Send Orders for Reprints to [email protected] Anti-Cancer Agents in Medicinal Chemistry, 2016, 16, 75-88 75 Therapeutic Potential of Targeting PAK Signaling

William Senapedis*, Marsha Crochiere, Erkan Baloglu and Yosef Landesman

Karyopharm Therapeutics, Inc., Newton, Massachusetts, USA

Abstract: The therapeutic potential of targeting -Activated (PAK1 – 6) for the treatment of cancer has recently gained traction in the biotech industry. Many pharmaceutically-viable ATP competitive inhibitors have been through different stages of pre-clinical development with only a single compound evaluated in human trails (PF-3758309). The best studied functional roles of PAK are control of cell adhesion and migration. PAK proteins are known downstream effectors of Ras signaling with PAK expression elevated in cancer (pancreatic, colon, breast, lung and other solid tumors). In addition altered PAK expression is a confirmed driver of this disease, especially in tumors harboring oncogenic Ras. However, there are very few examples of gain-of-function PAK , as a majority of the cancer types have elevated PAK expression due to amplification or transcriptional modifications. There is a substantial number of known substrates affected by this aberrant PAK activity. One particular substrate, β-catenin, has garnered interest given its importance in both normal and development. These data place PAK proteins between two major signaling pathways in cancer (Ras and β -catenin), making therapeutic targeting of PAKs an intriguing approach for the treatment of a broad array of oncological malignancies. Keywords: Inhibitors, oncology, p21-activated , Ras, Wnt; β-catenin.

INTRODUCTION conserved throughout evolution from yeast and worms to mammals [88]. The topic of this review focuses on the potential therapeutic role of targeting p21-activated kinases (PAK1 – 6). This The two groups of PAK proteins are differentiated primarily by kinase family has garnered recent interest from the pharmaceutical their structural conformation and route of activation. The Group I industry, but only one compound has reached human clinical trials, PAK proteins form a homodimer in the inactive state when the PF-3758309 (compound 1 in Fig. 1 and Table 1) [1-3]. PAK mRNA AID of one monomer interacts with the KD of the other monomer and protein levels are elevated in breast, colorectal, lung, gastric, (Fig. 2) [85, 89]. Group I PAK activation is facilitated by the and other solid cancers and are confirmed as drivers of cancer downstream regulators of Ras (Rho Cdc42 or Rac1) progression; (see reviews [4, 5] for additional details) [6-64]. binding to the CRIB domain of PAK proteins. This binding leads to Although there are a few examples of gain-of-function PAK gene auto-phosphorylation of 423 of PAK1 with additional mutations, elevated mRNA and protein levels resulting from phosphorylation of PAK1 attributed to Janus kinase 2 (JAK2; increases in PAK transcription, translation, or gene amplification Tyrosines 153, 201, and 285), 3-phosphoinositide-dependent kinase are much more common in patient samples [27, 41, 59]. 1 (PDPK1; Threonine 423), and A (PKA; unmapped) [4, 90-92]. There are also protein-protein interactions with adapter In addition to abnormal PAK expression levels in cancer, there proteins that can influence Group I PAK activity [4]. Unlike the is evidence that PAKs are downstream effectors of the hard-to- Group I PAKs, the predominant structural models of the Group II target oncogene K-ras [65-75]. Elevated PAK expression is often PAKs show that Group II exist as monomers in which the AID linked to oncogenic Ras in various cancer types. Additionally, there folds and intra-molecularly binds to the KD (Fig. 2) [84, 85, 93, is growing evidence that β-catenin is a phosphorylation substrate of 94]. Auto-phosphorylation at 474 in PAK4 is thought to be PAK kinase activity [29, 76-80]. Since aberrant Ras or Wnt/β- constitutive regardless of the protein conformation [84, 85, 93, 94]. catenin signaling is a hallmark of cancer development, small Therefore, binding of Cdc42 to the CRIB domain is necessary to molecule inhibitors of PAK proteins have the potential to be dissociate the AID from the KD for full activation of Group II therapeutically beneficial for the treatment of a diverse group of PAKs [84, 85, 93, 94]. cancers [81-83]. Due to the high degree of sequence similarity between Group I p21-ACTIVATED KINASE FAMILY and Group II PAKs (especially in the kinase domains), the substrates for both groups are thought to overlap considerably. The The PAK gene family consists of six protein isoforms which, most well-studied roles of PAK kinase activity are phosphorylation based on structure and activation, are divided in two groups: Group I of substrates involved in rearrangement, focal (PAK1, PAK2, PAK3) and Group II (PAK4, PAK5, PAK6) [4, 84- adhesions, and cell migration (Fig. 3; i.e. LIMK, GEF-H1, and 87]. Structurally, all PAK proteins contain three primary domains: a paxillin) [10, 40, 71, 95, 96]. However, there is a growing list of highly conserved carboxy-terminal kinase domain (KD), an amino- substrates involved in , survival, cell cycle, and terminal Cdc42-Rac1 interaction/binding (CRIB) domain, and an apoptosis (see references [5, 97] for detailed review) [58, 65, 67-72, autoinhibitory domain (AID) (Fig. 2). There is ~50% sequence 77, 79, 89, 98-174]. homology between all six PAK kinase domains with ~80% similarity within each group [88]. The PAK proteins are highly There are also data that some PAK functions are independent of kinase activity. PAK proteins can bring other interacting proteins together without the need for PAK kinase activity. For example,

*Address correspondence to this author at the Karyopharm Therapeutics, PAK4 kinase activity is required for repression of caspase 3- Inc. Address: 85 Wells Avenue, Newton, Massachusetts, USA, 02459; mediated apoptosis induced by serum deprivation [106]. However, Tel: 1-617-658-0524; Fax: 1-617-658-0601; a kinase-dead form of PAK4 retains the ability to inhibit caspase-8- E-mail: [email protected] and Bid-facilitated apoptotic activity in cells treated with tumor

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Table 1. The biochemical and in vitro properties of select PAK inhibitors from Fig. 1.

Compound Kinase Activity (IC50) Best Cellular Name Source Type Target References Number PAK1 PAK4 Activity (EC50)

1 PF-3758309 Pfizer ATP competitive Pan-PAK 36 nM 15 nM 800 nM [1-3, 205, 229] 2 Staurosporine Omura et al. (1977) ATP competitive Pan-PAK 0.6 nM 6 nM 200 nM [202, 203] 3 Λ-FL172 Staurosporine Analog ATP competitive Pan-PAK 130 nM NT NT [204] 4 - Pfizer ATP competitive PAK4 NT 30 nM 32 nM [206] 5 - Pfizer ATP competitive PAK4 NT 75 nM 4 nM [206] 6 LCH-7749944 Zhang et al. (2012) ATP competitive PAK4 NA 15 µM 10 µM [33, 207] 7 FRAX486 Afraxis ATP competitive Group I PAK ~60 nM ~800 nM NT [208] 8 FRAX597 Afraxis ATP competitive Group I PAK 8 nM >10 µM 70 nM [61, 219] 9 - AstraZeneca ATP competitive Group I PAK 18 nM 550 nM 870 nM [220] 10 AZ-PAK-36 AstraZeneca ATP competitive Group I PAK 1 nM 450 nM 140 nM [220] 11 - Genentech ATP competitive Group II PAK 5.4 nM 7.5 nM 10 µM [221] 12 KY-04031 Ryu et al. (2014) ATP competitive Unknown NT 790 nM 15 µM [222] 13 IPA-3 Deacon et al. (2008) Allosteric PAK1 NT NT 50 µM [223, 224, 228] 14 - Novartis Allosteric Group I PAK 5 nM >40 µM 100 nM [88] 15 KPT-7523 Karyopharm Therapeutics, Inc. Allosteric PAK4 NT NT 40 nM [225-227] 16 Glaucarubinone Pierre et al. (1980) Unknown Unknown NT NT 58 nM [230-232]

Best cellular activity refers to either cell-based signaling or proliferation assays. NA = not active, NT = not tested. necrosis factor (TNFα) [106]. Both PAK kinase-dependent and - Ras-PAK-β-CATENIN SIGNALING IN CANCER independent activity will be important to consider when targeting PAK for therapeutic intervention. An intriguing aspect of PAK function in normal and cancer development is that PAK proteins appear to be positioned between the Ras and Wnt/β-catenin signaling pathways (Fig. 4). PAKs are PAK EXPRESSION IN DEVELOPMENT AND CANCER one of the key downstream effector families of Ras, regulated The tissue expression patterns of mRNA and protein from the through the binding of Rho GTPases (Rac1 and Cdc42) [74, 85]. two PAK groups can be distinguished during embryonic, adult, and Studies show that Cdc42 is activated by Ras especially in the cancer development [4, 88]. Both PAK1 and PAK2 mRNA and context of oncogenic Ras-driven cancer development [178-180]. protein levels are high in embryonic and adult tissues such as the Oncogenic Ras is present in 30% of all cancers including some of , muscle, and spleen (PAK1), and are elevated in endothelial cells the most aggressive, such as pancreatic, colon, and lung [181, 182]. (PAK2). PAK3, PAK5, and PAK6 tend to localize to embryonic Cdc42 is overexpressed in cancer and is required for Ras-driven and adult neuronal tissues [175]. PAK4 mRNA and protein are transformation [183-185]. Activated Cdc42 interacts with the CRIB ubiquitously expressed in most cell types and are higher during domain of PAK proteins in order to achieve full activation of all six embryonic development and lower in adult tissues [176]. Mice with PAK proteins [84, 85, 93, 94]. In turn, Serine 675 of β-catenin is a a single genetic knockout of PAK2 or PAK4 are embryonically phosphorylation site of PAK kinase activity [29, 79, 80]. This site is lethal while all other single PAK gene knockouts are viable and particularly important for the transactivation of β-catenin [29, 79, fertile with various levels of cognitive impartment [176, 177]. 80]. Wnt/β-catenin signaling is also linked to cancer development and progression [186]. Numerous Wnt/β-catenin inhibitors are in Expression of PAK mRNA and protein is also linked to cancer different stages of development and are covered in detail elsewhere development (see reviews [4, 5] for additional details) [6-64]. [187]. Interestingly, there are very few reports of gain-of-function PAK mutations in cancer. One study describes a PAK4 Both Ras and β -catenin are well-established in normal and (E329K) in colon cancer, while another details a PAK5 mutational cancer cell development [83, 187, 188]. Ras (K-ras, N-ras, and H- ras) proteins are highly mutated in many different cancer types (T538N) link to lung cancer [27, 41]. Non-mutated PAK mRNA or including multiple myeloma, melanoma, pancreatic, colon, and lung protein activity is elevated in a variety of cancers through either cancers [189-194]. In pancreatic ductal adenocarcinoma (PDAC), gene amplification or through aberrant transcriptional or oncogenic K-ras is present in >90% of patient samples and is translational control [4, 5]. For example, there are post-translational considered the earliest genetic alteration [191]. PAK4 genomic modifications of PAK1 that cause aberrant PAK activity in amplification is quite common in pancreatic tumors with K-ras glioblastoma, breast, and kidney cancers [9-11, 36, 38]. There is mutations and is a driver of the disease [28, 52, 53]. In colon also evidence that PAK genomic amplification is linked to T-cell cancer, adenomatous polyposis coli (APC), the negative regulator lymphoma [64]. However, PAK status in hematological malignancies of β -catenin, is the most common protein rendered inactive by is underrepresented in the current body of literature. This may be mutations [83, 186, 195]. Individuals over the age of 40 with APC due to the fact that cytoskeletal rearrangement and cell migration is mutations have a high incidence (almost 100%) of developing the predominant activity ascribed to PAK proteins. These PAK . A mutation in another oncogene such as K-ras is activities are thought to be unimportant in hematological cell necessary for colon cancer progression in the mutant APC lineages. Of all the PAK , increases in PAK1 and PAK4 background [195]. In lung cancer, overexpression of constitutively mRNA or protein through transcriptional elevation or gene active β-catenin does not produce tumors. However in the context amplification in various solid tumors remain the best characterized. of oncogenic K-ras, lung tumors are able to form when β-catenin PAK Inhibitors Anti-Cancer Agents in Medicinal Chemistry, 2016, Vol. 16, No. 1 77

O F

O O H HN N N H N N N N N N HN O H O NH N Ru N N N N S N O Cl H O HO

1; PF-3758309 2; Staurosporine 3; !-FL 172 NH

O O F N O H N N N N HN H R Cl N N N N NH H Cl N N N O H N 4; R = O 6; LCH-7749944 7; FRAX486 5; R =

N HO N HN N N N NH N N N N N O HN N HN N O O NH N N Cl Cl Cl O NH2 S 8; FRAX597 9 10; AZ-PAK-36 11 N F F F O H HO N HN NN N S N N S N N N OH H NH H H N N Cl O 12; KY-04031 13; IPA-3 14

OH Cl HO O HO O HO H N H 2 O O O H N H N N O O O O NH H H

15; KPT-7523 16; Glaucarubinone Fig. (1). Select ATP competitive and allosteric PAK inhibitors. activity is elevated [186, 194]. Activated N-ras and β-catenin can appear in about 20% of patient samples [190, 197, 198]. Recent also induce melanoma with short latency [186, 193]. data has shown that N-ras but not K-ras is linked to reduced sensitivity to bortezomib treatment for myeloma patients [190]. Ras mutations are prevalent in hematological malignancies such as multiple myeloma, myelodysplastic syndrome, acute Mice lacking β-catenin in their hematopoietic stem cells (HSCs) myelogenous leukemia (AML), and acute lymphocytic leukemia have difficulty maintaining stem cells, although they do not fail to (ALL) [196]. In multiple myeloma, K-ras and N-ras mutations form HSCs [199]. Additionally, these β -catenin knockout mice 78 Anti-Cancer Agents in Medicinal Chemistry, 2016, Vol. 16, No. 1 Senapedis et al. Group I Group II Inactive Inactive

Rac1 Cdc42

Thr423/Ser474

CRIB AID Kinase (KD) Active Fig. (2). Group I and II PAK structure and activation. Group I PAK proteins form a homodimer in the inactive state. The auto-inhibitory domain (AID) of a monomer interacts intermolecularly with the kinase domain (KD) of the second monomer. Group II PAK proteins are inhibited by intramolecular inhibitions between AID (also referred to as pseudo substrate) and the KD. Full activation of both groups is achieved by binding to Rac1 or Cdc42 and phosphorylation of threonine 423 (PAK1) or serine 474 (PAK4).

Fig. (3). PAK signaling pathways. PAK proteins are influenced by small GTPases Rac1 and Cdc42 upstream. PAK signals to numerous substrates downstream that are in involved in survival (NF-κB), focal adhesion (GEF-H1, paxillin), cell cycle (β-catenin), apoptosis (caspase-8), cell migration (LIMK, cofilin) and proliferation (Akt, ERK). See references [5, 97] for detailed review of substrates and function. have a reduction in the development of chronic myelogenous Considering the predominant role of mutant Ras and β-catenin leukemia (CML) induced by BCR-ABL translocation [199]. In in hematological malignancies there is a lack of explicit research another study of leukemia stem cells (LSC) from mice with AML, that explores the function of PAK in HSC maintenance and the data shows the need for Wnt/β-catenin signaling in the self- hematological malignancies. This critical research on hematopoietic renewal of LSCs derived from HSCs or granulocyte macrophage stem cells and PAK would help inform and potentially expand the progenitors (GMP) [200]. Since β-catenin signaling is not normally population of individuals who might benefit from PAK targeted active in GMP and is therefore needed for transformation, therapies. inhibition of β-catenin represents an area of therapeutic intervention in AML [200]. In the development of mixed lineage leukemia ATP COMPETITIVE PAK INHIBITORS (MLL), LSCs display hyper-activation of the β -catenin pathway [201]. Inhibition or deletion of β -catenin blocks the growth and Staurosporine (compound 2; refer to Table 1 and Fig. 1 for oncogenic potential of MLL cell lines [201]. MLL cells resistant to select PAK inhibitor in vitro properties and structures, respectively) inhibitors of GSK-3β (a negative regulator of β -catenin) are re- is a natural product that inhibits not only the PAK proteins, but also sensitized by β-catenin inhibition [201]. many other kinases in the Sterile 20 (STE20) family [202, 203]. PAK Inhibitors Anti-Cancer Agents in Medicinal Chemistry, 2016, Vol. 16, No. 1 79

Fig. (4). PAK signaling is at the intersection of Ras and Wnt signaling. Both Ras and Wnt activation lead to inhibitory phosphorylation of GSK-3β. This phosphorylation leads to the loss of the APC/Axin/GSK-3β complex that degrades β-catenin. Loss of the degradation complex leads to stabilization and activation of β-catenin. PAK proteins can phosphorylate β-catenin at Serine 675 which enhances β-catenin nuclear localization and transcriptional activity of cell cycle drivers such as D1.

Because of its lack of selectivity, staurosporine has undesirable lines with upregulated epithelial markers (CLDN2, CDH1) are toxicities that limit its usefulness in the clinical setting. However, more resistant [205]. Moreover, when mesenchymal genes are analogs of staurosporine (Λ-FL172; compound 3) utilizing octahedral knocked down, the CRC cells become more resistant to PF- ruthenium show improved selectivity to PAK1 with some loss of 3758309 [205]. This data suggests that a patient population that has potency. Since there is no published in vitro or in vivo data on a higher expression of epithelial to mesenchymal transition (EMT) Λ-FL172, cellular and pharmacokinetic activity are unknown while markers would be more sensitive to PAK inhibition. PF-3758309, concerns of toxicity remain [204]. however, failed to be clinically beneficial in a single human trial. Poor oral bioavailability (~1 %) in humans and gastrointestinal The PAK inhibitor, PF-3758309 (compound 1), developed by toxicities coupled with no tumor response led to removal of PF- Pfizer, comes from a high throughput screen measuring the 3758309 from clinical investigation [3]. inhibition of PAK4 kinase activity [2]. This compound series arose from a positive hit in the initial PAK4 kinase screen which also Recently, Pfizer identified compound 4 which shows improved inhibits the phosphorylation of Serine 810 on GEF-H1 (a PAK4 PAK4 selectivity over PF-3758309 [206]. Because of its similarity phosphorylation site) in a cell-based assay [1, 2]. This compound is to PF-3758309, compound 4 is a substrate of the efflux transporters a pan-PAK inhibitor with less than ideal selectivity against other potentially reducing bioavailability (Caco-2 BA/AB: 326; a no kinases such as AKT and CHK2 (off-target proteins that could efflux ratio: 1). Attempts to decrease the efflux led to the discovery influence cellular activity) [2]. Nonetheless, this compound displays of a series of molecules with improved oral bioavailability that also broad anti-tumor activity in cellular proliferation and apoptosis maintain potency and reasonable PAK4 kinase selectivity (for assays in vitro and represses human Ras-driven xenograft models in example compound 5; Caco-2 BA/AB: 9.9) [206]. In mouse met mice (Colo-205, MDA-MB-231, A549, and M24 ) [2]. xenograft models of the human cell lines HCT116 and M24met, A follow-up in vitro study predicts patient populations that may compound 5 shows lower efficacy (52% and 68% TGI, respectively) be sensitive to PF-3758309 [205]. In this study researchers show when compared to PF-3758309 [206]. Despite this lower efficacy, that colorectal cancer (CRC) cell lines with upregulated mesenchymal these new compounds continue to hold promise for further markers (CALD1, VIM, ZEB1) are the most sensitive, while cell development. 80 Anti-Cancer Agents in Medicinal Chemistry, 2016, Vol. 16, No. 1 Senapedis et al.

A laboratory at the China Medical University in Shenyang (AZ-PAK-36, compound 10) has better cell permeability than described a quinazoline diamine molecule, LCH-7749944 compound 9 and leads to cellular EC50 of 140 nM (phos-PAK1 (compound 6), that inhibits PAK4 with minimal activity against assay). However, additional in vitro as well as in vivo efficacy and PAK1 [33]. Although it has not been crystallized, LCH-7749944 is pharmacology results are not available. predicted to bind the ATP binding pocket of PAK4. The compound A PAK inhibitor recently reported by Genentech shows is mainly characterized against gastric cancer cells where LCH- significant Group II selectivity (compound 11) [221]. Selectivity 7749944 treatment suppresses cell proliferation most likely through results for a panel of >222 kinases shows that compound 11 only a reduction in S phase and G1 cell cycle arrest. This compound is inhibited 60% of the Ephrin type-B receptor 1 (EphB1) at 100 nM. also able to inhibit migration and invasion while reversibly Compound 11 suppresses the migration, invasion, and viability of inhibiting filopodia formation. In cells, LCH-7749944 inhibits two triple negative (MDA-MB-436 and MCF10A PAK4 phosphorylation as well as the downstream effectors, PIK3A) cell lines in vitro; however, high doses (up to 50 µM) are phospho-c-Src and cyclin D1. Data from this study predict that required. The discrepancy in cellular activity between PF-3758309 SCG210 is involved in PAK4 mediated metastasis of gastric cancer (compound 1; cellular MDA-MB-436 EC : 800 nM) and cells [207]. There are no in vivo data reported for this compound 50 compound 11 (cellular EC : 10 µM) may be due to the lack of and further development is unknown. 50 selectivity of PF-3758309 against other kinases that drive tumor Afraxis, Inc, is developing another series of PAK inhibitors. growth. In a recent publication, FRAX486 (compound 7) is studied as a Lastly, KY-04031 (compound 12) is an early stage compound possible treatment of fragile X syndrome (FXS) [208]. FXS results isolated from a >8000 compound library using a high-throughput from the silencing of the fragile X mental retardation 1 (Fmr1) gene screen of PAK4 kinase activity [222]. This compound inhibits on the X and is one of the most common inherited PAK4 kinase activity with an IC : 790 nM. However, the forms of autism and mental disabilities in humans [208]. In the 50 selectivity against Group I PAKs as well as other kinases is normal setting, the structural integrity of dendritic spines and unknown. The EC viability data for KY-04031 is ~15 µM for synapses depends on PAK function for maintenance of the actin 50 LNCap and ~50 µM for PC-3. This series is in the early stages of cytoskeleton [209-213]. Fmr1 knockout (KO) mice recapitulate the development and will need to be improved in order to increase FXS human disorder which includes hyperactivity, repetitive potency and evaluate the other pharmacokinetic properties. behaviors, and seizures [214, 215]. Fmr1 KO mice also have impaired PAK signaling in the hippocampus [216]. In mouse ALLOSTERIC PAK INHIBITORS fibroblasts, the Fmr1 protein interferes with the activator of Group I PAKs, Rac1 [217]. There is also evidence that dominant negative One way to improve PAK inhibitor selectivity is to utilize PAK activity can suppress the Fmr1 knockout (KO) phenotype unique binding pockets in the individual PAK proteins through the [218]. FRAX486 is more potent against Group I PAK proteins discovery and development of allosteric inhibitors. In order to (IC50:~ 60 nM) than Group II PAK proteins (IC50: ~800 nM) [208]. increase specificity, one such approach by Deacon et al. utilizes a FRAX486 has high brain penetration and is able to reverse FXS PAK1 activation assay. This assay measures the concentration of phenotypes in adult Fmr1 KO mice in just one administration [208]. ATP remaining after Cdc42-activated PAK1 phosphorylation of the An advanced compound from the same chemical series, substrate, myelin basic protein (MBP) [223, 224]. This study FRAX597 (compound 8), shows activity in several cancer models, describes a novel compound, IPA-3 (compound 13), that only specifically in neurofibromatosis type 2 (NF2)-deficient schwannoma inhibits PAK1 in the inactive state through covalently binding to [219] and squamous cell carcinoma [61]. Neurofibromatosis type 1 PAK1-Cysteine 360. If PAK1 is pre-activated by Cdc42, IPA-3 can (NF1; neurofibromin) and NF2 (merlin) gene disorders are no longer bind and inhibit PAK activity. This activity is selective dominantly-inherited cancers that develop benign nerve sheath for Group I PAKs with almost no inhibition of Group II PAKs tumors of the peripheral nerves [44]. Deletion of neurofibromin observed. Cells pre-treated with IPA-3 have less PDGFR, Akt, and leads to increased Ras in the GTP bound state, whereas merlin Erk phosphorylation following serum starvation and activation with functions as an inhibitor of Rac1 signaling [219]. RNAi of Group I PDGF. In B-SC-1 cells exogenously expressing PAK1, IPA-3 PAKs suppresses cell transformation resulting from loss of NF2 treatment reverses PMA-induced membrane ruffling [224]. Despite activity [219]. FRAX597 is able to suppress cell proliferation of this promising data, metabolic instability of IPA-3 precludes its use transformed Schwann cells and Nf2-null SC4 cells [219]. Cells are in vivo and makes further development of this compound very arrested in the G1 phase of the cell cycle with decreases in S and challenging. G2/M phases. There is an absence of the sub-G1 phase, suggesting In a recent meeting, Novartis described a dibenzodiazepine a lack of apoptosis when using FRAX597. In an orthotopic model derivative Group I PAK allosteric inhibitor (compound 14) [88]. where luciferase-expressing Nf2-null schwannoma cells are However, the in vivo status and animal stability are currently injected into the myelinated nerve of mice, FRAX597 is able to unknown for this series [88]. slow the rate of tumor growth as compared to the negative vehicle control. Additionally, in vivo data show that FRAX597-treated mice Karyopharm Therapeutics is working on a novel chemical have lower average tumor weight as compared to vehicle treatment series of PAK4 inhibitors, characterized as allosteric modulators. (550 vs 1870 mg, respectively) [219]. One of the analogs in this family, KPT-7523 (compound 15), binds specifically to PAK4 with minimal binding to PAK5 and PAK6 and In a K-ras-driven mouse model of squamous cell carcinoma no discernible binding to Group I PAKs [225]. Advanced PAK4 (KrasG12D) skin cancer, FRAX597 is able to cause tumor allosteric modulator (PAM) analogs from this series are orally regression with a concomitant loss of Erk and Akt activity [61]. bioavailable and demonstrate anti-tumor cell selectivity. They show FRAX597 inhibition of PAK activity shows that Mek but not Akt is broad anti-cancer activity in vitro and in vivo across both solid and responsible for most of the K-ras-driven activity [61]. Considering hematological malignancies [226, 227]. Development of this the selectivity and the promising pre-clinical data, this series of compound series is currently ongoing. compounds could be developed further. Another Group I PAK selective chemical series is being TREATMENT COMBINATIONS WITH PAK INHIBITORS developed by AstraZeneca [220]. A 7-azaindole derivative PAK1 Based on what is known regarding PAK signaling, there are inhibitor (compound 9) shows preference for PAK1 kinase activity several logical combination treatments that have proven beneficial (IC50: 18 nM) over PAK4 (IC50: 550 nM). A more potent analog pre-clinically. In a synthetic lethal screen of Saccharomyces PAK Inhibitors Anti-Cancer Agents in Medicinal Chemistry, 2016, Vol. 16, No. 1 81 cerevisiae, knockouts of the two PAK homologues (Cla4 and potentially opening new avenues for future PAK inhibitor SKM1) show increased sensitivity to farnesyltransferase inhibitor development. FTI-277 [228]. In this study, the combination of FTI-277 and IPA-3 In terms of ATP competitive inhibitors, there is evidence that (compound 13) inhibits the proliferation of A375MM (melanoma), specificity and selectivity continue be major impediments to the A549 (lung), and HT29 (colon) cancer cell lines, but has limited progression of PAK inhibitors. Due to the 50% effect on HeLa and MCF7 cells [228]. Further elucidation of this between Group I and II PAKs and the 80% similarity within the mechanism shows that both HeLa and A375MM have increased groups, as well as a considerable amount of similarity to other nuclear localization of PAK after FTI-277 treatment, but only HeLa STE20 kinase domains, PAK selective ATP competitive inhibitors has elevated PAK protein levels [228]. This study does not examine will continue to prove difficult to develop. Additional approaches to the individual PAK isoforms to determine whether there might be a identifying allosteric inhibitors such as IPA-3, compound 14, and benefit of inhibiting Group II PAKs. Regardless, this data suggests KPT-7523 will need to be utilized in order to improve selectivity. It the promise of combining FTIs with PAK inhibitors for the should also be appreciated that not all PAK protein activity is treatment of melanoma, lung, and colon cancer. attributed to the kinase function (i.e. Caspase 8 inhibition) [106]. Cisplatin is successfully used for the treatment of gastric For these reasons, inhibitors that are isoform-specific, disrupt PAK cancer. However, like many , drug resistance binding partners, or reduce the level of PAK proteins should reduces cisplatin efficacy in patients over time. One particular study continue to garner the most attention. of cisplatin-resistant gastric cancer cells shows that PAK4 is elevated while RNAi of PAK4 causes re-sensitization of these cells CONFLICT OF INTEREST [229]. In addition, treatment with PF-3758309 (compound 1) plus cisplatin in these resistant cells reduces gastric cell viability [229]. William Senapedis, Marsha Crochiere, Erkan Baloglu, and Cisplatin-resistant cells treated with PF-3758309 alone show a Yosef Landesman are all current employees of Karyopharm marked reduction in Erk and Akt signaling. By using specific Therapeutics, Inc. inhibitors for either pathway, the authors demonstrate that inhibition of Akt can repress PAK4 activity through a reciprocal relationship. ACKNOWLEDGEMENTS These results are recapitulated in xenograft models using the William Senapedis is the principle author of this manuscript. same cells and treatment conditions [229]. Taken together, PAK4 Yosef Landesman, Marsha Crochiere and Erkan Baloglu confers cisplatin resistance in gastric cancer through activation of contributed to critical reviews of the manuscript as well as Erk and Akt pathways, making PAK4 an attractive target in this intellectual input to the content. Erkan Baloglu provided Fig. 1 setting. containing chemical structures. Gemcitabine is the primary line of defense against difficult-to- treat pancreatic cancer. In one study, gemcitabine treatment is REFERENCES combined with the natural product glaucarubinone (compound 16). [1] Guo, C.; Zhang, J.; McAlpine, I.; Johnson, C.; Marakovits, J.; Glaucarubinone is isolated from the seeds of Simarouba glauca tree Dong, L.; Kephart, S.; Yang, A.; Tikhe, J.; Li, H.; Guo, L.; [230, 231]. Treatment of pancreatic cell lines with glaucarubinone Bouzida, D.; Deng, Y.L.; Knighton, D.; Piraino, J.; Lee, J.; Smeal, alone shows a reduction of PAK1 and PAK4 phosphorylation. 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Received: January 21, 2015 Revised: April 04, 2015 Accepted: May 16, 2015