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Cancer stem-like cells (CSC) contribute to therapy resistance and recurrence. Focal adhesion kinase (FAK) has a role in CSC regulation. We determined the effect of FAK inhibition on breast CSC activity alone and in combination with adjuvant therapies. FAK inhibition reduced CSC activity and self-renewal across all molecular subtypes in primary human breast cancer samples. Combined FAK and paclitaxel reduced self-renewal in triple negative cell lines. An invasive breast cancer cohort confirmed high FAK expression correlated with increased risk of recurrence and reduced survival. Co-expression of FAK and CSC markers was associated with the poorest prognosis, identifying a high-risk patient population. Combined FAK and paclitaxel treatment reduced tumour size, Ki67, ex-vivo mammospheres and ALDH+ expression in two triple negative patient derived Xenograft (PDX) models. Combined treatment reduced tumour initiation in a limiting dilution re-implantation PDX model. Combined FAK inhibition with adjuvant therapy has the potential to improve breast cancer survival.

Purpose:Uveal melanoma is the most common eye cancer in adults. Approximately 50% of patients with uveal melanoma develop metastatic uveal melanoma (mUM) in the liver, even after successful treatment of the primary lesions. mUM is refractory to current chemo- and immune-therapies, and most mUM patients die within a year. Uveal melanoma is characterized by gain-of-function mutations in GNAQ/GNA11, encoding Gαq proteins. We have recently shown that the Gαq–oncogenic signaling circuitry involves a noncanonical pathway distinct from the classical activation of PLCβ and MEK–ERK. GNAQ promotes the activation of YAP1, a key oncogenic driver, through focal adhesion kinase (FAK), thereby identifying FAK as a druggable signaling hub downstream from GNAQ. However, targeted therapies often activate compensatory resistance mechanisms leading to cancer relapse and treatment failure.Experimental Design:We performed a kinome-wide CRISPR-Cas9 sgRNA screen to identify synthetic lethal gene interactions that can be exploited therapeutically. Candidate adaptive resistance mechanisms were investigated by cotargeting strategies in uveal melanoma and mUM in vitro and in vivo experimental systems.Results:sgRNAs targeting the PKC and MEK–ERK signaling pathways were significantly depleted after FAK inhibition, with ERK activation representing a predominant resistance mechanism. Pharmacologic inhibition of MEK and FAK showed remarkable synergistic growth-inhibitory effects in uveal melanoma cells and exerted cytotoxic effects, leading to tumor collapse in uveal melanoma xenograft and liver mUM models in vivo.Conclusions:Coupling the unique genetic landscape of uveal melanoma with the power of unbiased genetic screens, our studies reveal that FAK and MEK–ERK cotargeting may provide a new network-based precision therapeutic strategy for mUM treatment.See related commentary by Harbour, p. 2967

PURPOSE Inhibition of focal adhesion kinase has been shown to selectively kill mesothelioma cells that express low levels of moesin-ezrin-radixin-like protein (merlin). On this basis, we designed a randomized, phase II trial to investigate whether defactinib as maintenance therapy after standard first-line chemotherapy could improve progression-free survival (PFS) in patients with malignant pleural mesothelioma (MPM). METHODS This global, double-blind, randomized, placebo-controlled trial was conducted in patients with advanced MPM and disease control after at least four cycles of first-line chemotherapy. Patients were stratified for merlin and then randomly assigned (in a 1:1 fashion) to receive either oral defactinib or placebo until disease progression, unacceptable toxicity, or withdrawal occurred. The coprimary end points were PFS and overall survival (OS). Quality of life (QoL) was assessed using the Lung Cancer Symptom Scale for Mesothelioma tool. RESULTS Three hundred forty-four patients were randomly assigned to receive either defactinib (n = 173) or placebo (n = 171). The median PFS was 4.1 months (95% CI, 2.9 to 5.6 months) for defactinib versus 4.0 months (95% CI, 2.9 to 4.2 months) for placebo. The median OS was 12.7 months (95% CI, 9.1 to 21 months) for defactinib versus 13.6 months (95% CI, 9.6 to 21.2 months) for placebo (hazard ratio, 1.0; 95% CI, 0.7 to 1.4). Although shorter survival for both defactinib- and placebo-treated patients was observed, in the patients who had merlin-low MPM compared with the patients who had merlin-high MPM, there were no statistical differences in response rate, PFS, OS, or QoL between the treatment groups. The most common grade 3 or worse adverse events were nausea, diarrhea, fatigue, dyspnea, and decreased appetite. CONCLUSION Neither PFS nor OS was improved by defactinib after first-line chemotherapy in patients with merlin-low MPM. Defactinib cannot be recommended as maintenance therapy for advanced MPM.

Background Breast Cancer Stem-like Cells (BCSCs) have been associated with tumour development, metastasis and recurrence1. Focal Adhesion Kinase (FAK) is a non-receptor tyrosine kinase classically known for its role in metastasis, proliferation and survival. We have previously shown FAK plays a role in regulating CSC activity in DCIS2. We aimed to investigate FAK and CSC marker expression in a retrospective patient cohort. We aimed to evaluate the effects of FAK inhibition on CSC activity in Invasive Ductal Carcinoma. Methods Using a retrospective case-control cohort of 244 patients across a range of molecular phenotypes we evaluated FAK Immunohistochemical expression alongside CSC markers; Aldehyde Dehydrogenase 1 (ALDH1) and Integrin Alpha 6 (ITGa6). FAK expression was measured in IDC cell lines and ALDEFLUOR high expressing cells. FAK was inhibited using 0.5μM VS4718 or SiRNA and CSC activity evaluated in 5 cell lines and 25 patient samples. We determined the effects of 50mg/kg VS4718 for 4 weeks as single agent or in combination with Paclitaxel 7.5mg/kg in a ER-/PR-/HER- Patient Derived Xenograft model (PDX). Results Total FAK expression was associated with reduced breast cancer survival. Co-expression of FAK and either BCSC marker was associated with the poorest survival. FAK and CSC marker expression pFAKtFAKALDH1ITGα6tFAK and ALDH1tFAK and ITGα6Recurrence Risk0.58 (0.31-1.08) p = 0.0842.05 (1.23-3.43) p = 0.0062.21 (1.20-4.05) p=0.0111.54 (0.92-2.23) p=0.107 Breast Cancer Death0.41 (0.12-1.51) p=0.1824.84 (2.33 -10.04) p = pFAK was higher in ALDEFLUOR expressing cells and triple negative cell lines. SiRNA knockout of FAK reduced mammosphere formation, self-renewal and ALDEFLUOR expression from 1.2% to 0.2% (p= FAK inhibition reduces MFE Primary Breast Cancer samples ER negative cell lines ER+/PR+/ Her2-ER-/PR-/Her2+ER-/PR-/Her2-SKBr3MDA-MB-231SUM159Primary mammosphere formation51.1% (n=17)53.2% (n=4)49.6% (n=5)74.1%84.8%67.6%Secondary mammosphere formation45.8% (n=8) 43.9% (n=3)42.1%57.9%47.5%Percentage of mammospheres formed given relative to control. Cell line work, minimum n=6. All above significant p= VS4718 reduced tumour growth, Ki67 staining and CSC activity in our triple negative PDX model. VS4718 administration reduced ex-vivo mammosphere formation, tumour initiating capacity and prevented ALDEFLUOR enrichment when used in combination with Paclitaxel. Conclusions FAK, ALDH1 and ITGa6 are associated with increased breast cancer mortality in early breast cancer. Inhibition of FAK reduces CSC activity in vitro and in vivo in cell lines and patient samples. This data suggest that FAK inhibition may be used to reduce CSC activity in triple negative carcinoma. 1. Williams et al, Stem cells 2015. Citation Format: Timbrell S, Aglan H, Cramer A, Foden P, Weaver D, Pachter J, Farnie G, Clarke R, Bundred N. Investigating the role of focal adhesion kinase in regulating CSC activity in invasive ductal carcinoma [abstract]. In: Proceedings of the 2018 San Antonio Breast Cancer Symposium; 2018 Dec 4-8; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2019;79(4 Suppl):Abstract nr P2-06-07.

COVID-19 affects vulnerable populations including elderly individuals and patients with cancer. Natural Killer (NK) cells and innate-immune TRAIL suppress transformed and virally-infected cells. ACE2, and TMPRSS2 protease promote SARS-CoV-2 infectivity, while inflammatory cytokines IL-6, or G-CSF worsen COVID-19 severity. We show MEK inhibitors (MEKi) VS-6766, trametinib and selumetinib reduce ACE2 expression in human cells. Chloroquine or hydroxychloroquine increase cleaved active SP-domain of TMPRSS2, and this is potentiated by MEKi. In some human cells, remdesivir increases ACE2-promoter luciferase-reporter expression, ACE2 mRNA and protein, and ACE2 expression is attenuated by MEKi. We show elevated cytokines in COVID-19- (+) patient plasma (N=9) versus control (N=11). TMPRSS2, inflammatory cytokines G-CSF, M- CSF, IL-1a, IL-6 and MCP-1 are suppressed by MEKi alone or in combination with remdesivir. MEKi enhance NK cell (but not T-cell) killing of target-cells, without suppressing TRAIL-mediated cytotoxicity. We generated a pseudotyped SARS-CoV-2 virus with a lentiviral core but with the SARS-CoV-2 D614 or G614 SPIKE (S) protein on its envelope and used VSV-G lentivirus as a negative control. Our results show infection of human bronchial epithelial cells or lung cancer cells and that MEKi suppress infectivity of the SARS-CoV-2-S pseudovirus following infection. We show a drug class-effect with MEKi to promote immune responses involving NK cells, inhibit inflammatory cytokines and block host-factors for SARS-CoV-2 infection leading also to suppression of SARS-CoV-2-S pseudovirus infection of human cells in a model system. MEKi may attenuate coronavirus infection to allow immune responses and antiviral agents to control COVID-19 disease progression and severity.

The RAS/RAF/MEK/ERK pathway is the most mutated oncogenic pathway in cancer, and RAS pathway mutations often present with an overall worse prognosis. Although RAF and MEK have been validated as anticancer targets and several BRAF and MEK inhibitors (MEKi) are FDA approved, acquired resistance develops in most patients. Preclinically, inhibition of RAF or MEK has been found to activate focal adhesion kinase (FAK) signaling which may bypass RAS pathway blockade by driving tumor growth through activation of downstream pathways such as RhoA and YAP. VS-6766 is a unique dual RAF/MEK inhibitor which allows VS-6766 to block MEK signaling without the compensatory MEK activation that limits the efficacy of other MEK inhibitors. Defactinib is a selective FAK inhibitor (FAKi). Clinical studies are ongoing evaluating VS-6766 and defactinib for the treatment of various solid tumors. In 3D proliferation assays in vitro, defactinib was synergistic with VS-6766 or trametinib (MEKi) in reducing viability of several human tumor cell lines, including KRAS mutant (mt) ovarian cancer (TOV-21G) and KRAS-G12V mt non-small cell lung cancer (NSCLC; H441). We next investigated whether FAKi augments the efficacy of VS-6766 in solid tumor models. Combination of a FAKi with VS-6766 in a KRAS mt ovarian xenograft model (TOV21G) induced >30% tumor regression in 9/10 mice, whereas each agent alone induced mainly tumor stasis (>30% tumor regression with FAKi monotherapy or VS-6766 monotherapy in 1/10 and 3/10 mice, respectively) following 11 days of treatment. Similar results were observed in KRAS mt NSCLC (H2122) and GNAQ mt uveal melanoma (92.1) models in which the combination of FAKi with VS-6766 or trametinib induced tumor regression. In several patients with KRAS mt tumors, sequential biopsies showed that treatment with VS-6766 induced FAK activation (pY397) as a potential resistance mechanism, and this increased FAK activation was reversed in the presence of the defactinib/VS-6766 combination. Accordingly, the combination of VS-6766 with defactinib showed clinical activity in low grade serous ovarian cancer (LGSOC; ORR = 56% in KRAS-G12 mt and ORR = 41% in all 17 LGSOC patients; 8/17/20 data cut off). Importantly, the combination of defactinib with VS-6766 also induced responses in patients who had progressed on previous MEK inhibitor regimens. VS-6766 with defactinib also showed clinical activity in KRAS-G12V mt NSCLC. Furthermore, this combination regimen of VS-6766 with defactinib exhibited a manageable safety profile with no patients discontinuing for adverse events (NCT03875820). These preclinical and clinical data support the recent initiation of two registration-directed studies evaluating VS-6766 ± defactinib for the treatment of recurrent LGSOC with or without a KRAS mutation (NCT04625270) and recurrent NSCLC with KRAS-G12V or other KRAS mutation (NCT04620330). Citation Format: Silvia Coma, Justine S. Paradis, J Silvio Gutkind, Jonathan A. Pachter. Synergistic antitumor efficacy of the dual RAF/MEK inhibitor VS-6766 with FAK inhibition for treatment of RAS-dependent solid tumors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1425.

Despite remarkable clinical efficacy, CAR-T therapy has been limited by life-threatening toxicities in over 30% of patients (Maude, NEJM 2014 and Davila, SciTransMed 2014). Toxicities primarily manifest as Cytokine Release Syndrome (CRS) characterized by an early phase with fever, hypotension, and elevations of cytokines including IFNγ, GM-CSF, TNF, IL-10, and IL-6. Using a protein kinase inhibitor library containing 644 independent compounds, we aimed to identify compounds that could block CRS-related cytokine production without inhibiting CAR-T function. We identified, duvelisib (kindly provided by Verastem Oncology, Needham, MA), a novel and selective dual PI3K-δ,γ inhibitor as a potent inhibitor of CRS in vitro and in vivo without attenuating CAR-T function. Duvelisib (Copiktra) is approved for the treatment of relapsed/refractory CLL after 2 prior therapies and follicular lymphoma after 2 prior systemic therapies; the latter gained accelerated approval status based on overall response rate and continued approval may be contingent on confirmatory trials. To assess the ability of duvelisib to inhibit CAR-T mediated CRS, we performed an in vitro CRS assay (Singh, Cytotherapy, 2017). CART19 (19-28BBζ, 25,000 cells), Ramos (CD19+, 50,000 cells), and immature dendritic cells (iDC, 2,500 cells) were co-cultured in a 96 well plate for 48hrs in the presence of varying concentrations of duvelisib (0.3nM-1000nM). Secreted IL-6, a surrogate marker of CRS, was determined using a human IL-6 ELISA (R&D Systems). Duvelisib reduced IL-6 levels in a dose-dependent manner with 30nM duvelisib reducing IL-6 secretion more than 10-fold (Fig 1a). To confirm that duvelisib did not inhibit CAR-T function, we performed in vitro killing assays, in which CAR-T efficacy was determined using BLI imaging of luciferase labeled CD19+ Ramos targets. At clinically relevant therapeutic doses (C max of 200 to 500nM) of duvelisib, there was no effect on CAR-T function in vitro. Treatment with 10nM duvelisib resulted in a statistically insignificant ~20% reduction of CART19 efficacy (p>0.05) (Fig 1b). Of interest, although selective inhibitors of either or PI3Kδ (GSK2292767) or PI3Kγ (IPI549) had modest effects on blocking CAR-T induced IL-6 production in this in vitro model, the effect of combining both inhibitors had a more dramatic effect similar to the dual PI3K-δ,γ inhibitor, duvelisib. Next we assessed the ability of duvelisib to block IL-6 secretion in vivo using a fully immunocompetent murine model of CRS. Six-week old BALB/c mice were injected with the mitogenic anti-CD3ε antibody, 145-2C11 (10 µg/mouse). Duvelisib (450µg/mouse) was administered daily intraperitoneally (I.P), with the first dose of duvelisib injected 24 hours prior to injection of 145-2C11. Plasma IL-6 levels were determined using a mouse IL-6 ELISA (R&D Systems). Injection of 145-2C11 acutely elevated plasma IL-6 >32 fold relative to non-treated controls (4hrs; Control 34.4 pg/mL versus vs. 145-2C11 1088±99.6 pg/mL). Duvelisib significantly reduced mean plasma IL-6 >54% at 4hrs (Vehicle 1088±99.6 pg/ml vs duvelisib 492±99.6 pg/ml, p≤ 0.01) and >78% at 24hrs (Vehicle 220±101 pg/ml vs. duvelisib 49.3±16.1, p≤ 0.01) (Fig 1c) consistent with the effect of duvelisib in our in vitro CAR-T-induced CRS model described above. These studies demonstrate that duvelisib can inhibit CAR-T induced IL-6 production from iDC while having no inhibitory effect on CAR-T. Experiments are currently in progress to further characterize PI3K-δ,γ inhibition in humanized mouse models of CRS and CAR-T efficacy. Our preclinical data suggest that dual PI3K-δ,γ inhibition with duvelisib may represent an attractive alternative to IL-6 receptor antagonists, such as tocilizumab, for the treatment of CAR-T-associated cytokine release syndrome in the clinic which warrants further clinical evaluation. Figure 1. Dose dependent effect of duvelisib on IL-6 secretion in co-culture of CART19, Ramosluc, and iDC after 48 hours (a) Dose dependent inhibition of CART19 mediated cytotoxicity using duvelisib (b) Duvelisib effectively lowers IL-6 plasma level in an immunocompetent BALB/cJ mouse model of CRS (c). (** p≤ 0.01 and ns p>0.05). Figure 1 Disclosures Cooper: Wugen: Consultancy, Current equity holder in private company, Patents & Royalties. Pachter:Verastem Therapeutics: Current Employment. DiPersio:Magenta Therapeutics: Membership on an entity's Board of Directors or advisory committees.

TPS5603 Background: VS-6766 is a unique small molecule inhibitor that blocks MEK kinase activity and RAF phosphorylation of MEK. This mechanism of blockade has been shown to limit compensatory MEK activation, thereby potentially enhancing efficacy of MEK inhibition. Defactinib, (VS-6063), an orally active small molecule, is a potent adenosine 5'- triphosphate (ATP) competitive, reversible inhibitor of focal adhesion kinase (FAK). Defactinib has shown synergistic activity with BRAF and MEK inhibitors in both in vitro and in vivo solid tumor models. Prior molecularly unselected studies with single agent MEK inhibitors have shown response rates up to 26% in recurrent LGSOC. A third of patients with recurrent LGSOC harbor somatic KRAS mutations. FAK inhibition has been shown to induce tumor regression when combined with RAF, MEK or RAF/MEK inhibitors in in vivo models of KRAS mutant ovarian cancer. The combination of VS-6766 and defactinib is currently being evaluated in the ongoing Investigator Sponsored FRAME study (NCT03875820). In this proof of concept study, durable objective responses have been reported in recurrent LGSOC patients, particularly those with KRAS mutations including patients who have had a prior MEK inhibitor (Banerji et al AACR 2020). Based on preclinical studies demonstrating efficacy of both VS-6766 and the VS-6766/defactinib combination and preliminary results of the FRAME study, the phase II ENGOT-ov60/GOG3052 has been developed in recurrent LGSOC. Methods: This is a Phase II, adaptive, two-part, multicenter, parallel cohort, randomized, open label study designed to evaluate the efficacy and safety of VS-6766 versus VS-6766 in combination with defactinib (NCT04625270). The study will be conducted in two parts. Part A will determine the optimal regimen based on confirmed overall response rate (independent radiology review) in KRAS-mutated LGSOC. Part B will determine the efficacy of the optimal regimen identified in Part A in KRAS-mutated and KRAS wild-type LGSOC. The minimum expected enrollment is 52 subjects with KRAS-mutated tumors (32 subjects in Part A and 20 in Part B) and 36 with KRAS wild-type tumors in Part B. Patients will be randomized to receive VS-6766 (4.0 mg PO, twice weekly 3 weeks on, 1 week off) or VS6766 with defactinib (VS-6766 3.2 mg PO, twice weekly + defactinib 200 mg PO BID 3 weeks on, 1 week off) till progression. Key inclusion criteria include histologically confirmed LGSOC, presence of KRAS mutation (Part A), prior systemic therapy for metastatic disease and up to 1 prior line of MEK/RAF inhibitor therapy permitted. This international study is open to enrollment. Clinical trial information: NCT04625270.

// Yuichi Murakami 1, 2, * , Kahori Sonoda 1, * , Hideyuki Abe 3 , Kosuke Watari 1 , Daiki Kusakabe 1, 4 , Koichi Azuma 5 , Akihiko Kawahara 3 , Jun Akiba 3 , Chitose Oneyama 6 , Jonathan A. Pachter 7 , Kazuko Sakai 8 , Kazuto Nishio 8 , Michihiko Kuwano 2 and Mayumi Ono 1 1 Department of Pharmaceutical Oncology, Graduate School of Pharmaceutical Sciences, Kyushu University, Fukuoka, Japan 2 Cancer Translational Research Center, St. Mary’s Institute of Health Sciences, Fukuoka, Japan 3 Department of Diagnostic Pathology, Kurume University Hospital, Fukuoka, Japan 4 Physical Chemistry for Life Science Laboratory, Faculty of Pharmaceutical Sciences, Kyushu University, Fukuoka, Japan 5 Division of Respirology, Neurology and Rheumatology, Department of Internal Medicine, Kurume University School of Medicine, Fukuoka, Japan 6 Division of Microbiology and Oncology, Aichi Cancer Center Research Institute, Nagoya, Japan 7 Verastem Inc., Cambridge, MA, USA 8 Department of Genome Biology, Kinki University Faculty of Medicine, Osaka, Japan * These authors have contributed equally to this work Correspondence to: Mayumi Ono, email: mono@phar.kyushu-u.ac.jp Keywords: afatinib resistance, SRC family kinase, focal adhesion kinase, non-small cell lung cancer Received: February 28, 2017 Accepted: July 18, 2017 Published: August 07, 2017 ABSTRACT Second- and third-generation inhibitors of epidermal growth factor receptor (EGFR) tyrosine kinase activity (EGFR-TKIs) are improving the treatment of patients with non-small cell lung cancer. Here we established two sublines (BR1-8 and BR2-3) resistant to a second-generation inhibitor, afatinib, from the human lung cancer cell line HCC827 that harbors a mutation that activates the tyrosine kinase activity of EGFR. These afatinib-resistant sublines were resistant to first-generation EGFR-TKIs, gefitinib and erlotinib, and a third-generation EGFR-TKI, osimertinib. These resistant sublines showed markedly reduced levels of multiple EGFR family proteins, including the activated mutant EGFR, and complete loss of EGFR amplification as compared with their parental HCC827 cells harboring amplification of EGFR gene. Treatment with the multikinase inhibitor dasatinib or transfection with a SRC small interfering RNA inhibited cell survival and AKT phosphorylation in drug-resistant sublines to a greater extent compared with HCC827 cells. Further, the migration of drug-resistant cells was greater compared with that of HCC827 cells and was inhibited by dasatinib or an FAK inhibitor. These findings indicate that compensatory activation of SRC family kinases (SFKs) and FAK supports the survival and migration of afatinib-resistant cells when the expression of multiple EGFR family proteins was mostly abrogated. Combinations of potent drugs that target SFKs and FAK may overcome the resistance of lung cancer cells to second-generation TKIs.

Because cancer stem cells (CSCs) have been implicated in chemo-resistance, metastasis and tumor recurrence, therapeutic targeting of CSCs holds promise to address these clinical challenges to cancer treatment. VS-4718 and VS-6063 are potent inhibitors of focal adhesion kinase (FAK), a non-receptor tyrosine kinase that mediates cell signals transmitted by integrins and growth factor receptors. We report here that inhibition of FAK kinase activity by VS-4718 or VS-6063 preferentially targets CSCs, as demonstrated by a panel of orthogonal CSC assays in cell line models and surgically resected primary breast tumor specimens cultured ex vivo. Oral administration of VS-4718 or VS-6063 to mice bearing xenograft models of triple-negative breast cancer (TNBC) significantly reduced the proportion of CSCs in the tumors, as evidenced by a reduced tumor-initiating capability upon re-implantation in limiting dilutions of cells prepared from these tumors. In contrast, the cytotoxic chemotherapeutic agents, paclitaxel and carboplatin, enriched for CSCs, consistent with previous reports that these cytotoxic agents preferentially target non-CSCs. Importantly, VS-4718 and VS-6063 attenuated the chemotherapy-induced enrichment of CSCs in vitro and delayed tumor regrowth following cessation of chemotherapy. An intriguing crosstalk between FAK and the Wnt/β-catenin pathway was revealed wherein FAK inhibition blocks β-catenin activation by reducing tyrosine 654 phosphorylation of β-catenin. Furthermore, a constitutively active mutant form of β-catenin reversed the preferential targeting of CSCs by FAK inhibition, suggesting that this targeting is mediated, at least in part, through attenuating β-catenin activation. The preferential targeting of cancer stem cells by FAK inhibitors provides a rationale for the clinical development of FAK inhibitors aimed to increase durable responses for cancer patients.

Gene copy number alterations, tumor cell stemness, and the development of platinum chemotherapy resistance contribute to high-grade serous ovarian cancer (HGSOC) recurrence. Stem phenotypes involving Wnt-β-catenin, aldehyde dehydrogenase activities, intrinsic platinum resistance, and tumorsphere formation are here associated with spontaneous gains in Kras, Myc and FAK (KMF) genes in a new aggressive murine model of ovarian cancer. Adhesion-independent FAK signaling sustained KMF and human tumorsphere proliferation as well as resistance to cisplatin cytotoxicity. Platinum-resistant tumorspheres can acquire a dependence on FAK for growth. Accordingly, increased FAK tyrosine phosphorylation was observed within HGSOC patient tumors surviving neo-adjuvant chemotherapy. Combining a FAK inhibitor with platinum overcame chemoresistance and triggered cell apoptosis. FAK transcriptomic analyses across knockout and reconstituted cells identified 135 targets, elevated in HGSOC, that were regulated by FAK activity and β-catenin including Myc, pluripotency and DNA repair genes. These studies reveal an oncogenic FAK signaling role supporting chemoresistance., eLife digest Ovarian cancer is one of the deadliest types of cancer in women. There are two main reasons for the aggressiveness of this cancer. First, ovarian cancer cells can spread to other parts of a woman’s body before she has been diagnosed, where the cells grow as tiny clumps or spheres of tumor cells, also called tumorspheres. Second, in the majority of patients, some ovarian cancer cells will develop resistance to the chemotherapy used. It is not clear exactly how these tumor cells become resistant to therapy. One way in which cells could do this is by gaining extra copies of genes that remove toxic substances or repair DNA, which help them withstand the therapy. Here, Osterman, Ozmadenci, Keinschmidt, Taylor, Barrie, Jiang, Bean, Sulzmaier et al. set up a new experimental method to study how some ovarian cancer cells resist chemotherapy. Comparing ovarian cancer cells from mice at early and late stages of the disease showed that the later-stage, more aggressive cells had more genetic changes. One of these changes affected the gene for a protein called FAK, which was found to have more copies than normal. The FAK protein is an enzyme that helps cancer cells move around. In cells from mice with late-stage cancer, FAK was over-active and present at high levels. When these cells grew as tumorspheres, the tumors were more resistant to chemotherapy than their early-stage counterparts. In patients who have received chemotherapy, surviving tumor cells also exhibit high levels of FAK activity. Human ovarian cancer cells that are resistant to chemotherapy can be grown into tumors in mice, where they retain their resistance to chemotherapy. However, if chemotherapy is combined with a drug that targets the FAK enzyme, the tumors shrink. This experiment highlights a possible weak spot of these tumor cells. To understand how FAK makes ovarian cancer cells resistant to chemotherapy, Osterman et al. deleted the gene for FAK from the cells and then looked at how this changed the levels of activation of different genes. They found that, in addition to its effects on cell movement, FAK also activated a group of genes that increase resistance to chemotherapy and repair damaged DNA. This better understanding of how ovarian cancer cells resist chemotherapy could lead to new therapies. In particular, there is now a clinical trial for women with chemo-resistant ovarian cancer in which standard chemotherapy is combined with an inhibitor of the FAK protein.

Intro: Relapsed/refractory diffuse large B cell lymphoma (r/r DLBCL) is associated with poor outcomes, and remains an area of unmet clinical need. Emerging data indicate that a key macrophage immune checkpoint termed CD47 can be effectively targeted in r/r DLBCL. CD47 is a potent "don't eat me" signal and impairs tumor cell phagocytosis by engaging the inhibitory macrophage receptor, SIRPα. Importantly, early clinical studies demonstrate that blocking CD47-SIRPα interactions with an anti-CD47 antibody (magrolimab) yields promising activity in r/r DLBCL patients, particularly when combined with rituximab, which further potentiates lymphoma cell phagocytosis via its Fc domain. Moving forward, it will be crucial to identify combinatorial strategies that improve the efficacy of anti-CD47 therapy in r/r DLBCL. Tumor-associated macrophages (TAMs) contribute to an immunosuppressive environment in cancer through secretion of cytokines, metabolites, and expression of immune checkpoints. Sustained PI3K-γ signaling is critical to maintain immune suppressive TAM functions, and PI3K-γ inhibition polarizes TAMs into a pro-inflammatory state that culminates in potent anti-tumor T cell responses, sensitizing cancers to PD-1 blockade therapy. We hypothesized that activating TAMs through PI3K-δ/γ inhibition would also augment their inherent phagocytic capacity and synergize with phagocytosis-targeting treatments such as CD47 blockade. Thus, we investigated the impact of the dual PI3K-δ/γ inhibitor, duvelisib, on expression of key phagocytic receptors and pathways in human macrophages, as well as its effect on enhancing lymphoma cell phagocytosis with the anti-CD47 antibody, magrolimab. Results: RNA sequencing of duvelisib-treated human monocyte-derived macrophages revealed that PI3K-δ/γ inhibition induced broad transcriptional changes suggestive of an activated macrophage state (Fig 1A), including upregulation of interferon response genes (IRF9) and costimulatory molecules (CD86), while decreasing expression of immune suppressive cytokines (IL10) and inhibitory ligands (PDL1). Moreover, duvelisib upregulated transcription of canonical pro-phagocytic receptors (LRP1, FCGR2A,CD44), lysosomal degradation components (LYZ, CTSS) and phosphatidylserine-bridging genes (MFG-E8, GAS6), while inhibiting metabolic pathways, most notably mTOR, each of which has been associated with increased and more efficient phagocytosis. Given these findings, we evaluated the effect of dual PI3K-δ/γ inhibition on macrophage phagocytosis of DLBCL cells in the context of CD47 blockade in vitro. Across a broad panel of human DLBCL cell lines and multiple monocyte donors, pre-treatment of macrophages with duvelisib significantly increased lymphoma cell phagocytosis by magrolimab (Fig 1B-C). This effect reflected a direct increase in the phagocytic capacity of treated macrophages, as lymphoma cells were not exposed to duvelisib. In separate experiments, we found that duvelisib rarely induced full apoptosis of DLBCL cells, but did promote the expression of "eat me" signals, such as calreticulin and phosphatidylserine on many of the cell lines examined (Fig 1D). Collectively, these results suggest that PI3K-δ/γ inhibition enhances the inherent phagocytic capacity of macrophages, while also sensitizing lymphoma cells for engulfment. Similarly, potent effects were not observed with PI3K-δ- or PI3K-γ-specific inhibitors, suggesting that combined PI3K-δ/γ inhibition is critical to mediating these beneficial effects. Finally, to determine whether duvelisib impacted the efficacy of magrolimab in vivo, we treated human DLBCL xenografts and observed that while duvelisib had minimal impact on lymphoma growth, the combination of duvelisib and magrolimab induced complete tumor rejection in a high proportion of mice, leading to increased survival compared to mice treated with magrolimab alone (Fig 1E). Conclusion: Collectively, our findings suggest that targeting the PI3K-δ/γ axis favorably induces key macrophage phagocytic pathways, while simultaneously upregulating important "eat me" signals on lymphoma cells. Together, these alterations lower the phagocytic threshold of TAMs and dramatically increase the efficacy of anti-CD47 therapy in DLBCL xenograft models. This work paves the way for early-phase studies of duvelisib and magrolimab in r/r DLBCL and other lymphomas. Disclosures Godfrey: Verastem: Research Funding; Merck: Research Funding; Gilead: Research Funding. Coma:Verastem Oncology, Inc: Current Employment, Current equity holder in publicly-traded company. Maute:Gilead Sciences, Inc.: Current Employment. Chao:Gilead Sciences: Current Employment. Pachter:Verastem Therapeutics: Current Employment. Kline:Kite/Gilead: Speakers Bureau; Merck: Research Funding; Verastem: Membership on an entity's Board of Directors or advisory committees, Research Funding; Karyopharm: Membership on an entity's Board of Directors or advisory committees; Seattle Genetics: Membership on an entity's Board of Directors or advisory committees.

Blockade of immune checkpoints, such as PD-1, has provided meaningful clinical benefit for patients with a broad range of malignancies. However, limited response rates and resistance after initial response suggest a need for additional strategies. Duvelisib is an oral, dual PI3K-δ,γ inhibitor that is FDA-approved as monotherapy for chronic lymphocytic leukemia (CLL)/small lymphocytic lymphoma (SLL) and follicular lymphoma (FL) after ≥2 prior systemic therapies. In addition to targeting malignant B cells, duvelisib regulates key immune cells in the tumor microenvironment, making it a promising candidate for combination with PD-1 blockade. Using T cells from human donors or CLL patients, we now show that duvelisib induced a 3 to 4-fold increase in T cell expansion ex vivo, and that dual PI3K-δ,γ inhibition was necessary for maximal effect. Furthermore, duvelisib reduced expression of T cell exhaustion markers (Tim-3, Lag-3) and enriched early memory T cell populations. We have previously shown that duvelisib is more effective than inhibitors of only PI3K-δ or PI3K-γ in reducing Tregs and immunosuppressive myeloid cells when combined with an OX40 agonist antibody in a syngeneic tumor model. Accordingly, dual PI3K-δ,γ inhibition may represent an advantage over agents that inhibit only PI3K-δ or PI3K-γ for combination with PD-1 blockade. We next investigated whether duvelisib augments the efficacy of a PD-1 antibody (anti-PD-1) in solid tumor and lymphoma syngeneic models. In mice bearing CT26 colorectal tumors, duvelisib or anti-PD-1 alone induced tumor growth inhibition (TGI) of 59 and 49%, respectively, whereas duvelisib plus anti-PD-1 resulted in enhanced TGI (81%) and survival. Sixty percent of mice treated with duvelisib plus anti-PD-1 were tumor free, whereas only 10% of the mice treated with anti-PD-1 alone were tumor free by 60 days after tumor inoculation. In the A20 B-cell lymphoma model, reductions in tumor volume were observed with duvelisib or anti-PD-1 alone (TGI = 50 and 54%, respectively). Duvelisib plus anti-PD-1 was associated with enhanced antitumor activity (TGI = 85%) and survival (median survival of 37 days for the combination group compared to 16.5 and 18 days for duvelisib or anti-PD-1 monotherapy groups, respectively). Reductions in Tregs, M2 macrophages, and M-MDSCs in tumors were observed in the duvelisib plus anti-PD-1 combination group compared to anti-PD-1 alone. Taken together, these data indicate the value of dual PI3K-δ,γ inhibition for combination with PD-1 blockade and support clinical evaluation of duvelisib with anti-PD-1 in patients with solid tumors or hematologic malignancies. Clinical studies are underway investigating the combination of duvelisib plus nivolumab in aggressive lymphomas (Richter's transformation or transformed FL; NCT03892044) and duvelisib plus pembrolizumab in head and neck squamous cell carcinoma. Citation Format: Silvia Coma, Christopher R. Funk, Shuhua Wang, Edmund K. Waller, David T. Weaver, Jonathan A. Pachter. Synergistic anti-tumor efficacy of the dual PI3K-δ/PI3K-γ inhibitor duvelisib with PD-1 blockade in solid tumor and lymphoma models [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr CT045.

Uveal melanoma (UM) is characterized by gain-of-function mutations in GNAQ or GNA11, encoding Gα proteins from the Gq/11 family. UM is the most common eye malignancy in adults. Approximately 50% of UM patients develop liver metastasis (mUM) within 5-10 years after diagnosis, independently of the successful treatment of the primary lesions. mUM is refractory to cytotoxic, targeted, and immunotherapies, with most mUM patients dying within a year. Recent information suggests that GNAQ-oncogenic signaling involves a non-canonical pathway distinct from the activation of PLCβ and PKC-MEK-ERK, which may explain the failure of MEK inhibitors (MEKi) in increasing mUM patient survival. Instead, we found that GNAQ promotes the activation of YAP1, a key oncogenic driver, by a mechanism involving the activation of RhoA by the direct association of Gαq to TRIO, a Rho-GEF (Cancer Cell, 2014). In turn, YAP1 is essential for uveal melanoma cell growth, however no effective and safe YAP1 inhibitors are currently available. Using a novel bioinformatics pipeline, we recently found that PTK2, encoding Focal Adhesion Kinase (FAK), is a synthetic lethal gene with GNAQ activation, and uncovered that GNAQ controls YAP1 through FAK (Cancer Cell, 2019). This study identified FAK as a druggable signaling hub downstream from GNAQ in UM. However, activation of compensatory pathways often results in resistance to targeted agents. Here, we combined the use of CRISPR-Cas9 sgRNA screens with a recently described Cancer Signaling Toolkit approach to identify synthetic lethal interactions enhancing the response to FAKi and signaling networks mediating drug resistance, respectively. Remarkably, both approaches converged to reveal that co-targeting FAK and the MEK-ERK pathway would be a promising combination for treatment of UM. Indeed, MEK-ERK pathway inhibition by multiple approved MEKis (e.g., trametinib), combined with FAK inhibition (VS-4718 or defactinib), showed remarkable synergistic growth inhibitory effects in UM cells. Additionally, the novel RAF/MEK inhibitor RO5126766 also showed synergistic anti-proliferative effects with defactinib. Accordingly, FAKi combination with MEKi exerted cytotoxic effects (apoptotic death) leading to rapid tumor shrinkage in UM xenografts, whereas single drugs were primarily cytostatic. Furthermore, the FAKi/MEKi combination was successful at reducing tumor burden in recently developed liver metastasis UM models. By coupling the unique genetic landscape of UM with the power of unbiased computational pipelines and systems biology genetic screens, our studies revealed that FAK and MEK-ERK co-targeting may provide a new network-based precision therapeutic strategy for mUM treatment. Indeed, the combination of defactinib and RO5126766 is currently being evaluated in patients with various solid tumors (NCT03875820), and could be explored in mUM based on these preclinical findings. Citation Format: Justine S. Paradis, Monica Acosta, Nadia Arang, Robert Saddawi-Konefka, Ayush Kishore, Takahito Sugase, Xiaodong Feng, Kris C. Wood, Silvia Coma, Mizue Terai, Takami Sato, Jonathan A. Pachter, J. Silvio Gutkind. FAK and MEK co-targeting: A new multimodal precision therapy for GNAQ-driven uveal melanoma [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 6406.

Background: Preclinical experiments in-vitro show MEK inhibitors can cause upregulation of p-FAK in KRASM cells. The combination of a MEK and FAK inhibitor could overcome this in-vitro and in-vivo. Methods: We are conducting an open label phase I dose escalation with expansion study (NCT03875820). Dose escalation evaluated a twice a week schedule (Mon/Thurs) CH5126766/VS-6766 (CH) 3 weeks out of 4 with a twice a day dosing schedule of defactinib (Def) administered 3 weeks out of 4. The dose levels explored were cohort 1 (CH 3.2 mg, 200 mg Def), cohort 2a (CH 4 mg Def 200 mg) and cohort 2b (CH 3.2 mg and Def 400 mg). Pharmacokinetic data were collected at cycle 1 d15 when both drugs were administered. In a subset of patients consenting to multiple biopsies, 3 biopsies were performed (baseline, after a single dose of CH run in and after 8-15 days of the combination therapy). At the recommended phase 2 dose (R2PD), expansion cohorts in patients with low grade ovarian cancer (LGSOC, n=20), KRASM non-small cell lung cancer (NSCLC, n=20) and KRASM colorectal cancer (CRC, n=10) will be treated. Results: Forty two patients have been treated on the trial so far. There were no dose limiting toxicities (DLTs) in the first 3 patients in cohort 1 and thus patients were recruited to cohort 2a and 2b. The cohort 2b dose level was deemed intolerable because of 2/3 patients experiencing DLTs of grade 2 rash not allowing dosing of 75% of the planned dose. No DLTs were seen in the first 6 patients treated on schedule 2a. However, given chronic grade 2 toxicities in patients on treatment > 6 months, the RP2D was deemed to be cohort 1. The most common side effects in the study were rash, CK elevation, AST elevation, hyperbilirubinemia and nausea, most of which were NCI CTC grade 1-2. All changes were reversible. The mean AUC of CH and Def at R2PD was 6179 ng*hr/ml and 2071 ng*hr/ml, respectively, which is in the range of what was seen in the single agent dose escalation studies. Sequential biopsies were obtained in 7 patients and 6/7 showed an increase in p-FAK following single agent CH which was reduced in 5/7 with combined CH and Def therapy. The response rate was 67% (4/6) or 50% (4/8) in KRASM LGSOC or all LGSOC patients treated to date, respectively. A range of KRASM were found in tumors of LGSOC patients who responded (G12V, G12A and G12D). Of the 4 patients with LGSOC who have responded to treatment, 3 have had a prior MEK inhibitor and are currently are on study for a median of 20.5 months (range 7-23 months). Expansion cohorts in LGSOC and KRASM NSCLC and CRC are ongoing and will be presented. Conclusion: We report a novel intermittent schedule of the combination of a RAF-MEK and FAK inhibitor with very promising clinical activity in patients with KRASM LGSOC including those who had been previously treated with a MEK inhibitor. Citation Format: Rajiv Shinde, Angelika Terbuch, Martin Little, Reece Caldwell, Roopa Kurup, Ruth Riisnaes, Mateus Crespo, Ruth Ruddle, Bora Gurel, Adam Stewart, Jenny King, Mona Parmar, Alison Turner, Florence Raynaud, Muneeb Mahmud, Christina Yap, Jonathan A. Pachter, Gordon B. Mills, Anna Minchom, Juanita Lopez, Susana N. Banerjee, Johann S. de Bono, Matthew Krebs, Udai Banerji. Phase I study of the combination of a RAF-MEK inhibitor CH5126766 and FAK inhibitor defactinib in an intermittent dosing schedule with expansions in KRAS mutant cancers [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr CT143.

Gene copy number alterations, tumor cell stemness, and development of platinum chemotherapy resistance contribute to high-grade serous ovarian cancer (HGSOC) recurrence. Stemness phenotypes involving Wnt-beta-catenin, aldehyde dehydrogenase activities, intrinsic platinum resistance, and tumorsphere formation are here associated with spontaneous genetic gains in KRAS, MYC, and FAK (KMF) genes, in a new aggressive murine model of ovarian cancer. Noncanonical signaling via FAK sustained KMF and human tumorsphere proliferation as well as resistance to cisplatin cytotoxicity. Platinum-resistant tumorspheres can acquire a dependence on FAK for growth. Accordingly, increased FAK tyrosine phosphorylation was observed within HGSOC patient tumors surviving neoadjuvant chemotherapy. Combining a FAK inhibitor with platinum overcame chemoresistance, triggering tumor cell apoptosis. FAK transcriptomic analyses across knockout and reconstituted cells identified 135 genes elevated by a FAK activity-dependent, beta-catenin, and Myc signaling axis including pluripotency and DNA repair genes. Identified target increases in HGSOC tumors may reflect oncogenic FAK signaling. Citation Format: Carlos J. Díaz Osterman, Duygu Ozmadenci, Elizabeth G. Kleinschmidt, Kristin N. Taylor, Allison M. Barrie, Shulin Jiang, Lisa M. Bean, Florian J. Sulzmaier, Jian Li, Xiao Lei Chen, Guo Fu, Marjaana Ojalill, Pekka Rappu, Jyrki Heino, Adam A. Mark, Guorong Xu, Kathleen M. Fisch, David T. Weaver, Jonathan A. Pachter, Balázs Győrffy, Michael T. McHale, Denise C. Connolly, Alfredo Molinolo, Dwayne G. Stupack, David D. Schlaepfer. FAK activity sustains intrinsic and acquired ovarian cancer resistance to platinum chemotherapy [abstract]. In: Proceedings of the AACR Special Conference on Advances in Ovarian Cancer Research; 2019 Sep 13-16, 2019; Atlanta, GA. Philadelphia (PA): AACR; Clin Cancer Res 2020;26(13_Suppl):Abstract nr A61.

High-grade serous ovarian cancer (HGSOC) is the leading cause of death from gynecologic malignancies. Many patients initially respond well to surgery followed by chemotherapy, yet ~80% of patients recur with disease that is frequently recalcitrant to chemotherapy. Across a number of chemoresistant cancers, immunotherapies have shown great promise. Correlative studies in patients with HGSOC support a role for immune system in patient outcome. Yet the HGSOC tumor microenvironment (TME) is highly immunosuppressive, which constitutes a major barrier to immunotherapy success. Understanding the molecular signals in HGSOC that promote resistance to chemo- or immunotherapies is required for identification of actionable targets within HGSOC; yet, there are limited models that recapitulate the TME in ovarian cancer. We have generated a new syngeneic C57Bl6 mouse model of ovarian cancer that exhibits genomic copy number gains in KRAS, MYC, and (PTK2) FAK genes (termed KMF) and aggressive malignant phenotypes commonly observed in HGSOC. The KMF TME, like HGSOC, is populated by immunosuppressive myeloid-derived suppressor cells (MDSC) and T-regulatory (Treg) cells but lacks CD8+ T cell infiltration. We find that tumor-associated FAK expression and kinase activity are essential for KMF tumor growth. Mechanistically, by knockout and cell reconstitution approaches, FAK promotes expression of a select group of proteins mediating chemo- and immune-resistance. Inhibiting FAK modulates checkpoint inhibitor protein expression, limits MDSC and Treg recruitment, and enhances CD4 and CD8 T cell infiltration. Pharmacologic FAK inhibition reduced CD112/CD155 expression on KMF cells and, together with anti-TIGIT immunotherapy, significantly increased mouse survival from 28 to 60+ days. The chemoresistant KMF model recapitulates key aspects of the aggressive HGSOC TME including the generation of an immunosuppressive milieu, and may be used as a preclinical model to identify new therapeutic targets for HGSOC. Citation Format: Duygu Ozmadenci, Jayanth S. Shankara Narayanan, Jacob R. Andrew, Allison M. Barrie, Shulin Jiang, Esra Bilir, Thomas Bertotto, Rebekah R. White, Vijay K. Kuchroo, Jonathan A. Pachter, Dwayne G. Stupack, David D. Schlaepfer. Modeling the ovarian cancer immune response and tumor microenvironment [abstract]. In: Proceedings of the AACR Special Conference on the Evolving Landscape of Cancer Modeling; 2020 Mar 2-5; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2020;80(11 Suppl):Abstract nr A26.

Richtersyndrome (RS) is defined as the transformation of chronic lymphocytic leukemia (CLL) into an aggressive lymphoma, typically a diffuse large B cell lymphoma (DLBCL) with poor prognosis and without effective therapies. Chemotherapy, as well as novel inhibitory agents, show only partial and temporary responses, highlighting the need for alternative therapies that can overcome drug resistance. Duvelisib (DUV) is an oral phosphatidylinositol 3-kinase (PI3K)-δ and γ dual inhibitor showing efficacy in patients with refractory CLL. Venetoclax (VEN) is a Bcl-2 selective inhibitor, able to induce apoptosis in CLL cells. The lack of RS cell lines and murine models has limited understanding of the molecular mechanisms contributing to the pathogenesis of this disease, impacting also on the development of effective therapies. In our lab, 4 different RS patient-derived xenografts (PDXs) were established, representing useful tools to study the biology of the disease and to test novel therapeutic strategies. This work was undertaken to investigate expression and activity of the PI3K pathway and Bcl-2, and to test the ex-vivo and in vivo efficacy of DUV and VEN, alone or in combination, in our RS-PDX models. The 4 RS-PDX models are characterized by heterogeneous PI3K expression. RS1316 and IP867 expressed high levels of both p110δ and p110γ catalytic subunits. In contrast, RS9737 expressed low levels of both the PI3K catalytic subunits, while RS1050 expressed the p110δ subunit alone, but lacked p110γ and Bcl-2. We started by assessing the ex-vivo efficacy of DUV and VEN on RS-PDX-derived cells. Cells from the 4 RS-PDX models were obtained from disrupted tumor masses and immediately treated with DUV (5µM), VEN (25nM) or their combination. The DUV/VEN combination induced the highest levels of apoptosis in RS1316, RS9737 and IP867/17 cells at 24 and 48 h after treatment. Western blot confirmed the downregulation of Bcl-2 and Mcl-1, along with Caspase-3 and PARP-1 cleavage. In contrast, no effect was observed on RS1050 cells, which lack p110γ and Bcl-2, making this model intrinsically resistant to DUV and VEN treatment. Next, the effects of PI3K-δ/γ and Bcl-2 inhibition were examined in vivo on the RS1316-PDX model, selected as the best ex-vivo responder. NOD/SCID/gamma chain-/-(NSG) mice were subcutaneously injected with RS1316 cells. When tumor masses were palpable, animals were treated daily for 7 days including 2 holidays treatment with DUV (100 mg/kg p.o.), VEN (50 mg/kg p.o.) or their combination. In mice that received the combination, tumors were of smallest volume (4 out of 6 tumors were completely absent) and had the least viable cells, as highlighted by increment of cleaved Caspase-3 immunohistochemistry signal. In a second set of experiments, tumor masses were left to grow after 10 consecutive treatments and monitored daily. Used as single agents, DUV and VEN significantly reduced tumor growth, prolonging survival for 5 days compared to controls (mean survival 42 days in the control condition vs 47 in the single treatments). The combination of the two drugs was even more efficacious, leading to a complete remission in all the mice during treatment (Fig. 1). Tumor masses eventually grew back after cessation of treatment, but the combination significantly prolonged survival compared to vehicle or single-treatment groups (mean survival 56 days, p In conclusion, DUV and VEN treatment combination, simultaneously inhibiting PI3K-δ/γ and Bcl-2, induced RS tumor regression, offering a promising treatment combination, at least for RS patients expressing high levels of PI3K gamma and delta subunits and Bcl-2. These results pave the way for clinical testing of duvelisib and venetoclax combination for patients with RS. Figure 1 Disclosures Furman: AstraZeneca: Consultancy; Oncotracker: Consultancy; Verastem: Consultancy; Abbvie: Consultancy; Acerta Pharma: Consultancy; Janssen: Consultancy; Incyte: Consultancy; Genentech: Consultancy; Sunesis: Consultancy; Beigene: Consultancy; Pharmacyclics: Consultancy; TG Therapeutics: Consultancy. Allan:Acerta Pharma: Consultancy; Genentech: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Verastem Oncology, Inc.: Consultancy, Membership on an entity's Board of Directors or advisory committees; Bayer: Consultancy; AbbVie, Inc: Consultancy, Membership on an entity's Board of Directors or advisory committees; Pharmacyclics LLC, an AbbVie company: Consultancy; Janssen: Consultancy, Honoraria; Sunesis Pharmaceuticals: Consultancy, Membership on an entity's Board of Directors or advisory committees. Coma:Verastem Inc: Employment. Pachter:Verastem Inc: Employment. Deaglio:VelosBio Inc.: Research Funding; iTeos Therapeutics: Research Funding; Verastem Inc: Research Funding. Vaisitti:Verastem Inc: Research Funding; VelosBio Inc.: Research Funding. OffLabel Disclosure: Duvelisib: PI3K gamma and delta inhibitor Venetoclax: bcl2 inhibitor

Cancer stem cells (CSCs) are an underlying cause of tumor progression and metastasis. In breast cancer, CSCs can be identified by Aldehyde Dehydrogenase 1 (ALDH) or CD44-high/CD24-low expression. Neoadjuvant chemotherapy has been shown to lead to an increase in CSCs in locally advanced breast cancer (Alamgeer et al., 2014, Br. Can. Res. R14). In addition, the presence of CSCs in residual axillary disease is associated with a significantly worse prognosis following neoadjuvant chemotherapy and surgery (Sakakibara et al. 2011, Cancer 3899, 2011). Currently, there are no approved therapies that effectively target and kill CSCs. VS-6063 and VS-4718 are orally bioavailable small molecules that kill cancer stem cells through the inhibition of Focal Adhesion Kinase (FAK). Both VS-6063 and VS-4718 have demonstrated preferential targeting of CSCs in preclinical models and are currently in clinical development. Here we report that VS-6063 and VS-4718 effectively kill CSCs in multiple models of breast cancer. In an ex vivo model, biopsies from human breast tumors were obtained and cultured as primary explants within 24 hours of surgery. The primary explants were incubated with VS-6063, VS-4718 or paclitaxel for 4 days. Treatment with either VS-6063 or VS-4718 decreased the proportion of CSCs in contrast to paclitaxel. VS-6063 and VS-4718 diminished the self-renewal capacity of primary cultures from established TNBC patient-derived xenografts as measured by tumorsphere assays. In a MDA-MB-231 mouse xenograft model, treatment with VS-6063 decreased CSC more than 6-fold in an in vivo limiting dilutions assay. Similarly, using an imaging-based 4T1-luciferase TNBC orthotopic model, both VS-6063 and VS-4718 diminished the size of metastatic nodules within two weeks. CSCs are readily detectable in primary breast cancers at surgery, yet methods to detect these populations are still developing. A multiplex assay for the CSC markers, ALDH1, CD44 and CD24, was explored with biopsies of primary tumor and matched lymph node, and primary tumors taken pre- and post-neoadjuvant chemotherapy. Consistent with previously reported data, elevated levels of ALDH were observed at higher levels post-treatment, and in lymph nodes. In addition, zones of ALDH+ and CD44-high/CD24-low tumor cells can be mapped with the multiplex assay for the potential detection of CSCs in breast cancer biopsies. In summary, VS-6063 and VS-4718 diminish the CSC subpopulation in vitro, ex vivo and in xenograft models using a number of functional and biomarker assays. This critical subpopulation of CSCs is detectable in residual tumor following neoadjuvant therapy. Potentially, multiplex assays of CSC markers will be an improved means to monitor CSCs in clinical specimens. CSC-targeted agents such as VS-6063 or VS-4718 should be clinically tested in the neoadjuvant setting to potentially delay time to relapse and improve patient outcome. Citation Format: Vihren N Kolev, Sean McDermott, Max Wicha, Jonathan A Pachter, Qunli Xu, David T Weaver. VS-6063 (defactinib) and VS-4718 reduce cancer stem cells in models of breast cancer: Implications for clinical trials in the neoadjuvant setting [abstract]. In: Proceedings of the Thirty-Seventh Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2014 Dec 9-13; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2015;75(9 Suppl):Abstract nr P6-11-06.

Focal adhesion kinase (FAK) is a non-receptor tyrosine kinase that orchestrates cellular signaling through integrins and growth factor receptors and functions in multiple steps of tumorigenesis. Both VS-6063 and VS-4718 are potent, selective, and orally active FAK inhibitors. In a Phase 1 clinical trial, VS-6063 was well tolerated and demonstrated preliminary clinical activity. VS-6063 is currently being tested in multiple clinical studies both as a single agent and in combination with paclitaxel. VS-4718 was shown to inhibit tumor growth and metastasis in preclinical tumor models including triple negative breast cancer and is currently under evaluation in a Phase 1 clinical trial. We report here that the FAK inhibitors VS-6063 and VS-4718 effectively abrogate cancer stem cells (CSCs) in vitro and in vivo in triple negative breast cancer models. VS-6063 and VS-4718 were evaluated in multiple cancer stem cell assays in vitro. Pre-treatment of SUM159 and MDA-MB-231 breast cancer cells with FAK inhibitors in matrigel reduced the percentage of ALDH-positive cells. The proportion of side population (SP) cancer stem cells was also reduced by FAK inhibition. The expression of ALDH1 in human breast tumor samples has previously been shown to correlate with poor prognosis (Ginestier et al 2007 Cell Stem Cell 1, 555). Significantly, ex vivo treatment of primary human breast tumor tissue with these FAK inhibitors also effectively reduced the proportion of ALDH-positive CSCs. Similar inhibitory effects on CSCs were observed in ovarian cancer cell lines OVCAR-5 and OVCAR-8. In direct contrast, cytotoxic agents such as paclitaxel and carboplatin increased the percentage of cancer stem cells in vitro, suggesting that these agents do not effectively target CSCs. Importantly, combination of either VS-6063 or VS-4718 with cytotoxic agents attenuated the enrichment of cancer stem cells induced by these cytotoxic agents. The in vivo effect of a FAK inhibitor on cancer stem cells was evaluated in an MDA-MB-231 human breast cancer orthotopic model. Oral administration of VS-4718 significantly reduced the presence of ALDH1-positive cells in tumors as assessed by immunofluorescence. Following dissociation of cells from tumors, CSCs were found to be reduced in VS-4718-treated tumors as measured by multiple assays. Importantly, cells isolated from VS-4718-treated tumors also showed reduced tumor-initiating capability upon re-implantation into immunodeficient mice in limiting dilutions. Our results provide clear demonstration that while FAK inhibitors significantly inhibit the growth of human breast tumor xenografts, they also preferentially target breast cancer stem cells in direct contrast to standard-of-care agents. These data provide a strong rationale for the clinical development of Verastem's FAK inhibitors in the treatment of breast cancer. Citation Information: Cancer Res 2013;73(24 Suppl): Abstract nr P2-09-14.

Introduction: Indolent NHL (iNHL) remains largely incurable, with the majority of patients (pts) eventually becoming refractory to standard therapies, necessitating new effective and tolerable treatments. Duvelisib is an oral dual PI3K-δ,γ inhibitor being developed for the treatment of hematologic malignancies, including CLL/SLL and follicular lymphoma (FL). In the Phase 2 DYNAMO trial of duvelisib monotherapy for relapsed/refractory iNHL(NCT01882803), the FL subgroup (n=83) had a median progression-free survival (mPFS) of 8.3 months (mo) and an overall response rate (ORR) of 43%. Although adverse events (AEs) observed with other PI3K-inhibitors also occurred with duvelisib, they infrequently led to discontinuation of treatment. An understanding of prognostic factors that can inform duvelisib responsiveness would be valuable for guiding patient selection treatment options. This study presents updated data on prognostic factors potentially associated with duvelisib efficacy and safety. Methods: To identify FL risk subgroups, clinical parameters were assessed according to the FL Prognostic Index (FLIPI). Incidences of AEs (per combined preferred term: neutropenia, diarrhea, colitis, pneumonia, pneumonitis, transaminase elevation, rash, and infections) were also evaluated for associations with clinical responses. Blood collected at baseline (Cycle1 Day1) was analyzed by a central laboratory for a 7 gene expression signature to generate the combined M7-FLIPI, and biopsies were evaluated at screening for del(6q) cytogenetics. In addition, baseline immune cell counts (subgrouped low [L] or high [H] by flow cytometry), and levels of key chemokines, cytokines, and serum factors were centrally assessed. Multivariate regression models were developed via a stepwise procedure from a selection of these parameters identified by individual univariate analysis for the efficacy endpoints of mPFS and ORR. Results: In the DYNAMO study, there were no significant differences in mPFS, ORR, or lymph node response rate (LNRR) identified for FL pts between high and low risk subgroups stratified by prognostic indexes. Efficacy responses were similar using the FLIPI (FLIPI Prognostic factors selected via univariate analyses for mPFS or ORR were advanced into multivariate models. None of these factors reached significance in a multivariate mPFS model, and only CD3H was significant in a stepwise ORR model (odds ratio H/L=2.69, 95% CI [1.20,6.06]). The FL pt subgroups having AEs were evaluated for mPFS (mo [95% CI] as follows: neutropenia, 8.3 mo [3.5,11.8], n=25; diarrhea 11.0 mo [8.3,16.4], n=40; colitis 10.0 mo [8.3,24.0], n=5; rash 8.3 mo [1.9,28.0], n=26; transaminase elevation, 11.8 mo [1.4,24.0], n=12; pneumonitis 13.0 mo [9.5,16.4], n=4; pneumonia 12.0 [4.2,28.0], n=8; and infection 9.0 [3.7,12.0], n=38. Conclusion In the Phase 2 DYNAMO study, mPFS and ORR were similar for duvelisib-treated FL pts regardless of poor prognostic indicators, including FLIPI, M7-FLIPI, and chromosome 6q deletions. Elevated levels of CXCL11 and IL2RA correlated with shorter mPFS. This analysis also shows that AEs, when managed, allow continued treatment with duvelisib and increased benefit as demonstrated by mPFS. Overall, these data suggest that duvelisib as a single-agent is effective in FL patients, including pts with poor prognostic factors. Disclosures Zinzani: SERVIER: Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Merck: Honoraria, Membership on an entity's Board of Directors or advisory committees; Celgene: Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Gilead: Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Celltrion: Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Roche: Honoraria, Membership on an entity's Board of Directors or advisory committees; Merck: Honoraria, Membership on an entity's Board of Directors or advisory committees; TG Pharmaceuticals: Honoraria, Membership on an entity's Board of Directors or advisory committees; Bayer: Membership on an entity's Board of Directors or advisory committees; Janssen: Honoraria, Speakers Bureau; Takeda: Membership on an entity's Board of Directors or advisory committees; Bayer: Membership on an entity's Board of Directors or advisory committees; MSD: Honoraria, Speakers Bureau; PFIZER: Honoraria, Membership on an entity's Board of Directors or advisory committees; PFIZER: Honoraria, Membership on an entity's Board of Directors or advisory committees; TG Pharmaceuticals: Honoraria, Membership on an entity's Board of Directors or advisory committees; Verastem: Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Astra Zeneca: Speakers Bureau; BMS: Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau. Flinn:Novartis: Research Funding; Infinity: Research Funding; Takeda: Research Funding; Gilead: Research Funding; Trillium: Research Funding; Constellation: Research Funding; Curis: Research Funding; Verastem: Research Funding; TG Therapeutics: Research Funding; Verastem: Consultancy, Research Funding; BeiGene: Research Funding; Calithera: Research Funding; Constellation: Research Funding; Celgene: Research Funding; Seattle Genetics: Research Funding; Forty Seven: Research Funding; Merck: Research Funding; Gilead: Research Funding; Takeda: Research Funding; Pharmacyclics: Research Funding; Verastem: Research Funding; Trillium: Research Funding; Portola: Research Funding; TG Therapeutics: Research Funding; Forma: Research Funding; Forma: Research Funding; Genentech: Research Funding; ArQule: Research Funding; Genentech: Research Funding; Incyte: Research Funding; Agios: Research Funding; ArQule: Research Funding; Pfizer: Research Funding; Portola: Research Funding; Novartis: Research Funding; BeiGene: Research Funding; Celgene: Research Funding; Kite: Research Funding; Kite: Research Funding; Merck: Research Funding; Forty Seven: Research Funding; Pfizer: Research Funding; Agios: Research Funding; Seattle Genetics: Research Funding; Verastem: Consultancy, Research Funding; Calithera: Research Funding; Infinity: Research Funding; Janssen: Research Funding; Curis: Research Funding; Incyte: Research Funding; Janssen: Research Funding; Pharmacyclics: Research Funding. Miller:CTI: Research Funding; Gilead: Honoraria, Research Funding; Incyte: Consultancy, Honoraria, Research Funding, Speakers Bureau; Novartis: Honoraria, Speakers Bureau. Ardeshna:Celgene: Membership on an entity's Board of Directors or advisory committees; ADC Therapeutics: Membership on an entity's Board of Directors or advisory committees, Research Funding; Takeda: Membership on an entity's Board of Directors or advisory committees; Roche: Membership on an entity's Board of Directors or advisory committees, Other: Conference expenses, Research Funding. Le:Verastem Oncology: Employment. Pachter:Verastem Inc: Employment, Other: Stockholder. Sprott:Verastem Oncology: Employment. Lustgarten:Verastem Oncology: Employment. Weaver:Verastem Oncology: Employment, Other: Stockholder; Agios Pharmaceuticals: Employment; Femto Dx: Equity Ownership. Wagner-Johnston:Novartis: Research Funding; ASTEX: Research Funding; Celgene: Research Funding; JUNO: Honoraria, Membership on an entity's Board of Directors or advisory committees; Janssen: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees; Merck: Research Funding; ADC Therapeutics: Honoraria, Membership on an entity's Board of Directors or advisory committees.

Introduction In CLL, the timing and selection of therapy is largely informed by disease stage, prognostic molecular features, tumor load, and patient (pt) performance status. Duvelisib (DUV) is a novel, oral, dual PI3K-δ,γ inhibitor in clinical development for the treatment of CLL/SLL, FL, and other hematologic malignancies. Results from the Phase 3 DUO study (NCT02004522) for relapsed/refractory CLL/SLL showed that DUV monotherapy resulted in statistically significant improvement over ofatumumab (OFA) monotherapy for median progression-free survival (mPFS; 13.3 vs 9.9 months (mo); p Methods Of the 319 pts enrolled in the DUO study, 158 were treated with DUV and 155 with OFA. Tumor burden was assessed at baseline using target lymph node diameter and absolute lymphocyte count. Baseline blood samples from Cycle1 Day1 were analyzed for cytogenetic abnormalities (17p, 11q, 6q deletions; trisomy 12); immune cell counts by flow cytometry; and serum chemokine, cytokine, and serum factor levels. Univariate analysis of these markers was conducted for PFS and ORR. The statistically significant univariate markers were tested in a stepwise multivariate regression (MVR) model using binary high [H] or low [L] thresholds based on the median. Results Tumor Burden: The mPFS (in mo) for DUV monotherapy did not appear to be impacted by baseline tumor burden: high (n=20), 16.6; medium (n=88), 12.7; and low (n=47), 15.1. Across these tumor burden levels, DUV maintained its PFS advantage over OFA. Cytogenetics: DUV-treated pts with del(11q), a marker associated with poor CLL outcomes, had a longer mPFS (in mo) and ORR compared to pts without del(11q) (mPFS: 24.8, n=25 vs 12.7, n=81; HR 0.56, 95% CI [0.30,1.06]; ORR: 80.0% vs 69.1%, odds ratio, 0.56). In contrast, mPFS was shorter in OFA-treated del(11q) pts relative to those without this deletion (5.3, n=24 vs 11.3, n=71). Median PFS was significantly extended in the del(17p) subgroup for DUV vs OFA (16.6, n=27 vs 9.2, n=27; HR 0.42, [0.21,0.85]), and, to a lesser degree, in pts without del(17p) (13.1, n=83 vs 11.1, n=74; HR 0.55 [0.37,0.83]). For the 6 DUV pts with del(11q/17p), mPFS was 17.4 mo. A shorter mPFS was observed for DUV-treated pts with trisomy 12 compared with trisomy 12-negative pts (9.1, n=16 vs 16.5, n=73). For the 10 DUV pts with trisomy 12 as well as del(11q/17p), mPFS was more similar to the trisomy 12 group, at 9.1 mo. Immune cell profiles and serum factors: Several immune cell profiles correlated with longer mPFS (in mo) for DUV, including Treg (16.4H vs 12.9L, HR 0.69 [0.43,1.12]) and monocytes (15.1L vs 12.7H, HR 0.82 [0.54,1.24]). Baseline chemokine and serum factor levels associated with longer mPFS with DUV included CCL3 (22.1L vs 12.2H, HR 0.60 [0.37,0.99]), IL2RA (16.3L vs 11.6H, HR 0.73 [0.47,1.14]), and TNFα (16.6L vs 12.2H, HR 0.78 [0.49,1.24]). Multivariate regression model: The MVR model showed that the biomarker profile of trisomy 12-negative (HR -/+, 0.15), CCL22L (HR H/L, 2.23), TNFαL (HR H/L, 3.66), TregH (HR H/L, 0.31), and monocytesL (HR H/L, 2.45), was associated with longer PFS; TNFαL and TregH were the largest associations, with estimated 73% and 69% reductions in risk of progression or death, respectively. Similarly, stepwise models yielded TNFαL (odds ratio H/L, 0.37) and CXCL11H (odds ratio H/L, 2.99) as biomarkers for improvement in ORR. Based on these analyses, pts with TNFαL tend to have better efficacy profiles for PFS and/or ORR. Conclusions: DUV monotherapy was highly efficacious in CLL/SLL pts with markers of poor response, including high tumor burden, del(17p), and del(11q) as shown by univariate analyses of tumor burden, cytogenetics, immune cell profiles, and serum factors. Multivariate analyses revealed a biomarker profile of CCL22L, TNFαL, TregH, monocytesL, and trisomy 12-negative that correlated with improved mPFS and/or ORR. Additional analyses are underway to characterize the predicted PFS for the biomarker profile. Disclosures Brown: Verastem: Consultancy, Research Funding; Janssen: Consultancy; Morphosys: Membership on an entity's Board of Directors or advisory committees; Pharmacyclics: Consultancy; Sun Pharmaceutical Industries: Research Funding; TG Therapeutics: Consultancy; Sunesis: Consultancy; Gilead: Consultancy, Research Funding; Roche/Genentech: Consultancy; Genentech: Consultancy; Celgene: Consultancy; Boehringer: Consultancy; Beigene: Membership on an entity's Board of Directors or advisory committees; Acerta / Astra-Zeneca: Membership on an entity's Board of Directors or advisory committees; Abbvie: Consultancy; Invectys: Membership on an entity's Board of Directors or advisory committees; Loxo: Consultancy. Flinn:Novartis: Research Funding; Merck: Research Funding; Infinity: Research Funding; Forty Seven: Research Funding; Janssen: Research Funding; Trillium: Research Funding; Pharmacyclics: Research Funding; Verastem: Research Funding; Pfizer: Research Funding; Kite: Research Funding; Takeda: Research Funding; Forma: Research Funding; BeiGene: Research Funding; Curis: Research Funding; Seattle Genetics: Research Funding; TG Therapeutics: Research Funding; Verastem: Consultancy, Research Funding; Portola: Research Funding; Calithera: Research Funding; ArQule: Research Funding; Celgene: Research Funding; Constellation: Research Funding; Genentech: Research Funding; Incyte: Research Funding; Gilead: Research Funding; Agios: Research Funding. Davids:Surface Oncology: Research Funding; Merck: Consultancy; Roche/Genentech: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Gilead: Membership on an entity's Board of Directors or advisory committees; AbbVie, Inc: Consultancy, Membership on an entity's Board of Directors or advisory committees; Janssen: Consultancy, Membership on an entity's Board of Directors or advisory committees; Astra-Zeneca: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; MEI Pharma: Consultancy, Research Funding; Verastem: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Celgene: Consultancy; Pharmacyclics: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; BMS: Research Funding; TG Therapeutics: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding. Hillmen:F. Hoffmann-La Roche Ltd: Research Funding; Celgene: Research Funding; Alexion Pharmaceuticals, Inc: Consultancy, Honoraria; Abbvie: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Gilead Sciences, Inc.: Honoraria, Research Funding; Pharmacyclics: Research Funding; Janssen: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Novartis: Research Funding; Acerta: Membership on an entity's Board of Directors or advisory committees. Montillo:Gilead: Consultancy, Honoraria, Speakers Bureau; Janssen: Consultancy, Honoraria; Roche: Consultancy, Honoraria, Research Funding; AbbVie: Consultancy, Honoraria, Speakers Bureau. Tam:Janssen: Consultancy, Honoraria, Research Funding; Pharmacyclics: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees; Abbvie: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Gilead: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees; BeiGene: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding. Jaeger:GSK: Membership on an entity's Board of Directors or advisory committees; Gilead: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; AOP Orphan: Membership on an entity's Board of Directors or advisory committees; Bioverativ: Membership on an entity's Board of Directors or advisory committees; Roche: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Takeda-Millenium: Membership on an entity's Board of Directors or advisory committees; Janssen: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees; Takeda-Millenium: Membership on an entity's Board of Directors or advisory committees; Amgen: Membership on an entity's Board of Directors or advisory committees; AbbVie: Consultancy, Honoraria; Infinity: Membership on an entity's Board of Directors or advisory committees; Mundipharma: Membership on an entity's Board of Directors or advisory committees; Novartis: Membership on an entity's Board of Directors or advisory committees, Research Funding; Celgene: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees; MSD: Research Funding. Ghia:Sunesis: Honoraria, Research Funding; Novartis: Honoraria, Research Funding; Janssen: Honoraria, Research Funding; BeiGene: Honoraria, Research Funding; Acerta: Honoraria, Research Funding; Gilead: Honoraria, Research Funding; AbbVie, Inc: Honoraria, Research Funding. Stilgenbauer:Genzyme: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Novartis: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Gilead: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Celgene: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Boehringer-Ingelheim: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; GSK: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Pharmcyclics: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Genentech: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Amgen: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Sanofi: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Janssen: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; AbbVie: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Hoffmann La-Roche: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Mundipharma: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding. Moreno:Janssen: Consultancy; AbbVie: Consultancy; Pharmacyclics LLC, an AbbVie Company: Consultancy. Le:Verastem Oncology: Employment. Lustgarten:Verastem Oncology: Employment. Sprott:Verastem Oncology: Employment. Pachter:Verastem Inc: Employment, Other: Stockholder. Weaver:Agios Pharmaceuticals: Employment; Verastem Oncology: Employment, Other: Stockholder; Femto Dx: Equity Ownership.

Focal adhesion kinase (FAK) is a cytoplasmic tyrosine kinase that mediates signal transduction by integrins as well as growth factor receptors. FAK has been implicated in multiple steps of carcinogenesis including tumor initiation, growth and metastasis. Amplification and overexpression of FAK have been observed in aggressive human cancers including breast cancer. We have now observed that VS-4718, a selective FAK kinase inhibitor, exhibits preferential inhibitory effects on breast cancer stem cells. VS-4718 is a potent and selective FAK kinase inhibitor that blocks fibronectin-stimulated FAK autophosphorylation at Tyr397 at low nanomolar concentrations. To determine if FAK plays a role in the biology of breast cancer stem cells, VS-4718 was evaluated in a multitude of cancer stem cell assays both in vitro and in vivo. In parallel, treatment of SUM159 triple negative breast cancer cells in vitro with FAK shRNA inhibits tumorsphere formation. These data taken together indicate a role of FAK in breast cancer stem cell renewal. Similarly, pre-treatment of SUM159 cells with VS-4718 in matrigel attenuated secondary tumorsphere formation. Furthermore, VS-4718 reduced the side population (SP) and the percentage of ALDEFLUOR+ cancer stem cells in SUM159 breast cancer cells in vitro. In direct contrast, standard-of-care agent paclitaxel increased the percentage of ALDEFLUOR+ cancer stem cells in all three assays. The in vivo effect of VS-4718 on cancer stem cells was evaluated in SUM159 and MDA-MB-231 human triple negative breast cancer xenograft models. Following systemic administration, VS-4718 induced significant reduction of cancer stem cells in tumors as assessed by a decrease in ALDEFLUOR+ cells and tumorsphere-forming efficiency relative to vehicle-treated tumors. In summary, our results indicate the importance of FAK in the self-renewal of breast cancer stem cells in vitro and in vivo, and support the clinical development of the selective FAK inhibitor VS-4718 to target cancer stem cells for the treatment of triple negative breast cancer. Citation Information: Cancer Res 2012;72(24 Suppl):Abstract nr P6-11-09.

7579Background: Duvelisib (DUV), an oral dual inhibitor of PI3K-d,g, is clinically active in hematologic malignancies, including follicular lymphoma (FL), CLL, and T cell lymphoma (Flinn, 2017). PI...

12048Background: Duvelisib (IPI-145) (DUV) is an oral dual inhibitor of phosphoinositide 3-kinase (PI3K)-δ and PI3K-γ being developed to treat B-cell malignancies. PI3K-δ inhibition directly target...

33 Background: Duvelisib is a dual inhibitor of PI3K-δ and PI3K-γ which has shown clinical activity as monotherapy in CLL/SLL, FL and T cell lymphoma (O’Brien; Flinn; Horwitz, ASH 2014). Recent publications have demonstrated that PI3K-δ inhibition reduces immunosuppressive Tregs (Ali, Nature, 2014) whereas PI3K-γ inhibition reduces immunosuppressive myeloid cells (Kaneda, Nature, 2016; De Henau, Nature, 2016). Hence, we postulated that duvelisib may augment the efficacy of immune checkpoint or co-stimulatory antibodies. Methods: Mice bearing syngeneic A20 B cell lymphoma tumors (60-90 mm3) were treated with vehicle, duvelisib, anti-PD1, anti-PD1 + duvelisib, anti-OX40, or anti-OX40 + duvelisib. Tumor volumes were measured by caliper. Tregs, macrophages and MDSCs were quantified by flow cytometry from mice bearing A20 tumors after 8 days of treatment. Results: In the A20 model, duvelisib, anti-PD-1 and anti-OX40 treatments each induced tumor growth delay. When duvelisib and anti-PD-1 were combined in mice with pre-existing A20 tumors, strong anti-tumor synergy was observed. When anti-OX40 and duvelisib were combined, tumor regression was observed which correlated with strong reduction of tumor Tregs, M2 macrophages and MDSCs. To assess immune memory, tumor-free mice following anti-OX40 alone or anti-OX40 + duvelisib were injected with A20 cells in the contralateral flank with no further treatment. Whereas mice that had received anti-OX40 alone grew new tumors upon A20 re-challenge, all tumor-free mice that had received anti-OX40 + duvelisib did not grow tumors upon re-challenge and showed elevated memory T cells in blood and spleen. These findings indicate that anti-OX40 + duvelisib treatment established immune memory. Conclusions: The dual inhibition of PI3K-δ and PI3K-γ appears to make duvelisib especially effective in reducing both lymphoid and myeloid immuno-suppressive populations, enhancing the anti-tumor efficacy of immune checkpoint and co-stimulatory antibodies. These data support potential clinical exploration of duvelisib in combination with checkpoint or co-stimulatory antibodies.

Most ovarian cancer patients respond well to surgical debulking and platinum-taxol chemotherapy. However, ~80% of patients with a complete response to therapy will exhibit tumor recurrence, and in time develop platinum and chemotherapy-resistant disease. It is hypothesized that recurrent disease emanates from the regrowth of microscopic clusters of chemotherapy-resistant tumor cells - also termed cancer stem cells (CSCs). Here, we find that cisplatin chemotherapy elevates focal adhesion (FAK) tyrosine kinase phosphorylation (Y397 FAK) in 3D ovarian tumor spheroids. Notably, FAK is not known as a DNA-damage sensing kinase. Instead, FAK is linked to integrin adhesion-, migration-, and CSC-promoting signaling pathways. In mouse tumors, CP-taxol chemotherapy increases FAK Y397 phosphorylation and aldehyde dehydrogenase (ALDH-1A1, a known markers of CSCs) in tumor areas not associated with necrosis. FAK Y397 phosphorylation is constitutively-elevated in CP-resistant ovarian cancer cells and nanomolar levels of FAK inhibitor (VS-4718) block 3D colony cell growth. Oral VS-4718 administration to mice reduces CP-resistant orthotopic tumor burden with a concomitant decrease in tumor-associated ALDH activity and secondary tumor-initiating capacity. CRISPR-mediated FAK knockout or VS-4718 treated ovarian carcinoma cells exhibit diminished ALDH-1A1 expression. Importantly, co-administration of VS-4718 enhances the anti-tumor effectiveness of CP-taxol chemotherapy in an orthotopic CP-resistant mouse tumor model. As CP activates FAK and FAK signaling sustains ovarian carcinoma CSC phenotypes, our results support the future testing of FAK inhibitors in combination with CP to prevent recurrent and chemo-resistant ovarian cancer. Citation Format: Lisa M. Bean, Florian J. Sulzmaier, Kristen M. Anderson, Isabelle Tancioni, Cheyenne R. Butcher, Sean Uryu, Christine Jean, Christine Lawson, Xiao Lei Chen, Elizabeth G. Kleinschmidt, Vihren N. Kolev, Jonathan A. Pachter, Dwayne G. Stupack, and David D. Schlaepfer. FAK INHIBITION RE–SENSITIZES PLATINUM–RESISTANT SEROUS OVARIAN CANCER [abstract]. In: Proceedings of the 11th Biennial Ovarian Cancer Research Symposium; Sep 12-13, 2016; Seattle, WA. Philadelphia (PA): AACR; Clin Cancer Res 2017;23(11 Suppl):Abstract nr NTOC-106.

Although durable responses to single agent immune checkpoint inhibitors have been reported, additional approaches are needed to extend this therapeutic benefit to a greater proportion of patients. Combination of immunotherapy agents with tumor microenvironment modulators has the potential to overcome barriers that tumor cells develop to evade the immune system, and provide benefit to a greater proportion of patients. Focal Adhesion Kinase (FAK) and the family member PYK2 are potentially valuable targets due to their roles in regulating key cellular populations in the tumor microenvironment. The FAK/PYK2 dual inhibitors, VS-6063 and VS-4718, have previously been shown to inhibit monocyte-derived macrophages, reduce tumor-associated macrophages in xenograft models, and promote a CD8+ T cell-mediated anti-tumor response in squamous cell carcinoma models. We now report the combination of VS-4718 with an anti-PD-1 mAb shows substantially improved efficacy over anti-PD-1 mAb alone in MC38 syngeneic tumor-bearing animals. Analysis of MC38 tumors at day 12 of treatment revealed a significant increase in the CD8+ T cell/Treg ratio in tumors in the VS-4718 + anti-PD-1 combination group, providing a mechanistic understanding for the enhanced efficacy of this combination. To explore additional combination options, we tested the combination of VS-4718 with anti-4-1BB in the MC38 model. Consistent with what was observed with the anti-PD-1 combination, addition of VS-4718 also enhanced the efficacy of an anti-4-1BB mAb inducing full tumor regression. To further delineate the effect of FAK inhibition, an in vitro T cell proliferation assay was conducted. VS-6063 and VS-4718 dose-dependently stimulated proliferation of CD8+ cytotoxic T cells isolated from healthy donors. This is in distinct contrast to other protein kinase inhibitors, such as the SRC inhibitor dasatinib which potently impaired the proliferation of CD8+ cytotoxic T cells. In addition, both VS-4718 and VS-6063 decreased CD8+ T cell exhaustion markers (LAG3 and PD-1), and increased T cell-mediated tumor cell killing in vitro. These data provide additional rationale for ongoing clinical trials testing the FAK/PYK2 inhibitor VS-6063 in combination with pembrolizumab (anti-PD-1) plus gemcitabine in pancreatic cancer or in combination with avelumab (anti-PD-L1) in ovarian cancer. Citation Format: Jonathan A. Pachter, David T. Weaver. FAK/PYK2 inhibition enhances antitumor efficacy of anti-PD-1 and anti-4-1BB antibodies [abstract]. In: Proceedings of the Second CRI-CIMT-EATI-AACR International Cancer Immunotherapy Conference: Translating Science into Survival; 2016 Sept 25-28; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2016;4(11 Suppl):Abstract nr B031.

Although durable responses to single agent immune checkpoint inhibitors have been reported, additional approaches are needed to improve upon this therapeutic benefit. Combinations of immunotherapy agents with tumor microenvironment modulators have the potential to overcome barriers that tumor cells develop to evade the immune system, and provide benefit to a greater proportion of patients. Focal Adhesion Kinase (FAK) and its family member, PYK2, are potentially valuable targets due to their roles in regulating key cellular populations in the tumor microenvironment. In addition to targeting cancer stem cells, the FAK/PYK2 dual inhibitors, VS-6063 and VS-4718, have been shown to inhibit monocyte-derived macrophages, reduce tumor-associated macrophages in xenograft models, and promote a CD8+ T cell-mediated anti-tumor response in squamous cell carcinoma models. We now report that the combination of VS-4718 with an anti-PD-1 mAb shows improved efficacy over anti-PD-1 mAb alone and extends survival of MC38 syngeneic tumor bearing animals. Analysis of MC38 tumors at day 12 of treatment revealed a significant increase in the CD8+ T cells/Treg ratios in tumors in the VS-4718 + anti-PD-1 combination group, providing a mechanistic understanding for the enhanced efficacy of this combination. To explore additional combination options, we tested the combination of VS-4718 with anti-4-1BB in the MC38 model. Consistent with what was observed with the anti-PD-1 combination, VS-4718 also enhanced the efficacy of an anti-4-1BB mAb. To further delineate direct effect of FAK inhibition on human T cells, in vitro T cell proliferation assays were conducted. VS-6063 and VS-4718 dose-dependently stimulated proliferation of CD8+ cytotoxic T cells isolated from healthy donors. This is in distinct contrast to other protein kinase inhibitors, such as the SRC inhibitor dasatinib which impaired the proliferation of CD8+ cytotoxic T cells. In addition, both VS-4718 and VS-6063 decreased CD8+ T cell exhaustion markers, and increased T cell-mediated tumor cell killing in vitro. These data provide a rationale for clinical trials in cancer patients to test whether a FAK/PYK2 inhibitor in combination with an immune checkpoint inhibitor could increase the breadth of responsive tumor types, increase the number of responders, and confer more durable anti-tumor responses. Citation Format: Yan Wang, Jennifer E. Ring, Kam Sprott, David T. Weaver, Jonathan A. Pachter. FAK/PYK2 inhibition enhances immune checkpoint inhibitor efficacy. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 568.

Platinum and taxol administration is standard of care chemotherapy for serous ovarian cancer. Tumor recurrence occurs in a high percentage of patients, and this is directly related to poor overall survival. Ovarian cancer stem cell (CSC) resistance to chemotherapy treatment can give rise to tumor recurrence. Focal adhesion kinase (FAK), an intracellular tyrosine kinase, has been linked to mesothelioma and breast CSC survival. Here, we find that FAK activation is elevated in platinum (CP)-resistant ovarian cancer cells and that FAK tyrosine phosphorylation is increased after CP treatment of CP-sensitive ovarian cancer cells. Nanomolar levels of FAK inhibitor (VS-4718) selectively blocked CP-resistant ovarian carcinoma methylcellulose colony growth via cell cycle inhibition but not apoptosis. Oral VS-4718 administration to mice reduces CP-resistant orthotopic tumor burden with a concomitant decrease in tumor-associated aldehyde dehydrogenase (ALDH) activity, a marker of ovarian CSCs. Residual ovarian tumor cells from VS-4718-treated mice exhibit reduced ALDH activity and secondary tumor initiating capacity. CRISPR-mediated FAK knockout or VS-4718 treated ovarian carcinoma cells exhibit diminished Oct-4 transcription factor and ALDH-1A1 CSC-associated protein marker expression. Co-administration of VS-4718 with CP-taxol chemotherapy reduced CP-resistant tumor burden and exhibited additive inhibitory effects on ovarian carcinoma spheroid growth. As we find that CP activates FAK and that FAK activity sustains ovarian carcinoma CSC phenotypes, our results support the testing of FAK inhibitors in combination with CP to prevent recurrent and chemo-resistant ovarian cancer. Citation Format: Lisa M. Bean, Florian J. Sulzmaier, Isabelle Tancioni, Sean Uryu, Christine Jean, Christine Lawson, Xiao Lei Chen, Elizabeth G. Kleinschmidt, Kristen M. Anderson, Edward A. Cordasco, Joshua Axelrod, Vihren N. Kolev, Jonathan A. Pachter, Dwayne G. Stupack, David D. Schlaepfer. FAK inhibition resensitizes platinum-resistant serous ovarian cancer. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 3812.

Introduction: BCR-ABL1+ B-progenitor acute lymphoblastic leukemia (Ph+ B-ALL) is a highly aggressive disease that is often refractory to currently available therapies. Our previous genomic profiling studies have identified loss-of-function or dominant negative mutations in IKZF1, encoding the lymphoid transcription factor Ikaros, in over 80% of Ph+ ALL. In addition, deletion of CDKN2A, which encodes the INK4A and ARF tumor suppressors, is observed in approximately half of all cases (Mullighan et al., 2008). Alterations of IKZF1 are associated with poor outcome despite the use of tyrosine kinase inhibitors (TKIs). Ikzf1 alterations, including Ikaros isoform 6 (IK6), result in the acquisition of stem cell-like features, enhanced self-renewal, expression of adhesion molecules, and transcriptional upregulation of focal adhesion kinase (FAK), resulting in increased adhesion in vitro and in vivo, and decreased sensitivity to TKIs (Churchman, Cancer Cell, in press). VS-4718 is a potent, selective, and orally bioavailable FAK inhibitor currently under evaluation in a phase 1 clinical trial in subjects with various solid tumors, however in vivo efficacy in hematological malignancies had not been evaluated. Targeting FAK with VS-4718 is an attractive approach to abrogate the adhesive phenotype of IKZF1-altered leukemic cells potentially enhancing the effects of dasatinib in the treatment of high-risk BCR-ABL1 B-ALL. Methods: We examined the efficacy and mechanisms of FAK inhibition using VS-4718 as a single agent and in combination with dasatinib in vitro and in vivo in a range of xenograft and genetically engineered mouse models of BCR-ABL1 ALL. Each model had concomitant deletion of Arf which is observed in approximately 50% of human cases. Results: A pre-clinical in vivo trial of dasatinib and VS-4718 combination therapy in a murine C57Bl/6 Arf-/- BCR-ABL1 pre-B cell model resulted in a marked increase in survival in both IK6-expressing and non-IK6 cohorts of mice, and one complete long-term remission in the IK6-expressing group. Further, we showed increased efficacy of VS-4718 and dasatinib, compared to either agent alone, against two highly aggressive human Ph+ IK6-expressing B-ALL xenografts in vivo, with decreased infiltration of leukemic cells in bone marrow and spleens demonstrating a synergistic effect of the VS-4718/dasatinib combination. In vitro cell viability was reduced with induction of apoptosis at increasing concentrations of VS-4718 as a single agent, and further potentiated the effects of dasatinib in cytotoxicity assays using human xenografted and murine leukemic cells. VS-4718 profoundly diminished the ability of BCR-ABL1-expressing cells to form cell-matrix adhesions in vitro, as evident by the reduced adherence to fibronectin monolayers and bone marrow stromal cells. VS-4718 almost completely abolished the colony-forming potential of BCR-ABL1-expressing murine pre-B cells with and without Ikzf1 alterations at drug concentrations that do not affect cell viability suggestive of a reduction in self-renewal. Calvarial imaging of mice transplanted with Ikzf1-altered BCR-ABL1 leukemic cells and treated with VS-4718 alone in vivo revealed a discernible reduction in adhesion in the intact bone marrow niche of Prrx1-Cre; LSL-tdTomato recipient mice. VS-4718 treated leukemic cells localized to Prrx1-expressing perivascular endothelial cells and exhibited round morphology in contrast to the typical spindle-like appearance of Ikzf1-altered pre-B cells adhering to the bone marrow stroma, suggesting that VS-4718 treatment abolished the aberrant leukemic cell-stromal adhesion induced by Ikaros alterations in vivo. Conclusions: Direct inhibition of FAK with VS-4718 attenuates the adhesive, stem-like properties of IKZF1-altered BCR-ABL1 leukemic cells that contribute to the poor prognosis of patients treated with currently available therapies. Targeted FAK inhibition is thus a promising avenue for improving the response of BCR-ABL1 ALL to dasatinib, particularly in refractory cases harboring IKZF1 alterations. These data support the clinical development of VS-4718 in combination with dasatinib in Ph+ B-ALL. Disclosures Shapiro: Verastem: Employment, Equity Ownership. Pachter:Verastem: Employment, Equity Ownership. Weaver:Verastem: Employment, Equity Ownership. Mullighan:Amgen: Honoraria, Speakers Bureau; Cancer Science Institute: Membership on an entity's Board of Directors or advisory committees; Loxo Oncology: Research Funding; Incyte: Consultancy, Honoraria. Off Label Use: The FAK inhibitor VS-4718 for the treatment of BCR-ABL1 acute lymphoblastic leukemia in preclinical models.

Targeting cancer stem cells (CSCs) holds promise to address key challenges of cancer treatment: chemo- and radiation therapy resistance, metastasis, and recurrence. Increased CSC abundance after neoadjuvant chemotherapy has been associated with a significantly worse outcome. Combining CSC-targeted agents with chemotherapy may lead to more durable response with increased overall survival. Focal adhesion kinase (FAK), a non-receptor tyrosine kinase, mediates signal transduction by integrins and growth factor receptors to regulate cellular adhesion, proliferation, migration, and survival. Several studies have demonstrated that FAK expression and kinase activity are necessary for survival and maintenance of CSCs. FAK is also upregulated in many epithelial tumors and associated with poor patient prognosis and therefore has been pursued as a promising therapeutic target for cancer. VS-6063 and VS-4718 are orally bioavailable small molecules that impede CSCs through the inhibition of FAK. Both VS-6063 and VS-4718 preferentially kill CSCs in multiple cancer models, including models of breast, ovarian, SCLC, and mesothelioma, and these agents are in clinical development. We demonstrate that treatment of cancer cell lines or mice bearing xenograft tumors with VS-6063 or VS-4718 decreases CSCs as assessed by decreased tumor-initiating capability in limiting dilution assays. Both FAK inhibitors decrease significantly CSCs to a varying degrees across multiple tumor models. Furthermore, treatment of mice with either FAK inhibitor following cessation of chemotherapy delayed tumor regrowth in xenograft and PDX models of TNBC, SCLC, and mesothelioma. A mechanistic investigation in breast cancer cell lines revealed an important crosstalk between FAK and the Wnt/β-catenin pathway, where FAK inhibition blocked tyrosine-654 phosphorylation and β-catenin activation. Importantly, a constitutively active mutant form of β-catenin “rescued” CSCs, suggesting that preferential targeting of CSCs by FAK inhibitors is mediated, at least in part, through attenuation of downstream β-catenin activation. In breast cancer, CSCs are identified by the expression of aldehyde dehydrogenase 1 (ALDH1) or CD44-high/CD24-low markers. To probe the CSC populations in breast cancer patients, we developed a multiplex immunofluorescence assay with three CSC markers (ALDH1, CD44, and CD24) and an epithelial marker (pan-cytokeratin) for FFPE tissue. Automated image analysis with pathology review was used to evaluate single CSCs within the tumor. This assay may provide a means of monitoring a neoadjuvant CSC-targeted treatment in breast cancer FFPE samples in clinical trials. In summary, our results show that FAK inhibitors preferentially target cancer stem cells and delay the regrowth of tumor cells post standard-of-care treatment in mouse models providing rationale for the clinical development of FAK inhibitors to achieve more durable responses for cancer patients. Citation Format: Vihren Kolev, Yan Wang, Kam Sprott, Irina Shapiro, Jennifer Ring, Jonathan Pachter, David Weaver. FAK inhibition targets cancer stem cells. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2015 Nov 5-9; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2015;14(12 Suppl 2):Abstract nr C29.

Checkpoint immunotherapeutics are promising agents with potential to improve patient outcomes. Unfortunately, to date, single agent immunotherapy has achieved limited clinical benefit in patients with pancreatic ductal adenocarcinoma (PDAC). This may be due to the presence of the uniquely immunosuppressive tumor microenvironment present in PDACs that creates a barrier to immune surveillance by T cells. Critical obstacles to immune surveillance in PDAC tumors include a dense desmoplastic stroma that acts as a barrier to T cell infiltration and high numbers of tumor-associated immunosuppressive cells, such as tumor-associates macrophages, MDSCs, and regulatory T cells (Tregs). To understand which signaling pathways in pancreatic tumor cells might drive this suppressive tumor microenvironment, we analyzed the correlation between hyper-activated signaling molecules and tumor infiltrating leukocytes using 50 human PDAC tumor tissues. Of the pathways evaluated, we found that focal adhesion kinase (FAK) activity is elevated in human PDAC which correlates with highly fibrotic tumors with poor CD8+ T cell infiltration. The oral FAK kinase inhibitor VS-4718, currently in phase 1 clinical evaluation, was tested to determine if it could overcome the immunosuppressive tumor microenvironment of PDAC. Single agent VS-4718 dramatically limited tumor progression resulting in a doubling of survival in the p48-CRE/KrasG12D/p53flox/+ PDAC mouse model (KPC mice). This alteration in tumor progression was associated with dramatically reduced tumor fibrosis, decreased numbers of Tregs and tumor-infiltrating myeloid cells, and anti-tumor polarization of tumor-associated macrophages. We postulated that the desirable effects of FAK inhibition on the tumor microenvironment might render PDAC tumors more sensitive to immunotherapy. Accordingly, we found that VS-4718 significantly potentiated the efficacy of anti-PD-1 and anti-CTLA4 antibodies in KPC mouse models with a near tripling of survival. We next assessed the mechanism of this potentiation. We found that FAK in tumor cells regulated pro-inflammatory and pro-fibrotic cytokine secretion. Furthermore, we found that shRNA knockdown of FAK in PDAC cells resulted in failure of PDAC cells to induce pro-fibrotic programs in fibroblasts. Importantly, the FAK inhibitor VS-4718 and FAK shRNA in the tumor cells were each effective in increasing CD8+ cytotoxic T cell infiltration into the PDAC tumors in vivo. These data suggest that FAK inhibition increases immune surveillance programs in PDAC tumors by overcoming the fibrotic and inflammatory microenvironment rendering tumors more responsive to immunotherapy. These data provide rationale for clinical evaluation of a FAK inhibitor in combination with a PD-1 or PD-L1 antibody in patients with pancreatic and other cancers. Citation Format: Hong Jiang, Brett L. Knolhoff, Samarth Hedge, Yu Zhu, John Herndon, Irina M. Shapiro, David T. Weaver, Jonathan A. Pachter, David G. Denardo. Focal adhesion kinase inhibition enables efficacy of checkpoint immunotherapy in pancreatic cancer. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2015 Nov 5-9; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2015;14(12 Suppl 2):Abstract nr B197.

Although durable responses to single agent immune checkpoint inhibitors have been reported, additional approaches are needed to extend this therapeutic benefit to a greater proportion of cancer patients. Accordingly, substantial efforts are ongoing to identify agents that can augment T cell-mediated killing of tumor cells and potentiate the effects of checkpoint inhibitors. Focal Adhesion Kinase (FAK) and the closely related family member PYK2 are potentially valuable targets due to the roles of these protein kinases in regulating key cellular populations in the tumor microenvironment. In addition to the potency of the small molecule FAK/PYK2 inhibitors defactinib (VS-6063) and VS-4718 to target cancer stem cells, we have also reported that these agents inhibited monocyte-derived macrophages, decreased IL-6 production from macrophages in vitro, and reduced tumor-associated macrophages in xenograft models. We now report that defactinib and VS-4718 dose-dependently stimulate proliferation of CD8+ cytotoxic T cells isolated from healthy donors. This is in distinct contrast to other protein kinase inhibitors, such as the SRC inhibitor dasatinib and the MEK inhibitor trametinib, which potently impair the proliferation of CD8+ cytotoxic T cells. Based on the observed enhancement of CD8+ T cells and previously noted inhibition of tumor-associated macrophages, we investigated whether FAK/PYK2 inhibitors would potentiate the anti-tumor efficacy of an anti-PD-1 monoclonal antibody in syngeneic mouse tumor models. Mice bearing established MC38 colorectal tumors were treated with VS-4718 in combination with an anti-PD-1 antibody. Combination of VS-4718 with anti-PD-1 extended the median overall survival (OS) to 42 days relative to 21, 25 and 28 day median OS with vehicle control, single agent anti-PD-1 and single agent VS-4718, respectively. Moreover, on day 56, 30% of mice treated with the VS-4718/anti-PD-1 combination were alive in contrast to the vehicle control, single agent VS-4718, and single agent anti-PD-1 groups in which no mice survived. Analysis of MC38 tumors at day 12 of treatment revealed a significant increase in CD8+ T cells and a decrease in Tregs in the VS-4718/anti-PD-1 combination group, relative to vehicle control, providing a mechanistic understanding for the enhanced efficacy of this combination. Assessment of immune biomarkers in tumors from patients treated with defactinib is in progress. These data provide a rationale for clinical trials in cancer patients to test whether a FAK/PYK2 inhibitor in combination with an immune checkpoint inhibitor could increase the breadth of responsive tumor types, increase the number of responders, and confer a more durable anti-tumor response. Citation Format: Jennifer E. Ring, Yajuan Li, Irina M. Shapiro, Yan Wang, David T. Weaver, Jonathan A. Pachter. FAK/PYK2 inhibitors defactinib and VS-4718 enhance immune checkpoint inhibitor efficacy. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2015 Nov 5-9; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2015;14(12 Suppl 2):Abstract nr A191.

This report describes the investigation of a series of 5,7-disubstituted imidazo[5,1-f][1,2,4]triazine inhibitors of insulin-like growth factor-1 receptor (IGF-1R) and insulin receptor (IR). Structure−activity relationship exploration and optimization leading to the identification, characterization, and pharmacological activity of compound 9b, a potent, selective, well-tolerated, and orally bioavailable dual inhibitor of IGF-1R and IR with in vivo efficacy in tumor xenograft models, is discussed.

Cancer stem cells (CSCs), a subpopulation of tumor cells characterized by tumor-initiating capability and resistance to chemotherapy, are an underlying cause of tumor progression and metastasis. In breast cancer, CSCs can be identified by aldehyde dehydrogenase 1 (ALDH) or CD44+/CD24- expression separately, although combination of ALDH, CD44+, and CD24- together may better describe the CSC population in breast cancer. Neoadjuvant chemotherapy has been shown to lead to an increase in CSCs in locally advanced breast cancer (Alamgeer et al., 2014, Br. Can. Res. 16:R14) which is associated with a significantly worse prognosis. It is also evident that stem-like features of tumor cells are present in metastatic breast cancer (Yu et al., 2013, Science 339:580) indicating that targeting of CSCs may be valuable in metastatic disease. VS-6063 and VS-4718 are orally bioavailable, potent and selective inhibitors of Focal Adhesion Kinase (FAK). These agents preferentially target CSCs in preclinical models and are currently in clinical development. Here we report that VS-6063 and VS-4718 preferentially target CSCs in multiple breast cancer models. Ex vivo treatment of explants from human breast tumors with either VS-6063 or VS-4718 decreased the proportion of CSCs in contrast to paclitaxel treatment which increased the proportion of CSCs. In an MDA-MB-231 mouse xenograft model, in vivo treatment with VS-6063 decreased CSCs by more than 6-fold as demonstrated by limiting dilution transplantation assay. Consistent with the notion that CSCs are responsible for cancer relapse after chemotherapy, VS-6063 and VS-4718 substantially delayed tumor growth following cessation of paclitaxel or cisplatin treatment in models of TNBC, including a patient-derived xenograft. Furthermore, using an imaging-based 4T1-luciferase TNBC orthotopic model, both VS-6063 and VS-4718 inhibited metastatic outgrowth or induced regression of metastases after primary tumor resection, whereas metastases progressed in all animals in the vehicle control group. A multiplex assay for multiple CSC markers (ALDH1, CD44 and CD24) was developed and validated in biopsies of primary patient tumors and matched lymph node biopsies taken pre- and post-neoadjuvant chemotherapy. CSCs, represented by the ALDH+ and/or CD44+/CD24- populations were present at detectable levels in a high fraction of triple negative breast cancer primary tumors. In summary, CSCs are readily detectable in primary breast cancers at surgery, and VS-6063 and VS-4718 diminish the CSC subpopulation in vitro, ex vivo and in xenograft models. This critical subpopulation of CSCs is detectable in residual tumors after neoadjuvant therapy, and may also be present in metastatic disease. CSC-targeted agents such as VS-6063 or VS-4718 should be clinically tested in these breast cancer settings to potentially delay time to relapse and improve patient outcome. Citation Format: Vihren N. Kolev, Kam Sprott, Qunli Xu, Jonathan A. Pachter, David T. Weaver. FAK inhibitors VS-6063 and VS-4718 target cancer stem cells: Implications for TNBC sequential and combination therapies. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 974. doi:10.1158/1538-7445.AM2015-974

Deletions and dominant-negative mutations in IKZF1, the gene encoding Ikaros transcription factor, are found in ∼85% of Ph+ B-ALL and in some cases of Ph− B-ALL, and are associated with poor prognosis. Genomic studies of high-risk Ph− or “Ph-like” B-ALLs have revealed frequent mutation and activation of TK genes and signaling pathways. While ABL1 tyrosine kinase inhibitors (TKIs) such as dasatinib and ponatinib have been incorporated into chemotherapy regimens for Ph+ B-ALL, the majority of patients still relapse, which correlates with residual bone marrow disease following induction therapy. New therapeutic strategies are needed for patients with Ikaros-mutant, high-risk Ph+ and Ph− B-ALL. Using mice with a conditional Ikzf1 mutation (Ike5fl) that mimics the dominant-negative Ik6 mutant found in human B-ALL, we demonstrated that loss of Ikaros DNA-binding function arrests B-lymphoid development at a large pre-B cell stage that can give rise to B-ALL. Survival and proliferation of Ikaros mutant pre-B cells is dependent on increased integrin-mediated stromal adhesion and activation of focal adhesion kinase (FAK). FAK is a non-receptor TK downstream of integrins and growth factor receptors that plays important roles in cancer stem cell biology and the tumor microenvironment. Here, we show that dysregulated tyrosine kinases, including BCR-ABL1, cooperate with Ikaros mutation to accelerate the development of B-lymphoblastic leukemia in mice, correlated with a striking (∼30-fold) increase in the frequency of engrafting leukemia-initiating or leukemic stem cells (LSCs). Ikzf1-mutant BCR-ABL1+ lymphoblasts exhibit relative resistance to ABL1 TKIs including dasatinib that is dependent on the presence of stroma. VS-4718 is a potent, orally bioavailable FAK inhibitor that inhibits tumor growth and metastasis in preclinical models, and is currently under evaluation in clinical trials in patients with various solid tumors. VS-4718 treatment abolished stromal adhesion and induced apoptosis of Ikzf1-mutant B-ALL and synergized with dasatinib, but had little effect on Ikzf1 WT B-ALL cells. Primary human Ph+ B-ALL samples showed a correlation between IKZF1 mutation status, stromal adherence, and sensitivity to FAK and ABL inhibitors in vitro, and BM tropism in vivo in xenografted NSG mice. Results from ongoing in vivo therapy experiments will be presented. Together, these results suggest a new model for the pathogenesis of high-risk B-ALL and its resistance to conventional therapy. B-ALLs with IKZF1 mutations may be resistant to TKIs and to chemotherapy by virtue of their stromal adhesion phenotype, resulting in failure to eliminate BM leukemic stem cells. Inhibition of FAK signaling in Ph+ or Ph− IKZF1-mutant B-ALL may reverse the stromal-mediated resistance to ABL1 TKIs and/or chemotherapy. FAK inhibitors represent a promising new approach for the treatment of high-risk IKZF1-mutant B-ALL patients. Citation Format: Nilamani Jena, Ila Joshi, Toshimi Yoshida, Zhihong Zhang, Zhong-Ying Liu, Prasanthi Tata, Irina M. Shapiro, Jonathan A. Pachter, David T. Weaver, Katia Georgopoulos, Richard A. Van Etten. Targeting focal adhesion kinase is a novel approach to therapy of high-risk, Ikaros-mutant acute B-cell lymphoblastic leukemia. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 4202. doi:10.1158/1538-7445.AM2015-4202

Malignant pleural mesothelioma (MPM) is an aggressive tumor in the lining of the lung often resulting from prior exposure to asbestos. Median overall survival with standard of care chemotherapy is only 12 months from diagnosis. This poor prognosis may be attributable at least in part to cancer stem cells (CSCs) which are resistant to chemotherapy and can mediate cancer recurrence and progression. Focal adhesion kinase (FAK) has been shown to play an essential role in the survival, self-renewal and tumor-initiating capability of CSCs. Accordingly, the FAK inhibitor VS-6063 is currently being tested in patients with MPM following disease control on standard pemetrexed/platinum chemotherapy (COMMAND, ClinicalTrials.gov NCT01870609). Aldehyde dehydrogenase (ALDH) activity was validated as a CSC marker in MPM by assessment of tumor-initiating capability in mice. As compared to ALDH-negative cells, sorted ALDH-positive MM87 MPM cells showed 35-fold greater tumor initiating capacity when implanted in limiting dilutions into immunodeficient mice. Indeed, 50 ALDH-positive cells were sufficient to generate sizeable tumors in 3 weeks illustrating the aggressive nature of MPM CSCs. Treatment of a human MPM cell line with pemetrexed enriched ALDH-positive CSCs 6-fold, with similar CSC enrichment by cisplatin. In direct contrast, the FAK inhibitor VS-6063 markedly reduced the proportion of CSCs. The enrichment of MPM CSCs was similarly observed in samples from 11 tested patients following first line chemotherapy. Patient specimens post treatment with pemetrexed and cisplatin showed an elevated ALDH immunohistochemistry H-score and an increase in expression of CSC genes such as CD133 as compared to matched MPM biopsies taken from the same patients prior to chemotherapy. To assess drug effects on tumor-initiating capacity, MM87 merlin-low MPM and H28 merlin-high MPM cell lines were treated in vitro with VS-6063, pemetrexed or the combination and subsequently implanted into mice. While control and pemetrexed-treated MPM cells showed robust tumor initiation, cells treated with VS-6063 alone or VS-6063 plus pemetrexed showed little or no tumor initiating capacity. Accordingly, in tumor biopsies from MPM patients treated for 12 days with VS-6063, tumor pFAK (Y397) and expression of CSC genes such as CD133 were reduced. MPM patient-derived xenograft (PDX) tumors were employed to model the clinical scenario. Compared to control, tumor growth was blocked by 2-week treatment with pemetrexed/cisplatin. Tumors then grew rapidly upon cessation of pemetrexed/platinum treatment, whereas tumor growth was substantially delayed by FAK inhibitor treatment after cessation of chemotherapy. These data provide strong rationale for the current clinical testing of VS-6063 following treatment with pemetrexed plus platinum to potentially prolong time to progression in patients with mesothelioma. Citation Format: Jonathan A. Pachter, Vihren N. Kolev, Laurel Schunselaar, Irina M. Shapiro, Raphael Bueno, Paul Baas, Qunli Xu, David T. Weaver. FAK inhibitor VS-6063 (defactinib) targets mesothelioma cancer stem cells, which are enriched by standard of care chemotherapy. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 4236. doi:10.1158/1538-7445.AM2015-4236

Small cell lung cancer (SCLC) is an extremely aggressive cancer with poor outcome. In most patients with SCLC tumors initially respond to first line chemotherapy, but subsequently experience aggressive recurrence that may be attributed to the presence of a cancer stem cell (CSC) population with tumor-initiating and metastatic potential. Inhibitors of FAK and PI3K/mTOR have been shown to preferentially target CSCs in preclinical models of other cancers, where the evidence indicates that CSC populations are increased following chemotherapy. FAK and PI3K/mTOR inhibitors may be particularly valuable in a SCLC setting, where CSCs may be strong mediators of recurrence. Tumors of patients with SCLC are generally not resectable, such that tumor biopsies are not available for CSC evaluation. Therefore, it will be important to be able to monitor CSC markers by blood-based testing. A drug-resistant population of SCLC CSCs (side population, SP) can be monitored by enhanced ability to exclude Hoechst 33342 dye. Additionally, SCLC CD133+ cells have been demonstrated to possess CSC properties and presence of CD133+ cells in SCLC has been linked to poor prognosis. Our data indicate that cisplatin and etoposide, first line chemotherapy for SCLC, enrich for SP and CD133+ cells, suggesting that chemotherapy is not effective against CSCs and should be combined with anti-CSC agents for more durable response. Here we investigate the antitumor activity of FAK and PI3K/mTOR inhibitors in SCLC xenograft models in vivo. Both a FAK inhibitor (VS-4718) and PI3K/mTOR inhibitor (VS-5584) were found to enhance the antitumor activity of chemotherapy in an NCI-H69 SCLC xenograft model. Additionally, oral administration of VS-5584 reduced the proportion of CSCs in vivo in an NCI-H841 SCLC tumor model as evidenced by a decrease in the percentage of SP cells and approximately 70-fold reduction in in vivo tumor-initiating capability. The combination of chemotherapy and FAK or PI3K/mTOR inhibitors demonstrate an extended time to relapse after first line therapy. VS-5584 significantly delayed tumor regrowth following cessation of cisplatin treatment in H69 xenograft tumors and a SCLC patient-derived xenograft (PDX) model. Patients with relapsed SCLC have elevated circulating tumor cells, consistent with the poor prognosis of their disease status. Blood samples from these patients are under evaluation for CSC markers in the circulation. The preferential targeting of CSCs in preclinical SCLC models provides an important rationale for clinical development of FAK and PI3K inhibitors, where combination with chemotherapeutic agents or single agent activity post chemotherapy may potentially lengthen the time to relapse and improve outcome for patients with small cell lung cancer. Citation Format: Vihren N. Kolev, Qunli Xu, Jonathan A. Pachter, David T. Weaver. FAK and PI3K/mTOR inhibitors target cancer stem cells: Implications for SCLC treatment strategies. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 1525. doi:10.1158/1538-7445.AM2015-1525

Cancer stem cells (CSCs) have been implicated in resistance to anticancer therapies, disease recurrence, and metastasis, which represent major challenges in the clinical management of cancer. Therapeutic targeting of CSCs thus holds promise in addressing these challenges. VS-5584 is a selective dual PI3K/mTOR kinase inhibitor that potently inhibits mTORC1, mTORC2 and all four Class I PI3K isoforms. We report here that VS-5584 is up to 30-fold more potent at inhibiting the proliferation and survival of CSCs than non-CSCs in breast cancer cell lines using multiple orthogonal CSC assays. Moreover, VS-5584 preferentially induced apoptosis in Aldefluor-positive CSCs relative to Aldefluor-negative non-CSCs as measured by Annexin V and Caspase 3/7 assays. In contrast, paclitaxel induced more apoptosis in non-CSCs than CSCs cells. VS-5584 also preferentially diminished CSCs in human breast and small cell lung cancer xenograft models in vivo, as evidenced by marked reduction of tumor-initiating capacity in an in vivo limiting dilution re-implantation assay. Likewise, ex vivo VS-5584 treatment of surgically removed breast and ovarian patient tumors preferentially reduced CSC populations. In contrast, cytotoxic chemotherapeutic agents such as paclitaxel and cisplatin preferentially eliminate non-CSCs and thus enrich the CSC population. Interestingly, preferential targeting of CSCs requires inhibition of multiple components of the PI3K/mTOR pathway; simultaneous knockdown of PI3Kα, PI3Kβ, and mTOR by siRNA phenocopied the effect of VS-5584 and exhibited the strongest preferential targeting of CSCs, while knocking down of individual PI3K isoform or mTOR failed to do so. Consistent with its strong suppression of CSCs, VS-5584 effectively delayed tumor regrowth following chemotherapy in small cell lung cancer xenograft models including a patient-derived primary tumor model. The preferential targeting of CSCs thus prompts a new paradigm for testing VS-5584 in the clinic: clinical trials designed with CSC-directed endpoints may facilitate demonstration of the therapeutic benefit of VS-5584 at well-tolerated doses and thus enable an optimal therapeutic window. Furthermore, suppression of CSCs by VS-5584 in combination with other agents that target the bulk tumor may represent an appealing strategy to achieve more durable responses for cancer patients. Citation Format: Vihren N. Kolev, Quentin G. Wright, David T. Weaver, Mahesh V. Padval, Jonathan A. Pachter, Qunli Xu. Combined inhibition of PI3K isoforms and mTOR kinase is critical for cancer stem cell inhibition by VS-5584. [abstract]. In: Proceedings of the AACR Special Conference: Targeting the PI3K-mTOR Network in Cancer; Sep 14-17, 2014; Philadelphia, PA. Philadelphia (PA): AACR; Mol Cancer Ther 2015;14(7 Suppl):Abstract nr A36.

Current chemotherapy is effective in killing leukemic blasts in the periphery, but not leukemic stem cells (LSCs) in the bone marrow, which are thought to be responsible for the high relapse rate in leukemia. Thus, new therapies that effectively target LSCs are urgently needed to prevent cancer relapse. Accumulating evidence supports an essential role for adhesion pathways: particularly integrin beta 3 and its downstream focal adhesion kinase (FAK) in the maintenance of LSCs (Miller et al., Cancer Cell 2013; Despeaux et al., Stem Cells 2012). Previously, we have shown that FAK inhibitors preferentially target cancer stem cells in solid tumors (Shapiro et al., Sci Transl Med, 2014; Kolev et al., AACR 2013). We are extending our investigation of FAK inhibitors into hematological malignancies and report here that the FAK inhibitor VS-4718 displays anticancer activity in leukemia models both in vitro and in vivo. VS-4718 is a potent and orally bioavailable small molecule compound that targets cancer stem cells through inhibition of FAK kinase which is currently being tested in a Phase 1 clinical trial (NCT01849744). The anti-proliferative effect of VS-4718 was evaluated in a panel of 10 cell lines derived from patients with acute promyelocytic leukemia (APL), T-cell acute lymphocytic leukemia (T-ALL), B-cell acute lymphocytic leukemia (B-ALL), chronic myeloid leukemia (CML) and acute myeloid leukemia (AML) using a CellTiter-Glo cell viability assay on tissue culture or collagen coated plates. VS-4718 displayed anti-proliferative effects against most of these cell lines, with MV-4-11 AML being the most sensitive with an EC50 value of 200 nM. We further investigated the in vivo efficacy of VS-4718 against AML in both subcutaneous and disseminated models using the MV-4-11 cell line. Nude mice bearing MV-4-11 subcutaneous xenografts were treated orally twice daily with either vehicle control or VS-4718 for 14 days. 75 mg/kg VS-4718 caused 50% tumor growth delay and significantly extended median survival from 28 days to 48 days (p < 0.05). Moreover, tumor regression was observed in 4 out of 10 mice. We extended these observations to a disseminated MV-4-11 AML model to incorporate bone marrow stromal biology. When compared with vehicle control, VS-4718 dosed at 25 or 75 mg/kg, on a twice daily oral dosing schedule for 14 days, resulted in 40% and 76% increase in mouse life span, and significantly extended survival with p values < 0.05 and < 0.001 (log rank test), respectively. The effect of VS-4718 on leukemia stem cells and minimal residual disease (MRD) is currently under investigation. Taken together, results of our preclinical studies suggest that VS-4718 may have activity against leukemia that warrants further investigation. Disclosures Tam: Verastem: Employment. Ring:Verastem: Employment. Trombino:Verastem: Employment. Weaver:Verastem: Employment, Equity Ownership. Pachter:Verastem Inc.: Employment, Equity Ownership. Padval:Verastem: Employment. Xu:Verastem: Employment.

Triple negative breast cancer (TNBC) is defined phenotypically as the lack of ER, PR and HER2 expression, and comprises a heterogeneous group of breast cancers. Cancer stem cells (CSCs), identified as either ALDH1+ or CD44hi/CD24lo subpopulations in breast cancer, have been shown to be resistant to standard chemotherapy and associated with poor clinical outcome. In recent reports, the presence of ALDH1+ (but not ALDH1-) cells in axillary lymph nodes following neo-adjuvant chemotherapy was associated with poor overall survival. Focal adhesion kinase (FAK) is a non-receptor tyrosine kinase that orchestrates cell signaling through integrins and growth factor receptors. In addition to regulating the proliferation, survival, invasion and metastasis of cancer cells, FAK also plays a critical role in the self-renewal and survival of CSCs. VS-6063 (defactinib) is a potent, selective, and orally bioavailable FAK inhibitor with demonstrated tolerability and preliminary signs of clinical activity as a single agent and in combination with paclitaxel in phase I clinical trials. We report here that VS-6063 preferentially targets CSCs in preclinical models of TNBC. The effect of VS-6063 on CSCs was assessed in a panel of orthogonal assays. Treatment of human TNBC cells with VS-6063 in 3D matrigel reduced the percentage of Aldefluor+ CSCs, Hoechst dye-excluding side population (SP) CSCs, and tumorsphere-forming efficiency in a serial tumorsphere passaging assay, suggesting that VS-6063 preferentially inhibits CSCs. VS-6063 also reduced the proportion of Aldefluor+ CSCs in primary breast cancer tissue specimens cultured ex vivo. Consistent with the concept that the effect of VS-6063 on CSCs is mediated by FAK inhibition, siRNA-mediated knockdown of FAK in SUM159 TNBC cells recapitulated the inhibitory effect of VS-6063 on CSCs. In contrast, the standard-of-care (SoC) chemotherapeutic agents paclitaxel and doxorubicin enriched the percentage of CSCs, suggesting that these SoC agents preferentially target bulk tumor cells relative to CSCs. When administered in combination, VS-6063 attenuated the chemotherapy-induced enrichment of CSCs. Following oral administration of VS-6063 in the MDA-MB-231 human TNBC xenograft model in vivo, reduction of CSCs in tumors was evidenced by a decrease in secondary tumorsphere-forming efficiency. Importantly, cells dissociated from VS-6063-treated tumors displayed reduced tumor-initiating capability upon re-implantation into immunodeficient mice in limiting dilutions. In summary, our results indicate that the FAK inhibitor VS-6063 preferentially targets TNBC CSCs and support the clinical development of VS-6063 in combination with SoC agents, such as paclitaxel, to potentially improve clinical outcomes for patients with TNBC through simultaneous targeting of both CSCs and bulk tumor cells. Citation Format: Qunli Xu, Vihren N. Kolev, Quentin G. Wright, Jennifer E. Ring, Christian M. Vidal, Irina M. Shapiro, David T. Weaver, Mahesh V. Padval, Jonathan A. Pachter. Focal adhesion kinase (FAK) inhibitor VS-6063 (defactinib) preferentially targets cancer stem cells in triple negative breast cancer. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 3908. doi:10.1158/1538-7445.AM2014-3908

Malignant pleural mesothelioma (MPM) is an aggressive tumor in the lining of the lung often resulting from prior exposure to asbestos. Median overall survival with the standard of care chemotherapy is only 12 months from diagnosis. Thus, new therapeutic modalities are urgently needed to improve the prognosis of MPM patients. ∼50% of MPM patients exhibit biallelic inactivation of the NF2 tumor suppressor gene by mutation and/or deletion resulting in lack of expression of functional merlin protein. Merlin inactivation is a critical step in MPM tumorigenesis and is related, at least in part, to up-regulation of focal adhesion kinase (FAK) activity. FAK has also been demonstrated to play a role in maintenance of cancer stem cells (CSCs), a subpopulation of tumor cells associated with drug resistance and tumor recurrence. Therefore, therapeutic targeting of FAK using defactinib (VS-6063), a potent, orally active FAK kinase inhibitor, may benefit cancer patients through targeting of both CSCs and bulk tumor cells. Among a panel of MPM cell lines in 3-dimensional Matrigel culture, MPM lines lacking expression of merlin were found to be especially sensitive to defactinib. In contrast, MPM cell lines with wild-type merlin expression were less sensitive with average EC50 values of 5.1 μM. Oral dosing of defactinib induced significant tumor growth inhibition in a merlin-negative MPM model pre-implanted in the lungs of mice. Using Aldefluor activity as a marker for CSCs in MPM, we find that chemotherapeutic agents enriched for Aldefluor-positive CSCs, while the FAK inhibitor defactinib reduced the proportion of CSCs. When used in combination with pemetrexed or cisplatin, defactinib blocked the enrichment of CSCs cells by these chemotherapeutic agents. Disruption of cell-cell or cell-extracellular matrix (ECM) contacts with blocking antibodies allowed us to propose a molecular model to account for the observed merlin-FAK inhibitor synthetic lethal relationship. We find that weak cell-cell adhesions in merlin-negative MPM cells lead to a greater dependence of tumor cell survival and proliferation on cell-ECM-induced FAK signaling rendering merlin-negative cells especially vulnerable to FAK inhibitor treatment. Taken together, these data support the clinical development of defactinib in an ongoing Phase 2 registration-directed clinical trial evaluating the activity of defactinib in MPM patients (stratified by merlin status) with a confirmed response (PR or SD) following first line platinum/pemetrexed therapy. Citation Information: Mol Cancer Ther 2013;12(11 Suppl):C262. Citation Format: Irina M. Shapiro, Vihren N. Kolev, Yuwaraj Kadariya, Christian M. Vidal, Quentin G. Wright, Jennifer E. Ring, Craig Menges, Joseph R. Testa, Qunli Xu, Jonathan A. Pachter. Malignant mesothelioma lacking merlin shows enhanced sensitivity to the FAK inhibitor defactinib (VS-6063): Elucidation of the merlin-FAK relationship. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2013 Oct 19-23; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2013;12(11 Suppl):Abstract nr C262.

Malignant pleural mesothelioma (MPM) is an aggressive tumor in the lining of the lung often caused by asbestos exposure. With a median survival on standard of care chemotherapy of only 12 months from diagnosis, new therapeutic modalities and predictive biomarkers are urgently needed for MPM patients. The Neurofibromatosis 2 (NF2) tumor suppressor gene that encodes the protein merlin is disrupted in approximately 50% of MPMs. The role of merlin in cell adhesion, invasion and cell motility may be partially mediated through the focal adhesion kinase (FAK), an important signaling node downstream of integrin cell adhesion proteins. An examination of 15 mesothelioma cell lines grown in 3-dimensional Matrigel culture demonstrates that low merlin by immunoblotting correlated with increased sensitivity to the FAK inhibitor, defactinib (VS-6063). An immunohistochemistry (IHC) test for merlin may be an optimal clinical tumor specimen measurement for merlin loss, provided that merlin reduction or loss is correlated with other indicators of NF2 change. The presence or absence of merlin and NF2 was evaluated using MPM patient-derived xenograft (PDX) tumor models using merlin IHC and an NF2 Fluorescence In situ Hybridization (FISH), as well as Western blotting to determine merlin levels in the tumor lysates. The PDX MPM-0235 tumor had low merlin by Western blotting, merlin IHC, and NF2 FISH, whereas PDX MPM-0237 showed high merlin levels by Western blotting, merlin IHC, and diploid chromosome 22 by FISH. In addition to PDX analysis, Merlin IHC and NF2 FISH optimized by these conditions was evaluated with in patient MPM Tissue Microarrays (TMAs) of 64 primary tumors. Chromosome 22 monosomy was significantly more frequent than NF2 regional deletion of chromosome 22. We observed that 42% of the tumors had a low merlin IHC score (merlin-low) that correlated with loss of NF2 (unpaired t-test, p Citation Information: Mol Cancer Ther 2013;12(11 Suppl):A31. Citation Format: David T. Weaver, Irina Shapiro, Christian Vidal, Qunli Xu, Mahesh Padval, Jonathan Pachter, Daniel Paterson, Jullianne Barlow, William Richards, Sabina Signoretti, Raphael Bueno. Merlin loss as a biomarker for defactinib (VS-6063) sensitivity: High frequency in malignant mesothelioma tumors. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2013 Oct 19-23; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2013;12(11 Suppl):Abstract nr A31.

Focal adhesion kinase (FAK) is a non-receptor tyrosine kinase that orchestrates cell signaling through integrins and growth factor receptors and plays important roles in tumorigenesis. Amplification and overexpression of FAK have been observed in aggressive human cancers including ovarian and breast cancers. Defactinib is a potent, selective, and orally active FAK inhibitor with demonstrated tolerability and preliminary clinical activity as a single agent in a Phase 1 clinical trial. In an ongoing Phase 1/1b clinical trial (NCT01778803), we have demonstrated that defactinib can be combined with paclitaxel and have observed preliminary signs of clinical activity. We report here preclinical data supporting the clinical testing of defactinib in combination with paclitaxel in patients with ovarian cancer. In human ovarian cancer cell lines TOV-21G and OV-7, defactinib was found to enhance the efficacy of paclitaxel. Combination Index analyses demonstrated synergistic inhibition of tumor cell proliferation/survival with combination of defactinib and paclitaxel. We find that FAK inhibitors preferentially target breast and ovarian cancer stem cells (CSCs) relative to bulk tumor cells as evidenced by a diminished proportion of CSCs in multiple orthogonal CSC assays. In direct contrast, the standard-of-care cytotoxic agents, paclitaxel and carboplatin, increased the percentage of CSCs, indicating that these agents do not effectively target CSCs. Importantly, combination of FAK inhibitors with cytotoxic agents attenuated the enrichment of CSCs induced by the cytotoxic agents. We subsequently determined if defactinib reduces CSCs in tumor specimens from ovarian cancer patients who were previously treated with taxane and platinum. Ex vivo treatment of ovarian tumor tissue fragments with defactinib reduced the percentage of CSCs as assessed by CD24hi/CD117hi CSC markers and the Aldefluor assay. To assess drug effects on tumor-initiating capability, the ‘gold standard’ for CSC activity, TOV-21G cells were pretreated in vitro with the FAK inhibitor, paclitaxel or the combination of both agents and 1,000 of the resulting cells were injected into immunodeficient mice. The FAK inhibitor alone or in combination with paclitaxel prevented tumor initiation, while paclitaxel alone did not reduce tumor initiation, suggesting that treatment with a FAK inhibitor could effectively eliminate tumor initiating CSCs. In summary, our data indicate that defactinib preferentially targets cancer stem cells and enhances the activity of paclitaxel in preclinical models of ovarian cancer. These results provide additional support for the current clinical development of defactinib in combination with paclitaxel for the treatment of ovarian cancer. Citation Information: Mol Cancer Ther 2013;12(11 Suppl):C271. Citation Format: Christian M. Vidal, Vihren N. Kolev, Quentin G. Wright, Winnie F. Tam, David T. Weaver, Mahesh V. Padval, Qunli Xu, Jonathan A. Pachter. FAK inhibitor defactinib (VS-6063) enhances the efficacy of paclitaxel and preferentially targets ovarian cancer stem cells. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2013 Oct 19-23; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2013;12(11 Suppl):Abstract nr C271.

Focal adhesion kinase (FAK) is a non-receptor tyrosine kinase that orchestrates cell signaling through integrins and growth factor receptors and plays important roles in tumorigenesis. Amplification and overexpression of FAK have been observed in aggressive human cancers including ovarian and breast cancers. VS-6063 and VS-4718 are potent, selective, and orally active FAK inhibitors. In a Phase 1 clinical trial, VS-6063 was well tolerated and demonstrated preliminary clinical activity. In addition, we have demonstrated that VS-6063 can be combined with paclitaxel and preliminary signs of clinical activity, including CA-125 reduction to normal levels, were observed with this combination in patients with ovarian cancer. VS-4718 was shown to inhibit tumor growth and metastasis in preclinical tumor models including an ovarian cancer xenograft model and is currently under evaluation in a Phase 1 clinical trial. We have shown previously that inhibition of FAK preferentially targets cancer stem cells in breast cancer models. We extended these studies and report here that the FAK inhibitors VS-6063 and VS-4718 effectively abrogate cancer stem cells (CSCs) in ovarian cancer models. VS-4718 and VS-6063 were first evaluated in several orthogonal cancer stem cell assays in vitro. Treatment of OVCAR-8 or TOV-21G human ovarian cancer cells with these FAK inhibitors in matrigel reduced the percentage of Aldefluor+ CSCs and attenuated tumorsphere forming efficiency. Accordingly, in a side population (SP) assay assessing CSCs by Hoechst dye exclusion, these FAK inhibitors reduced SP CSCs in the OVCAR-5 ovarian cancer cell line. Similar effects were observed with the breast cancer cell lines SUM159 and MDA-MB-231. In direct contrast, the standard-of-care cytotoxic agents, paclitaxel and carboplatin, increased the percentage of CSCs, indicating that these agents do not effectively target CSCs. Importantly, combination of VS-6063 or VS-4718 with cytotoxic agents attenuated the enrichment of CSCs caused by cytotoxic agents as measured by the Aldefluor assay and in vivo tumor initiating potential. To assess drug effects on tumor-initiating capability, the clearest arbiter of CSC activity, TOV-21G cells were pre-treated in vitro with various compounds and 1,000 of the resulting cells were injected into immunodeficient mice. VS-4718 alone or VS-4718 in combination with paclitaxel prevented tumor initiation, while paclitaxel alone did not reduce tumor initiation, suggesting that treatment with VS-4718 could effectively eliminate tumor initiating CSCs. Furthermore, ex vivo treatment of primary human ovarian cancer tissue with VS-4718 or VS-6063 also reduced the percentage of CSCs as assessed by CD24hi/CD117hi CSC markers and the Aldefluor assay. In addition to targeting CSCs, we also observed that FAK inhibitors enhanced the activity of paclitaxel to reduce bulk tumor cells both in vitro and in vivo. In summary, our data indicate that the FAK inhibitors VS-6063 and VS-4718 preferentially target cancer stem cells and enhance the activity of paclitaxel. These results provide rationale for the clinical development of Verastem's FAK inhibitors for the treatment of ovarian cancer. Citation Format: Vihren N. Kolev, Quentin G. Wright, Christian M. Vidal, Winnie F. Tam, Mahesh V. Pavdal, Qunli Xu, Jonathan A. Pachter. FAK inhibitors VS-6063 and VS-4718 preferentially target ovarian cancer stem cells. [abstract]. In: Proceedings of the AACR Special Conference on Advances in Ovarian Cancer Research: From Concept to Clinic; Sep 18-21, 2013; Miami, FL. Philadelphia (PA): AACR; Clin Cancer Res 2013;19(19 Suppl):Abstract nr A39.

e18541 Background: Malignant pleural mesothelioma (MPM) is an aggressive tumor in the pleural lining of the lung often resulting from prior exposure to asbestos. MPM patients are usually diagnosed at an advanced stage of disease and the prognosis is poor. Median survival after diagnosis is 9 to 12 months and standard-of-care agents such as cisplatin and pemetrexed have only a modest impact on median survival time for MPM patients. New therapeutic modalities are urgently needed to improve the prognosis of MPM patients. 40-50% of MPM patients exhibit homozygous disruption of the NF2 tumor suppressor gene by mutation and/or deletion resulting in lack of expression of functional merlin protein. Methods: Proliferation of drug-treated mesothelioma cell lines in 3-dimensional (3D) Matrigel culture was assessed by MTS, and MPM xenograft growth was measured in vehicle- vs. FAK inhibitor-treated SCID mice. Since absence of merlin expression can theoretically result from several mechanisms including NF2 mutation and chromosome 22 abnormalities, we assessed NF2 gene deletion by FISH and merlin protein levels by IHC in the same human mesothelioma tumor samples. Results: Among a panel of mesothelioma cell lines in 3D culture, MPM lines lacking expression of merlin protein were found to be especially sensitive to the selective FAK inhibitor VS-6063. In contrast, MPM cell lines with wildtype merlin were less sensitive with EC50 values greater than 1 μM. Accordingly, oral dosing with a FAK inhibitor induced significant tumor growth inhibition in a merlin-negative mesothelioma model in mice. To enable the planned stratification of MPM patients by merlin status, an immunohistochemistry (IHC) assay has been optimized to quantify merlin protein levels. A merlin IHC H-score below the defined cutoff was associated with loss of at least one copy of chromosome 22, indicating that chromosomal deletion is an important mechanism of merlin loss in mesothelioma patients. Conclusions: These data support the clinical development of VS-6063 for treatment of malignant pleural mesothelioma patients stratified by merlin/NF2 status. A potentially pivotal mesothelioma trial is set to initiate in 2013.