mTOR Inhibition With Chemotherapy

C H A P T E R

14 Combining PI3K/Akt/mTOR Inhibition With Chemotherapy Albert A. De Vera*, Sandra E. Reznik*,†,‡ *

Department of Pharmaceutical Sciences, College of Pharmacy and Health Sciences, St. John’s University, Queens, NY, United States † Department of Pathology, Albert Einstein College of Medicine, Bronx, NY, United States ‡ Department of Obstetrics and Gynecology and Women’s Health, Albert Einstein College of Medicine, Bronx, NY, United States

Abstract Since the discovery of rapamycin in the 1970s, much has been learned of the role of the PI3K/Akt/mTOR axis in cell survival. This vital pathway plays a central role in cell metabolism, growth, differentiation, proliferation, and survival in physiological and pathological conditions. It serves as a signaling pathway that is activated by growth factor receptor tyrosine kinases, G-protein coupled receptors, cytokine receptors, and integrin receptors. Dysregulation of this pathway has been associated with diseases such as diabetes, autoimmune diseases, and cancer. This pathway is one of the most frequently altered pathways in cancer, enabling cancer cells to survive and proliferate under stressful conditions. The PI3K/Akt/mTOR and insulin-like growth factor (IGF) signaling pathways have been shown to be hyperactivated in response to chemotherapy. Many cancers have been associated with aberrant IGF signaling, including prostate cancer, melanoma, osteosarcoma, pancreatic cancer, colon cancer, and childhood cancers. Activation of the PI3K/Akt/mTOR pathway has been associated with resistance to chemotherapeutic agents doxorubicin, daunorubicin, etoposide, cisplatin, and paclitaxel. Targeting the PI3K/ Akt/mTOR signaling axis in cancer has shown promising results in preclinical studies in which combining PI3K/ Akt/mTOR inhibitors with standard chemotherapy has been shown to attenuate chemotherapy resistance. In vitro and in vivo studies have led to clinical trials and FDA approval of rapalogs in the treatment of specific types of cancer. Because monotherapeutic strategies of signal-targeting or conventional chemotherapy frequently lead to resistance, numerous phase-I and phase-II clinical trials are underway combining PI3K/Akt/mTOR inhibitors with conventional chemotherapy. This chapter outlines the rationale and current status of clinical trials combining PI3K/Akt/mTOR inhibitors with conventional chemotherapy.

Abbreviations 4E-BP 4E binding protein AGC kinase family AMP/GMP kinases and protein kinase C Akt protein kinase B

Protein Kinase Inhibitors as Sensitizing Agents for Chemotherapy https://doi.org/10.1016/B978-0-12-816435-8.00014-6

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# 2019 Elsevier Inc. All rights reserved.

230 eIF4E EUDRACT FDA FKBP12 FOLFOX6 GBM GEJ HCC HER HER2/neu HNSCC IGF-1 IGF-1R IRS-1 LAM LST8 mLST8 mSIN1 mTOR mTORC1 mTORC2 Nab NCT NEC PDK-1 PI3K PIKK PIP2 PIP3 PKB PKC PNET PRAS40 Protor 1/2 PTEN Raptor RCC Rheb Rictor RTK S6K1 SEGA SGK-1 TACE TNBC TOP TSC1/TSC2 VEGFRs

14. COMBINING PI3K/AKT/mTOR INHIBITION WITH CHEMOTHERAPY

eukaryotic initiation factor 4E European Clinical Trials Database U.S. Food and Drug Administration FK506-binding protein 12 folinic acid/5-fluorouracil/oxaliplatin glioblastoma multiforme gastroesophageal junction hepatocellular carcinoma human epidermal growth factor receptor human epidermal growth factor receptor-2/proto-oncogene neu head and neck squamous cell cancer insulin-like growth factor-1 insulin-like growth factor receptor insulin receptor substrate-1 lymphangioleiomyomatosis lethal with SEC13 protein 8 mammalian lethal with SEC13 protein 8 mammalian stress-activated map kinase-interacting protein 1 mammalian target of rapamycin mammalian target of rapamycin complex 1 mammalian target of rapamycin complex 2 nanoparticle albumin-bound National Clinical Trials neuroendocrine carcinomas phosphoinositide-dependent kinase-1 phosphoinositide 3-kinase phosphoinositol kinase-related protein kinase phosphatidylinositol-4,5-bisphosphate phosphatidylinositol-3,4,5-triphosphate protein kinase B or Akt protein kinase C progressive neuroendocrine tumors proline-rich Akt1 substrate 40 protein observed with Rictor 1 and Rictor 2 phosphatase and tensin homolog regulatory-associated protein of mTOR renal cell carcinoma Ras homolog enriched in brain rapamycin-independent companion of mTOR receptor tyrosine kinase p70S6 kinase subependymal giant cell astrocytoma glucocorticoid-regulated kinase 1 transarterial chemoembolization triple-negative breast cancer 50 -terminal oligopyrimidine tract tuberous sclerosis complex 1 and 2 vascular endothelial growth factor receptors

Conflict of Interest No potential conflicts of interest were disclosed.

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INTRODUCTION The PI3K/Akt/mTOR signaling pathway plays a central role in cell metabolism, growth, differentiation, proliferation, and survival in physiological and pathological conditions. It serves as a signaling pathway that is activated by growth factor receptor tyrosine kinases, G-protein coupled receptors, cytokine receptors, and integrin receptors. Dysregulation of this pathway has been associated with diseases such as diabetes, autoimmune diseases, and cancer. This pathway is one of the most frequently altered pathways in cancer, enabling cancer cells to survive and proliferate under stressful conditions. Aberrant activation of this pathway has also been implicated in resistance to chemotherapy. Preclinical studies combining PI3K/Akt/mTOR inhibitors with standard chemotherapy have been shown to attenuate chemotherapy resistance. Numerous clinical trials combining PI3K/Akt/mTOR inhibitors with chemotherapy are currently underway. This chapter describes the rationale and strategies of combining PI3K/Akt/mTOR signal-targeted inhibitors with conventional chemotherapy.

PI3K/AKT/mTOR SIGNALING Phosphoinositide 3-kinases (PI3Ks) are a family of lipid kinase proteins that phosphorylate the 30 -OH group of inositol phospholipids. PI3Ks are divided into three classes (I–III) which vary in structure and substrate specificity. Mutations or alterations of the class-IA subgroup are frequently found in cancer [1–5]. Class-IA PI3Ks are composed of catalytic (p110) and regulatory (p85) subunits of four isoforms (alpha, beta, gamma, and delta). The alpha, beta, and delta isoforms are activated by receptor tyrosine kinases (RTKs). The class-IB gamma isoform is activated by G-protein coupled receptors. Activation of growth factor receptor tyrosine kinases, such as insulin-like growth factor-1 (IGF-1), human epidermal growth factor receptor (HER), and vascular endothelial growth factor receptors (VEGFRs) triggers autophosphorylation of tyrosine residues [6–9]. PI3K is recruited to the cell membrane by binding to the phosphotyrosine residues. PI3K activation leads to the phosphorylation of substrate phosphatidylinositol-4,5-bisphosphate (PIP2) into phosphatidylinositol-3,4,5triphosphate (PIP3). PIP3 then recruits phophoinositide-dependent kinase-1 (PDK-1) and protein kinase B(PKB)/Akt to the cell membrane for activation (Fig. 1). Akt is a family of highly conserved cellular homologues, AKT1, AKT2, and AKT3. Akt kinases are serine/threonine kinases of the AGC kinase family (AMP/GMP kinases and protein kinase C (PKC)). Akt controls cellular transcription, translation, cell cycle progression, apoptosis, autophagy, and metabolism [10, 11]. Akt is recruited to the cell membrane by PI3K production of PIP3. Akt becomes phosphorylated and partially activated by PDK-1 in its activation loop on threonine 308 (T308). Akt becomes fully activated after phosphorylation at serine 473 (S473) of its regulatory domain. Activated Akt phosphorylates and inhibits the tuberous sclerosis complex 1 and 2 (TSC1/TSC2). Inhibition of TSC1/TSC2 by Akt allows the GTPase of Ras homolog enriched in brain (Rheb) to switch from the inactive GDP state to the active GTP state and activates the regulatory-associated protein of mTOR (Raptor) of the mTORC1 complex [12].

232

14. COMBINING PI3K/AKT/mTOR INHIBITION WITH CHEMOTHERAPY RTK

IGF-1

IRS-1

PI3K PIP3 PIP2 PTEN

mTOR inhibitors Rapamycin (sirolimus)

PI3K inhibitors Buparlisib (BKM120)

Everolimus (RAD001)

Pictilisib (GDC-0941)

Temsirolimus (CCI779)

Copanlisib (BAY80-6946)

Ridaforolimus (MK8669)

Alpelisib (BYL719)

S6

P70S6K

mTOR

PIP3 PTEN

T308

P Akt

PIP2

Dual PI3K/mTOR inhibitors

Akt inhibitors

Dactolisib (BEZ235)

Ipatasertib (GDC-0068)

Voxtalisib (XL765, SAR245409)

MK-2206

P S473 Rictor

TSC2/ TSC1

mLST8

mTOR

mTORC2

Proctor

Raptor PRAS40

IRS-1

PI3K PDK1

mSIN1

mTORC1 Rheb

Dual mTORC1/mTORC2 inhibitors Vitusertib (AZD2014)

eIF4E

4EBP1

mLST8

Sapanisertib (MLN0128, TAK-228)

FIG. 1 Red lines represent inhibition. Green arrows represent activation.

The mammalian or mechanistic target of rapamycin (mTOR) is an atypical serine/threonine protein kinase that acts as a central hub of signal integration [13]. It is found in two large multiprotein complexes referred to as mTORC1 and mTORC2. mTORC1 controls anabolic pathways, including protein synthesis, ribosome production, lipid synthesis, and nucleotide synthesis needed for cell growth, proliferation, and survival. mTORC1 also suppresses the production and activation of lysosomes and their catabolic autophagic effects. The mTORC1 complex includes mTOR, mammalian lethal with SEC13 protein 8 (LST8), proline-rich Akt1 substrate 40 (PRAS40), and regulatory-associated protein of mTOR (Raptor) [14]. Upon activation of Raptor by Akt, mTORC1 phosphorylates downstream the effector ribosomal protein p70S6 kinase (S6K1) and the eukaryotic initiation factor 4E (eIF4E) inhibitory proteins (4E-BPs). Activation of S6K1 leads to translation of mRNAs containing 50 -terminal oligopyrimidine tract (TOP), while phosphorylated inhibition of 4E-BPs allows ribosomal release of eIF4E to allow binding of mRNAs to the 40S ribosomal subunit for translation [14]. Much less is known about mTORC2 signaling and activation compared with mTORC1. mTORC2, unlike mTORC1, is not directly activated by Akt phosphorylation of the TSC1/ TSC2 complex. mTORC2 was initially thought to be rapamycin resistant, but long-term treatment with rapamycin was shown to inhibit mTORC2 signaling [15]. mTORC2 has also been shown to act as the kinase of Akt serine-473 in Akt activation, placing mTOR both upstream and downstream of Akt [15a]. mTORC2 is made of mTOR, mLST8, mammalian stress-activated map kinase-interacting protein 1 (mSIN1), protein observed with Rictor 1 and Rictor 2 (Protor 1/2), and rapamycin-independent companion of mTOR (Rictor), [16]. mTORC2 controls the actin cytoskeletal functions of the cell and cell survival [17]. Downstream effectors of mTORC2 include Akt (protein kinase B), glucocorticoid-regulated kinase 1 (SGK-1), and PKC, [11].

PI3K/AKT/mTOR PATHWAY ACTIVATION AND CHEMOTHERAPY RESISTANCE

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FEEDBACK LOOPS: S6K AND PTEN In order to maintain normal cell growth and homeostasis, negative feedback loops have evolved to control excessive signal activation. S6K is the most researched downstream effector of mTOR1 acting as a feedback mechanism to the pathway. After phosphorylation and activation by mTORC1, S6K is recruited to the membrane where it phosphorylates IRS-1 at multiple sites to prepare it for proteosomal degradation and subcellular relocalization to stop pathway activation [18]. The phosphatase and tensin homolog (PTEN) acts as an upstream regulator to the PI3K/Akt/ mTOR pathway. PTEN dephosphorylates PIP3 back to PIP2 to terminate signaling. Mutations and deletions of PTEN, which allow aberrant signal activation, are frequently found in cancer [19].

PI3K/AKT/mTOR PATHWAY ACTIVATION AND CHEMOTHERAPY RESISTANCE The PI3K/Akt/mTOR and IGF signaling pathways have been shown to be hyperactivated in response to chemotherapy and the pathways have been implicated in chemotherapy resistance [20]. In a brief summary of the signaling pathway, the insulin-like growth factor receptor (IGF1R) is activated through autophosphorylation by IGF ligand binding which then phosphorylates insulin receptor substrate 1 (IRS-1) [20]. Phosphorylation of IRS-1 activates PI3K which phosphorylates substrate PIP2 into second messenger PIP3. PIP3 phosphorylates and activates Akt. Akt is also partially activated by mTORC2. Activation of Akt leads to phosphorylation and inhibition of the TSC2/TSC1 complex. Inhibition of TSC2/TSC1 by Akt allows activation of mTORC1. mTORC1 activates downstream the effector ribosomal protein S6 kinase (S6K) which serves as a feedback messenger to IRS1 by causing phosphorylation of IRS1 at multiple sites to inactivate it by proteosomal degradation. In a parallel signaling pathway, IGF-1R also promotes cell proliferation by activation of the Ras/ MAPK/ERK pathway [9]. Many cancers have been associated with aberrant IGF signaling, including prostate cancer, melanoma, osteosarcoma, pancreatic cancer, colon cancer, and childhood cancers [21]. Aberrant signaling of IGF leads to activation of downstream PI3K/Akt/mTOR signaling. Activation of the PI3K/Akt/mTOR pathway has been associated with resistance to chemotherapeutic agents doxorubicin and daunorubicin (anthracyclins), etoposide (a topoisomerase inhibitor), cisplatin (a DNA damaging agent), and paclitaxel (a microtubule inhibitor) [22]. This opened the door to strategies of combining signal-targeted therapy of PI3K/Akt/mTOR inhibitors with traditional cytotoxic chemotherapeutic agents [23]. In vitro and in vivo preclinical results have led to clinical trials and FDA approval of rapalogs in the treatment of specific types of cancer. Numerous phase-I and phase-II clinical trials are underway, and this review outlines the rationale and current status of clinical trials combining PI3K/Akt/mTOR inhibitors with conventional chemotherapy.

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PI3K/AKT/mTOR INHIBITORS AS CHEMOSENSITIZERS Rapamycin and the Rapalogs mTOR proteins are evolutionarily conserved serine/threonine kinases of the phosphoinositol kinase-related protein kinase (PIKK) family. Rapamycin (sirolimus) and the rapamycin analogs, or rapalogs (everolimus, temsirolimus, ridaforolimus), are the first generation of mTOR inhibitors. Rapamycin was serendipitously discovered in 1965 during a Canadian Medical Mission expedition to the island of Rapa Nui (Easter Island) in the southeastern Pacific Ocean. The bacterium Streptomyces hygroscopicus was isolated in a soil sample and was found to produce the secondary metabolite, rapamycin, first isolated in the 1970s [24]. Preclinical studies found rapamycin to have potent antifungal, antitumor, and immunosuppressive activity [25, 26]. Rapamycin acts as an allosteric inhibitor of mTOR by forming a complex with the immunophilin FK506-binding protein 12 (FKBP12) which then binds to a site on the N-terminal side of the kinase domain. In vitro studies have shown rapamycin to have synergistic effects in combination with paclitaxel, carboplatin, and vinorelbine and additive effects with doxorubicin and gemcitabine. Rapamycin also enhanced the sensitivity to paclitaxel and carboplatin in resistant HER2/neu-overexpresssing cells. In vivo, rapamycin combined with paclitaxel significantly reduced tumor size compared to either agent alone in rapamycinsensitive cells [27]. Rapamycin has been shown to resensitize paclitaxel-resistant cervical cancer cells to paclitaxel in vitro [28]. In xenograft models rapamycin combined with cisplatin significantly inhibited growth of esophageal squamous cell carcinoma tumors [29]. Sirolimus (Rapamycin, Rapamune) has been approved by FDA as an immunosuppressant to prevent organ rejection in kidney transplant patients and as an eluting agent in stents for use in coronary angioplasty procedures. It has also been approved by FDA for the treatment of lymphangioleiomyomatosis (LAM), a condition of the lungs, kidneys, and lymphatic system (fda.gov). It is currently under clinical trials in combination with mitoxantrone, etoposide, and cytarabine for high-risk acute myelogenous leukemia [National Clinical Trials (NCT) 01184898, NCT00780104], with docetaxel for advanced stage malignancies (NCT01054313), with vinblastine in pediatric patients with recurrent or refractory solid tumors (NCT01135563), and with gemcitabine and cisplatin for high-risk cholangiocarcinoma after liver transplant or surgery (NCT01888302). Because of rapamycin’s poor water solubility limiting its bioavailability, rapalogs as prodrugs of rapamycin were developed. Rapalogs (everolimus, temsirolimus, ridaforolimus) are water soluble and have been approved by FDA for treating specific cancers and noncancerous conditions. Everolimus (RAD001) is an orally administered rapamycin derivative with an O-(2hydroxyethyl) chain at the C-40 position which becomes converted to rapamycin. In human xenograft models of prostate cancer in mice, combination of everolimus with docetaxel significantly reduced tumor volume compared to either agent alone [30]. In similar human xenograft prostate cancer models, everolimus alone or in combination with docetaxel significantly suppressed tumor growth and tumor vasculature [31]. Everolimus in combination with cisplatin significantly suppressed cell proliferation and increased cell apoptosis in hepatocellular carcinoma (HCC) cells in vitro and increased apoptosis in tumor tissues in vivo [32].

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Unresectable HCC is commonly treated by transarterial chemoembolization (TACE) in which embolic particles coated with chemotherapeutic agents such as cisplatin are injected into the hepatic artery. This blocks the tumor blood supply and delivers the chemotherapeutic drug directly to the tumor avoiding systemic side effects. Everolimus in combination with cisplatin in TACE enhanced the chemotherapeutic efficacy of TACE by inhibiting cell proliferation, promoting apoptosis, and inhibiting angiogenesis in vitro and inhibited tumor growth and neoangiogenesis in vivo [33]. Everolimus has been approved by FDA for the treatment of advanced renal cell carcinoma, subependymal giant cell astrocytoma (SEGA), advanced hormone receptor-positive, HER2negative breast cancer, and progressive neuroendocrine tumors of pancreatic origin (PNET) (cancer.gov). A phase-II clinical trial combining everolimus with carboplatin for patients with triple negative metastatic breast cancer was found to be well tolerated with continuing patient responses ([34]; NCT01127763). Everolimus is currently under numerous clinical trials in combination with chemotherapeutic agents including phase-I/II trials with cisplatin for metastatic or unresectable neuroendocrine carcinomas (NEC) of extrapulmonary origin (NCT02695459), with liposomal doxorubicin for localized triple-negative breast cancer (TNBC), (NCT02456857), with temozolomide for advanced gastroenteropancreatic NEC (NCT02248012), with doxorubicin for liver metastases from gastroenteric endocrine tumors (NCT01678664), and with 5-fluorouracil, leucovorin, and oxaliplatin for metastatic stomach or esophageal cancer (NCT01231399). Temsirolimus (CCI779, Torcel) is a dihydroxymethyl propionic acid C-40 ester rapamycin prodrug, allowing greater water solubility, intravenous administration, and rapid conversion to rapamycin upon injection. It has been approved by FDA for the treatment of advanced renal cell carcinoma (cancer.gov). Temsirolimus in combination with docetaxel or 5-fluorouracil showed greater efficacy in human prostate or breast cancer cells, respectively, than either agent alone in in vitro or in vivo models [35]. Temsirolimus in combination with cisplatin showed synergistic inhibition of mTOR downstream signaling, increased growth inhibition, and increased apoptosis in vitro of human malignant pleural mesothelioma (MPM) cells. Temsirolimus also displayed antitumor activity in xenograft mouse models of MPM both as a single agent and in combination with cisplatin [36]. Temsirolimus is currently under numerous phase-I/II clinical trials in combination with chemotherapeutic agents such as with capecitabine for advanced stage cancers (NCT01050985), with paclitaxel and carboplatin for recurrent or metastatic head and neck squamous cell cancer (HNSCC), (NCT01016769), with docetaxel for prostate cancer (NCT01206036), with vinorelbine for unresectable or metastatic solid tumors (NCT01155258), with irinotecan for resistant metastatic colorectal cancer (NCT00827684), and with irinotecan and temozolomide for relapsed or refractory solid tumors (NCT01141244). Ridaforolimus (MK8669, AP23573, formerly deforolimus) is a C-40 phosphine oxide substituted parental nonprodrug formulation of rapamycin which is in the development for the treatment of metastatic soft tissue or bone sarcomas. A phase-IB trial of ridaforolimus combined with capecitabine showed good tolerability and antitumor activity [[37]; European Clinical Trials Database (EUDRACT) 200400296056]. Another phase-IB trial of ridaforolimus combined with paclitaxel showed antiangiogenic effects and antitumor activity [38]. Under phase-I clinical trials, ridaforolimus in combination with carboplatin and paclitaxel for endometrial, ovarian, and other solid tumors showed no unanticipated toxicities and had anticancer activity ([39]; NCT01256268).

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PI3K Inhibitors PI3K inhibitors are divided into pan-PI3K inhibitors (buparlisib, pictilisib, copanlisib) and isoform-specific PI3K inhibitors (alpelisib). Pan-PI3K inhibitors target all four isoforms (alpha, beta, gamma, and delta) of the class-IA PI3K p110 catalytic subunit. Buparlisib (BKM120) is one of the furthest developed of PI3K inhibitors in combination with chemotherapeutic agents. It is a pan-PI3K inhibitor also found to inhibit VEGF-induced neovascularization in vivo, suggesting antiangiogenic effects [40]. In vitro buparlisib decreases the cellular levels of phosphorylated Akt in cancer cell lines. In vivo buparlisib combined with temozolamide or docetaxel caused robust tumor shrinkage in glioblastoma multiforme (GBM) and prostate cancer tumors, respectively [40]. Numerous phase-I/II clinical trials have been completed or are currently underway combining buparlisib with chemotherapeutic agents (Table 1, clinicaltrials.gov). TABLE 1 Buparlisib (BKM120) Combination Strategies Clinical Trial Number

Intervention + Buparlisib

Condition

Phase

NCT02194049

Cisplatin + etoposide

Advanced solid tumors or small-cell lung cancer

Phase I

NCT01934361

Carboplatin or lomustine

Recurrent glioblastoma multiforme

Phase Ib/II

NCT02439489

Cisplatin or carboplatin

Advanced solid tumors

Phase I

NCT01852292

Paclitaxel

Recurrent or metastatic head and neck cancer previously pretreated with a platinum therapy

Phase II

NCT02113878

Cisplatin and radiotherapy

High-risk locally advanced squamous cell cancer of head and neck

Phase I

NCT01816594

Trastuzumab + paclitaxel

HER2-positive primary breast cancer

Phase II

NCT02000882

Capacitabine + trazstuzumab

Metastatic breast cancer with brain metastasis

Phase II, recruiting subjects

NCT01723800

Carboplatin + pemetrexed

Stage-IV nonsmall-cell lung cancer

Phase I

NCT01571024

mFOLFOX6 (oxaliplatin + leucovorin + 5fluorouracil)

Advanced solid tumors including metastatic pancreatic cancers

Phase I

NCT01540253

Docetaxel

Advanced solid tumors, locally advanced, that cannot be removed by surgery or are metastatic

Phase I

NCT01473901

Radiotherapy and temozolomide

Newly diagnosed glioblastoma

Phase I

NCT01304602

Irinotecan

Previously treated advanced colorectal cancer

Phase I

NCT01297452

Carboplatin + paclitaxel

Advanced solid tumors

Phase I

NCT01285466

Dactolisib (BEZ235) + paclitaxel with or without trastuzumab

Determine the maximum tolerated dose

Phase I

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Pictilisib (GDC-0941), a pan-PI3K inhibitor, has been tested in combination with docetaxel in vitro and in vivo in human breast cancer cells. Pictilisib enhanced the antitumor activity of docetaxel in xenograft models and increased the rate of apoptosis with docetaxel cotreatment [41]. Results are pending for pictilisib in clinical trials combined with paclitaxel for metastatic breast cancer (NCT01740336, NCT00960960), and with either paclitaxel, carboplatin, or cisplatin for advanced nonsmall-cell lung cancer (NCT00974584, NCT01493843). Copanlisib (BAY80-6946) is a pan-PI3K inhibitor with preferential inhibitory activity against both PI3K-alpha and PI3K-delta isoforms. In a first-in-human phase-I study in patients with advanced solid tumors and non-Hodgkin’s lymphomas, copanlisib as monotherapy achieved complete and partial responses for endometrial carcinoma, metastatic breast cancer, follicular lymphoma, and diffuse large B-cell lymphoma ([42]; NCT00962611). In a phase-I study on the treatment of advanced solid tumors, copanlisib combined with either gemcitabine alone or gemcitabine plus cisplatin showed favorable responses, most notably in two patients with biliary tract cancer ([43]; NCT01460537). This led to a further study now recruiting patients for copanlisib in combination with gemcitabine and cisplatin to treat advanced cholangiocarcinoma (NCT02631590). Results are pending for copanlisib in combination with paclitaxel for advanced breast cancer (NCT01411410). A phase-III study is now recruiting for copanlisib in combination with standard immunochemotherapy (rituximab/bendamustine) and rituximab with cyclophosphamide, doxorubicin, vincristine, and prednisone/prednisolone (R-CHOP) in patients with relapsed indolent non-Hodgkin’s lymphoma (NCT02626455). Alpelisib (BYL719), a PI3K alpha-specific inhibitor, was developed with the intention of avoiding the side effects of pan-PI3K inhibitors, such as hyperglycemia. It has been tested in combination with ifosfamide (mafosfamide for in vitro experiments), a conventional chemotherapeutic agent in the treatment of osteosarcoma. The addition of alpelisib had a significant inhibitory effect on tumor growth as compared to mafosfamide alone [44]. Results are pending for alpelisib tested with paclitaxel in the treatment of breast cancer and head-andneck cancer (NCT02051751). There are several active studies and studies in recruitment using alpelisib in combination with capecitabine for metastatic breast cancer (NCT01300962), with nanoparticle albumin-bound (Nab)-paclitaxel for metastatic HER2-negative breast cancer (NCT02379247), with gemcitabine and Nab-paclitaxel for metastatic pancreatic cancer (NCT02155088), with capecitabine and radiation for rectal cancer (NCT02550743), with cetuximab and cisplatin for oropharyngeal squamous cell carcinoma (NCT02298595), and with cisplatin and radiation for squamous cell head and neck cancer (NCT02537223).

Dual PI3K/mTOR Kinase Inhibitors Dual PI3K/mTOR kinase inhibitors (dactolisib, voxtalisib) represent the third generation of PI3K/mTOR inhibitors. They were developed with the intention of overcoming the activation of the IRS-1/PI3K/Akt response to loss of mTORC1/S6K feedback caused by the rapalogs and to cause more complete inhibition of the pathway at multiple signaling points. Dual PI3K/mTOR inhibitors inhibit the catalytic subunits of both PI3K and mTOR. Dactolisib (BEZ235), a dual PI3K/mTOR kinase inhibitor, has been tested in vitro and in vivo with doxorubicin in the treatment of leiomyosarcoma. Dactolisib alone caused a 42% decrease in tumor volume while dactolisib in combination with doxorubicin caused a

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68% decrease in tumor volume [45]. Dactolisib was shown to provide additive antiproliferative effects in combination with nab-paclitaxel in experimental gastric cancer cells and extended median animal survival in xenograft models [46]. Dactolisib combined with cisplatin or carboplatin induced synergistic antitumor effects in cervical cancer cells in vitro [47]. Dactolisib in combination with docetaxel inhibited growth of xenograft prostate cancer models compared to monotherapy of either agent alone [48]. Dactolisib in combination with temozolomide synergistically inhibited glioma cell growth and induced apoptosis, and in xenograft in vivo models the combination significantly reduced tumor growth rates and prolonged median survival [49]. Results are pending for dactolisib tested in phase-I/II clinical trials in combination with paclitaxel and trastuzumab (HER2 antibody) for metastatic or locally advanced solid tumors and HER2-positive metastatic or locally advanced breast cancer (NCT01285466), and a phase-Ib/II trial of dactolisib with paclitaxel for locally advanced or metastatic breast cancer (NCT01495247). Voxtalisib (XL765, SAR245409), a dual-PI3K/mTOR kinase inhibitor, is able to efficiently penetrate the blood-brain-barrier. The combination of voxtalisib with temozolomide caused a 16-fold decrease of intracranial GBM xenograft tumor size compared to temozolomide alone, and increased the survival times in mouse models [50]. Voxtalisib and temozolomide synergistically inhibited growth and induced apoptosis of pituitary adenoma cells in vitro, and synergistically inhibited tumor growth in vivo [51]. In a phase-I dose-escalation trial, voxtalisib in combination with temozolomide had a favorable safety profile while inhibiting the PI3K/mTOR pathway with and without radiation in adults with malignant gliomas ([52]; NCT00704080).

Dual mTORC1/mTORC2 Inhibitors Catalytic mTORC1/2 kinase inhibitors (vitusertib, sapanisertib) inhibit the catalytic subunit of both mTORC 1 and 2 and were developed with the goal of suppressing the activation of Akt by mTORC2 caused by rapalog inhibition of mTORC1. Vitusertib (AZD2014), an mTORC1/2 kinase inhibitor, showed higher efficiency than allosteric mTORC1 inhibitor rapamycin in inhibiting renal cell carcinoma (RCC) cell survival and growth. Vitusertib showed higher efficiency in inhibiting RCC xenograft growth than rapamycin in preclinical models [53]. Vitusertib is currently under phase-II clinical trials in combination with paclitaxel for nonsmall-cell lung cancer (NCT02403895) and advanced gastric adenocarcinoma (NCT02449655). Sapanisertib (MLN0128, INK128, TAK-228), a dual mTORC1/2 kinase inhibitor, is undergoing further clinical trials after showing a suitable safety profile and preliminary antitumor activity in combination with paclitaxel, with and without trastuzumab, in a range of advanced solid tumor types ([54]; NCT01351350)

Akt Inhibitors Inhibition of Akt is also being considered in combination with chemotherapeutic agents as it plays a significant role in the signaling pathway. Akt inhibitors can act by competing for the ATP-binding site (ipatasertib) or as an allosteric inhibitor (MK-2206).

REFERENCES

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Ipatasertib (GDC-0068) is an Akt inhibitor that has been shown to improve progression-free survival (PFS) in women with metastatic triple-negative breast cancer (mTNBC) when given in combination with standard chemotherapeutic paclitaxel in a phase-II trial ([55]; NCT02162719). Phase-II trials are also underway for ipatasertib in combination with oxaliplatin, 5-fluorouracil, and leucovorin (modified FOLFOX6) chemotherapy for advanced or metastatic gastric or gastroesophageal junction (GEJ) cancer (NCT01896531). MK-2206 is an allosteric Akt inhibitor that has been shown to increase chemosensitivity and apoptosis in gastric cancer cells in vitro in combination with 5-fluorouracil and doxorubicin by inhibiting the phosphorylation of Akt [56]. In combination with carboplatin and paclitaxel, MK-2206 showed synergistic antiproliferative effects on gastric cancer cell lines [57]. MK-2206 showed synergistic effects when dosed in combination with doxorubicin, camptothecin, gemcitabine, 5-fluorouracil, docetaxel, and carboplatin in lung and ovarian cancer cells in vitro. The same drug combinations in vivo showed more potent effects than when administered as monotherapy [58]. MK-2206 has been tested in clinical trials with paclitaxel and trastuzumab for HER2-overexpressing solid tumors (NCT01235897). MK-2206 in combination trials with carboplatin/paclitaxel, docetaxel, or erlotinib in patients with advanced solid tumors gave complete and partial responses in patients with melanoma, endometrial, neuroendocrine, prostate, nonsmall-cell lung cancer, and cervical cancers ([59]; NCT00848718). MK-2206 is currently being tested in combination with paclitaxel to treat metastatic solid tumors or metastatic breast cancer (NCT01263145).

CONCLUSION Since the discovery of rapamycin in the 1970s, much has been learned of the role of the PI3K/Akt/mTOR axis in cell survival. Activation of the pathway by chemotherapy drugs leads to therapeutic resistance. Targeting the PI3K/Akt/mTOR signaling axis in cancer has shown promising results in preclinical studies. Rapamycin and the rapalogs, everolimus and temsirolimus, have been approved by FDA for treating cancerous and noncancerous conditions. Because monotherapeutic strategies of signal-targeting or conventional chemotherapy frequently lead to resistance, the rationale of combining such strategies has reached phase-I and phase-II clinical trials with drugs targeting various nodes of this vital pathway.

Acknowledgment We are very grateful to Dr. Zhe-Sheng Chen for assisting us through the entire manuscript preparation practice.

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