P21 activated kinase signaling in cancer

Accepted Manuscript Title: P21 activated kinase signaling in cancer Authors: Chetan K. Rane, Audrey Minden PII: DOI: Reference:

S1044-579X(17)30249-3 https://doi.org/10.1016/j.semcancer.2018.01.006 YSCBI 1437

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Seminars in Cancer Biology

Received date: Revised date: Accepted date:

1-11-2017 4-1-2018 8-1-2018

Please cite this article as: Rane Chetan K, Minden Audrey.P21 activated kinase signaling in cancer.Seminars in Cancer Biology https://doi.org/10.1016/j.semcancer.2018.01.006 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

P21 activated kinase signaling in cancer Chetan K. Ranea and Audrey Mindena

Susan Lehman Cullman Laboratory for Cancer Research, Department of Chemical Biology,

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Ernest Mario School of Pharmacy, Rutgers, The State University of New Jersey, 164

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Frelinghuysen Road, Piscataway, NJ 08854, United States

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Abstract

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The p21 Activated Kinases (PAKs) are a family of serine threonine kinases, that consist

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of 6 members, PAKs 1-6, which are positioned at an intersection of multiple signaling pathways implicated in oncogenesis. The PAKs were originally identified as protein kinases that function

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downstream of the Ras related Rho GTPases Cdc42 and Rac. PAK1 and PAK4, which belong

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to Group I and Group II PAKs, respectively, are most often associated with tumorigenesis. On account of their well characterized roles in cancer, several small molecule inhibitors are being

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developed to inhibit the PAKs, and there is interest in investigating their efficacy as either first line or adjuvant treatments for cancer. Studies to delineate PAK regulated signaling pathways

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as well as the long term effects of PAK overexpression on gene expression are beginning to shed light on the mechanism by which PAK proteins may lead to cancer when they are

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overexpressed or activated. This review will describe the association between PAK expression in cancer, with a focus on PAK1 and PAK4, which are most often associated with the disease. The current understanding of the molecular mechanisms by which the PAKs operate in cancer will be discussed. We will also review some of the potential drug candidates, and discuss which of them are currently being tested for their efficacy in cancer treatments.

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A. INTRODUCTION The PAKs belong to a family of serine threonine kinases that were originally identified as

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downstream effectors of Cdc42 and Rac, which are members of Rho family, all of which are part of the Ras superfamily of small GTPases. The PAKs are broadly classified into two groups based on their structural differences and sequence homologies. The two groups are the Group I PAKs, consisting of PAKs 1-3, and Group II PAKs, consisting of PAKs 4-6 [1-3]. The PAKs all contain a GTPase binding domain (GBD) at the amino terminus, which binds to Cdc42 and Rac,

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and they contain a serine/threonine kinase domain at the amino terminal part of the protein. The

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serine/threonine kinase domain shares sequence similarity to yeast Ste20 which is also a

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Cdc42 binding protein [4]. The group I PAKs have several domains that are not present in the

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group II PAKs, such as an autoinhibitory domain, proline rich domains, and binding sites for PIX, an exchange factor for Rho GTPases [5, 6]. The group I PAKs have strong sequence similarity

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to each other, while the group II PAKs are highly similar to each other in the GBD and kinase

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domains. Overall, the group I PAKs and group II Paks have approximately 50% amino acid identity in the GBD and kinase domains, but differ throughout their other domains [7].

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One factor that differentiates the different PAK family members is their expression

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patterns. PAK4 is expressed in all tissues, and is expressed at particularly high levels during embryogenic development [8, 9]. PAK1 is also expressed at high levels in embryogenesis, and

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in some adult tissues including brain, muscle and spleen [9, 10]. PAK2 is also high in endothelial cells, and the other PAK family members are seen at high levels in the nervous system [10]. PAK2 and PAK4 are essential for embryonic development, as mouse knockouts of these PAKs are embryonic lethal. Mouse knockouts of the other PAKs are not embryonic lethal, but lead to various abnormalities, including cognitive defects [10, 11]. In adult tissues,

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overexpression of PAK kinases is often linked to cancer. PAK1 and PAK4 levels in particular, are frequently found to be elevated in various types of cancer either at the DNA, RNA, or protein levels, although there are also numerous examples where other members of the PAK family have been linked to cancer [12]. Monitoring the expression levels of the PAKs in adult tissues

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can therefore potentially be used as a diagnostic biomarker for cancer.

Neither the Group I or Group II PAKs are frequently mutated in human cancers, but their dysregulated expression, particularly overexpression, are frequently associated with cancer [12]. The expression levels of the PAKs may be regulated via different mechanisms, but it is

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significant that both PAK1 and PAK4 genes are found on chromosomal regions that are frequently amplified in cancer [12, 13]. The PAKs are known to have central roles in several

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biological functions such as cell growth, cell survival, cytoskeletal organization, cell migration,

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and cell morphology. Aberrant modulation of these cellular processes are known to be

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associated with cancer. In cancer, the PAKs have been linked to uncontrolled cell proliferation,

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altered cell signaling, increased metastasis, drug resistance, and regulation of the immune system [10, 12, 14], some of which are described in more detail in this review. Here, we

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describe the functions of PAKs 1 and 4 that are most closely linked to cancer, we describe what

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is known about the molecular mechanisms by which the PAKs operate, and we provide examples of specific types of cancer which appear to be linked to PAK1 or PAK4. Since several

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excellent reviews on PAK kinases and cancer have been published previously [9, 15, 16], this

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review will focus primarily on the most recent literature on PAK biology.

B. PAK MEDIATED SIGNALING PATHWAYS ASSOCIATED WITH CANCER

PAK mediated cell migration in cancer

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Protein tyrosine phosphatase N (PTPN)-12 is a protein phosphatase that plays a role in many cellular activities, including development and cytoskeletal organization, bone resorption and immune responses. Importantly, it is also considered to be a tumor suppressor. Reduction in PTPN-12 levels is linked to poor prognosis in a number of different cancers, including breast,

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lung, and colorectal cancer [17]. PTPN12 exhibits its anti-tumor activity by dephosphorylating and thereby inactivating oncogenic proteins such as HER2, especially in triple negative breast cancer. Phosphorylation of Tyr-1196 on Her2 is associated with its activation, and this site can be dephosphorylated by PTPN12. Activation of Her2 is linked to PAK1 signaling, because active Her2 leads to PAK1 recruitment and phosphorylation on Ser-423, which in turn leads to

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increased cell migration. PTPN12 negatively regulates Her2, by dephosphorylation on Tyr-1196

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on Her2. PTPN12 activity itself is regulated by phosphorylation of Ser-19. In MDA-MB-231 cells,

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a human triple negative breast cancer cell line, phosphorylation of PTPN12 on Ser 19 was

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increased in response to cyclin-dependent kinase 2 (CDK2), and this impaired PTPN12's ability to dephosphorylate HER2 on Y1196. In turn, Her2-pY1196 phosphorylated PAK1 at Ser-423,

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thus leading to PAK1 activation and migration. PTPN12 is thus proposed to be an intermediary

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between CDK2, Her2, and PAK1 in cancer. Interestingly, it was shown that PAK1 is implicated in regulating cytoskeletal dynamics and is frequently associated with invasiveness of several

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metastatic cancer cell lines. Thus, a CDK2-PTPN12-HER2-PAK1 signaling cascade orchestrates a crosstalk between the CDK2 and HER2 oncogenic pathways, and the proteins in

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this signaling pathway may be good targets for treating metastatic cancer.

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Another example of the role for PAKs in migration comes from studying PAK4 in

melanoma. PDZ-RhoGEF, a RhoA activator, is a protein that is associated with invadopodia, which are actin rich invasive protrusions that play an active role in promoting metastasis [18]. PAK4 was shown to be overexpressed in invasive melanoma cell lines and in patient samples of melanoma. In turn, PAK4 promotes invadopodia maturation by binding to and inhibiting PDZ-

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RhoGEF activity [19]. Conversely, PAK4 depletion in invasive melanoma cells was associated with increased RhoA activity, which resulted in increased actin stress fibers, associated with enhanced cell rigidity and reduced cell invasion. Interestingly, a kinase-inactive mutant of PAK4 could not inhibit PDZ-RhoGEF induced RhoA activation, suggesting that PAK4 kinase activity is

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required for regulating RhoA activity. Thus, a specific PAK4:PDZ-RhoGEF interaction inhibits RhoA activity thereby promoting invadopodia maturation and inducing metastasis in melanoma

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cells [19].

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The PAK-PI3K/AKT signaling pathway

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Phosphatidylinositide 3-kinase (PI3K) is known to be important for several types of cancer [20], and recent studies have shown that there is a connection between PI3 Kinase and

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PAKs. PI3K (which consists of a regulatory domain p85, and a catalytic domain p110) is a lipid

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kinase that is often activated downstream to oncogenic Ras [20]. PI3K phosphorylates the membrane lipid phoshatidylinoside 4,5 bisphosphate (PIP2) to produce phoshatidylinoside

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3,4,5-triphosphate (PIP3). This causes proteins including PDK1 and AKT to be recruited to PIP3 via their pleckstrin homology (PH) domains. As a result, PDK1 phosphorylates a site on AKT,

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while another kinase phosphorylates a second site on AKT, and AKT becomes activated. AKT activation in turn promotes cell survival, and consequently unregulated AKT activation is often

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associated with cancer. PTEN, a phosphatase, is a tumor suppressor that is frequently mutated in cancer. It functions by downregulating the AKT pathway. This occurs by dephosphorylation of PIP3 by PTEN, and subsequent formation of PIP2, thus blocking the activation of the AKT via its interaction with PIP3.

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Recent studies have shown that there are several possible links between the PAK kinases and the PI3K pathway. PI3K, for example, activates Rac [20], and the PAKs were specifically identified as effectors for Rac and Cdc42. Rac also activates AKT, and this has been shown to occur at least in part via activation of PAK1. Interestingly, however, AKT activation in

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response to Rac is not dependent on PAK1's kinase activity, because even a kinase inactive mutant of PAK1 is sufficient for AKT activation in response to Rac. This suggests that one way PAK1 may activate AKT is by functioning as a scaffold, to facilitate the connection between AKT and PDK1 [21]. A connection between PAK1 and AKT has been observed consistently in multiple types of cancer. In a mouse model of Kras driven squamous cell carcinoma, deletion of

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the PAK1 gene resulted in reduced tumor progression and an almost total reduction of AKT

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activity. Treatment with two small molecule PAK inhibitors (PF-3758309 and FRAX597) led to

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significant regression of Ras-driven tumors and reduced AKT activity, indicating that PAK1

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function is required for Kras mediated activation of the PI3K/AKT pathway in vivo [22]. In colorectal cancer cells, shRNA mediated PAK1 knockdown resulted in a decrease in AKT

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phosphorylation and activity, and this was associated with a reduction in cell growth, cell

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survival and invasiveness [23]. In oral squamous cell carcinomas, PAK1 amplification was frequently associated with activation of PI3 Kinase [24]. In breast cancer cells, treatment with a

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non-specific PI3K pathway inhibitor LY294002 not only reduced PI3K activity, but also reduced PAK1 phosphorylation. This result lends further support to a connection between PI3K and

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PAK1, and suggests the presence of a feedback loop [25].

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A recent study alludes to a connection between PAK4 and PI3K in breast cancer. PAK4

levels were shown to be up regulated in a large number of human breast cancer cells, and the levels of PAK4 was linked to metastasis and tumor size and advanced stage disease. The presence of PAK4 was inversely correlated with disease free survival. [26]. Further overexpression of PAK4 in MDA-MB-231 cells, a PAK4 positive triple negative breast cancer

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cell line, led to increased proliferation, migration, and invasion, whereas knockdown of PAK4 using siRNA had the opposite effect. Interestingly, PAK4 overexpression in these cells was also associated with increased PI3K expression and increased levels of phospho-AKT and mammalian target of rapamycin (mTOR), a downstream effector of AKT [26]. Knockdown of

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PAK4 had the opposite effect. Studies using a PAK4 kinase inactive mutant, suggest that PAK4 leads to AKT phosphorylation by both kinase dependent and kinase independent mechanisms. By using a PI3K inhibitor, PI3K was shown to be essential for tumor invasiveness and migration in response to PAK4, indicating that PI3K plays an important role in the PAK4 signaling pathway

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in breast cancer cells.

In gastric cancer, there is a correlation between PAK4 and cancer metastasis [27],

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where PAK4 may operate via an interacting protein DGCR6L and LIMK1 to promote migration

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[28-30]. Since metastasis and chemo-resistance are closely linked, the role for PAK4 in

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resistance to cisplatin (CDDP) was assessed [31]. PAK4 levels were shown to strongly

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correspond to CDDP resistance, while PAK4 knockdown could reverse resistance. By using a combination of inhibitors, PAK4 induced resistance to CDDP was shown to be mediated by both

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the MEK/ERK pathway and the PI3K AKT pathway. In the gastric cancer cells, while PAK4

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could activate PI3K, PI3K could also activate PAK4, indicating a reciprocal regulation between the two. This suggests that PAK4 and the PI3K/AKT pathway might function via a feedback loop

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similar to that of PAK1 [31]. One way that PAK4 can regulate the PI3K pathway is by interacting with the p85 subunit

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of PI3K. This has been demonstrated in a pancreatic cancer cell line where PAK4 was shown to function downstream to HGF and c-MET, and to mediate cell migration [32]. Specifically, the proline rich region of PAK4 was shown to bind to the SH3 domain containing subunit of p85, most likely independent of PAK4's kinase activity. p85 is the regulatory subunit of PI3K, and by binding to this subunit, PAK4 can modulate the inhibitory interaction between p85 and the

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p110 catalytic subunit of PI3K. The result is increased PI3K activity and consequently increased AKT phosphorylation. This PAK4-PI3K/AKT interaction has been shown to be crucial for regulating invasiveness in a pancreatic cancer cell line. Further studies to investigate the

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benefits of targeting this nexus in pancreatic cancer are warranted. Recently a connection has been demonstrated between PAK4 and Hypoxia Inducible Factor (HIF-1) translation in colon and cervical cancer cells. This has important implications in cancer because hypoxic regions are associated with many solid tumors [33]. HIF-1 is an important transcription factor that regulates gene expression under hypoxic conditions. In

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normoxia, HIF-1 is targeted for proteosomal degradation, while under hypoxic conditions, HIF-

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1 is stabilized so that it can translocate to the nucleus and upregulate transcription of genes

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involved in cell survival under oxygen-deficient conditions. Under hypoxic conditions, siRNA

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mediated PAK4 knockdown depleted HIF-1 protein levels and also downregulated HIF-1 downstream target genes. The effect of PAK4 on HIF1 was independent of changes in HIF-1

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transcription or protein degradation, but rather mediated by regulating HIF-1 protein levels.

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Interestingly, the connection between PAK4 and HIF-1 appears to be mediated by AKT and

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cancer.

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mTOR downstream to AKT, thus providing another important link between PAK4, AKT, and

The PAK -Wnt/-catenin signaling pathway

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Aberrant activation of the canonical Wnt/-catenin signaling pathway is associated with

several cancers including colorectal tumors and hepatocellular carcinomas [34, 35]. The Wnt/catenin pathway is activated upon binding of Wnt ligands to Frizzled Receptors (Fz or Fzd) and its co-receptor, low density lipoprotein receptor-related proteins (LRP5 and LRP6). In the absence of ligand binding, -catenin is phosphorylated and targeted for proteosomal

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degradation [36] by a multi-protein complex, called as the “destruction complex” that includes the scaffolding protein Axin, the tumor suppressor adenomatous polyposis coli gene product (APC), casein kinase 1 (CK1), and glycogen synthase (GSK3). This destruction complex involves phosphorylation of -catenin by the kinase GSK3, at the amino terminus, and targets -

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catenin for destruction by ubiquitination. Activation of the Wnt signaling pathway prevents catenin phosphorylation and degradation, allowing it to accumulate and translocate to the nucleus [37], where it can interact with transcription factors of the T-cell factor (TCF) and lymphoid enhancer factor (LEF) families. This leads to activation of Wnt target genes such as cmyc and cyclin D1 [38], genes that are associated with increased cell proliferation and

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frequently linked to cancer. Thus, stabilization and activation of the -catenin pathway plays an

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important role in driving cellular processes that mediate tumorigenesis. PAK4 has known links to the Wnt pathway. In the cytoplasm, PAK4 phosphorylates -

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catenin at Serine 675, a carboxyl terminus site [39], and this is thought to prevent -catenin from

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getting ubiquitinated and consequently targeted for proteosomal degradation. In the nucleus,

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PAK4 upregulates -catenin protein expression and is associated with increased TCF/LEF transcriptional activity. PAK4 can shuttle between the cytoplasm and the nucleus to exert these

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dual effects on the Wnt pathway. This nucleo-cytoplasmic shuttling is important for mediating catenin stability and activity, and hence PAK4 plays an important role in activating Wnt/-

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catenin signaling pathway.

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Recently, PAK4 was also shown to be connected to the Wnt pathway via methylation.

PAK4 has been shown to be modified by methylation, an important post-translation modification that is less frequently studied than other modifications, such as phosphorylation, but which is thought to have important roles in cellular function [40]. PAK4 was shown to be methylated by SETD6, a family of protein lysine methyltransferases (PKMTs) [41, 42]. SETD6 was found to

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bind to and methylate PAK4 both in vitro and on the chromatin. Interestingly, this cross-talk is associated with the regulation of the Wnt/-catenin pathway, and subsequent regulation of Wnt target genes in MDA-MB-231 breast cancer cells [40]. SETD6 binding and methylation of PAK4 at the chromatin enhances the physical interaction between PAK4 and -catenin, thereby

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stabilizing it and increasing its nuclear localization, which results in increased Wnt/-catenin transcriptional activity. PAK4 is also important for SETD6--catenin association, acting as a physical linker between SETD6 and -catenin to form the three protein SETD6-PAK4--catenin complex,

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enhances

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stability.

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formation

of

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SETD6-PAK4--catenin complex at chromatin is a new link in the Wnt/-catenin pathway,

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leading to enhanced transcription of several Wnt/-catenin target genes known to have cardinal

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roles in cancer.

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Kinase-Independent and Cdc42 independent PAK functions

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While the PAKs have important functions as protein kinases, they also have functions that are independent of their catalytic activity. Kinase-independent functions are known for the

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various members of the PAK family [43-46]. PAK4, for example, promotes cell survival by both kinase dependent and independent mechanisms [43, 44, 47]. Recent work has indicated that

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the regulation of cell adhesion and migration by PAK4 can also occur by a mechanism that is independent of PAK4 kinase activity and even its ability to bind to Cdc42 [48]. The regulation of

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adhesion and migration are important processes in cancer as they are critical for invasion and metastasis. PAK4 has often been linked to cell adhesion and migration, most likely due to its role in regulating the cytoskeleton and focal adhesions. PAK4 was found to be highly expressed in breast cancer, with the highest levels found in the carcinomas with a severe grade and high invasiveness. PAK4 knockdown studies in breast cancer cells illustrate a critical role for PAK4 in

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breast cancer motility, specifically in migration and the regulation of adhesion turnover. Interestingly, this was found to be independent both of Cdc42 binding to PAK4, and of PAK4 kinase activity. Instead, regulation of adhesion turnover was associated with PAK4 binding to RhoU, a nonconventional Rho GTPase that is constitutively active. Binding of PAK4 to RhoU

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was found to stabilize RhoU and to prevent its destruction by ubiquitination. Furthermore, the complex formed between PAK4 and RhoU leads to phosphorylation of paxillin, which in turn plays key roles in focal adhesion turnover, and hence is important for cell migration and presumably metastasis. Interestingly, however, this interaction and phosphorylation of paxillin appears to be independent of PAK4's kinase activity, because even a kinase inactive mutant of

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PAK4 can promote the phosphorylation of paxillin and consequent changes in cytoskeletal

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dynamics. Thus, the PAK4:RhoU complex most likely serves as a scaffold which links RhoU

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and PAK4 with Paxillin and with a yet to be identified paxillin kinase, but this kinase does not

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appear to be PAK4 or other PAK family members [48]. This study illustrates that PAK4 has important kinase independent functions. Hence effective cancer treatments may require PAK4

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inhibitors that inhibit total PAK4 levels, rather than blocking PAK4 catalytic activity alone.

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C. TYPES OF CANCER THAT HAVE BEEN LINKED TO PAK KINASES

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Pancreatic cancer

Pancreatic ductal adenocarcinoma (PDAC) is the most common type of pancreatic

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cancer. Current treatments for pancreatic cancer have limited effectiveness, and therefore identification of biomarkers and potential druggable targets for the disease is critical. PAKs are frequently upregulated in pancreatic cancer, especially PAK1 and PAK4. In one study where 202 PDACs were analyzed, 82% of them showed increased levels of PAK1 [49, 50]. In another study, PAK1 was shown to be overproduced in primary pancreatic tumors, but less

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overexpression was seen in cancer cells that had metastasized to the liver [49-51]. In in vitro overexpression studies, PAK1 overexpression in a PDAC cell line was associated with aggressiveness, including increased proliferation and migration [50]. In KRAS wild-type pancreatic cell lines, PAK1 overexpression resulted in increased colony formation, while its

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depletion significantly reduced cell migration and invasion [52].

Pancreatic cancer almost always has a poor prognosis, regardless of treatment. One hypothesis is that this may be because of the stroma, which has been proposed to encourage tumor growth and cause resistance to therapy [53]. The stroma is a heterogeneous region made

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up of several cell types, including the pancreatic stellate cells (PSCs) which play key roles in stromal regulation [54]. Although the stroma is often considered to be an important factor in poor

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prognosis for pancreatic cancer, its role is complex, and recent work indicates that it may have

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both tumor promoting and tumor inhibiting properties. Interestingly, PAK1 was shown to be

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highly expressed in the pancreatic cancer stroma and in isolated PSCs [55]. PAK1 was shown

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to induce resistance to hypoxia in pancreatic cancer through regulation of Hypoxia Inducible

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Factor (HIF-1. In response to hypoxia, both PAK1 expression and activity were shown to be increased in human pancreatic stellate cells (hPSC1). Normal mouse pancreatic stellate cells

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(nmPSCs) were isolated from both wild-type and PAK1 knockout mice, and the knockout cells grew significantly more slowly than the wild-type cells. Furthermore, when cells from a

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pancreatic cell line, Pan02, known to be PAK1 positive, were injected into the head of the pancreas of either PAK1 knockout or wild-type mice, the resulting tumors in the PAK1 knockout

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mice had significantly less PAK1 and lower PAK1 activity, and consequently, the PAK1 knockout mice had a greater survival time. The PAK1 knockout mice also exhibited a decrease in proliferation of the nmPSCs. These results suggest that knockout of PAK1 in the stroma led to a decrease in PAK1 levels and activity in the associated tumors, and a corresponding decrease in tumor growth. Interestingly, this role of PAK1 appears to be bidirectional, as PAK1

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in tumors was also shown to have an effect on PAK1 levels in the stroma. This was illustrated by generating a PAK1 shRNA knockdown and control of the human pancreatic cancer cell line PANC-1, and isolating conditioned media from both the wild-type and PAK1 knockdown cells. hPSC1 cells that were incubated with the conditioned media from the PAK1 knockdown cells

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showed reduced levels and activity of PAK1 compared with those incubated with the control PANC-1 cells. This was associated with a decrease in migration and invasion. Thus, PAK1 appears to have a role in signaling from the stroma to the tumor, and from the tumor to the stroma as well.

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PAK4 upregulation in pancreatic cancer is also common, and is often associated with DNA amplification at specific amplicons [50, 56], but it is also overexpressed at the mRNA level

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[50, 56-58]. PAK4 appears to have significant roles in anchorage independent growth and

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migration in PDAC cells [50, 59]. PAK4 is often overexpressed in pancreatic cancer cells,

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although its levels are low in normal pancreas. PAK4 silencing in pancreatic cancer cell lines

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using interfering RNA led to apoptosis resistance, decreased cell growth, and decreased cell cycle progression [59]. This was associated with a reduction of NF-kB accumulation in the

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nucleus. Since NF-kB is known to have a role in cell growth and survival and to be upregulated

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in cancer, this study suggests that PAK4 induced tumorigenesis may be mediated by NF-kB. Further studies indicate that the mechanism by which PAK4 regulates NF-kB in pancreatic cells

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is through activation of both the ERK MAP Kinase pathway and the AKT pathway [59]. Another mechanism by which PAK4 may regulate cell growth in pancreatic cancer is via

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Signal Transducer and Activator of Transcription 3 (STAT3). Pancreatic cancer initiating cells/cancer stem cells (CSCs) have been reported to have high PAK4 expression levels as compared to non-CSCs [60]. shRNA mediated PAK4 knockdown in PC cells significantly reduced expression of several stemness-associated markers. Unlimited self-renewal and the ability to maintain an undifferentiated state along with resistance to chemotherapy are key

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characteristics of CSCs, and high PAK4 expression in pancreatic CSCs was associated with increased sphere-forming potential (representative of self-renewal ability) and chemoresistance. Interestingly, PAK4 silencing led to a decreased nuclear STAT3 levels and a corresponding decrease in STAT3 transcriptional activity. Furthermore, high nuclear levels of

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STAT3 were associated with high PAK4 levels in pancreatic cancer cells, while the opposite was true in pancreatic cancer cells with lower levels of PAK4. Restoring STAT3 in the cells that had silenced PAK4, restored the expression of stemness markers and sphere forming ability. Consequently, PAK4 was proposed to mediate stem-cell-like phenotype of PC cells through

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activation of STAT3 signaling [60].

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Prostate Cancer

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Prostate Cancer is one most common types of cancer and a leading cause of deaths among men in the United States. Hormonal therapy is the prevailing therapy for prostate cancer,

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but resistance eventually develops leading to androgen-independent metastatic prostate cancer.

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At this stage the cancer is difficult to treat [61]. In prostate cancer, high PAK1 levels were seen specifically in invasive cancer cells, while its levels were lower in non-invasive prostate cancer

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cells. This is in contrast to PAK6, which was shown to be high in both invasive and non-invasive

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prostate cancer cells [62], and which is known to be implicated in cell growth. Knockdown studies showed that both PAK1 and PAK6 are necessary for prostate cancer cell proliferation,

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but only PAK1 was predominant in regulating micro invasion of prostate cancer cells and prostate tumor growth. PAK1 mediated invasiveness was shown to operate via the cytoskeletal network that enhances directional migration. Gene expression studies have identified several candidate genes whose expression levels could account for PAK1's role in invasiveness. These include Transforming Growth Factor  (TGF, whose expression levels increased in PAK1

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knockdown cells, and Matrix Metallopeptidase 9 (MMP-9), whose levels decreased in PAK1 knockdown cells. TGF was also found to inhibit motility in prostate cancer cells, suggesting a direct connection between PAK1 levels, TGF levels, and prostate cancer cell motility [62].

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Interestingly, TGFsignaling is known to promote epithelial-mesenchymal transition (EMT) in advanced stages of prostate cancer. Upon long-term stimulation of TGFprostate the cancer cell line PC3, exhibited increased expression of EMT markers such as Snail and N-cadherin, and they also exhibited enhanced cell scattering. This was shown to be mediated through tumor necrosis factor receptor-associated factor-6 (TRAF6) activation of RAC1/PAK1 pathway. siRNA

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mediated PAK1 knockdown or PAK1 inhibition by the inhibitor iPA-3 reversed TGF-induced prostate cancer cell EMT and inhibited expression of mesenchymal markers [63]. This indicates

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that a feedback loop exists between PAK1 and TGF where PAK1 inhibition leads to

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increased TGF expression and reduces motility of prostate cancer cells. Prolonged TGF

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signaling however, leads to activation of the RAC1/PAK1 pathway, thereby promoting

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invasiveness and metastasis.

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PAK4 has also been implicated in prostate cancer. An important aspect of prostate cancer is metastasis, and PAK4 has been shown to be play a necessary role in regulating

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prostate cancer cell migration, via its role in regulating the actin cytoskeleton and focal adhesion. This in turn is important for prostate cancer cell migration and metastasis [64]. Protein

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kinase A (PKA) was shown to phosphorylate and activate PAK4 in prostate cancer cells. Activated PAK4 was in turn shown to regulate the transcriptional activity of CREB, a

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phosphorylation substrate of PKA and key regulator of cell survival, proliferation and differentiation. This resulted in increased expression of CREB target genes such as Bcl-2. PAK4 mediated upregulation of Bcl-2 was shown to promote cell survival of prostate cancer cells, while its knockdown induced apoptosis and increased sensitivity to chemotherapeutic drugs. PAK4 was also shown to promote neuroendocrine (NE) differentiation in pancreatic

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cancer cells, via a cAMP dependent manner. NE is known to promote tumor progression in pancreatic cancer cells, and it is responsible for androgen-independence and poor prognosis. PAK4 was also shown to be important for prostate tumor formation in a mouse model. This was illustrated by shRNA mediated PAK4 knockdown in PC3 and DU145 prostate cancer cells,

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following xenograft transplantation into athymic mice. The result was drastic reduction in tumor formation [65].

Melanoma

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Melanoma is often linked to BRAF mutation, and some melanoma patients carrying the

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oncogenic BRAF mutation have had good responses to existing therapies [66]. However,

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treatment options for patients with non-BRAF melanoma are more limited. The identification of

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other melanoma biomarkers is hence critical. PAK1 DNA copy number, mRNA, and protein levels were also found to be upregulated in human melanoma [67]. Interestingly, dysregulated

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PAK1 expression had a negative correlation with BRAF mutation. This raises the possibility that,

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targeted PAK1 inhibition may serve as a pharmacologically target for treating melanomas that do not contain the oncogenic BRAF1 mutation. In fact, treatment of PAK1 positive melanoma

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cells with PAK1 inhibitors reduced cell proliferation and migration, by a mechanism that was

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mediated by the ERK pathway, and the inhibitors also reduced tumorigenesis in mouse xenograft models.

A

While the study above indicates that PAK inhibition is important in wild-type Raf

melanoma cells, inhibition of PAK signaling is also important for BRAF mutant cells. This is because although BRAF inhibitors are initially effective in melanoma, melanoma cells often eventually become resistant to BRAF inhibitor therapy. However, in experimental models, targeting PAK1 has been shown to be effective in mutant BRAF melanomas that are resistant to

16

other therapies [68]. One way that PAK is regulated in melanoma cells is by the small GTPase RhoJ. RhoJ is upregulated in about half of stage III and IV BRAF mutant melanomas [69]. RhoJ binds to group I PAKs via the GBD, similar to Cdc42 or Rac. Importantly, PAK inhibitors reduce the growth of RhoJ positive melanoma cells that are also BRAF mutant. The role for PAK in

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tumorigenesis in the RhoJ positive cells is thought to be mediated by phosphorylation of the proapoptotic protein Bad. PAK phosphorylation of Bad has already been documented, as a mechanism by which PAKs can lead to cell survival [70]. Phosphorylation of BAD blocks its ability to block the function of the anti-apoptotic protein Bcl2. Blocking PAK4 thus blocks cell survival and blocks tumorigenesis in the RhoJ positive cells via blocking BAD phosphorylation.

U

Thus, inhibiting PAK is a promising therapy not only for BRAF wild-type melanoma, but also for

N

BRAF mutant melanomas that have become resistant to standard anti BRAF therapies.

A

Another approach to treating BRAF mutant cells that are resistant to BRAF inhibition, is

M

the use of combination therapy. BRAF operates via the ERK MAP Kinase pathway, and

D

therefore combination therapies which block both BRAF and MEK, an activator of the ERK pathway, are often used. However, even the combination therapies can eventually lead to

TE

resistance. Interestingly, resistance to treatment was shown to be associated with increased

EP

activations and levels of PAK1 and PAK2. Inhibition of PAK1 or even PAK4, led to decreased cell viability in resistant cells. The PAKs were shown to operate via different mechanisms, to

CC

confer resistance to BRAF or combination therapy. In cancer cells resistant to BRAF inhibition, the PAKs circumvent BRAF inhibition by phosphorylating CRAF and MEK, and thereby leading

A

to ERK activation. In the cells resistant to combination treatment, the PAKs are thought to confer resistance by activating several pathways that can promote cell proliferation and survival. These include the JNK, AKT-mTOR, and -catenin pathways, as well as promoting phosphorylation and thereby inhibition of the pro-apoptosis protein Bad [71].

17

Hepatocellular Carcinoma Hepatocellular carcinoma (HCC) is one of the most prevalent type of cancer in the world, with late stage cancer being non-responsive to curative treatments such as hepatic resection

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and liver transplantation [72]. PAK4 mRNA and protein expression levels were found to be high in HCC cells [73]. IHC staining indicated a strong cytoplasmic staining pattern for total PAK4 and a strong nuclear staining pattern for phospho-PAK4 (Ser474), which is phosphorylated at a site associated with its activation. This suggests that the activated PAK4 preferentially localizes to the nuclei of HCC cells. PAK4 overexpression was associated with venous invasion, liver

U

invasion, poor tumor cell differentiation and the late pTMN stage, suggesting a more aggressive tumor behavior and a higher incidence of metastasis. Overexpression of PAK4 in an HCC cell

N

line with lower levels of PAK4 promoted cell migration and invasiveness, while shRNA mediated

A

PAK4 knockdown significantly reduced cell migration. PAK4 was also shown to interact with and

M

phosphorylate p53 on Ser215, in HCC cells. PAK4 mediated p53 phosphorylation resulted in

D

significant reduction of p53 tumor suppressive, trans-activating, and DNA binding activities, with the nuclear localization of PAK4, augmenting p53 inhibition. This suggests that PAK4 is an

TE

inhibitory kinase of p53. PAK4 mediated p53 inhibition is likely to be responsible for protecting

EP

HCC cells from DNA damaging drug-induced cell death. Unphosphorylated p53 could suppress HCC cell invasiveness, but was abolished by PAK4 phosphorylation, suggesting that p53

CC

mediated inhibition of cell migration is also PAK4 and Ser215 phosphorylation dependent in HCC cells. A qRT-PCR analysis suggested that PAK4 mediated p53 phosphorylation resulted in

A

significant down regulation of three metastasis suppressors, cadherin 6 (CDH6), cyclindependent kinase inhibitor 2A (CDKN2A), and kisspeptin-1 receptor (KISS1R), while levels of two metastasis enhancers, fibroblast growth factor receptor 4 (FGFR4) and vascular endothelial growth factor A (VEGFA), were up regulated [73]. The finding that PAK4 is linked to p53 activity

18

in HCC provides new insight into the mechanism of action of PAK4 in cancer and can open up new avenues for drug development. PAK5, a member of the group II family of PAKs, was shown to be responsible for

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mediating cisplatin resistance by inhibiting apoptosis and cell cycle arrest and promoting cell growth in HCC cells [74]. PAK5 was shown to downregulate p-chk2, a key protein involved in regulation of cell growth and cisplatin induced cell cycle arrest, thereby leading to G1 arrest prevention and cell proliferation. The exact mechanism is however, poorly understood, and further studies to investigate the role of PAK5 in HCC are warranted. PAK6, also a group II

U

PAK, was also found to be up regulated in HCC patient samples as opposed to non-cancerous tissues. Patients with high PAK6 expression had an overall poor survival rate with a positive

N

correlation observed between PAK6 expression and Ki-67 immunoreactivity, a prognostic

A

marker of tumor proliferative activity [75]. In contrast, another study showed that PAK6 might

M

exhibit a tumor-suppressive function in HCC. In this study, decreased PAK6 expression was

D

associated with tumor node metastasis, stage progression and poor survival in HCC patients. PAK6 overexpression was shown to promote cell death and inhibit cell proliferation, colony

TE

formation and cell migration of HCC cells. This function was shown to be dependent on PAK6

EP

kinase activity and the ability to translocate to the nucleus. Intriguingly, PAK6 overexpressing HCC cells displayed reduced tumor growth in vivo, hinting at a tumor suppressive role for PAK6

CC

in HCC [76]. Thus, PAK6 has been shown to have both tumor suppressive and tumor promoting functions in HCC, and it will be important to determine how PAK6 functions in different patients

A

and under different conditions.

Breast cancer

19

PAK4 protein and mRNA levels are elevated in a number of breast cancer cells and primary human breast cancer tumor samples [26, 48, 77-81]. In a study of tumors from 80 breast cancer patients, PAK4 protein levels increased as the disease progressed, with cells at the most advanced stage having the highest levels of PAK4 [80]. In a study of 93 patients with

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invasive breast carcinoma, high levels of PAK4 correlated with advanced stage disease, large tumor size, lymph node metastasis, and poor survival [26]. In another panel of 300 human breast cancers, PAK4 protein was also highly expressed in the more severe grade invasive carcinomas [48]. In a study of basal like breast cancer, a subset that is frequently triple negative, DNA analysis revealed that the chromosomal region containing the gene for PAK4

U

was frequently amplified [82]. In a mouse model, PAK4 overexpression in a mouse mammary

N

epithelial cell line (iMMECs) led to growth characteristics that were reminiscent of oncogenic

A

transformation in vitro, and led to tumor formation in vivo [78]. In contrast, blocking PAK4 with

M

siRNA inhibited tumor formation of a human breast cancer cell line in a xenograft model system [78, 83]. Further evidence for the importance of PAK4 in breast cancer comes from studying the

D

microRNA, mir-199a.b-3p. This microRNA can function as a tumor suppressor by

TE

downregulating PAK4 and was shown to inhibit cell proliferation, cell migration and invasiveness of breast cancer cells [84]. PAK4 is involved not only in proliferation and migration in breast

EP

cancer, but also appears to have a role in drug resistance. Specifically, PAK4 was found to have a role in tamoxifen resistance in Estrogen Receptor (ER) positive breast cancer [81], and

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blocking PAK4 could sensitize tamoxifen resistant cells to the drug. This was shown to be associated with a feedback loop, where ER could bind to the promoter of the PAK4 gene, and

A

PAK4 in turn stabilized and activated ER. PAK4 also appears to have an important role in triple negative breast cancer, and a new family of PAK4 inhibitors including KPT-9274 and KPT-8752, described in more detail below, can block triple negative breast cancer cell growth in vitro and in vivo [85].

20

PAK1 also has an important role in breast cancer, and has been shown to function as an oncogene in many breast cancers [86]. One important link between PAK1 and breast cancer was illustrated by studying miRNAs. miR-494 is a microRNA that is frequently downregulated in cancer. In a study examining miR-494 in breast cancer, miR-494 was found to be decreased in

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breast cancer cells and tumors [87]. Ectopic expression of this microRNA in breast cancer cells that had particularly low levels, could suppress proliferation and migration in vitro, and suppress metastasis in vivo. Importantly, miR-494 was shown to block the levels of the PAK1 gene, and

M

A

N

U

PAK1 was shown to be a direct mediator of oncogenesis in response to reduction of miR-494.

D

Delineating the PAK4 transcriptome profile in a breast cancer model

Many previous studies have focused largely on identifying PAK substrates, but the long

TE

term effects of overexpression of the PAKs on gene expression patterns are still not well

EP

understood. A better understanding of the transcriptome changes triggered by overexpression of the PAKs will help to clarify how their expression may lead to cancer. This will be instrumental

CC

for the development of more efficient compounds that can target PAK regulated signaling in cancer cells. This was addressed in PAK4 overexpression studies in a breast cancer model, by

A

using RNA Sequencing.

PAK4 is highly expressed in breast cancer cells and in primary breast tumors [26, 48,

77-81], and blocking PAK4 reduces tumorigenesis [83, 85]. Immortalized mouse mammary epithelial cells (iMMECs) are a mouse model of normal mammary cells [88]. While these cells do not form tumors, if they are stably transfected with PAK4, they form tumors when implanted

21

into the mammary fat pads of mice [78]. These studies indicate that PAK4 is sufficient to cause tumorigenesis in mammary cells, and these cells provide a model for understanding the mechanism of action of PAK4 in cancer. To get a better understanding of PAK4 signaling in breast cancer, high throughput sequencing analysis was performed on RNA samples collected

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from non-transformed iMMECs (WT iMMECs) and iMMECs overexpressing PAK4. RNAsequencing (RNA-seq) analysis from this study generated a list of genes that were previously unknown to be regulated by PAK4 [89]. For example, the gene for ParvB was found to be downregulated in the PAK4 overexpressing cells. Interestingly, ParvB has been shown to be associated with suppression of cell growth in breast cancer cells. In fact, ParvB is sometimes

U

considered to be a tumor suppressor gene in breast cancer [90, 91]. Another gene that was

N

regulated by PAK4 overexpression is FoxC2. FoxC2 was upregulated in the PAK4

A

overexpressing cells. FoxC2 codes for a protein that has been associated with oncogenesis,

M

epithelial/mesenchymal transition, and metastasis in breast and other cancers [92-94]. The finding that FoxC2 is upregulated in response to PAK4 overexpression suggests a role for this

D

protein as a mediator of PAK4 induced oncogenesis. Further work will be required to determine

TE

the mechanism by which PAK4 leads to regulation of genes such as ParvB and FoxC2, whether these genes have key roles in breast tumorigenesis in response to PAK4, and which PAK4

CC

EP

target genes may be good drug targets for cancers that overexpress PAK4.

A

D. PAK INHIBITORS Since PAK kinases are strongly linked to human cancer, there has been a considerable

amount of interest in developing PAK inhibitors for disease treatment. PAK inhibitors are described in more detail in other reviews [95], but the sections below outline some of the PAK inhibitors that have promise for research and disease treatment.

22

Inhibitors of Group I PAKs

Among the PAK families, the rationale for targeting Group I PAKs has been established due to their regulatory roles in multiple signaling pathways involved in cancer. The ATP-binding

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pocket of PAK1 has been extensively studied and hence has helped to design structure based inhibitors. Several approaches for designing Group I PAK inhibitors have been explored and some of them have been successful. Several fine-design ATP competitive inhibitors have been identified that lock the movements of the active kinase-site in Group I PAKs. Given the diversity and overlapping nature of PAK regulators and effectors, some of the inhibitors that have been

U

developed lack specificity, resulting in the delayed use of these inhibitors in clinical studies. One

N

problem with the development of PAK kinase inhibitors is the fact that the catalytic pockets of

A

the PAKs are similar to those of other protein kinases. One new approach therefore, was to

M

carry out a high throughput screen to identify a new family of inhibitors that would be allosteric inhibitors [96]. Full-length PAK1 protein activated in vitro with recombinant Cdc42-GTPxS was

D

targeted by screening a library of 33,000 compounds, which led to identification of iPA-3. iPA-3

TE

functions by selectively stabilizing the PAK1 autoinhibitory conformation. It can prevent PAK1mediated auto-phosphorylation and activation, and is quite specific to the Group I PAKs. While

EP

this specificity is encouraging, iPA-3 thus far has limited potential as a therapeutic inhibitor

CC

because of its unstable nature [97]. A high-throughput screen of a kinase focused compound library that was optimized for

A

PAK1 specificity led to the development of FRAX597 by the pharmaceutical company Afraxis, FRAX597 is a small-molecule pyridopyrimidinone, and strongly inhibits the group I PAKs [22, 98]. In addition to FRAX567, Afraxis also developed the related group I inhibitors, FRAX486 and FRAX1036 [99-102]. FRAX567 was shown to have anti tumorigenic activity towards NF2 deficient Schwann cells in vitro and in vivo, suggesting that group I PAK inhibition may be a promising treatment for Neurofibromatosis (NF), which is consistent with previous findings that 23

the PAKs play important roles in both Neurofibromatosis type 1 and 2 ( NF1 and NF2) [98]. FRAX597 has subsequently been shown to have an inhibitory effect on a number of cancer models including inhibition of pancreatic stellate cell activation [55], as described above, pancreatic cancer cells [52, 103], malignant mesothelioma [104] and a skin cancer model [22],

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The related compound FRAX1036, was shown to act synergistically with a MEK inhibitor, to reduce the growth of peripheral nerve sheath tumors in vitro and in vivo [105], illustrating the importance of blocking multiple synergizing pathways in cancer treatment. FRAX1036 was also shown to be effective at blocking the growth of oncogenic Rac1 mutant melanoma cells, where

U

it had a strong anti-proliferative effect [106].

Research focused on minimizing toxic effects of conventional chemotherapeutic agents,

N

has resulted in seeking natural ingredients possessing anti-cancer properties. Frondoside A

A

(FRA), is a sulfated saponin extracted from sea cucumber Cucumaria frondosa and nymphaeols

M

(geranylated flavonoids) obtained from Okinawa propolis (a bee product from Okinawa, Japan).

D

FRA inhibited PAK1 in vitro selectively over LIMK and AKT, and blocked cell growth of A549

TE

lung cancer cells and pancreatic cancer cells [107]. However, these studies are limited to in vitro

EP

results and further research is warranted to test their in vivo efficacy.

CC

Inhibitors of Group II PAKs Group II PAKs, while they share some sequence homology with group I PAKs, are very

A

different from the group I PAKs [4]. PAK4 is structurally distinct from PAK1 in terms of its regulatory and catalytic domain. PAK4, unlike PAK1, exists as a monomer instead of a dimer, in its inactive state. While PAK1 is activated by a direct interaction with Rho GTPases, GTPase interactions with PAK4 appears to have a role primarily in directing the cellular location of PAK4, rather than being essential for its kinase activity. PAK4 also phosphorylates different substrates

24

than that of PAK1, even though there is some overlap in substrate specificity. Importantly, PAK4 has been shown to have both kinase-dependent and –independent functions in promoting cell growth [43, 44, 47]. Consequently, the mechanisms by which PAK1 inhibitors work might not be as effective at targeting PAK4. Taken together, structural and regulatory differences between

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PAK1 and PAK4 indicate that the strategy for development for PAK4 inhibitors should be different from that of PAK1 inhibitors.

One of the first PAK inhibitors to be generated was PF-3758309, by Pfizer. PF-3758309 is an ATP- competitive inhibitor of the PAK4 kinase domain. PF-3758309 inhibited PAK4

U

dependent phosphorylation of its substrate GEFH1. It strongly inhibited cell proliferation, cell survival and anchorage independent growth of HCT116 cells. PF-3758309 showed robust tumor

N

inhibition in five of the seven models tested: HCT116, A549, MDA-MB-231, M24met, and

A

colo205. Although it was designed as a PAK4 inhibitor, it turned out to be broadly active against

M

both Group I and Group II PAKs, and also other kinases, including AMPK (AMP-dependent

D

kinase) and RSK (Ribosomal S6 kinase) [108, 109]. Human clinical trials were terminated due to undesirable PK characteristics of the drug (<1% bioavailability), adverse side effects, and

TE

consequent lack of tumor responses [110, 111]. A second PAK4 kinase inhibitor, LCH-779944,

EP

inhibits PAK4 kinase activity more modestly [28, 112]. It also displays inhibitory activity towards PAKs 1, 5 and 6 and reduces EGFR and c-Src phosphorylation. LCH-779944 reduces

CC

proliferation and invasion of gastric cancer cells in vitro, and reduces filopodia formation and cell

A

elongation, but it has not been tested for in vivo studies. A third compound, Compound 17, generated by Genentech, displays good biochemical

potency and enhanced Group II selectivity. This is due to the presence of an open back pocket, which is more energetically accessible in PAK4 (and other group II PAKs) than it is in PAK1. Compound 17 reduces the viability of breast cancer cell lines and decreases tumor cell migration and invasion in two human triple negative breast cancer cell lines [113], and it

25

enhances tamoxifen sensitivity in MCF7 cells [81]. However, it has poor bioavailability due to poor permeability and/or high efflux [97]. Glaucarubinone, a natural product isolated from the seeds of the tree Simarouba glauca,

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has been recently shown to demonstrate anti-cancer properties. Glaucarubinone was originally developed as an anti-malarial agent, but was shown to inhibit both PAK4 and PAK1 levels [114]. Glaucarubinone inhibited cell proliferation and migration of human pancreatic cells in vitro, and reduced tumor growth in vivo. Glaucarubinone can also synergize with gemcitabine, to result in a more significant reduction of PAK4 and PAK1 protein levels and consequently, tumor growth

U

inhibition in vivo. Being a natural agent, Glaucarubinone is likely to have pleiotropic effects

N

which can complicate its development as a therapeutic agent.

A

A novel PAK4 inhibitor, KY-04031 was identified using a high throughput screen [115].

M

Unfortunately, KY-04031 had low PAK4 binding affinity and required high drug concentration to inhibit cell proliferation in a PAK4-dependent manner. GL-1196 is another small molecule

D

inhibitor that inhibits PAK4 kinase activity and suppresses cell proliferation through

TE

downregulation of the PAK4/c-Src/EGFR/cyclin D1 pathway and CDK4/6 expression, and inhibits invasiveness by blocking the PAK4/LIMK1/cofilin pathway in human gastric cancer cells

EP

[30].

CC

KPT-9274 belongs to a family of inhibitors called PAK4 Allosteric Modulators that were first identified as small molecules which are able to interact with PAK4 [9, 85, 116-120]. This

A

series of PAK4 inhibitors consists of several analogues, including KPT-8752 and KPT-9274, with KPT-9274 being currently under phase I clinical trial. These compounds are different from some of the other PAK4 inhibitors because they reduce steady state PAK4 protein levels, instead of inhibiting PAK4 catalytic activity. They most likely function by binding specifically to, and destabilizing, the PAK4 protein, but the exact mechanism of action of these inhibitors is not

26

yet completely understood. Since PAK4 has been shown to have kinase independent functions, as described above, inhibitors such as KPT-9274 that block PAK4 levels rather than kinase activity might be more effective in antagonizing PAK4 function in cancer.

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In addition to PAK4 inhibition, KPT-8752 and KPT-9274 were also shown to inhibit synthesis of NAD (nicotinamide adenine dinucleotide), by blocking activity of the enzyme NAMPT (nicotinamide phosphoribosyltransferase), a rate-limiting enzyme in the NAD biosynthesis salvage pathway [116]. NAD is an essential metabolite required for sustaining energy production (TCA cycle) and regulating cellular processes, including DNA repair (PARP),

U

epigenetics (sirtuins), and cell signaling, in rapidly proliferating cancer cells [121]. Inhibition of NAMPT results in a significant depletion of NAD, making NAMPT an attractive potential drug

A

M

successful therapeutic strategy in cancer.

N

target. Thus, a dual inhibition of PAK4 signaling and NAD biosynthesis has the potential for a

Recent studies have shown that KPT-9274 can block the growth of several types of

D

cancer cells, both in vitro and in vivo. These include PDAC, Renal Cell Carcinoma (RCC),

TE

multiple myeloma, triple negative breast cancer, and B cell acute lymphoblastic leukemia (BALL) [85, 116-120]. The compound is also effective against autosomal dominant polycystic

EP

kidney disease which although not cancer, shows many similarities to malignant disease [122]. Future work is warranted to determine whether the strong anti-proliferative effect of the

CC

compound is due primarily to PAK4 inhibition or NAD inhibition, or to the dual specificity of this inhibitor. KPT-9274 has thus far proven to be a promising clinical compound and it is currently in

A

phase I clinical trial for patients with advanced solid malignancies and non-Hodgkin lymphoma (NHL; NCT02702492).

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EP

1.

A

18.

19.

20. 21.

28

27.

28.

29.

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32.

EP

31.

TE

D

30.

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26.

U

25.

N

24.

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