzumab, and pertuzumab,75 an antibody that binds to a different HER2 epitope (the dimerization domain) than trastuzumab and results in impaired dimer formation, is also approved for breast cancer.76–78

Although the introduction of trastuzumab has resulted in a significant improvement in the survival of patients with HER2overexpressing breast cancers, drug resistance remains a major clinical problem. Potential resistance mechanisms include concomitant overexpression of other HER kinase family members and/or ligands, PTEN loss, and the expression of a truncated HER2 protein lacking the extracellular antibody binding site.79 Additional HER2-directed agents include the tyrosine kinase inhibitors lapatinib and neratinib (see Table 2.1). Lapatinib is FDA approved for use in combination with capecitabine in patients with HER2-overexpressing advanced or metastatic breast cancer that has progressed on prior therapy with trastuzumab and certain classes of chemotherapy.80 The combination

32 Part I: Science and Clinical Oncology

of lapatinib and trastuzumab is also FDA approved in HER2-amplified breast cancer.81–83 Lapatinib also received accelerated approval for use in combination with the aromatase inhibitor letrozole.84 Clinical efficacy has been reported with lapatinib in HER2-mutant NSCLC.85,86 The irreversible pan-HER kinase inhibitor neratinib has shown promising clinical activity in patients with ERBB2-amplified and ERBB2-mutant breast tumors, but also other cancer types.46,87–93

alectinib and ceritinib that are either more potent or more selective for ALK have significant clinical activity in patients with acquired resistance to crizotinib and are FDA approved for this indication.116,117 In addition, brigatinib, a dual inhibitor of ALK and EGFR, was granted accelerated FDA approval in 2017 for patients with metastatic NSCLC and in patients with ALK alterations who progressed on crizotinib.118

Insulin, Insulin-Like Growth Factor-1 Receptor Signaling, ALK, and ROS1

Platelet-Derived Growth Factor Receptor, KIT, and FLT-3 Signaling

The insulin and insulin-like growth factor 1 (IGF1) receptor family is dysregulated in multiple malignancies.94 The insulin receptor exists as two isoforms encoded by splice variants of the same gene.95 Each isoform can dimerize with the other (forming hybrid dimers) or with itself.96 The IGF1 receptor (IGF1R) can dimerize with either of the insulin receptor isoforms or with itself, resulting in six different dimer combinations.96 The insulin receptor is stimulated by insulin or insulin-like growth factor 2 (IGF2), whereas IGF1R can be activated by either IGF1 or IGF2. Both of these latter ligands can stimulate IGF1R in an autocrine fashion or can be elaborated from distant sites.97,98 Circulating IGF binding proteins have a similar affinity for IGF1 and 2 as IGF1R does and therefore compete for binding to both ligands, thus titrating the amount of free ligand available for IGF1R stimulation.99 IGF binding protein proteases provide an additional mechanism for controlling ligand levels by increasing the half-life of free ligand available for receptor binding.100 After ligand binding, IGF1R dimerizes and undergoes transphosphorylation, leading to activation of downstream signaling pathways, including both the Ras-Raf-MAPK and the PI3 kinase-AKT-mTOR cascades (see individual sections later in the chapter; see also Fig. 2.1).101 Specifically, insulin receptor substrate 1 (IRS1) binds to a phosphotyrosine motif on IGF1R via its SH2 domains and is phosphorylated by IGF1R.102 It subsequently recruits PI3 kinase to the plasma membrane, which converts phosphatidylinositol-4,5-bisphosphate (PIP2) to phosphatidylinositol-3,4,5-triphosphate (PIP3), subsequently resulting in AKT and mTOR pathway activation. Activating mutations of IGF1R do not appear to be common in human cancer. However, amplification of the IGF1R gene locus has been identified in some colon, pancreas, and lung cancers. Sarcomas often exhibit either increased expression of the IGF1 and IGF2 ligands or decreased IGFBP-3 expression (Ewing sarcoma), which results in increased IGF1 levels in the tumor microenvironment.103 Gastrointestinal stromal tumors (GISTs) lacking c-KIT and platelet-derived growth factor receptor (PDGFR) mutations also commonly harbor IGF1R amplification.104 AMG479, a monoclonal human antibody targeting IGF1R, has shown promising antitumor activity in patients with Ewing sarcoma.103 Activating kinase domain point mutations and gene rearrangements of the insulin receptor family members anaplastic lymphoma kinase (ALK) and ROS1 play driving roles in many cancers, most notably lymphomas, neuroblastoma, NSCLC, and thyroid cancer.105–108 Chromosomal translocations involving ALK and at least 22 5′ fusion partners have been identified,108 which dictate spatial and temporal expression of the ALK fusions, and likely their function and tumorigenic potential.105 In NSCLC, the EML4 gene is the preferred translocation partner, resulting in the expression of an EML4-ALK fusion protein in 4% to 6% of patients.109,110 Notably, EML4-ALK fusions are found in a mutually exclusive pattern with EGFR kinase domain mutations, suggesting that they have overlapping downstream effects. ROS1 gene rearrangements are also found in a minority of NSCLC patients with binding partners including SLC34A2 and CD74.106,111,112 Crizotinib, an inhibitor of the ALK, ROS1, and MET tyrosine kinases (see Table 2.1), is now FDA approved for use in NSCLC patients with ALK or ROS1 fusions (see Table 2.1),113–115 although acquired resistance mutations in ALK (of note, C1156Y and the gatekeeper mutation L1196M) commonly develop. Newer ALK inhibitors including

Platelet-derived growth factor (PDGF) is the ligand for PDGFRs, which stimulate the proliferation and migration of mesenchymal cells, such as oligodendrocyte precursors, vascular smooth muscle cells, and pericytes during embryonic development.119 PDGF signaling is also implicated in organ development, including lung and intestinal epithelial folding and glomerular capillary tuft formation. Furthermore, PDGFs promote angiogenesis, wound healing, and erythropoiesis.120 Aberrations in the PDGFR pathway result in uncontrolled proliferation and enhanced angiogenesis. Four isoforms of PDGF have been identified: PDGFA, PDGFB, PDGFC, and PDGFD.121 These isoforms are activated by proteolytic cleavage and assemble into five homodimeric or heterodimeric combinations that bind to and stimulate either PDGFRα or PDGFRβ. PDGFRα homodimers inhibit chemotaxis, whereas PDGFRβ homodimers and α/β heterodimers stimulate chemotaxis within fibroblasts and smooth muscle cells.122 Angiogenic endothelial cells recruit PDGFRβ-positive pericytes to cover blood channels and aid in their maturation and stabilization through secretion of PDGFβ.123 Following dimerization and transphosphorylation, PDGFRs activate signal transduction pathways through recruitment of adaptor proteins containing SH2 domains, most notably the Grb2 protein, which in turn binds the guanine nucleotide exchange factor (GEF) Sos, which subsequently activates Ras.124,125 In addition, phosphorylated tyrosine residues serve as docking sites for SH2 domain–containing kinases, including PI3 kinase, phospholipase-Cγ, and Src, as well as the tyrosine phosphatase SHP2 and the STAT transcription factor family.125 Alterations in PDGFR signaling in cancer include excess autocrine secretion of PDGF (glioblastoma, sarcomas), gain-of-function mutations that cause constitutive tyrosine kinase activation (GISTs),126 translocation of either the PDGF or PDGFR gene (dermatofibrosarcoma protruberans, chronic myelomonocytic leukemia, hypereosinophilic syndrome),127–129 and PDGFR gene amplification (glioblastoma).130 PDGFRα mutations are found in approximately 10% of KIT wild-type GISTs and are sensitive to imatinib, a tyrosine kinase inhibitor of KIT, BCR-ABL, and PDGFRs, which is standard of care in this setting (see Table 2.1).122,126,131 The D842V mutation comprises approximately two-thirds of PDGFRα activating mutations, and confers resistance to imatinib. Notably, the second-generation inhibitor dasatinib is effective in preclinical models of imatinib-resistant GIST.126,132,133 Dermatofibrosarcoma protuberans is a rare, low-grade cutaneous sarcoma that harbors a chromosome 17;22 translocation that fuses portions of the COL1A1 (collagen 1A1) gene and PDGFB, resulting in overexpression of PDGF-β and subsequent stimulation of PDGFR signaling.122 Twenty other fusions partners have been identified in PDGFβ rearrangements, including ETV6 and EBF1. Imatinib has shown significant benefit in patients with recurrent or metastatic dermatofibrosarcoma protuberans, myelodysplasia, and myeloproliferative neoplasms and is FDA approved for these indications.134–138 The KIT gene is a member of the type III RTK family, which includes PDGFR and FLT3 (see later).139–141 It was first identified as the human homologue of the viral oncogene v-Kit responsible for the Hardy-Zuckerman IV feline sarcoma virus.142 Mutation of KIT or its ligand, stem cell factor (SCF),143–146 in mice induces coat color abnormalities (“white spotting”), anemia, and mast cell deficiencies, suggesting that it plays a role in hematopoiesis and melanogenesis.147,148 Furthermore, the KIT protein was discovered as a cell surface receptor

Intracellular Signaling  •  CHAPTER 2 33

in acute myeloid leukemia (AML).149 KIT expression is mainly restricted to mast cells, hematopoietic cells, germ cells, melanocytes, and the interstitial cells of Cajal (ICCs) in the gut.150,151 SCF and KIT integrate signals that lead to mitogen-activated protein kinase (MAPK), PI3K, and SRC pathway activation, and mediate critical survival and proliferation cues to distinct hematopoietic lineages, including the bone marrow and progenitor cells. Hot-spot mutations in exons 9 and 11 of KIT have been identified in several tumor types, including GIST, melanomas, and germ cell tumors.146,152–156 In GIST, 85% of tumors have activating KIT mutations that drive the transformation of precursors of ICCs.153 Imatinib157–161 and sunitinib158,162,163 inhibit KIT and PDGFR, among other kinases, and are FDA approved for use in patients with KIT mutant GIST (see Table 2.1). Regorafenib is approved for patients with imatinib- or sunitinib-refractory GIST.164 Imatinib has also been shown to induce tumor regression in patients with KIT-mutant or KIT-amplified melanoma.165,166 Second site mutations in KIT, typically in exon 17, are a mechanism of acquired resistance to imatinib therapy in patients with GIST.167 Novel agents that retain activity in the setting of an exon 17 KIT mutation are now in clinical testing (clinical trial NCT02401815).168 New areas of investigation into mutant KIT therapies in GIST include blocking mutant KIT subcellular localization to the Golgi150 and combination of FGFR3 and KIT inhibitors to quench pathway cross talk.169 The FMS-like tyrosine kinase 3 receptor (FLT3), a third member of the RTK class that includes PDGFR and KIT, is involved in the development of normal hematopoietic cells. It contains an extracellular region composed of five immunoglobulin (Ig) domains, TM and juxtamembrane domains, and two cytoplasmic tyrosine kinase domains that transmit proliferative signals through the RAS/MAPK, PI3K/AKT, and STAT5 pathways.170 Two main FLT3 alterations are common in hematopoietic malignancies, namely in approximately 30% of AMLs.171 First, internal tandem duplication (ITD) within exons 14 and 15 of the FLT3 gene (FLT-ITD) interferes with the negative regulatory function of the juxtamembrane segment.172 This duplication results in ligand-independent activation of FLT3 and is associated with a poor prognosis in patients with AML. Second, kinase domain mutations at or near D835 in the activation loop of FLT3 disrupt autoinhibitory interactions and render the kinase open and active.173 The clinical activity of FLT3 inhibitors has been modest to date, although responses appear to be more common in patients with FLT3/ITD AML.171 TAK-659, a reversible dual Syk/Flt inhibitor, showed early clinical activity in numerous lymphoma subtypes and AML.174,175 Sorafenib has been shown in preclinical in vitro studies, mouse models, and in a phase I study of AML patients to reduce leukemia burden and block signaling selectively in FLT3-ITD versus FLT-wt settings.176 Interesting to note, resistance to FLT3 inhibition in such patients is associated with selection for secondary mutations within the tyrosine kinase domain of FLT3, suggesting a central role of FLT3 in AML pathogenesis.171

Fibroblast Growth Factor Receptor Signaling Fibroblast growth factor receptors (highly conserved FGFR1, FGFR2, FGFR3, and FGFR4; and FGFRL1/FGFR5, which lacks a kinase domain) comprise a family of RTKs that regulate cell proliferation, differentiation, and migration as well as selective apoptosis during embryogenesis. The FGFRs are composed of an extracellular ligandbinding domain, a hydrophobic TM region, and an intracellular tyrosine kinase domain.177 The extracellular domain is organized into three Ig domains; differential splicing of the second half of the third Ig domain dictates tissue-specific expression of the receptor. Fibroblast growth factors (FGFs) are protein ligands that bind to the extracellular domain of the FGFRs in combination with specific heparan sulfate glycosaminoglycans inducing FGFR dimerization and transphosphorylation of intracellular tyrosine residues. Eighteen FGFs have been identified, and specificity for FGFRs is based on numerous

factors, including tissue-specific FGF ligand and receptor expression, the presence of cell surface molecules that facilitate the interaction between individual FGF ligands and receptors, and the differential binding capability of the ligands themselves for specific FGFRs.178 Subsequent stimulation of the tyrosine kinase domain leads to phosphorylation and activation of multiple downstream signaling proteins in the same manner as described earlier for other RTKs. Unique to the FGFR signaling complex is FGFR substrate 2 (FRS2), an adaptor protein that binds to specific phosphotyrosines on the intracellular domain of active FGFR dimers.179 FRS2 is itself phosphorylated by FGFRs and serves as a docking site for the Grb2-Sos adaptor complex, which activates the Ras/Raf/MAPK pathway. Phosphorylated FRS2 also recruits Grb2-associated binding protein 1 (GAB1), which activates PI3 kinase. In addition, phospholipase-Cγ binds to phosphorylated FGFR dimers via an SH2 domain, leading to its activation and the cleavage of phosphatidylinositol 4,5-bisphosphate (PIP2) to form inositol 1,4,5-triphosphate (IP3) and diacylglycerol (DAG). Germline mutations of the FGFR genes are the basis of a spectrum of skeletal developmental disorders that are thought to derive from premature differentiation and growth restriction of chondrocytes resulting from dysregulated FGFR pathway activation.180,181 FGFR signaling is dysregulated in cancer by multiple mechanisms including mutational or translocation-induced activation of FGFRs, gene amplification of receptors, and abnormal ligand regulation.182 For example, autocrine and paracrine FGF ligand secretion with resultant pathway activation has been reported to occur in a subset of melanomas and prostate cancers, respectively.183,184 FGFR1 amplification occurs in approximately 17% of squamous cell lung cancers and 6% of small cell lung cancers (SCLCs).185 Approximately 10% of diffuse-type, aggressive gastric cancers display FGFR2 gene amplification, and cell lines with this amplification show ligand-independent pathway activation and sensitivity to selective FGFR inhibitors.186,187 Whereas FGFR1 mutations are rather rare, FGFR2 mutations are found in approximately 10% of endometrial cancers.188,189 FGFR3 mutations occur in up to 75% of non–muscle invasive bladder cancers and 15% of patients with advanced urothelial tumors.44,179,189,190 Activating mutations within FGFR3 result in constitutive receptor dimerization and subsequent signaling. Unlike EGFR-activating mutations, which predominantly affect the tyrosine kinase domain of the receptor, FGFR3 mutations are commonly located within the extracellular domain (R248, S249) and TM segment (G370, Y373) and promote ligand-independent receptor dimerization through formation of an aberrant disulfide bridge between two receptor monomers.191 Chromosomal rearrangement of FGFRs have been identified using next-generation sequencing. Up to 15% of multiple myelomas harbor an intergenic 4;14 translocation between the FGFR3 gene and the Ig heavy chain locus, which places FGFR3 expression under the highly active heavy chain promoter.192,193 More recently, translocations involving FGFR2 have been reported in cholangiocarcinoma and more rarely in other cancers, whereas FGFR3 fusions are most common in glioblastoma and bladder cancers but also are found rarely in other solid tumor types.194,195 The FGFR3-TACC3 constitutively active fusion protein has been characterized to induce aneuploidy by disrupting proper chromosomal segregation.196 Multiple FGFR inhibitors are currently being tested in early-phase clinical trials, but the majority of these compounds are multitargeted tyrosine kinase inhibitors, many of which also potently inhibit members of the VEGFR and PDGFR families. The close structural similarity between these RTKs has made development of FGFR-selective inhibitors challenging, although several such drugs are now in early clinical testing, such as BGJ398, AZD4547, JNJ-42756493, and Debio1347 (see Table 2.1).179,182,197–200 On-target hyperphosphatemia resulting from FGFR1 inhibition is a primary toxicity with this class of agents, suggesting that the development of isoform-selective FGFR inhibitors may be a more rational approach for patients whose tumors are driven by mutations or translocations in FGFR2 and FGFR3. FGFRs are located on the cell surface and thus may also be susceptible to monoclonal antibody

34 Part I: Science and Clinical Oncology

mediated inhibition similar to trastuzumab-mediated inhibition of HER2. FGFR ligand traps are also in development.182

RET Signaling The RET (Rearranged During Transfection) gene encodes three alternatively spliced isoforms (RET9, RET43, and RET51) which are TM RTKs that contain four cadherin-like extracellular repeats important for dimerization, a cysteine-rich juxtamembrane region critical for ligand binding and conformation, and an intracellular kinase domain.201 RET9 and RET51 are highly conserved in all vertebrates and play important roles in the normal development and maintenance of many tissues, including the kidney, spermatogonial stem cells, and the enteric nervous system.201–203 RET is expressed predominantly on the surface of neural crest tissues, and glial-derived neurotrophic factors (GDNFs) such as neurturin, artemin, and persephin serve as ligands for RET. GDNFs initially bind to their cognate coreceptors, the GDNF receptors (GFPα1 to GFPα4), on the cell surface, which recruits RET into lipid raft membrane domains and causes conformational changes via the cadherin-like moieties, dimer formation, and then subsequent transphosphorylation of tyrosine residues and kinase activation.204 Y1062 is the common docking site for all three RET isoforms and serves to recruit many adapters including SHC1, FRS2, IRS1/2, DOK, and JNK.201 Phosphorylation of Y752 and Y928 binds STAT3, whereas other phosphorylated residues are recognized by Src, resulting in activation of focal adhesion kinase (FAK), which promotes cell migration and metastatic spread. In addition, the MAP kinase, PI3 kinase/AKT, and phospholipase-Cγ pathways can be activated by RET to promote cellular proliferation and survival.204 Germline loss-of-function RET mutations occur in Hirschsprung and CAKUT (congenital anomalies of the kidney and urinary tract) disease, which causes abnormalities of the developing gut and kidneys, respectively. Conversely, germline activating RET mutations are the basis for the multiple endocrine neoplasia type 2 (MEN2) syndromes. Patients with MEN2 develop familial medullary thyroid carcinomas and other cancers.205 MEN2A is mainly driven by mutations in six cysteine resides in the RET extracellular domain (C609, C611, C618, C620, C630, and C634), whereas the kinase domain mutations M918T or A883F are associated with MEN2B. Sporadic medullary thyroid carcinomas are much more common, and up to 60% of such tumors harbor somatic mutations in RET, notably G691S, which are thought to be a driver alteration in this disease.206 Furthermore, RET gene rearrangements with numerous fusion partners, including CCDC6 and NCOA4, termed RET-PTC1 and RET-PTC3, respectively, occur in 20% to 40% of papillary thyroid carcinomas (PTCs) and often occur as a consequence of high doses of radiation.207 RET inhibitors have shown significant antitumor activity in patients with medullary thyroid cancer. Vandetanib, an oral inhibitor of RET, EGFR, and VEGFR, is FDA approved for the treatment of patients with advanced medullary thyroid cancer.208 A randomized, placebocontrolled phase III study of cabozantinib, an oral, multitargeted TKI that inhibits RET, VEGFR2, and MET, was also recently conducted in patients with unresectable, locally advanced, or metastatic medullary thyroid carcinoma.209 This trial documented a statistically significant improvement in median progression-free survival with cabozantinib as compared with placebo (11.2 months versus 4.2 months in placebo arm, P < .0001). More recently, cabozantinib was FDA approved for the treatment of renal cell carcinomas that progressed on antiangiogenic therapy, although the activity of cabozantinib in this context may not be attributable to its inhibition of RET. Several RET inhibitors have, however, shown promising clinical activity in patients with NSCLC treated with RET fusions (see Table 2.1).210

Vascular Endothelial Growth Factor Signaling Six vascular endothelial growth factor (VEGF) ligands have been identified: VEGF-A, VEGF-B, VEGF-C, VEGF-D, and placental

growth factors 1 and 2.211 VEGF-A has four isoforms produced by alternative gene splicing, with the 165–amino acid length isoform playing a central role in tumor angiogenesis.212 Specifically, VEGF-A enhances vascular permeability and stimulates endothelial cell proliferation, resulting in new blood vessel formation. Vascular endothelial growth factor receptors (VEGFR1 to VEGFR3) are RTKs that possess a modular structure consisting of an extracellular domain with seven Ig-like regions, a TM domain, and an intracellular tyrosine kinase domain.211 VEGF-A, VEGF-B, and placental growth factor all bind VEGFR1 (also known as FLT1), but the exact role of VEGFR1 in tumor angiogenesis has yet to be fully elucidated. In some settings it may act as a decoy receptor that prevents ligandmediated stimulation of VEGFR2 (also known as FLK1/KDR).213 VEGFR2 has been implicated in the development of vasculature during development and is considered the primary receptor through which VEGF exerts its angiogenic effects in endothelial cells.213,214 Binding of ligand to VEGFR2 results in receptor dimerization and transphosphorylation followed by activation of multiple mitogenic signal transduction cascades.215,216 More recently, VEGF has been implicated in many angiogenesis-independent roles including regulation of immune cells in the tumor microenvironment, fibroblasts in the tumor stroma, and cancer stem cells.217 VEGF can also bind and signal through a class of TM glycoprotein coreceptors called neuropilins (NRP1 and NRP2), which are found on tumor cells and can signal along many oncogenic axes including Hedgehog and JNK.217 The complex network of cross talk among VEGFs, VEGFRs, and canonic oncogenic pathways makes VEGF and VEGFR critical but elusive targets in cancer therapy. Targeted therapies that inhibit VEGF signaling include antibodies that bind circulating ligand and RTK inhibitors. The humanized monoclonal antibody bevacizumab binds to free VEGF, thereby preventing its association with VEGFRs. This antibody has been FDA approved for use in combination with chemotherapy for patients with several cancers, including metastatic colorectal218 and nonsquamous NSCLCs.211,219 Bevacizumab also has activity in patients with glioblastoma220 and metastatic renal cell carcinoma, where it is often used in combination with interferon-α (IFN-α).221 In addition, ramucirumab is a VEGFR2-directed antibody that has received FDA approval with or without chemotherapy in several cancers.222 Sorafenib, sunitinib, pazopanib, and axitinib are multitargeted tyrosine kinase inhibitors with nanomolar potency for VEGFR2. Sunitinib is used in the treatment of patients with metastatic renal cell carcinoma, GISTs, and pancreatic neuroendocrine tumors.223 Sorafenib has been approved for the treatment of liver and renal cell cancers.224,225 Although these agents inhibit multiple kinases, their antitumor effects have been attributed primarily to their antiangiogenic activity. More recently, the tyrosine kinase inhibitor pazopanib was approved for the initial treatment of metastatic renal cell carcinoma and in cytokine-pretreated patients,226 and axitinib227 was approved in the second-line setting following failure of prior systemic therapy. Lenvatinib, a multitargeted RTK inhibitor that inhibits VEGFR1, VEGFR2, and VEGFR3, has received recent FDA approval for both thyroid cancer (as monotherapy)228 and renal cell carcinoma in combination with everolimus.229 Despite widespread activity in preclinical models, antiangiogenic therapies have shown disappointing activity in several tumor types. A number of resistance mechanisms have been hypothesized to explain the lack of broader clinical activity, including the activation of redundant signaling pathways that promote angiogenesis; the recruitment by tumors of bone marrow–derived endothelial progenitor cells; increased pericyte density around existing blood vessels, which enhances vascular growth and survival; and the ability of tumor cells to invade surrounding stroma to co-opt additional blood supply.230 A better understanding of these resistance mechanisms may lead to the development of more effective antiangiogenic therapies in the future.

Intracellular Signaling  •  CHAPTER 2 35

Hepatocyte Growth Factor Receptor Signaling The hepatocyte growth factor receptor (HGFR or MET) is encoded by the MET gene.231,232 Both MET and its ligand hepatocyte growth factor/scatter factor (HGF/SF) are expressed as immature precursors that require proteolytic cleavage.233 The MET extracellular domain consists of an alpha subunit connected by a disulfide bridge to a TM beta subunit, and contains a Sema domain, a PSI domain, and four IPT domains.234,235 The intracellular portion of the receptor contains a juxtamembrane region that harbors a serine residue (Ser 975) that inhibits RTK activity on phosphorylation, as well as a tyrosine kinase domain with Y1234 and Y1235 acting as key sites of autophosphorylation required for activation.235 A tyrosine residue at position 1003, proximal to the tyrosine kinase domain, serves as an interaction site for the ubiquitin ligase CBL, which marks the receptor for endocytosis and degradation.236,237 C-terminal residues Y1349 and Y1356 represent docking sites for adaptor proteins.238 On binding of HGF/SF to the extracellular portion of MET, receptor dimerization occurs, followed by transphosphorylation. A number of adaptor proteins then bind to phosphorylated tyrosine residues, including Grb2 and GAB1, phospholipase-C (PLC), and SRC, which promotes the activation of the MAP kinase and PI3 kinase/AKT signaling pathways.239,240 MET can also activate RAC1/CDC42 and p21-activated kinase (PAK1), both of which regulate cytoskeletal proteins and integrin expression and activation, and thus cell migration.238,239 MET also plays an important role in driving epithelial-to-mesenchymal transition (EMT) cell migration during embryo development, and organ regeneration.238,241 Dysregulation of MET signaling can occur through multiple mechanisms, including activating point mutations (often in the kinase domain; prevalent in lung cancer), exon skipping events, receptor overexpression, and upregulation of HGF, which can activate MET in an autocrine and/or paracrine manner.238,240,242–246 Germline mutations of MET are found in patients with hereditary papillary renal cell carcinomas, and MET overexpression is observed in a significant proportion of sporadic papillary cancers as well as collecting duct carcinomas.247 Multiple other malignancies exhibit aberrations in MET signaling, including lung, breast, pancreatic, colon, and gastric cancers. Amplification of MET is associated with a worse prognosis in lung and gastric cancers, whereas expression of MET or HGF is an unfavorable prognostic biomarker in liver, kidney, colorectal, and gastric cancers.248 Recurrent somatic splice site alterations involving MET exon 14 (METex14) have been identified in lung cancer. These mutations result in exon skipping, loss of the juxtamembrane CBL E3-ubiquitin ligase-binding site, diminished receptor turnover, and ultimately, MET activation.243,249,250 These exon 14 MET mutations are mutually exclusive with activating mutations in EGFR and KRAS as well as ALK, ROS1, and RET fusions, and treatment of patients with exon 14 MET splice variants with a MET kinase inhibitor is now considered to be a standard treatment option (see Table 2.1).210 Several RTKs have also been shown to activate MET, including EGFR, HER2, and IGF1R. For example, EGFR activation can stimulate MET signaling, and resistance to EGFR inhibitors in some lung cancers has been shown to stem from coactivation of MET in the setting of gene amplification.251 Inhibitors of MET signaling have been in development for a number of years. Therapeutic strategies for targeting MET activation in cancer patients include antibodies that target the extracellular domain of the receptor, antibodies that bind to and thus sequester circulating HGF, and small-molecule tyrosine kinase inhibitors that selectively target MET or are multi-kinase MET inhibitors.248 Durable responses to crizotinib and cabozantinib, multikinase MET inhibitors, have been reported in patients with NSCLC with MET splice mutants or MET amplification (see Table 2.1).252,253 Cabozantinib is also now standard of care in MET-amplified renal cell carcinoma.254,255 Combination therapies to combat MET reactivation after EGFR kinase inhibitor therapy suggest an improvement in progression-free survival.256

Tropomyosin Receptor Kinases/Neurotrophic Tyrosine Kinase First cloned as an oncogenic fusion partner of the tropomyosin receptor kinases and subsequently characterized for their role in neural differentiation and survival in the peripheral and central nervous systems, the TRKA/B/C family of RTKs, encoded by the neurotrophic tyrosine kinase (NTRK) genes 1 to 3 (NTRK1/2/3), respectively, integrate ligand stimulation from nerve growth factor, brain-derived neurotrophic factor, neurotrophins 3 to 6 with downstream activation of PI3K, phospholipase-C (PLC)-gamma, and MAPK signaling.257–261 Chromosomal rearrangements involving the tropomyosin receptor kinases (TrkA/NTRK1, TrkB/NTRK2, and TrkC/NTRK3) were recently shown to occur at high frequency in several rare cancer types including mammary secretory carcinoma of the breast and congenital-infantile fibrosarcoma.262–264 NTRK fusions also occur at low frequency in a broad range of more common adult solid tumors.260,265 These NTRK fusions induce ligand-independent constitutive kinase activity, resulting in upregulation of canonic downstream signaling pathways involved in growth and survival. TPM3, LMNA, MPRIP, TRIM24, ETV6, and PPL, among others, have been identified as fusions partners with the NTRK genes.266,267 Dramatic and durable clinical responses have recently been reported in patients with NTRK fusions, with first- and secondgeneration TRK inhibitors such as larotrectinib (LOXO-101), entrectinib, and LOXO-195 (see Table 2.1).268–273 Notably, clinical activity was observed in both adult and pediatric patients and was independent of site of tumor origin.264

G PROTEIN–COUPLED RECEPTOR SIGNALING G protein–coupled receptors (GPCRs) are seven TM domain–containing proteins that transduce ligand-specific signals across the plasma membrane to mediate numerous physiologic processes including sensory perception, immunologic responses, neurotransmission, weight regulation, and cardiovascular activity.274 GPCRs also regulate basic cellular functions including growth, motility, differentiation, and gene transcription. The GPCR family comprises more than 800 receptors, which are the targets of over 30% of all FDA-approved drugs, although few to date have found a role as anticancer therapies.275,276 Given that GPCRs activate many of the signaling cascades that are deregulated in human cancer, it is not surprising that studies have implicated GPCRs in cancer initiation and progression.277–279 GPCRs can be categorized into five or six families, depending on the nomenclature used.280 In a more recent phylogenetic classification, the five major families are represented by the acronym GRAFS: Glutamate, Rhodopsin, Adhesion, Frizzled/Taste2, and Secretin.281 The Rhodopsin family of receptors (also referred to as class A GPCRs) is the largest class, with more than 670 members. Crystallization of the bovine rhodopsin receptor in 2000 provided the first high-resolution insight into the structure of GPCRs.282 In general, Rhodopsin family receptors have short N-termini. Included in this family are the α group (histamine, dopamine, serotonin, adrenoceptors, and muscarinic, prostanoid, and cannabinoid receptors), the β group (endothelin, gonadotropin-releasing hormone, and neuropeptide Y), the γ group (opioid, somatostatin, and angiotensin), and the δ group (P2RYs, glycoprotein-binding FSHR/TSHR/LHCGR, PARs, and olfactory receptors).283 The 15 Secretin receptors (class B) all have conserved cysteines in the first and second extracellular loop, and most have three cysteine bridges in the N-termini. This class includes the calcitonin-like, corticotrophin-releasing hormone, glucagon-like, gastric inhibitory polypeptide, growth hormone–releasing hormone, adenylate cyclase– activating polypeptide, parathyroid hormone, secretin, and vasoactive intestinal peptide receptors.284 The Adhesion family (also included in class B, according to another classification system) has distinctly long N-termini, and only 3 of 33 receptors in the family have known ligands (epidermal growth factor-like module containing mucin-like

36 Part I: Science and Clinical Oncology

receptors EMR2, EMR, EMR4).285 The Glutamate receptor family (class C) binds ligands in their N-termini, which have a complex two-domain folded structure bridged by disulfide bonds. Common receptors in this family of 22 include the glutamate, GABAB, calciumsensing, sweet and umami taste (TAS1R1–TAS1R3), and GPCR6 receptors.286,287 The Frizzled receptors (FZD1–FZD10 and SMO; see later for in depth discussion of signaling) bind Wnt ligands in the extracellular domain region containing nine conserved cysteine residues.288 The Taste2 receptors (25 members of T2R, bitter taste) have varying sequence homologies that likely allow the sensing of thousands of distinct bitter tastes.289,290 Overall, GPCRs share the seven–TM domain structure, but have different regions of conservation. All of the families include orphan receptors, which are related by sequence and structure but have no identified ligand to date. All GPCRs have seven α-helical domains that weave through the plasma membrane and are interconnected by flexible extracellular and intracellular segments, thus engendering the synonyms serpentine or heptahelical receptors. The specificity of biologic response initiated by each GPCR depends on (1) ligand recognition; (2) the distinct structure of each receptor class and subclass; and (3) ligand-directed binding of specific cytosolic enzymes and adapters that initiate a plethora of intracellular signaling cascades (general and cancer-specific examples are discussed further later). The GPCR Network was created in 2010 to tackle the challenge of delineating the structure and function of this diverse class of receptors.291 Operationally, ligand binding on the GPCR extracellular surface induces a conformational change in the receptor, mainly via TM helices TM3, TM5, and TM6, which creates a deep pocket in the intracellular face of the receptor.292–294 This cleft enables binding and activation of heterotrimeric G proteins, which consist of an inactive, GDP-bound Gα subunit and a Gβγ-subunit dimer, which act as a molecular switch.295,296 Activated GPCRs promote GDP for GTP exchange on the Gα nucleotide binding site.297 This GTP-bound Gα dissociates from Gβγ and the receptor, and then both activated subunits go on to initiate signaling cascades. There are four members of the Gα family, Gαs, Gαi/o, Gαq/11, and Gα12/13, which can be further subtyped and can each stimulate several downstream effectors. Moreover, each GPCR can couple to multiple Gα family members, thus generating a complex pattern of intracellular signaling. Classic GPCR activation of Gαs stimulates adenylyl cyclase, which generates the second messenger 3′-5′-cyclic adenosine monophosphate (cAMP).298,299 cAMP activates multiple downstream effectors including cAMP-gated ion channels; Epac, a GEF for Rap1/2 (which functions in cell adhesion and junction formation); and protein kinase A (PKA).274,295,300–303 cAMP binds to PKA regulatory subunits, releasing catalytic subunits and triggering the activation of cytosolic and nuclear substrates, including the transcription factor CREB (cAMP response element binding protein), which can induce proliferation and differentiation, among other phenotypes, depending on cell origin.304–306 Gαs also activates the Src tyrosine kinase and the GTPase activity of tubulin.295,300 GPCR-coupled Gαi/o typically works in opposition to Gαs by inhibiting adenylyl cyclase and decreasing cAMP levels.274,307 In addition, some Gαi/o isoforms can signal to K+ and Ca+ channels, increase cGMP phosphodiesterases, interact with Rap1GAP1, and cross talk to the MAP kinase pathway (as described later in the chapter).274 Members of the Gαq/11 family activate phospholipase-Cβ, which catalyzes the hydrolysis of PIP2 to yield IP3 and DAG.308 IP3 mobilizes calcium from intracellular stores, whereas DAG activates some isoforms of protein kinase C (PKC).309,310 Both Gαq/11 and Gα12/13 activate a variety of RhoGEFs (p115-RhoGEF, PDZ-RhoGEF, LARG, Lbc, AKAP-Lbc) and thus regulate Rho activity (mainly RhoA) and its contribution to actin stress fiber formation, cell shape and polarity, cell adhesion and migration, gene transcription, and cell cycle progression.311 In addition, Gα12/13 activates the Na+/H− exchanger, inducible nitric oxide synthase, phospholipase D, E-cadherin, radixin, and protein phosphatase 5. Gβγ signaling is equally complex, as the active dimer

stimulates G protein–regulated inwardly rectifying K+ channels adenylyl cyclase (types II, IV, VII), PLCβ, PI3Kγ, Src, and GPCR kinases (GRKs; see later). It inhibits adenylyl cyclase (type I), some Ca+ channels, and calmodulin; stabilizes Gα in the GDP-bound, inactive state; and helps specify coupling to the proper Gα member.274,295,296,300 Gα signaling is switched off by a family of GTPase activating proteins called Regulators of G-protein Signaling, or RGS proteins, which enhance the intrinsic rate of GTP hydrolysis by greater than 1000-fold.312,313 Some RGS proteins enhance signaling through RhoGEFs and act as scaffolds for other signaling cascades (e.g., tethering to Raf and MEK2). The GPCR effectors PKA and PKC also contribute to receptor inhibition by phosphorylating their cognate-activated GPCR and thereby uncoupling and inactivating Gαs/Gαq in a classic negative feedback loop.314,315 GPCRs can also function via G protein–independent mechanisms by binding to the arrestin family of cytosolic adapter proteins.316,317 GPCR-coupled arrestins have pleiotropic cellular roles including (1) dampening G-protein signaling by scaffolding enzymes that degrade G-protein second messengers; (2) desensitizing receptors by binding GPCR kinase (GRK)–phosphorylated GPCRs and sterically blocking access to further Gα subunits; (3) mediating GPCR trafficking and endocytosis to clathrin-coated pits; and (4) acting as a scaffold for multiple MAP kinase cascades. For example, MEK1 is engaged by a tethered complex of Raf-1, ERK2, and β-arrestin to facilitate mitogenic signaling.318,319 GPCRs are well established drug targets for antihistamine, antacid, cardiovascular, and antipsychotic drugs; pain suppressants; and antihypertension therapies.275,276 Although less well appreciated as drug targets in cancer, there is increasing evidence that dysregulated GPCR signaling contributes to cancer initiation and progression. For example, in 1986 the wild-type MAS1 gene, which encodes the MAS GPCR, was reported to induce transformation by coupling to the small G protein Rac.320,321 Large-scale deep sequencing efforts have revealed that GPCR mutations occur in approximately 20% of all cancers. Receptors for thyroid-stimulating hormone (TSHR), Hedgehog (Smoothened receptor [SMO]), glutamate (GRM), the adhesion family, lysophosphatidic acid (LPA), and sphingosine-1-phosphate (SIP) are the most frequent GPCRs altered in cancer.277,312,313,322 G proteins themselves are also mutated in cancer. In particular, recurrent mutations in GNAS (which encodes Gαs) have been identified in thyroid and pituitary tumors, as well as mutations in GNAQ and GNA11 in melanoma of the eye and skin, respectively.322 Hot spot resides that disrupt GTPase activity and render the protein constitutively active have been identified in GNAS at positions R201 and Q227, in GNAQ at R183, and in GNA11 at Q209. Numerous inhibitors of GPCR signaling are also being studied in the clinic including BKT-140/ BL-8040 in pancreatic adenocarcinoma and blood cancers (target: CXCR4 receptor), and CXCR2 ligands (target: CXCR2 receptor).278 Detailed later are a few specific examples of GPCRs that have been shown to play a role in cancer initiation and/or progression. A connection between GPCRs and EGFR signaling has been established in both normal cell physiology and in colon, lung, breast, ovarian, prostate, and head and neck cancer development.323–326 Ligand-bound GPCRs activate Src, PKC, Ca+ channels, and PKA intermediaries, which stimulate proteolytic cleavage and release of membrane-tethered growth factors that bind and thereby transactivate EGFR. Specifically, estrogen binding to the GPCR GRP30 facilitates matrix metalloproteinase–2 (MMP2) and MMP9-mediated cleavage of the growth factor precursor pro-heparin-binding-EGF (pro-HB-EGF), thus initiating HB-EGF–mediated EGFR transactivation.324 Through a related mechanism, LPA-, SIP- and thrombin-activated GPCRs transactivate EGFR in breast cancer cells via growth factor shedding of tumor necrosis factor–α (TNF-α) through the action of TACE/ ADAM17 zinc-dependent proteases.326 Thrombin-mediated N-terminal cleavage and activation of proteinase-activated receptor 1 (PAR1), which can act through EGFR, has been found to promote metastasis and invasiveness in melanoma, breast, colon, and prostate cancers.278,279

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Intriguingly, MMP1 was found to function similarly to thrombin in activating PAR1 and promoting breast cancer tumorigenesis and invasion.327,328 This cross talk between GPCRs and EGFR provides a rationale for the combinatorial use of GPCR agonists/antagonists and EGFR inhibitors in patients with EGFR-driven lung and colorectal cancers. Aberrant GPCR signaling through Gα12/13 also contributes to tumorigenesis by enhancing cancer cell migration, invasion, angiogenesis, and metastasis.329 Ligand-activated LPA, PAR1, SIP, thromboxane A2 (TP), CXC chemokine (CXCR4), and prostaglandin E2 (PGE2) receptors couple to Gα12/13 and RhoGEFs to hyperactivate RhoA (see earlier), which elicits these progression-associated phenotypes in glioma, melanoma, lung, breast, and ovarian cancers.278,279 Overexpression of Gα12/13 and RhoA in breast, prostate, and colon cancers also promotes metastasis by decreasing cell adhesion.311 RhoGEF inhibitors; RhoGTPase inhibitors; inhibitors of prenylation, which could indirectly impair proper Rho localization (statins, farnesyl/ geranylgeranyl transferase inhibitors); and inhibitors of kinases downstream of Rho (ROCK, LIMK, MRCK, PAK) have all been developed and tested in biochemical, cell line, and mouse experiments. However, a clear benefit to the use of such compounds in cancer patients has yet to be established.330 Two other prominent examples of dysregulated GPCR signaling in human cancer are the Hedgehog/Smoothened and Wnt/Frizzled signaling pathways.277,278,331 Both pathways, along with Notch, also play critical roles in cell fate and have been implicated in the underlying pathway cross talk that is key to cancer stem cells.332 Secreted Hedgehog (Hh) ligands (first identified based on their roles in normal development and stem cell homeostasis, with Sonic Hedgehog [SHH] being the most ubiquitous) bind to the 12-pass TM receptor Patched (PTCH), which relieves its repression of the GPCR Smoothened (SMO).333,334 Activated Smoothened couples to Gαi and Gα12, which regulate the glioma-associated oncogene homologue (GLI) transcription factors, which in turn regulate proliferative, survival, and differentiation signals involving cyclin D1, myc, BCL2 and the Forkhead transcription factors, to name a few.335,336 Mutations in SHH, PTCH, and SMO are found in patients with inherited and sporadic basal cell carcinomas337,338 and ameloblastomas,339 whereas overexpression of Hh ligands has been shown to result in hyperactivation of the pathway in breast, colon, prostate, and pancreatic ductal adenocarcinomas331,340 Vismodegib and sonidegib, both selective Smoothened receptor inhibitors, are FDA approved for the treatment of advanced basal cell carcinomas (see Table 2.1).341–344 Several other Hh/SMO inhibitors are currently being tested in patients with cancer, mostly for advanced and/or metastatic solid tumors.278,345,346 The secreted Wnt glycolipoprotein ligands activate the single TM low-density lipoprotein–related coreceptors LRP5/6 and the GPCR-like TM protein Frizzled (Fz), which are phosphorylated and likely couple to Gαq and Gαo, respectively, to activate the cytoplasmic scaffold Dishevelled.347–349 Dishevelled in turn inhibits the β-catenin degradation complex (which consists of APC, axin, CKIα and GSK-3β, and the E3 ubiquitin ligase β-TrCP).350 This results in accumulation of β-catenin, which translocates to the nucleus where it induces TCF/ LEF-mediated transcription of genes important for cell differentiation and proliferation, including myc, cyclin D1, VEGF, FGF4/18, E-cadherin, COX-2, and members of the Wnt cascade itself.349,351,352 Noncanonic Wnt/Fz pathways include signaling to the transcription factors NFAT via Gαq/i/Gβγ/PLC/PKC/Ca+ (Wnt-calcium pathway) and AP1 via Rho/Rac/JNK (planar cell polarity pathway).277,353 These pathways regulate cell polarity and migration and are implicated in cancer metastasis.353 Aberrations in canonical Wnt signaling promote tumorigenesis in melanoma and colon, liver, ovarian, and prostate cancers.354,355 Specifically, loss-of-function mutations or truncations in APC and AXIN1/2 and gain-of-functions mutations in β-catenin are found in almost all colorectal cancers, with APC alteration found in over 85%.349,356 Germline mutations in the APC gene are also the basis for the inherited cancer predisposition syndrome familial

adenomatous polyposis (FAP).357 Efforts to develop selective inhibitors of Wnt signaling are ongoing and will be aided by current endeavors to crystallize members of the Wnt cascade.354,358 Of note, nonsteroidal antiinflammatory drugs (NSAIDs) have shown some promise in modulating Wnt signaling, likely by inhibiting the Wnt-output gene COX-2 or by enhancing E-cadherin signaling.349,359 COX-2 inhibitors have shown efficacy in reducing the risk of polyps in patients with FAP.358,360

CYTOKINE RECEPTOR SIGNALING Cytokines are protein and glycoprotein ligands secreted by immune cells; they initiate diverse and often opposing effects based on target cell lineage. Processes regulated by cytokine signaling include cell proliferation, differentiation, survival, inflammation, angiogenesis, antiviral activity, and modulation of immune function. Cytokines signal in an autocrine and/or paracrine fashion and can be subclassified by protein structure into four families, which total over 100 members: hematopoietins, IFNs, chemokines, and the TNF superfamily. The hematopoietin family consists of interleukins (IL-1 to IL-31), growth hormone, prolactin, erythropoietin, thrombopoietin, leptin, granulocyte colony-stimulating factor and granulocyte-macrophage colony-stimulating factor, and a few others.361 The majority bind to either class I or II cytokine receptors, which are single TM glycoproteins that lack kinase activity and diverge in their extracellular domains in order to specify ligand binding.362,363 Class I receptors function as a cluster of two or three subunits that each have two sets of conserved cysteine pairs and a WSXWS motif in their external domains. Class II receptors lack the WSXWS motif and one of the class I conserved cysteine pairs, but contain conserved proline, tryptophan, and an additional two conserved cysteine residues. Ligand binding causes aggregation of the γ-chain (also called γc, CD132) which is common to many cytokines, and the β-chain (also known as IL-2Rβ, IL-15Rβ, or CD122).364,365 In the case of specific cytokines, such as IL-2, association with a third subunit, the α chain (also called IL-2Rα, CD25, or Tac antigen), allows for high-affinity ligand binding.366,367 The IFN family members are divided into type I, II, and III classes.368,369 Most IFNs are type I and can be further subtyped.370 All type I IFNs bind the type I IFN receptor, which consists of two subunits (IFNAR1 and IFNAR2). The sole type II IFN, IFN-γ, binds the type II IFN receptor, which is composed of the IFNGR1 and IFNGR2 subunits. Both types of IFN receptors belong to the class II cytokine receptor family. The IFN family also includes a third branch, the IFN-like molecules, IFN-λ1 (IL-29), IFN-λ2 (IL-28A), and IFN-λ3 (IL-28B), which display some structural overlap with ILs and the antiviral properties of IFNs, and bind a distinct receptor made up of IFNLR1/IL-28Rα and IL-10Rβ.371 Given that cytokine receptors are promiscuous and bind multiple cytokine ligands, there is a high degree of redundancy in the output profiles of individual cytokines. This redundancy serves to amplify and sustain signaling downstream of these transitory stimuli. Hematopoietin or IFN binding induces oligomerization of cytokine receptor subunits and autophosphorylation and activation of the JAK (Janus activated kinase) family of intracellular tyrosine kinases (TYK2, JAK1–JAK3), which are constitutively bound to box I and box II α-helix motifs in the receptor cytoplasmic tail.372 Activated JAK proteins phosphorylate tyrosine residues on the cytokine receptor chains, which classically bind the STAT (signal transducer and activator of transcription) family of transcription factors (STAT1–STAT6).361,373 Docking of STATs facilitates their phosphorylation by JAKs, which in turn causes STAT dimerization, nuclear translocation, and alterations in gene transcription.374 There is also cross talk between STAT signaling and the nuclear factor–κB (NF-κB) and SMAD signaling pathways.375 STATs are also activated by RTKs, SRC, and ABL, and STAT activation also plays a role in transformation initiated by these oncogenes.376 JAK/STAT activity is regulated by several posttranslational modifications as well as by the SOCS (suppressor of cytokine signaling) and PIAS

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(protein inhibitor of activated STAT) proteins.375 In addition to JAK-STAT signaling, cytokine receptors can transduce messages through LCK and SYK (Src-family kinases), through BCL-2, and via PI3 kinase/AKT and Ras/Raf mediated upregulation of Fos- and Jundependent transcription.376,377 The 29-member TNF superfamily of receptors (TNFSFR1) and their corresponding 19 ligands (TNFSF) induce inflammation in addition to prosurvival and proapoptotic phenotypes.378,379 TNF ligands are TM proteins that function as membrane-integrated or cleaved, soluble trimers that bind and activate preformed, single-TM TNF receptor trimers on the cell surface.380 Conserved cysteine residues in the TNF receptor external domains dictate ligand binding, and the presence of death domains (DDs), TRAF-interacting motifs (TIMs), or neither (decoy) dictate downstream signaling. For example, on apoptotic stimuli, ligand activation of the TNF-R1 and DR3 receptors recruits the adaptor TRADD (TNFR-associated death domain) via DDs, which in turn binds to FADD (Fas-associated protein with death domain).378,379,381 FADD binds procaspase-8 and procaspase-10 via death effector domains (DEDs), and induces their cleavage to form active enzymes, which cleave caspase-3 and induce apoptosis. TRADD binding is also capable of inducing the intrinsic apoptotic cascade (mitochondrial release of ROS, cytochrome C, and Bax, which leads to caspase-9 and caspase-3 activation and apoptosis).378,382 Under proliferative stimuli, TNF-α–dependent activation of TNF-R1/TRADD and TNF-R2 converges on the recruitment of TRAF2 (TNFR-associated factor 2). TRAF2 binding sequentially recruits the RIP (receptor interacting protein), TAK1 (TGF-β activated kinase 1), and IκB kinase (IKK) trimer; this functions to degrade IκB-α (inhibitor of NF-κB-α) and in turn facilitates the activation and translocation of NF-κB.378,379 NF-κB in turn induces mediators of inflammation and cytoprotective phenotypes.383 Alternatively, TAK1 can signal to MKK3/6 (MAP kinase kinase 3/6) and to two MAP kinases, ERK (proproliferation) and p38α (to activate the transcription factor AP-1 [activator protein-1]).384 Upstream, TRAF2 can additionally trigger MEKK1 (MAP/ERK kinase kinase 1), MKK7, and JNK (c-Jun activating kinase), the recruitment of which also converges to activate AP-1 and thus regulate proliferation and survival.385–387 There is an important avenue of cross talk between the TNF-directed NF-κB and JNK pathways, the balance of which ultimately decides cell fate.388 In cancer, dysregulation of cytokine signaling promotes chronic inflammatory signals and prevents the immune system from attacking cancer cells. Unfortunately, because of their pleiotropic effects, which are often cell type and microenvironment specific, therapeutic strategies that modulate cytokine signaling have demonstrated only modest clinical activity in patients with cancer. For example, the recombinant human IFN-α2a mimetic, Roferon-A, has been shown to inhibit tumor growth in patients with melanoma and hairy cell leukemia, but such treatments have now been supplanted by more active therapies.389 Moreover, focal delivery of TNF-α to limbs affected by soft tissue sarcomas and melanomas through isolated limb perfusion techniques has been more effective than systemic use, which is toxic.390,391 Proapoptotic, anti-TRAIL therapies, such as mapatumumab, are also in clinical testing.381 Downstream components of the cytokine signaling cascades are also being explored as targets for drug development. Gain-of-function mutations in the JAK2 (hot spot V617F) and MPL genes, the latter of which encodes the thrombopoietin receptor, are common in myeloproliferative neoplasms, and the selective JAK1/2 kinase inhibitor ruxolitinib has been approved for this indication (see Table 2.1).392–394 STAT3 is frequently hyperactivated in cancer, because it is a downstream effector of both cytokine receptors and mutated and amplified tyrosine kinases.376,395 Constitutive activation of STAT5 and STAT6 plays a critical role in BCR-Abl–driven chronic myelogenous leukemia (CML), and IL-13–driven lymphomas and leukemias.376 On the basis of these findings, direct inhibitors of JAK and STAT are currently in development.374,396 Inhibition of NF-κB has been shown to induce apoptosis in leukemias and lymphomas and enhance chemotherapy and radiation

response.383 JNK1 is upregulated in hepatocellular carcinoma and prostate cancer, whereas p38α activity is either lost (in hepatocellular carcinoma) or activated in numerous cancers, and targeted inhibitors are being developed.385,397

SERINE/THREONINE RECEPTOR SIGNALING Receptor serine/threonine kinases are exemplified by the TGF-β type I and II receptors.398,399 Ligands for these receptors include the TGF-β superfamily, which comprises the TGF-β1-3 isoforms, activins, inhibins, Nodal, and Lefty, and the more distantly related bone morphogenic proteins (BMPs), growth and differentiation factors (GDFs) and müllerian inhibitory substance (MIS). TGF-β ligands regulate a diverse array of physiologic processes including growth, proliferation, survival, hormone release, and differentiation.398,400 They thus have a central role in embryonic patterning, tissue development, and morphogenesis. Signaling mediated by TGF-β, the namesake and most studied ligand of the superfamily, will be used to exemplify the general structure of the serine/threonine kinase cascades. TGF-β is ubiquitously expressed and requires a multistep maturation and secretion process to be functional and bioavailable. Initially translated as an immature proprotein, the prodomain (called latencyassociated protein [LAP]) is cleaved and noncovalently bound to the remaining mature form of TGF-β.401 Covalently bound mature, active dimers are further bound to latent TGF-β binding protein (LTBP) such that this complex is sequestered by LTBP binding to the extracellular matrix (ECM) until appropriate signals initiate matrix metalloproteinase, plasmin, or thrombin-dependent cleavage of LTBP and subsequent release of active TGF-β dimers.401,402 TGF-β dimers bind constitutively active, single TM, homodimeric TGF-β type II receptors (TβRII). Once bound by ligand, TβRII forms a complex with TGF-β type I (TβRI) receptor homodimers, thus creating a receptor heterotetramer.398,400 TβRII receptors phosphorylate and activate the intrinsic kinase activity of TβRI, which in turn phosphorylates serine residues in the C-terminal–SSXS motif of receptor-activated SMAD proteins (R-SMAD2 and R-SMAD3 for TGF-β).403 Activated R-SMAD2 and R-SMAD3 then form a heterotrimer with SMAD4, which then localizes to the nucleus, where it both binds DNA and partners with other transcription factors (i.e., Forkhead, homeobox, zinc-finger, AP-Ets, and bHLH family transcription factors) and cofactors (e.g., p300, CBP).404,405 These SMAD-containing complexes then target selective promoter elements with high affinity, leading to the induction or repression of hundreds of genes, depending on cell context. For example, TGF-β induces the expression of 4EBP1 and the cyclin-dependent kinase inhibitors INK4B and p21 and represses Myc to elicit growth inhibitory effects.406 It also activates DAPK (death-associated protein kinase), GADD45β (growth arrest and DNA damage-inducible 45β), and BIM to promote apoptosis and PDGF in smooth muscle cells to enhance proliferation, among other effects.407 TGF-β also signals through many non-SMAD effectors including Shc, which can result in enhancement of Ras/ERK signaling, and TRAF6, which activates the TAK1/MKK3&6/JNK/p38 cascades.408 TGF-β, through indirect mediators, can also activate Src, Rho, and PI3 kinase, and through pathway cross talk the Wnt, Hedgehog, and Notch cascades.409 The outcome of TGF-β–directed transcriptional responses depends on access of TGF-β to particular signaling receptors and SMAD complexes, the availability of transcription factors, and the epigenetic status of the cell.410 Alterations in TGF-β signaling are common in cancer.407,411,412 For example, mutational inactivation of TβRII is seen in colorectal cancers with microsatellite instability.413 Mutations and loss of SMAD4 expression are common in colorectal, pancreas, and head and neck cancers.414,415 Conversely, increased TGF-β expression occurs in breast, prostate, and colorectal cancers and has been associated with cancer progression and the development of metastases.407 TGF-β has also been shown to play a role in maintaining the tumor-initiating cell or cancer stem cell population in gliomas, leukemias, and breast

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cancer.407 It has been proposed that TGF-β signaling promotes invasion and metastasis by promoting EMT.406 Interesting to note, in pancreatic ductal adenocarcinoma, wherein loss of SMAD4 is common, TGF-β can play a novel tumor suppressor role by inducing a lethal EMT through the lineage and progenitor regulators Klf5 and Sox4.416 Intense efforts are underway to develop therapeutic strategies that inhibit the tumorigenic properties of TGF-β signaling. These include the development of selective TGFβRI inhibitors, TGF-β blocking antibodies, soluble TGF-β antisense therapies, and selective kinase inhibitors.411,417,418 The development of inhibitors of TGF-β signaling has, however, been confounded by the ability of TGF-β to both promote and suppress tumor progression in a context-specific manner.419,420

NOTCH RECEPTOR SIGNALING The mammalian Notch receptors, Notch1 to Notch4, are single-pass TM receptors that are functionally unique with regard to receptor processing and signal activation and transduction.421–424 Notch receptors are translated as immature receptors that undergo S1 cleavage by furin-like convertase during Golgi trafficking.425 This generates two subunits that are retethered at the cell membrane by noncovalent bonds in the heterodimerization domain.423,426 The extracellular domain subunit consists of ligand-binding EGF-like repeats, a heterodimer domain, three negative regulatory LIN12 and Notch repeats (LNRs) containing numerous cysteines, and a hydrophobic TM-interacting region. The other subunit contains the TM domain and the intracellular domain, which has ankyrin repeats and a RAM domain central to the Notch receptor’s unusual activity as a direct transcription factor, and a PEST motif important for the quick termination of Notch activity and ubiquitin-mediated degradation. Delta-like ligand (DLL1, DLL3, DLL4) and Jagged (JAG1 and JAG2) are the five TM-protein ligands for the Notch receptors.427 A Notch receptor on one cell binds to DLL or JAG ligand on an adjacent cell. This results in a change in Notch receptor conformation, which facilitates a series of proteolytic cleavages required for receptor activation. First, the ADAM17/TACE (a disintegrin and metalloprotease-17/ TNF-α converting enzyme) metalloprotease performs S2 cleavage of the extracellular domain.428,429 This allows subsequent S3 cleavage of the TM domain by the γ-secretase complex and release of the Notch intracellular domain (NICD).430 The active NICD translocates to the nucleus, where it binds to and converts the repressor complex CSL (CBF1, Suppressor of Hairless, Lag-1) into a transcriptional activator that recruits coactivators including the Mastermind-like family proteins and p300. NICD target genes include the HES (hairy enhancer of split) and HRT/HEY (hair-related transcription factor) transcriptional repressors, as well as cyclin D1, myc, p21, NF-κB, and the Notch receptors and ligands themselves, among others.422,426,431 Notch is also heavily involved in cross talk with other pathways, including RTKs, PI3 kinase, Ras/ERK, JAK/STAT, Wnt, Hedgehog, TGF-β/SMAD, and p53. Overall, Notch plays a role in proliferation, cell fate determination in development, and survival. Notch’s causal role in tumorigenesis was established on its discovery in 1991, when a translocation event in T-cell acute lymphoblastic leukemia or lymphoma (T-ALL) was identified that fused the T-cell receptor-β promoter/enhancer elements to Notch, generating a truncated form of Notch1 that was constitutively nuclear and active.432 Activating NOTCH1 mutations have since been found to occur in approximately 60% of T-ALL patients.433 Subsequent studies have determined that Notch and its ligands have both oncogenic and tumor suppressor properties in several hematologic and solid tumor malignancies, including melanoma, glioblastoma, and breast, lung, colorectal, and pancreatic cancers, depending on cellular context.423,426 Thus far, efforts to develop inhibitors of Notch signaling have focused primarily on inhibiting cleavage, and thus activation of Notch. Specifically, selective γ-secretase inhibitors (GSIs), such as MK-0752, PF03084014, and BMS-906024, are currently being tested in early-stage

clinical trials.422,434–436 Newer avenues of drug development include monoclonal antibodies targeting Notch receptors or ligands.437

NUCLEAR HORMONE RECEPTOR SIGNALING The nuclear hormone superfamily is composed of hormone receptors and orphan receptors (receptors for which no ligand has yet been identified). The nuclear hormone receptors are characterized structurally by a ligand binding domain, a DNA binding domain, and a hinge region that connects the ligand and DNA binding domains.438 Nuclear hormone receptors are classified into four subtypes based on ligand specificity: steroid, retinoid X receptor (RXR), monomeric or tethered orphan receptors, and dimeric orphan receptors.439 The steroid receptor ligands include estrogen, progesterone, androgen, and growth hormone. Ligand binding occurs in the cytoplasm and results in receptor homodimerization followed by nuclear translocation. Once translocated into the nucleus, the ligand-bound receptor acts as a transcription factor that modulates the expression of several downstream proteins through binding to steroid response elements, which are conserved nucleotide sequences within the regulatory regions of genes.440 In contrast to the steroid receptors, the RXR receptors form heterodimeric complexes with other partners, including the retinoic acid receptor, the thyroid hormone receptor, and vitamin D receptors. Hormone receptor blockade is a cornerstone in the treatment of estrogen receptor (ER)– and progesterone receptor–expressing breast cancers. The selective estrogen receptor modulator (SERM) tamoxifen competes with estradiol for binding to ER. Notably, tamoxifen binding results in ER dimerization, nuclear translocation, and receptor binding to estrogen response elements in the promoter regions of estradiol-target genes. It is thought that the ER/tamoxifen complex recruits transcriptional corepressors, in contrast to ER/estradiol binding, which recruits transcriptional coactivators.440,441 Although tamoxifen has antiproliferative effects in ER-expressing breast cancer cells, it causes hypertrophy and neoplastic transformation of endometrial tissue, likely as a result of cell type and context-specific recruitment of transcriptional coactivators that are differentially expressed in these tissues.440 Recently, mutations in the ESR1 gene, which encodes the ER, have been shown to be a common mechanism of acquired resistance to hormonal therapy in patients with breast cancer.442 Retrospective studies suggest that patients with ESR1-mutant breast cancer may have more durable responses to the selective estrogen receptor downregulator (SERD) fulvestrant than those treated with the aromatase inhibitor exemestane (see Table 2.1).442,443 Dihydrotestosterone is the primary ligand of the androgen receptor (AR). AR blockade by antiandrogens such as bicalutamide is a commonly used therapeutic modality for patients with locally advanced or metastatic prostate cancer. Bicalutamide competes with dihydrotestosterone for binding to cytoplasmic AR.444 The mechanism by which bicalutamide-bound AR inhibits androgen-dependent gene transcription is unclear but may involve the recruitment of transcriptional corepressors as well as histone modifications that lead to tighter chromatin binding and therefore reduced access to promoter regions by transcription factors. Enzalutamide, a nonsteroidal small-molecule antagonist of the AR that inhibits AR nuclear translocation and DNA binding is FDA approved for the treatment of patients with metastatic prostate cancer that has progressed following treatment with bicalutamide and medical castration (see Table 2.1).445 Abiraterone acetate, an irreversible inhibitor of CYP17A1 that enables intratumoral and adrenal androgen depletion, also has significant clinical activity in patients with castrateresistant prostate cancer.446

INTEGRIN RECEPTOR SIGNALING The integrin receptor family regulates cell adhesion, migration, invasion, and cell survival.447,448 Integrin receptors are heterodimeric molecules consisting of combinations of alpha and beta subunits. Each combination dictates the spectrum of ECM components to which these receptors

40 Part I: Science and Clinical Oncology

bind. Once ligated to the ECM, the receptors recruit multiple proteins to the cell membrane, including cytoskeletal molecules such as paxillin and vinculin that form focal adhesions to ECM components.449 Unlike the RTK family, integrin receptors do not possess intrinsic kinase activity but rather promote signaling by facilitating the activation of kinases such as Src or FAK.450 Integrins are also unique in that they participate in bidirectional signaling. “Inside-out” signaling occurs when intracellular adapters, of which talin-1 and kindlin-1/2/3 are the best known activators, trigger a conformational change of the cytoplasmic tails of the alpha and beta subunits, which is transduced to the extracellular component of the receptor, resulting in increased affinity for portions of the ECM.451 Conversely, “outside-in” signaling involves binding of ligand to integrins, which stimulate the activation of multiple intracellular signaling pathways.452 Collectively, the term integrin adhesome is used to describe the site at which integrins initiate contact with the ECM and other cells and recruit all of the underlying intracellular machinery (e.g., cytoskeleton, scaffolds, signaling adapters— over 200 intrinsic and transient components) to generate diverse types of adhesions (e.g., focal complexes, focal adhesions, fibrillar adhesions, podosomes, invadopodia).453 Integrins are expressed on cancer cells and have been shown to promote disease progression.447,450,454 Integrins are also present on stromal cells, including pericytes (which promote endothelial cell growth and proliferation and thus angiogenesis) and fibroblasts, where they influence the surrounding microenvironment and thus indirectly stimulate tumor growth and proliferation. For example, vascular cell adhesion molecule 1 (VCAM1) is expressed on pericytes and binds to the integrin receptor α4β1, which is found on the surface of endothelial cells, resulting in pericyte recruitment to sites of vascular maturation.455 Integrin signaling also plays a role in the activation of matrix metalloproteinase 2,456 which promotes cell invasion, and has been shown to regulate cyclin D and cyclin-dependent kinase inhibitor expression, thereby controlling cell cycle progression.457 Finally, integrin receptor activation can lead to increased secretion of growth factors, which then stimulate tumor invasion through autocrine and paracrine mechanisms.456 Tumors that express integrin receptors include melanomas, glioblastomas, and breast cancers. In melanoma, the αvβ3 and α5β1 integrin receptors promote vertical growth and metastatic spread to lymph nodes.458,459 In glioblastoma, αvβ3 and αvβ5 are expressed mainly at the edge of tumors, suggesting a role in tumor invasion.460 The expression of the α6β4 and αvβ3 integrin receptors in breast cancer is associated with higher grade and tumor size,461 the development of bone metastases,462 and decreased survival.463 To date, drug development has targeted three integrins—αIIbβ3, α4β1, and α4β7—in the context of platelet activation and blood clotting, multiple sclerosis, and inflammatory bowel disease.451 Clinical trials of monoclonal antibodies that target integrin receptors are underway in several cancer types. For example, etaracizumab, a humanized monoclonal antibody targeting αvβ3 integrin, is being tested in solid tumors and has shown activity in patients with metastatic melanoma.464 Cilengitide, an inhibitor of the αvβ3 and αvβ5 integrins, inhibited angiogenesis and tumor cell proliferation in preclinical studies; however, a phase III trial in combination with temozolomide and radiation in patients with glioblastoma did not result in improved outcomes compared with chemoradiation alone.456,465

NON–RECEPTOR TYROSINE KINASE SIGNALING SRC Signaling The SRC family of intracellular, non-RTK proteins is composed of 11 members (SRC, FYN, YES, Blk, Yrk, Frk/Rak, Fgr, Hck, Lck, Srm, and Lyn).466 They share common structural features including the so-called SRC-homology (SH) domains 1 to 4. The SH1 domain includes the kinase domain. Only SRC, FYN, and YES are expressed

ubiquitously, whereas the tissue distribution of the latter six is more restricted.467 Together, SRC family kinases have pleiotropic roles in cellular proliferation, apoptosis, differentiation, motility, adhesion, angiogenesis, and immunity.468,469 SRC is by far the most intensively studied family member and was the first gene observed to have oncogenic potential.470 Peyton Rous was awarded the Nobel Prize for a series of experiments showing that a transmissible factor was present in avian sarcomas capable of initiating tumors in recipient birds. Five decades later the viral oncogene v-Src was identified as the oncogenic factor in the Rous sarcoma virus.471–473 Bishop and Varmus later showed that v-Src was a mutant form of the cellular proto-oncogene c-Src, and Hunter and colleagues showed that its transformative capacity was dependent on its tyrosine kinase activity.14,15,474 SRC is regulated in a number of ways. First, SRC has a myristoylation site in its N-terminus that is necessary for membrane localization and that promotes its interaction with nearby membrane-bound effectors.475 The SH2 and SH3 domains facilitate protein-protein interactions and conformational changes in the protein. Inactive SRC is maintained in a closed conformation with phosphorylated Y530 (mediated by CSK, C-terminal SRC, and Csk homology kinases) interacting with the folded-over SH2 domain.476 The closed, inactive confirmation of SRC is further stabilized by proline-rich segments of the kinase domain associating with the SH3 domain.477 SRC activation requires dephosphorylation of Y530, likely by PTPα/γ/1β (protein tyrosine phosphatase α/γ/1β) or SHP1/2 (SH-containing phosphatases), which allows the kinase to assume an open conformation.466,478 Autophosphorylation of Y419 in the activation loop of the kinase domain also promotes full activity,479 whereas binding of FAK and CRK-associated substrate (CAS) to the SH2 domain induces SRC activation and links SRC signaling to the regulation of focal adhesion, actin reorganization, and migratory phenotypes.480,481 SRC is a downstream mediator of numerous receptor families including RTKs, integrin receptors, hormone receptors, cytokine receptors, and GCPRs and promotes signaling through the PI3 kinase/AKT, Ras/MAP kinase, and JAK/STAT cascades, among others.466,467,469,478,482–486 More than two decades of research have uncovered numerous SRC substrates, including p85-cortactin, p110-AFAP1, p130Cas, p125FAK, and p120-catenin.487 Although mutations in SRC are rare in human cancers, SRC is frequently activated as a consequence of other mutational events in colorectal, breast, esophageal, gastric, pancreatic, hepatocellular, ovarian, and lung cancers.466 In colorectal and hepatocellular carcinomas, SRC activation occurs in the setting of concomitant loss of CSK.488–490 Newer signaling discoveries have identified roles for SRC in promoting tissue repair after intestinal inflammatory injury, as seen in inflammatory bowel diseases and colorectal cancer, via the IL-6 cytokine coreceptor gp130 and YAP.491 Furthermore, norepinephrine-mediated, β-adrenergic/PKA activation of SRC has recently been shown to enhance tumor cell migration, invasion, and growth.492 The tyrosine kinase inhibitor dasatinib, which is used in the treatment of CML and Philadelphia chromosome–positive acute lymphoblastic leukemia (ALL), inhibits SRC family kinases, in addition to BCR-ABL, KIT, Ephrin A2 receptor, and PDGFR (see Table 2.1).493–495 Additional dual SRC/ABL and SRC selective inhibitors are approved in CML (bosutinib, ponatinib; see later section on ABL signaling) or are in clinical testing, including saracatinib, XL-228, KX2-391, and DCC2036. Most have shown limited single-agent activity and are being developed as combination therapies.469,478,484–486

ABL Signaling The ABL gene was first identified in 1980 as the oncogene responsible for driving the Abelson murine leukemia virus, and later was found to be part of the translocations found in many types of human leukemias.496 ABL1 and ABL2 isoforms have both overlapping and divergent functions. ABL is found in an autoinhibited conformation dictated by clamping of the N-terminal hydrophobic region, SH3

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and SH2 domains onto the C-lobe of the kinase domain.497 Intermolecular interactions with adaptors such as RIN1 or phosphorylation by SRC, among others, promote stabilization of the open, active form of ABL 23842626. The ABL tyrosine kinase is found in both the cytoplasm and the nucleus, and its function varies based on subcellular localization.498 Cytoplasmic ABL has been implicated in G1/S checkpoint regulation and interaction with actin on C-terminal binding sites,499 whereas nuclear ABL inhibits binding of the DNA repair protein Rad51 to sites of DNA damage.500,501 Activated ABL kinases are now recognized to play an important role in tumor initiation by disrupting cell polarity and by promoting invasion and metastasis by regulating invadopodia.502 In cancer, translocation of the ABL gene on chromosome 9 with the breakpoint cluster region (BCR) gene on chromosome 22 results in the expression of a BCR-ABL fusion protein.503 This translocation, the Philadelphia chromosome, is found in almost all CMLs and represents the pathognomonic molecular lesion in this disease. BCR-ABL translocations are also found in approximately one-third of acute lymphoblastic leukemias (ALLs).504,505 In addition, at least eight other fusion partners of ABL have been discovered.502 The realization that the proliferation and survival of CML cells is critically dependent on the ABL fusion protein led to the development of imatinib, an inhibitor of the ABL tyrosine kinase.505 To date, there are five FDA-approved inhibitors for CML: imatinib, dasatinib, bosutinib, nilotinib, and ponatinib (see Table 2.1).506 Moreover, imatinib and dasatinib are approved for treatment of ALL.507–511 A secondary mutation in the gatekeeper site T315I is the most common reason for acquired resistance to ABL kinase inhibitors. Combination of ATP-competitive and allosteric inhibitors such as GNF-5 may be a novel strategy to combat resistance.512

RAS/MAP KINASE PATHWAY SIGNALING First identified over 30 years ago as the oncogenes responsible for the transforming potential of the Harvey and Kirsten murine sarcoma retroviruses (Ha-MSV and Ki-MSV), RAS proteins are guanine nucleotide binding proteins.513–517 Ras proteins have intrinsic GTPase activity and cycle between inactive GDP- and active GTP-bound states. GDP/GTP exchange thus allows Ras proteins to function as binary molecular switches. In the human genome, there are three RAS genes, which encode four homologous proteins (HRas, NRas, and the alternative splice variants KRAS4A and KRAS4B) with highly conserved N-terminal and variable C-terminal regions. Following stimulation of cells by serum growth factors, cytokines, hormones, and neurotransmitters, Ras undergoes a series of C-terminal (C186AAX) posttranslational modifications that result in its localization to specific membrane microdomains.518 Membrane localization is required for the transforming properties of Ras, because mutation of Ras at C186 results in cytosolic localization and protein inactivation whereas Ras activity can be rescued by myristoylation, which promotes membrane localization.519–521 GEFs promote Ras activation by binding to GDP-bound Ras and facilitating the release of GDP and the binding of GTP. SOS1, RAS-GRF (dual specificity GEFs for RAS and RAC), and RAS-GRP (stimulated by DAG/phorbol esters and Ca+) are the mostly highly characterized RAS-GEFs.522–524 Using RTK stimulation as an example, ligand binding induces RTK dimerization and autophosphorylation of tyrosine residues in the receptor cytoplasmic tail. Phosphotyrosine docking sites recruit scaffold proteins such as SHC and permit interaction with the SH2 domains of adaptor proteins such as Grb2.525 Grb2 in turn recruits SOS1 via its SRC homology 3 (SH3) domain, thereby positioning SOS near membrane-anchored RAS (see Fig. 2.1).525–529 SOS1 in turn activates RAS via its CDC25 homology (RASGEF) domain and N-terminal RAS exchanger motif (REM or RASGEFN domain). Ras inactivation is catalyzed by GTPase-activating proteins (GAPs), which enhance the intrinsic GTPase activity of Ras proteins by 100,000-fold.530 RAS GAPs, which include p120-RASGAP,

neurofibromin (NF1), GAP1IP4BP, and CAPRI, negatively regulate RAS activity and thus function as tumor suppressors.514,518 Binding of GTP to RAS induces conformational changes in the switch I (loop 2 residues 30–38) and switch II (helix 2 and loop 4 residues 60–76) domains, which facilitate the association of RAS with regulators and downstream effectors.531,532 RAS directly interacts with over 20 effector proteins, of which the RAF kinases, PI3 kinases, and RALGDS are the best characterized (see Fig. 2.1). The canonic RAS/ RAF/MEK/ERK (classic MAP kinase) cascade is by far the most extensively characterized RAS effector pathway. This prototype of a three-tiered kinase signaling cascade exemplifies numerous RASdependent mitogen-activated protein kinase (MAPK) cascades that respond to diverse signals, including cell stress and cytokine signaling (see section on cytokines for details of the JNK and p38 pathways).533 The RAF protein family (which represent the top-tier MAPK kinase kinases [MAPKKKs], or MEK kinases [MEKKs]) is composed of three differentially expressed isoforms, A-RAF, B-RAF, and C-RAF (RAF-1).534,535 RAF, via its RAS binding domain (RBD) and cysteine rich domain (CRD), interacts with GTP-bound Ras.536–539 Binding of RAF to GTP-bound RAS results in RAF localization to the plasma membrane and its subsequent phosphorylation and activation.540 The mechanisms of RAF-1 and B-RAF phosphorylation and activation are distinct and are derived from the summation of signaling inputs from small G proteins (RAS, RAC, CDC42, RAP-1), kinases (activating inputs: SRC/PAK/PKC, inhibitory inputs: PKA/AKT/SGK), isoform homodimerization and heterodimerization, phosphatases (PP2, PP2A), scaffolding proteins (KSR, RKIP, HSP90, and so on) and cofactors (14-3-3), with phosphorylation events regulating critical aspects of RAF activation.535,541 In brief, RAS binding to RAF-1 releases 14-3-3, a negative regulator that binds to basally phosphorylated residues S259 and S261 and sequesters RAF-1 in the cytosol in a closed, inactive conformation. Liberation from 14-3-3 exposes the PKA/ AKT/SGK-dependent inhibitory phosphorylation on S259, facilitating its dephosphorylation by protein phosphatase 2A.542–546 Loss of this inhibitory phosphorylation primes RAF-1 for RAS, PAK, SRC, growth factor, and integrin-stimulated activating phosphorylations on S338, Y341, T491, and S494.547–551 A-RAF is activated similarly to RAF-1. Notably, B-RAF activation requires fewer steps owing to its constitutive phosphorylation at S445, a site analogous to S338 in RAF-1.541,552 Binding to RAS-GTP stimulates B-RAF phosphorylation at critical residues in its activation loop, T599 and S602 (analogous to T491 and S494 in RAF-1).553,554 The B-RAF isoform is the most potent activator of ERK pathway output.535,552,554 Activated Raf proteins bind and phosphorylate MEK1 and MEK2 (mitogen-activated protein kinase/extracellular signal-regulated kinase kinases 1 and 2, or MAPKK, or MAP2K) on serines 217 and 221.555 Activated MEK, a dual-specificity threonine/tyrosine kinase, in turn phosphorylates MAPK/ERK-1/2 (mitogen-activated protein kinases/ extracellular signal-regulated kinases 1 and 2) on threonine 183 and tyrosine 185, inducing a conformational change, activation, and dissociation from MEK.556 Activated ERK then phosphorylates substrates in the cytosol (e.g., p90RSK) and in the nucleus (such as the transcription factors ELK-1, ETS-2, FOS, JUN, ATF-2, AP-1, MYC, CREB1), which promote proliferation and survival.557,558 Raf-1 has also been shown to mediate suppression of apoptosis through non–MEK-dependent interactions with ASK1, MST2, and MEKK1/NF-κB.559 RAS/RAF signaling triggers numerous regulatory mechanisms, including classic negative feedback loops, which serve to attenuate pathway output.560 The Sprouty and Sprouty-related EVH1-domaincontaining protein (Spred) proteins (encoded by the SPRY1–4 and SPRED1–4 genes, respectively) inhibit the cascade at the level of RAS and RAF activation.561–564 The dual-specificity phosphatases (MKPs/ DUSPs) dephosphorylate MAP kinases, including ERK.565,566 In addition, increased pathway activity induces ERK-dependent negative phosphorylations on B-RAF (at S151, S750, T401 and T753) and on RAF-1 (at S29, S43, S289, S296, S301 and S642) that abrogate interactions with RAS and homodimer and heterodimer formation.567,568

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RAS signaling is further regulated by RKIP (RAF kinase-inhibitory protein), which disrupts the RAF-1/MEK interaction and IMP (impedes mitogenic-signal propagation), which represses KSR-dependent scaffolding of RAF/MEK, among other functions.534,569–571 In its active, GTP-bound state, RAS alternatively binds to the p110α catalytic subunit of class I PI3 kinases,572,573 causing activation of its lipid kinase activity and thereby generating PIP3, which in turn stimulates the proproliferative and prosurvival kinases PDK1 and AKT (see section on PI3 kinase signaling for details). RAS-dependent activation of PI3 kinase can further stimulate RAC, a RHO family GTPase involved in regulation of actin and NF-κB.513,574,575 A third major class of RAS effectors is the group of GEFs for RALA and RALB, namely RALGDS, RGL, and RGL2.576–578 These RAL exchange factors stimulate phospholipase D and the CDC42/RAC-GAP RAL binding protein 1 (RALBP1) and inhibit Forkhead transcription factors to regulate transcription, vesicular trafficking, and cell cycle progression. Several additional RAS effectors have been identified. These include (1) PLCε, which generates IP3 and DAG and regulates calcium release and PKC activation; (2) T-cell lymphoma invasion and metastasis-1 (TIAM1), which facilitates actin reorganization via RAC; (3) AF6, which contributes to cytoskeletal changes; (4) RIN1, which regulates endocytosis; and (5) RASSF and NORE1, which have been shown to regulate apoptosis and cell cycle progression.513,518 Many other nondirect connections allow RAS to affect the cellular microenvironment, metabolic signaling, autophagy, inflammation, and immune responses. Overall, the complex effects of RAS activation result in a diversity of context-dependent phenotypes ranging from cell proliferation to cell death that are influenced by a wide variety of extracellular stimuli and intricately woven layers of regulation. The potent oncogenic effects of RAS signaling are highlighted by the high prevalence of mutations in the RAS genes and its proximal downstream effectors.518,579–581 RAS mutations are found in approximately 30% of all human tumors, the majority of which (85%) occur in the KRAS isoform. KRAS is frequently mutated in pancreatic cancers (58%), colorectal and biliary tree cancers (33 and 31%), and non–small cell lung adenocarcinomas (17%). Mutations in HRAS are most common in low-grade bladder cancers (11%), whereas mutation in NRAS is a frequent event in melanoma (18%) and biliary tree cancers (11%). Mutations that lock RAS in its GTP-bound state confer oncogenic potential. Point (missense) mutations in residues 12, 13, 61 (exons 2 and 3) generate a constitutively active RAS oncoprotein by abrogating intrinsic GTPase activity and inhibiting GAP binding.582 Other mutations, including those at residues 117 and 146, contribute to RAS activation by increasing GDP-to-GTP exchange.583,584 Heritable germline mutations of several RAS/MAPK pathway components have been shown to be the underlying cause of the so-called RASopathies neurofibromatosis type 1 (NF1); Noonan, Costello, and cardiofaciocutaneous syndromes; and other developmental disorders.579 More recently, deep sequencing efforts have identified recurrent somatic mutations in the RAS-GAP NF1 in glioblastoma585 and melanoma,586,587 as well as in A-RAF (hot spot at S214) in histiocytosis and lung cancer588–590 and in RAF-1 (hot spot S257).588 Mutations in BRAF are also common in human cancer and typically occur in a mutually exclusive pattern with RAS mutations. BRAF mutations have been identified in approximately 8% of all cancers, most notably in melanoma (50%–60%) and in papillary thyroid (30%–50%), biliary tree (14%), colorectal (10%), ovarian (12%), and lung cancers (3%) and hairy cell leukemia (100%).534,535,559,579,591,592 A single valine to glutamic acid substitution at residue 600 (V600E) accounts for more than 80% of all BRAF mutations and renders BRAF an active monomer in settings of low RAS activity, that is sensitive to RAF inhibition. Other nonV600E BRAF mutants have been recently identified and characterized to function as RAS-independent dimers that are insensitive to current RAF inhibitors, such as vemurafenib, that only effectively inhibit mutant monomers.593 Activating mutations in MEK1 (MAP2K1) and MEK2 (MAP2K2) are also present in approximately 1% of human tumors, more prominently in melanoma and lung cancer, and have

emerged as a mechanism of acquired resistance to RAF inhibition.594,595 Hot spots include mutations at MEK1 residues F53, K57, and P124 and at MEK2 residue F57, which is paralogous to MEK1 F53. ERK amplification and mutation, although equally rare (approximately 1% of all tumors), may also emerge as mechanisms of resistance to upstream MAPK inhibition, including alteration at the hot spot residue E322.596 Given the high prevalence of RAS pathway alterations in human cancers, significant attention has been directed toward the development of selective inhibitors of this pathway.560,580,597,598 To date, clinically effective direct inhibitors of oncogenic RAS have yet to be identified. The inability to directly target RAS has been attributed to the high affinity of the RAS-GTP interaction. Extensive efforts were thus directed toward inhibiting the posttranslation modifications required for RAS activation. Specifically, inhibitors of the enzyme farnesyltransferase, which regulates RAS localization, were tested in randomized phase III trials but were found to be inactive.599 The failure of farnesyltransferase inhibitors in KRAS-mutant tumors was predicted by the preclinical observation that geranylgeranyl modification can substitute for farnesylation in targeting KRAS and NRAS to the plasma membrane.600,601 Small molecules that irreversibly bind to the mutant cysteine in tumors with G12C K-RAS have been developed.602 Because this class of compounds is selective for the G12C mutant, they are a promising novel approach for a subset of patients with KRAS-mutant tumors. As an alternative, extensive efforts have focused on the development of selective inhibitors of key kinase effectors of RAS transformation. The selective RAF inhibitors vemurafenib and dabrafenib induce tumor regressions in most patients with BRAF V600E mutant melanoma and are FDA approved for this indication (see Table 2.1).603–612 Notably, these agents selectively inhibit RAF signaling in BRAF-mutant tumors and are thus inactive in RAS-mutant tumors.613 Several mechanisms of resistance to RAF inhibitors have emerged, including BRAF V600E splice variants or amplification; loss or mutation of NF1; parallel pathway activation of RTKs; mutation of PI3K/AKT components; mutations in NRAS, RAF1, and MEK1/2; and amplification of MITF.592,614 Sorafenib, a multikinase inhibitor of RAF, VEGFR2, and PDGFRβ, has been shown to have some clinical efficacy in ARAFmutant histiocytosis and NSCLC.588,589,615 The selective MEK inhibitor trametinib is also FDA approved for use in melanomas with BRAF mutation (see Table 2.1).605,616–619 Furthermore, the combination of a RAF and a MEK inhibitor has been shown to have greater activity than Raf inhibitor monotherapy in both preclinical models and in patients with melanoma, including the combinations of vemurafenib plus cobimetinib620 and dabrafenib plus trametinib.605,619,621,622 MEK inhibition is also emerging as a strategy to target NRAS-mutant melanoma,623 colorectal624 and thyroid cancers,625 NF1-mutant melanoma,587 GBM,626 and neuroblastoma.627 MEK inhibition in combination with immunotherapy, chemotherapy, radiation, and PI3K and CDK4/6 inhibitors is being investigated in KRAS-mutant tumors including colorectal and lung cancer (see Table 2.1).624,628 Furthermore, clinical evidence of activity of the MEK inhibitors cobimetinib, selumetinib, and trametinib has been revealed in cases and preclinical reports of MEK1-mutant histiocytosis,589 low-grade serous ovarian cancer,629 NSCLC,594 and melanoma.630 Alternative strategies for inhibiting MAP kinase signaling include HSP90 inhibitors that induce the degradation of RAF1 and mutant BRAF.631 Drug development of kinase- and dimerization-targeted ERK inhibitors (SCH772984, BVD-523, DEL-22379) is also underway and may provide a downstream alternative when upstream RAF or MEK inhibitors fail.632–635 Combinatorial approaches are also being actively pursued, given the modest activity of MEK inhibitors in patients with RAS-mutant tumors and the frequent co-occurrence of RAS and PI3 kinase pathway alterations in multiple cancer types.636–638 Given the recent success of immunotherapy in many cancers, preclinical evidence supports the triple combination of BRAF and MEK inhibitors with immunotherapy in BRAF V600E mutant melanoma, despite substantial liver toxicities described with initial trials with combined treatment of vemurafenib and ipilimumab.639 In addition, elevated

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antitumor immune responses in mouse models of triple negative breast cancer following combined MEK inhibition and immunotherapy suggest that hyperactivate MEK signaling may contribute to immune evasion.640 Given the prominent role of activated ERK in initiating the transcription and translation of cell cycle components such as cyclin D1, inhibitors of cell cycle kinase components have emerged as an alternative strategy to abrogate the output of MAPK signaling. CDK4, a critical serine/threonine kinase that functions in an active complex with cyclin D to phosphorylate RB and stimulate E2F release and S phase entry, is amplified in liposarcoma. Palbociclib, an inhibitor of CDK4, is FDA approved for use in postmenopausal women with ER+, HER2− metastatic breast cancer; a second drug, abemaciclib, has also show efficacy in this same patient population (see Table 2.1).641–644

PI3 KINASE/AKT/MTOR PATHWAY SIGNALING The PI3 kinase/AKT/mTOR pathway is a key regulator of growth factor–mediated proliferation and survival.645 Several extracellular growth factors stimulate PI3 kinase by binding to their cognate RTKs or GPCRs.646 Activated PI3 kinase phosphorylates the 3-OH group of the inositol ring of phosphatidylinositol, catalyzing the conversion of PIP2 to PIP3. PIP3 then binds to the pleckstrin homology domain (PH domain) of multiple proteins, facilitating their recruitment to the plasma membrane and thus regulating their function (see Fig. 2.2). PI3 kinases are grouped into class I, II, and III kinases based on their structure and substrate preferences, although class II and III kinases have been much less studied. Class I PI3 kinases are subdivided into two groups, IA and IB. Class IA comprises the PIK3CA, PIK3CB, and PIK3CD genes, which encode the catalytic p110α, p110β, and p110δ subunits, respectively. These p110 components heterodimerize with a regulatory subunit (p85), of which there are five isoforms and splice variants encoded by the PIK3R1-3 genes. Together, the p110/ p85 complex functions primarily in the generation of PIP3.647,648 The PIK3CG gene encodes the class IB p110γ isoform, which couples with p101 (PIK3R5) or p87 (PIK3R6) regulatory subunits. Notably, p110δ/γ catalytic subunits have specialized expression patterns mainly in leukocytes, whereas p110α/β are ubiquitous.649 Analogous to the activation of the Ras pathway detailed previously, the p85 regulatory subunit of PI3 kinase associates with phosphorylated tyrosine residues located on the intracellular domains of RTKs through an SH2 domain, resulting in allosteric activation of the p110 catalytic subunit. PI3 kinases can also be activated indirectly through the adaptor protein Grb2 following its association with the scaffolding protein GAB1. PI3 kinase pathway activity is negatively regulated by the tumor suppressor PTEN (phosphatase and tensin homolog), which is a dual lipid and protein phosphatase that dephosphorylates PIP3, converting it back to PIP2.650,651 The best characterized effectors of PI3 kinase are the three members of the serine/threonine kinase AKT family (AKT1, AKT2, and AKT3). Both AKT and phosphoinositide-dependent kinase 1 (PDK1) are recruited by PIP3 to the plasma membrane via their PH domain, where AKT is phosphorylated at Thr308 by PDK1, resulting in its activation.652–654 Phosphorylation at a second residue, Ser473, by mTOR complex 2 (mTORC2) further enhances AKT activity.655 Activated AKT promotes cellular proliferation, survival, and other phenotypes through activation of multiple downstream effectors. Proliferative effects are regulated through phosphorylation and inhibition of GSK3β, which phosphorylates cyclin D1 and marks it for degradation; FOXO4, a transcription factor that regulates the expression of the CDK inhibitor p27; and p21, a second CDK inhibitor that, on phosphorylation, translocates from the nucleus to the cytoplasm, where it regulates cell survival.656–659 In sum, these actions promote the expression of cyclin D1, which drives progression of cells through the cell cycle and the downregulation of cyclin-dependent kinase inhibitors that inhibit cell cycle progression.

AKT-mediated antiapoptotic effects occur through phosphorylation and inhibition of Bad, which negatively regulates the antiapoptotic protein Bcl-xL; caspase-9, a proapoptotic protease; and the FOXO1 transcription factor which regulates the expression of proapoptotic genes.660–662 AKT also controls cell survival by upregulating NF-κB activity through phosphorylation and activation of Iκb kinase, which marks Iκb, an inhibitor of NF-κB, for degradation.663,664 Once released from IκB, NF-κB translocates into the nucleus, where it regulates a multitude of genes involved in cell survival. AKT also phosphorylates and activates Mdm2, an E3 ubiquitin ligase that binds to the proapoptotic tumor suppressor p53 and directs it for proteasomal degradation.665 AKT-independent effectors of PI3 kinase activation have also been identified and likely play important roles in the development and progression of some cancers. These include CDC42 and Rac1, which are involved in motility and reorganization of the cytoskeleton, and the serum glucocorticoid kinase (SGK) family of serine/threonine kinases, which promote cell survival.666,667 Many of the canonic functions of AKT with regard to cell growth and proliferation are mediated through the mTOR pathway. mTOR is a serine/threonine kinase and a member of the phosphatidylinositol kinase-related kinase family.668 mTOR is a component of two complexes, the rapamycin-sensitive mTORC1 and the rapamycininsensitive mTORC2 complexes.669 Within mTORC1, mTOR associates with Raptor (regulatory associated protein of mTOR) and mLST8, whereas the mTORC2 complex consists of mTOR, Rictor (rapamycin insensitive companion of mTOR), SIN1, and mLST8.670 In addition to its role in activating AKT as described earlier, mTORC2 also controls cytoskeletal changes through regulation of paxillin, Rho, Rac, and PKCα.671 mTORC1 activation is regulated in part by AKT phosphorylation of TSC2. TSC2 forms a heterodimeric complex with TSC1 that acts as a GTPase-activating protein (GAP) for the small GTPase Rheb, causing accumulation of inactive Rheb-GDP.672,673 TSC2 phosphorylation results in suppression of this GAP activity and subsequent activation and accumulation of Rheb-GTP, which then activates mTORC1. mTORC1 serves as a central nexus for integration of multiple extracellular signals, including oxygen and amino acid levels, growth factors, and stress. Based on these input signals, mTORC1 activity influences cellular growth, metabolism, protein synthesis, and cell cycle progression. One such example is the energy sensing mechanism of the cell comprised of LKB1 and AMPK.674 Increasing levels of AMP, a marker of decreased nutrient levels, results in AMPK phosphorylation and activation by LKB1. AMPK phosphorylates TSC2, which, as described earlier, inhibits mTORC1 activity, leading to downregulation of protein synthesis and cell growth in response to low nutrient levels.675 In part, mTORC1 activation regulates these phenotypes via phosphorylation and activation of p70S6 kinase and inhibition of 4EBP1. The former protein stimulates mRNA translation, whereas the latter inhibits translation of mRNA transcripts with a 5′ cap.676 Both mTORC1 and S6 kinase also participate in a negative feedback loop in which both proteins activate insulin receptor substrate 1 (IRS1), which results in inhibition of insulin-mediated PI3 kinase activation.677 AKT can also activate mTORC1 in a TSC2-independent manner via phosphorylation of PRAS40, a protein that interacts with mTORC1. The mechanisms responsible for PI3 kinase pathway activation in cancer are diverse and include activating mutations and amplification of PIK3CA, AKT1, AKT2, and AKT3, and mTOR; deletion or loss of PTEN expression or function; mutation in PIK3R1; loss of TSC1 or TSC2 function; RAS mutation; and dysregulated growth factor receptor and integrin activation, as outlined later.645,648,678 In human tumors, activation of PI3 kinase is frequently a direct consequence of dysregulated RTK signaling secondary to mutation, amplification, or ligand overexpression. For example, ERBB2 amplification in breast and gastric cancer results in AKT activation.27,678 Similarly, kinase domain mutations of EGFR induce constitutive AKT activation in lung cancers and glioblastomas, and AKT activity in these tumors

44 Part I: Science and Clinical Oncology

is critical for EGFR-mediating transformation.637,679 Oncogenic Ras mutations also activate PI3 kinase, and PI3 kinase activation is required for Ras-mediated tumorigenesis in genetically engineered mouse models.572,680–682 Mutations in the PIK3CA gene, which encodes the p110α catalytic subunit, are frequently observed in tumors of the colon, breast, brain, stomach, and ovary and other cancers.648,678,683 The most frequent are E542K and E545K, located in the helical domain (exon 9), and H1047R (exon 20), located in the kinase domain.684 All three mutants demonstrate increased lipid kinase activity, induce phosphorylation of AKT and its downstream effectors, and can transform chicken embryo fibroblasts.685 Exon 9 helical domain mutations block the inhibitory interaction between the p85 regulatory subunit and the p110 subunit and result in constitutive kinase activation.686 Exon 20 catalytic domain mutations result in constitutive kinase activation.678 PIK3CA mutations commonly co-occur with KRAS mutations, and ERBB2 amplification in colon and breast cancers, respectively, and expression of mutant PI3 kinase in breast cell lines as well as fibroblasts causes neoplastic transformation.687 Unlike wild-type p110α which lacks oncogenic potential, wild-type overexpression of the other three isoforms can induce transformation of cultured cells.688 PIK3R1 encodes the p85 regulatory subunit of PI3 kinase. Alterations in within this gene have also been reported in multiple cancers, including glioblastomas and colorectal, endometrial, and ovarian cancers.585,689 A recurrent PIK3R1 mutation, PIK3R1(R348∗), has been shown to have neomorphic functions resulting in activation of MEK and JNK and sensitivity to inhibitors of these effector kinases.690 Furthermore, partial loss of the PIK3R1 gene product p85α was demonstrated to increase the proportion of p110α/p85 heterodimers bound to active receptors, indicating that targeting p110α-selective inhibitors may be effective in the setting of PIK3R1 loss.691 Loss of PTEN function is the most frequently observed PI3 kinase/ AKT pathway alteration in human malignancies and is common in tumors of the prostate, breast, ovary, lung, colon, and bladder as well as melanomas and glioblastomas.678 Loss of PTEN function in tumors is mediated by a diversity of mechanisms including mutation, deletion, posttranslational modification, and promoter hypermethylation.678 Dysregulated expression of microRNAs that target the 3′-untranslated region of PTEN has also been shown to induce cell survival and cisplatin resistance.692 As AKT activation enhances proliferation and suppresses apoptosis, its activation would be predicted to have strong oncogenic function. Indeed, AKT was initially identified as a proto-oncogene in the mouse leukemia virus AKT8.693 A recurring hot spot mutation in the PH-domain of AKT1 (E17K) occurs with low frequency in breast, colorectal, bladder, endometrial, and ovarian cancers.694–696 This mutation results in constitutive localization to the plasma membrane without the need for PIP3 recruitment.684 Amplification of the AKT2 gene has been reported in ovarian and pancreatic cancers, and gain-of-function AKT3 mutations have been reported to occur in melanoma.697 Given the significant proportion of malignancies that harbor mutations in the PI3 kinase/AKT/mTOR pathway, a concerted effort is ongoing to identify selective inhibitors of various PI3 kinase pathway components. These agents can be categorized as pan-selective or selective PI3 kinase inhibitors, dual PI3 kinase/mTOR kinase inhibitors, AKT inhibitors, and mTOR inhibitors. Both isoform-specific and pan-selective inhibitors of PI3 kinase are being explored in several clinical trials across cancers. Most notably, the PI3Kδ inhibitor idelalisib is FDA approved for non-Hodgkin lymphoma and certain types of leukemia.698,699 Promising clinical responses have also been reported with α-selective PI3 kinase inhibitors in patients with several cancer types, most notably breast cancer (see Table 2.1).700–703 Although clinical activity with AKT inhibitors has been modest in patients, a pan-cancer basket trial of the ATP-competitive pan-AKT kinase inhibitor AZD5363 demonstrated significant clinical responses in patients with AKT1 E17K mutant tumors.704

Temsirolimus and everolimus, analogues of rapamycin that inhibit the mTORC1 complex, are FDA approved for use in patients with renal cell carcinomas, and everolimus is FDA approved for the treatment of patients with pancreatic neuroendocrine tumors (see Table 2.1).705–708 Everolimus also has significant clinical activity in patients with subependymal giant cell astrocytomas that arise in the setting of tuberous sclerosis, an inherited cancer-predisposition syndrome resulting from germline mutations in the TSC1 and TSC2 genes.709,710 In metastatic renal cell carcinoma, patients that benefited from treatment with everolimus more commonly harbored TSC1/2 or mTOR mutations (see Table 2.1).711 A complete response to everolimus has been reported in a patient with metastatic bladder cancer that harbored loss-of-function mutations in TSC1 and NF2, suggesting that such agents can induce significant antitumor responses in genetically selected patients.712 Additional responses to everolimus were observed in TSC1-mutant tumors but not to the degree of the complete responder, indicating that coaltered genes likely confer resistance to mTOR inhibitory therapy even in the setting of a canonic predictive biomarker of response. In sum, mTOR inhibitors are likely most effective when used in genetically defined patient populations, with the majority of patients requiring combination therapies because of the presence of coaltered genes. As an example of the latter, everolimus in combination with exemestane was FDA approved for the treatment of advanced hormone receptor– positive, HER2− breast cancer.713 Resistance to mTOR inhibition is thought to derive from multiple mechanisms, including activation of parallel signaling networks such as the MAP kinase pathway.714 Furthermore, inhibiting mTORC1 can preferentially lead to an increase in mTORC2 signaling, and, as described earlier, mTORC2 enhances AKT activation via phosphorylation of the serine 473 residue.715 As an alternative approach, potent mTOR kinase inhibitors are being developed, including RapaLink-1, and have shown significant preclinical activity in cell lines resistant to rapamycin and in in vivo models of glioblastoma.716,717

TRANSLATIONAL IMPLICATIONS As outlined earlier, mutational and epigenetic alterations induce constitutive activation of a broad array of signaling pathways in human tumors. In some instances, the growth and survival of tumor cells have been shown to be dependent on a single signaling pathway activated by a mutated oncogene or tumor suppressor, a phenomenon referred to as oncogene addiction.718 In such instances, targeted inhibitors of such pathways have demonstrated unprecedented clinical activity in molecularly defined subsets of patients (see Table 2.1). Examples include imatinib in patients with CML, erlotinib in patients with EGFR-mutant NSCLC, and vemurafenib in patients with BRAF-mutant melanoma.30,505,511,603,612,719 Despite these dramatic successes, the majority of cancer patients have yet to benefit from this approach. Potential explanations for this lack of benefit include the redundant regulation of key downstream mediators of transformation by multiple signaling pathways, the lack of specificity of the drug for the driver alteration, and intrinsic and acquired drug resistance due to second site mutations in the target gene or by other mechanisms. Progress in this field has also been delayed by the continued practice of performing clinical trials of targeted inhibitors in unselected patient populations. Recent advances in sequencing methodology have made it feasible to now prospectively sequence all patients with advanced cancer with the goal of identifying potentially “actionable” genomic alterations.720 Such prospective sequencing efforts have highlighted several challenges that have slowed the application of targeted inhibitors in cancer patients. As one example, most mutations, even in well-characterized cancer genes, are likely inert passenger mutations. Furthermore, not all activating mutations respond similarly to targeted inhibitors. For example, exon 19 EGFR deletions are sensitive to the EGFR kinase inhibitor erlotinib, whereas exon 20 insertions are intrinsically resistant.721,722 Given the large number of somatic mutations present

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in each human tumor, and the variable biologic and clinical significance of individual mutant alleles, there is an urgent need for clinical support tools that will aid clinicians in interpreting molecular tumor profiling and guiding treatment selection. A second challenge is that many oncogenic alterations are rare. To address this challenge, novel clinical trial designs have been formulated to test the efficacy of targeted inhibitors in patients with defined molecular events independent of the primary site of disease. Eligibility for these “basket” studies is based on the presence of a particular molecular alteration (e.g., BRAF V600E, AKT1 E17K, or an NTRK fusion) rather than site of tumor origin.268,273,704,723 Although promising results from such trials highlight the importance of tumor mutational profile in dictating drug sensitivity, lineage-specific differences also play a role in determining clinical response to inhibitors of activated signaling pathways. As an example, most BRAF V600E melanomas

respond to vemurafenib, whereas colorectal tumors with the same mutation are intrinsically resistant. Resistance in the latter results from rapid adaptation of the cancer cell resulting in activation of EGFR.724 This phenomenon of adaptive resistance, defined as a rapid reactivation of parallel signaling pathways after relief of negative feedback signals, likely abrogates the effects of selective pathway inhibitors in many cancer types.592,725,726 Because significant drug development efforts are currently focused on the development of targeted inhibitors of oncogene-activated signaling pathways, a detailed understanding of normal physiologic pathways and their dysregulation in cancer will be critical for the optimal development and clinical application of targeted kinase inhibitors. The complete reference list is available online at ExpertConsult.com.

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46.e12 Part I: Science and Clinical Oncology

SELF-ASSESSMENT REVIEW QUESTIONS

ANSWERS

1. Next-generation sequencing has helped reveal numerous unusual genomic events, including chromosomal translocations. Which of the following genes is NOT found as oncogenic fusion partners in chromosomal re-arrangements? a. ABL b. PTEN c. PDGFRβ d. ALK e. NTRK 2. Which of the following is true of Hedgehog (SHH) signaling and targeted therapies? a. Secreted hedgehog ligands bind Smoothened receptors, which causes receptor dimerization and autophosphorylation of intracellular tyrosine residues. b. Erlotinib and gefitinib are FDA approved Smoothened receptor inhibitors. c. Sonic hedgehog (SHH) binds to the receptor Patched (PTCH), which relieves its repression of Smoothened (SMO). d. Mutations in SHH, PTCH, and SMO are found in patients with small cell lung cancer. e. Smoothened receptors couple directly to actin. 3. The Ras/MAP kinase and PI3 kinase/mTOR signaling cascades are parallel but interconnected pathways that are frequently mutated in human cancer. Which of the following has NOT been an effective approach in targeting these pathways? a. Allosteric inhibitors of MEK kinase b. Small-molecule inhibitors and kinase of mTOR c. Monoclonal antibodies that bind to EGFR d. Farnesyltransferase inhibitors of Ras e. Kinase inhibitors of mutant BRAF 4. Acquired resistance commonly occurs after treatment with targeted inhibitors. Which of the following are known mechanisms of resistance? a. Parallel pathway upregulation b. Gatekeeper mutation c. Amplification of specific RTKs d. Alternative splicing or amplification of the driver oncogene e. All of the above 5. Which of the following statements is FALSE? a. Chromosomal rearrangement of BCR and ALK to generate the Philadelphia chromosome is the driving event in CML. b. Recurrent somatic splice site alterations involving MET exon 14 (METex14) have been identified in lung cancer. c. Integrin receptors perform inside-out and outside-in signaling at the integrin adhesome. d. ESR1 mutations are a common mechanism of acquired resistance to hormonal therapy in patients with breast cancer. e. Extracellular and transmembrane FGFR3 mutations are recurrent in noninvasive and high-grade invasive bladder cancer.

1. (b) PTEN is a tumor suppressor of the AKT pathway and would not be a likely candidate fusion partner, because truncating mutations or deletions confer loss of PTEN function. All of the other genes listed have been found in patients with a variety of fusions partners. BCR-ABL, the Philadelphia chromosome, which is found in nearly all cases of chronic myelogenous leukemias (CML) and one-third of ALLs, is the classic example of an oncogenic fusion driving cancer. EML4-ALK fusions play a driving role in non–small cell lung cancer (NSCLC). COL1A1-PDGFRβ fusions occur in dermatofibrosarcoma protruberans, a rare sarcoma. TPM3-TRKA fusions were first identified in colon cancer. 2. (c)  The Smoothened receptor is a GPCR, not a receptor tyrosine kinase, and therefore it does not get activated by dimerization and transphosphorylation. Vismodegib and sonidegib are FDA-approved smoothened receptor inhibitors. Mutations in SHH, PTCH, and SMO are found in patients with inherited and sporadic basal cell carcinomas. Smoothened receptors couple to the G proteins Gαi and Gα12. 3. (d)  No direct inhibitors of Ras are available for clinical use. Farnesyltransferase inhibitors were tested but were found to be ineffective in tumors in which Ras mutations are common, likely because of the ability of geranylgeranyl modifications to substitute for farnesylation in effectively targeting Ras to the membrane. New drug development of allosteric inhibitors that specifically target KRAS G12C is underway. Trametinib is an FDA-approved allosteric inhibitor of MEK. Rapamycin and its analogues, as well as novel kinase inhibitors such as RapaLink, are efficacious mTOR inhibitors. Cetuximab and panitumumab are anti-EGFR monoclonal antibody therapies. Vemurafenib, an ATP-competitive inhibitor of BRAF, is approved for the treatment of patients with BRAF V600E mutant melanoma. 4. (e)  Several mechanisms of resistance to kinase inhibitors have emerged. For example, patients with BRAF V600E mutations that are treated with the specific RAF inhibitor vemurafenib often develop resistance mediated by BRAF V600E splice variants or amplification; loss or mutation of NF1; parallel pathway activation of RTKs; mutation of PI3K/AKT components; mutations in NRAS, RAF1 and MAP2K1/2 (MEK1/2); and amplification of MITF. 5. (a)  Translocation of the ABL gene on chromosome 9 with the breakpoint cluster region (BCR) gene on chromosome 22 results in the expression of a BCR-ABL fusion protein, called the Philadelphia chromosome. This event is the pathognomonic molecular lesion in almost all chronic myelogenous leukemias (CML). It is also found in approximately one-third of acute lymphoblastic leukemias (ALLs). The ABL tyrosine kinase inhibitor imatinib, as well as four other ABL inhibitors, are FDA approved in CML; imatinib and dasatinib are approved for ALL.