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Supplementary Figure S5. Representative Aldefluor FACS data of cells dissociated from MDA-MB-231 and MCF-7 tumors (Fig. 3B and 3D).

Supplementary Materials and Methods. More detailed Materials and Methods

Supplementary Figure Legends. Legends for Supplementary Figures S1-S5

Cancer stem cells (CSC) have been implicated in disease recurrence, metastasis, and therapeutic resistance, but effective targeting strategies for these cells are still wanting. VS-5584 is a potent and selective dual inhibitor of mTORC1/2 and class I PI 3-kinases. Here, we report that VS-5584 is up to 30-fold more potent in inhibiting the proliferation and survival of CSC compared with non-CSC in solid tumor cell populations. VS-5584 preferentially diminished CSC levels in multiple mouse xenograft models of human cancer, as evidenced by marked reduction of tumor-initiating capacity in limiting dilution assays. Likewise, VS-5584 treatment ex vivo preferentially reduced CSC in surgically resected breast and ovarian patient tumors. In contrast, chemotherapeutics such as paclitaxel and cisplatin were less effective in targeting CSC than bulk tumor cells. Mechanistic investigations revealed that preferential targeting of CSC required inhibition of multiple components of the PI3K–mTOR pathway: coordinate RNAi-mediated silencing of PI3Kα, PI3Kβ, and mTOR phenocopied the effect of VS-5584, exhibiting the strongest preferential targeting of CSC, while silencing of individual PI3K isoforms or mTOR failed to replicate the effect of VS-5584. Consistent with CSC ablation, VS-5584 delayed tumor regrowth following chemotherapy in xenograft models of small-cell lung cancer. Taken together, the preferential targeting of CSC prompts a new paradigm for clinical testing of VS-5584: clinical trials designed with CSC-directed endpoints may facilitate demonstration of the therapeutic benefit of VS-5584. We suggest that combining VS-5584 with classic chemotherapy that debulks tumors may engender a more effective strategy to achieve durable remissions in patients with cancer. Cancer Res; 75(2); 446–55. ©2014 AACR.

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.

Cancer stem cells (CSC) have been implicated in disease recurrence, metastasis, and therapeutic resistance, but effective targeting strategies for these cells are still wanting. VS-5584 is a potent and selective dual inhibitor of mTORC1/2 and class I PI 3-kinases. Here, we report that VS-5584 is up to 30-fold more potent in inhibiting the proliferation and survival of CSC compared with non-CSC in solid tumor cell populations. VS-5584 preferentially diminished CSC levels in multiple mouse xenograft models of human cancer, as evidenced by marked reduction of tumor-initiating capacity in limiting dilution assays. Likewise, VS-5584 treatment ex vivo preferentially reduced CSC in surgically resected breast and ovarian patient tumors. In contrast, chemotherapeutics such as paclitaxel and cisplatin were less effective in targeting CSC than bulk tumor cells. Mechanistic investigations revealed that preferential targeting of CSC required inhibition of multiple components of the PI3K–mTOR pathway: coordinate RNAi-mediated silencing of PI3Kα, PI3Kβ, and mTOR phenocopied the effect of VS-5584, exhibiting the strongest preferential targeting of CSC, while silencing of individual PI3K isoforms or mTOR failed to replicate the effect of VS-5584. Consistent with CSC ablation, VS-5584 delayed tumor regrowth following chemotherapy in xenograft models of small-cell lung cancer. Taken together, the preferential targeting of CSC prompts a new paradigm for clinical testing of VS-5584: clinical trials designed with CSC-directed endpoints may facilitate demonstration of the therapeutic benefit of VS-5584. We suggest that combining VS-5584 with classic chemotherapy that debulks tumors may engender a more effective strategy to achieve durable remissions in patients with cancer. Cancer Res; 75(2); 446–55. ©2014 AACR.

Purpose: The purpose of this study was to evaluate mechanisms controlling secretory IgA (SIgA) production, thereby ensuring maintenance of ocular surface health. Methods: To determine whether the presence of specific gut commensal species regulates SIgA levels and IgA transcripts in the eye-associated lymphoid tissues (EALT), specific-pathogen-free (SPF) Swiss Webster (SW) mice were treated with antibiotic cocktails, germ-free (GF) SW mice were reconstituted with diverse commensal gut microbiota, or monocolonized with gut-specific commensals. Proteomic profiling and quantitative real-time polymerase chain reaction (qRT-PCR) were used to quantify SIgA and IgA levels. 16S rDNA sequencing was carried out to characterize commensal microbiota. Results: Commensal presence regulated ocular surface SIgA levels and mRNA IgA transcripts in EALT. Oral antibiotic cocktail intake significantly reduced gut commensal presence, while maintaining ocular surface commensal levels reduced SIgA and IgA transcripts in EALT. Analysis of gut microbial communities revealed that SPF SW mice carried abundant Bacteroides organisms when compared to SPF C57BL6/N mice, with B. acidifaciens being the most prominent species in SPF SW mice. Monocolonization of GF SW mice with B. acidifaciens, a strict gut anaerobe, resulted in significant increase of IgA transcripts in the EALT, implying generation of B-cell memory. Conclusions: These data illustrated a “gut-eye” axis of immune regulation. Exposure of the host to gut commensal species may serve as a priming signal to generate B-cell repertoires at sites different from the gut, such as EALT, thereby ensuring broad protection.

IRAK4 is a central kinase in innate immunity, but the role of its kinase activity is controversial. The mechanism of activation for IRAK4 is currently unknown, and little is known about the role of IRAK4 kinase in cytokine production, particularly in different human cell types. We show IRAK4 autophosphorylation occurs by an intermolecular reaction and that autophosphorylation is required for full catalytic activity of the kinase. Phosphorylation of any two of the residues Thr-342, Thr-345, and Ser-346 is required for full activity, and the death domain regulates the activation of IRAK4. Using antibodies against activated IRAK4, we demonstrate that IRAK4 becomes phosphorylated in human cells following stimulation by IL-1R and Toll-like receptor agonists, which can be blocked pharmacologically by a dual inhibitor of IRAK4 and IRAK1. Interestingly, in dermal fibroblasts, although complete inhibition of IRAK4 kinase activity does not inhibit IL-1-induced IL-6 production, NF-κB, or MAPK activation, there is complete ablation of these processes in IRAK4-deficient cells. In contrast, the inhibition of IRAK kinase activity in primary human monocytes reduces R848-induced IL-6 production with minimal effect on NF-κB or MAPK activation. Taken together, these studies define the mechanism of IRAK4 activation and highlight the differential role of IRAK4 kinase activity in different human cell types as well as the distinct roles IRAK4 scaffolding and kinase functions play.

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.

The goal of targeted therapy is to match a selective drug with a genetic lesion that predicts for drug sensitivity. In a diverse panel of cancer cell lines, we found that the cells most sensitive to focal adhesion kinase (FAK) inhibition lack expression of the neurofibromatosis type 2 (NF2) tumor suppressor gene product, Merlin. Merlin expression is often lost in malignant pleural mesothelioma (MPM), an asbestos-induced aggressive cancer with limited treatment options. Our data demonstrate that low Merlin expression predicts for increased sensitivity of MPM cells to a FAK inhibitor, VS-4718, in vitro and in tumor xenograft models. Disruption of MPM cell-cell or cell-extracellular matrix (ECM) contacts with blocking antibodies suggests that weak cell-cell adhesions in Merlin-negative MPM cells underlie their greater dependence on cell-ECM-induced FAK signaling. This provides one explanation of why Merlin-negative cells are vulnerable to FAK inhibitor treatment. Furthermore, we validated aldehyde dehydrogenase as a marker of cancer stem cells (CSCs) in MPM, a cell population thought to mediate tumor relapse after chemotherapy. Whereas pemetrexed and cisplatin, standard-of-care agents for MPM, enrich for CSCs, FAK inhibitor treatment preferentially eliminates these cells. These preclinical results provide the rationale for a clinical trial in MPM patients using a FAK inhibitor as a single agent after first-line chemotherapy. With this design, the FAK inhibitor could potentially induce a more durable clinical response through reduction of CSCs along with a strong antitumor effect. Furthermore, our data suggest that patients with Merlin-negative tumors may especially benefit from FAK inhibitor treatment.

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.

Verastem is developing VS-5584, a potent and selective dual inhibitor of the mammalian target of rapamycin complexes 1 and 2 (mTOR) and Class I phosphatidylinositide 3-kinases (PI3K), for the treatment of advanced cancer. The PI3K/mTOR signaling pathway is a key regulator in cancer progression and in the survival of cancer stem cells (CSCs). VS-5584 has been shown to be an equipotent inhibitor of all four human Class I PI3K isoforms and the mTOR kinase, and PI3K signaling has been implicated in the maintenance of CSCs in solid tumors. In multiple orthogonal in vitro assays, VS-5584 has been shown to preferentially target CSCs and exhibited significant antiproliferative activity across multiple cancer cell lines. Furthermore, oral administration of VS-5584 has been shown to reduce CSCs in xenograft models. The in vivo antitumor efficacy of once daily and intermittent oral administration of VS-5584 was evaluated in multiple xenograft tumor models representing small cell (SCLC) and non-small cell lung cancer (NSCLC), triple negative breast cancer (TNBC) and mesothelioma. Once daily (QD) treatment with VS-5584 demonstrated potent anti-tumor activity, with mean percentage tumor growth inhibition (TGI) ranging from 40% to 97% (P < 0.05). In these models, TGI was dose-dependent with dosages at and above 15 mg/kg showing good antitumor activity (P < 0.05). Interestingly, tumor regression was observed in 33% (3/9) of mice bearing H69 (SCLC) tumors, 60% (6/10) of mice bearing MDA-MB-468 (TNBC) tumors and 50% (5/10) of mice bearing NCI-H226 mesothelioma tumors. This significant antitumor activity was generally observed at well-tolerated dosages. In studies exploring intermittent dosing schedules, efficacy and tolerability were similar or better with a QD 5 days on/2 days off or Monday, Wednesday, Friday schedule compared to continuous daily dosing. We also explored the efficacy of VS-5584 in combination with either cisplatin or docetaxel. In these studies, VS-5584 plus either cisplatin or docetaxel showed significant TGI compared to cisplatin alone in H69 SCLC and docetaxel alone in A549 NSCLC xenograft models (P < 0.05). This potent in vivo anti-tumor activity in xenograft models of NSCLC, SCLC, TNBC and mesothelioma suggests that VS-5584 has the potential for anticancer activity across a variety of cancer types. Intermittent dosing with VS-5584 was sufficient to achieve good efficacy while minimizing side effects, thus allowing a broader therapeutic window compared to QD dosing. VS-5584 in combination with either cisplatin or docetaxel had enhanced anti-tumor activity compared to either chemotherapeutic agent or VS-5584 alone in two in vivo models of lung cancer. VS-5584 is being evaluated in a Phase 1 clinical trial assessing intermittent dosing in subjects with advanced non-hematologic malignancies or lymphoma. Citation Format: Anthony F. Trombino, Vihren N. Kolev, Quentin G. Wright, Qunli Xu, Mahesh V. Padval. VS-5584 a dual mTORC1/2 and PI3K inhibitor has anti-tumor activity in multiple in vivo xenograft tumor models and enhanced efficacy in combination with cisplatin or docetaxel. [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 213. doi:10.1158/1538-7445.AM2014-213

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

Cancer stem cells (CSCs) represent a subpopulation of cancer cells that have tumor-initiating capability, are particularly resistant to chemotherapy, and can mediate tumor recurrence both locally and at metastatic sites. As such, these cells represent a critical challenge for effective treatment of cancer. High-throughput screening for small molecules that preferentially target CSCs identified dual inhibitors of the PI3K/mTOR pathway, suggesting the importance of this signaling pathway for CSC biology. Here we demonstrate that ablation of mTOR or individual PI3K isoforms by isoform-specific siRNA is not sufficient to reduce the proportion of CSCs. In contrast, combined knock down of PI3K isoforms and mTOR effectively reduced the proportion of CSCs in tumor cell lines. VS-5584 is a potent and selective pan-PI3K/TORC1/TORC2 inhibitor with equipotency against all four human Class I PI3K isoforms and the mTOR kinase. We demonstrate that VS-5584 preferentially targets CSCs in multiple orthogonal assays both in vitro and in human tumor xenograft models. Cancer stem cells express high levels of aldehyde dehydrogenase, and an Aldefluor assay that measures activity of this enzyme was used to identify CSCs. VS-5584 decreased the percentage of Aldefluor+ cells across multiple breast cancer cell lines. We demonstrated that VS-5584 preferentially induced apoptosis in Aldefluor+ SUM159 cells relative to Aldefluor- cells as measured by Annexin V and Caspase 3/7 assays. In contrast, paclitaxel induced more apoptosis in Aldefluor- than Aldefluor+ cells and enriched the percentage of CSCs. Another characteristic of CSCs is their enhanced ability to efflux cytotoxic agents. This drug-resistant CSC population, called side population (SP), can be monitored by exclusion of Hoechst dye. VS-5584 effectively eliminated the SP CSCs across multiple cancer types, while cisplatin and etoposide increased this subpopulation. Furthermore, ex vivo treatment of primary breast and ovarian tumor specimens with VS-5584 decreased the proportion of CSCs as measured by the Aldefluor assay and cell surface markers. Significantly, VS-5584 also targets CSCs in vivo in MDA-MB-231 triple negative and MCF7 ER+ breast cancer xenograft models as evidenced by decreases in the percentage of Aldefluor+ cells, tumorsphere-forming efficiency, and tumor-initiating capability in an in vivo limiting dilution re-implantation assay. Consistent with the notion that combined inhibition of PI3K isoforms and mTOR is critical for exerting a strong anti-CSC effect, the mTORC1-selective inhibitor everolimus did not reduce CSCs in the MCF7 xenograft model. The potent anti-CSC activities in primary patient cancer tissue and in xenograft models provide strong rationale for the clinical development of VS-5584 in combination with agents targeting the bulk tumor to potentially achieve durable clinical responses for cancer patients. Citation Format: Vihren N. Kolev, Quentin G. Wright, David T. Weaver, Jennifer E. Ring, Christian M. Vidal, Mahesh V. Pavdal, 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 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 3906. doi:10.1158/1538-7445.AM2014-3906

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.

Focal adhesion kinase (FAK) is a non-receptor tyrosine kinase which mediates integrin and growth factor receptor signaling to regulate tumor growth, metastasis and angiogenesis. FAK is also required for the proliferation/survival of cancer stem cells (CSCs). Protein tyrosine kinase 2 (PYK2) is the most closely related protein kinase to FAK, sharing 60% amino acid identity in the kinase domain and a similar role in cellular signaling pathways. Several lines of evidence suggest that dual targeting of FAK and PYK2 should confer greater antitumor efficacy than an agent which only inhibits FAK. Inhibition of tumor angiogenesis or mammary stem cells by FAK inhibition can be bypassed by PYK2 activity, suggesting advantages of dual FAK/PYK2 inhibition. Accordingly, a critical role of PYK2 in CSC regulation and NSCLC patients’ prognosis has recently been reported (Kuang et al. 2013, Br J Cancer). Furthermore, preclinical data suggest that PYK2 inhibition can prevent growth of metastatic tumor cells in bone. Results of PYK2 knockout in mice suggest that PYK2 inhibition is likely to be well tolerated. Based on the observation of reduced macrophage infiltration in PYK2 knockout mice, we investigated effects of PYK2 inhibition on tumor-associated macrophages (TAMs). Abundance of TAMs has been correlated with poor prognosis in multiple cancer types including mesothelioma and breast cancer. Defactinib is a potent and orally active inhibitor of FAK kinase activity. Preliminary clinical activity has been observed in a Phase 1 single agent trial and in an ongoing trial in combination with paclitaxel in patients with ovarian cancer. We find that in addition to potent FAK inhibition, defactinib also inhibits PYK2 kinase activity with nanomolar potency in both biochemical enzymatic and cellular autophosphorylation assays. Using both pharmacological FAK inhibitors and FAK siRNA, we have previously demonstrated that FAK inhibition effectively reduces CSCs. We report here that dual targeting of FAK and PYK2 by defactinib may more effectively suppress CSCs than inhibition of FAK alone. We find that differentiated THP-1 macrophages release IL-6 and IL-8, and these cytokines increase the proportion of ALDH-positive CSCs in mesothelioma and breast cancer cell lines. Defactinib dose-dependently inhibited production of IL-6 and IL-8, whereas a FAK-only reference inhibitor had no effect on these cytokines. Furthermore, in MM87 mesothelioma and MDA-MB-231 breast cancer xenograft models, defactinib substantially reduced the number of F4/80-positive TAMs in the tumors. These data support a model in which TAMs release IL-6 and IL-8 in a PYK2-dependent manner. Dual inhibition of FAK and PYK2 by defactinib effectively decreases both the presence of TAMs in tumors and the ability of TAMs to release cytokines that stimulate CSC proliferation and survival, and may therefore provide the opportunity for more durable clinical response than inhibition of FAK alone. Citation Information: Mol Cancer Ther 2013;12(11 Suppl):B283. Citation Format: Jennifer E. Ring, Vihren N. Kolev, Irina M. Shapiro, Quentin G. Wright, Joseph R. Testa, Mahesh V. Padval, Qunli Xu, Jonathan A. Pachter. Defactinib (VS-6063) targets cancer stem cells directly and through inhibition of tumor-associated macrophages and cytokine production. [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 B283.

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.

Cancer stem cells (CSCs) constitute a subpopulation of cancer cells with tumor-initiating capability. Because CSCs are generally resistant to chemotherapy and drive tumor metastasis and recurrence, they represent an important challenge in the clinical treatment of cancer. In a high-throughput screen for compounds that preferentially target CSCs, several inhibitors of the PI3K/mTOR pathway were identified, suggesting the importance of this signaling pathway for CSC biology. VS-5584 is a highly potent and selective mTORC1/mTORC2/PI3K inhibitor with equipotency against all four human Class I PI3K isoforms and the mTOR kinase. We demonstrate that VS-5584 preferentially targets CSCs in vitro and in vivo in multiple orthogonal assays based on known characteristics of CSCs. Cancer stem cells express higher levels of aldehyde dehydrogenase (ALDH) than bulk tumor cells, thus an Aldefluor assay which measures ALDH enzymatic activity is used to quantify CSCs. VS-5584 decreased the percentage of Aldefluor-positive cells across multiple breast cancer cell lines. In SUM159 triple negative breast cancer cells, VS-5584 dose-dependently induced apoptosis preferentially in Aldefluor-positive cells as measured by both Annexin V and Caspase 3/7 assays. In contrast, paclitaxel induced more apoptosis in Aldefluor-negative cells and as a consequence increased the proportion of CSCs. Another characteristic of CSCs is enhanced drug efflux compared to non-CSCs, thus a Hoechst 33342 dye-exclusion assay, also known as a side population (SP) assay, is used to identify CSCs. In the H69 small cell lung cancer model, VS-5584 effectively eliminated the CSC side population, while the cytotoxic agents cisplatin and etoposide increased the CSC side population. To further evaluate the effect of VS-5584 on CSCs, we utilized ex vivo culture of surgically excised primary cancer tissue. Ex vivo treatment of breast tumors with VS-5584 decreased the proportion of Aldefluor-positive and CD44hi/CD24lo CSCs. Similarly, VS-5584 reduced the proportion of Aldefluor-positive and CD44hi/CD117hi CSCs in primary ovarian cancer tumor specimens. Significantly, oral dosing with VS-5584 was found to substantially reduce CSCs in vivo in the MDA-MB-231 triple negative and MCF-7 ER+ breast cancer xenograft models as evidenced by decreases in the percentage of Aldefluor-positive cells, tumorsphere-forming efficiency, and tumor-initiating capability in an in vivo limiting dilution re-implantation assay. Consistent with its strong suppression of CSCs, VS-5584 effectively impeded tumor regrowth following cisplatin treatment in a H69 small cell lung cancer xenograft model. The preferential targeting of CSCs thus provides a strong rationale for the clinical development of VS-5584 with the goal of achieving a more durable response in cancer patients. Phase 1 testing of VS-5584 is planned to initiate in the fourth quarter of 2013. Citation Information: Mol Cancer Ther 2013;12(11 Suppl):B52. Citation Format: Vihren N. Kolev, Quentin G, Wright, Christian M. Vidal, Jennifer E. Ring, Irina M. Shapiro, David T. Weaver, Mahesh V. Pavdal, Jonathan A. Pachter, Qunli Xu. Dual mTORC1/2 and PI3K inhibitor VS-5584 preferentially targets 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 B52.

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.

e13523 Background: 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 I clinical trial, VS-6063 was well tolerated and demonstrated preliminary clinical activity. VS-4718 is in late stage preclinical development and has been shown to inhibit tumor growth and metastasis in tumor models. We report here that the FAK inhibitors VS-6063 and VS-4718 effectively abrogate cancer stem cells (CSCs). Methods: VS-6063 and VS-4718 were evaluated in the ALDEFLUOR and Side Population (SP) CSC assays in vitro and in xenograft tumors in vivo. Results: Treating SUM159 breast cancer cells with FAK inhibitors reduced the percentage of ALDEFLUOR+ and SP CSCs. Similar inhibitory effects on CSCs were observed in the ovarian cancer cell lines OVCAR-5 and OVCAR-8 and the mesothelioma cell line H2052. In direct contrast, cytotoxic agents paclitaxel, carboplatin and pemetrexed increased the percentage of CSCs, suggesting that these agents do not effectively target CSCs. Importantly, combination of either VS-6063 or VS-4718 with cytotoxic agents blocked induction of CSCs by these agents. The effect of a FAK inhibitor on CSCs in vivo was evaluated in an MDA-MB-231 human breast cancer orthotopic model. Oral administration of FAK inhibitor substantially reduced the number of ALDH1- positive cells in tumors as assessed by immunofluorescence. Following dissociation of cells from tumors, CSCs were found to be reduced in FAK inhibitor-treated tumors as measured by multiple assays. Furthermore, cells isolated from FAK inhibitor-treated tumors showed reduced tumor-initiating capability upon re-implantation into immunodeficient mice. FAK inhibitor also decreased ALDH1-positive CSCs in an orthotopic MM87 mesothelioma model. Conclusions: Our results provide clear demonstration that FAK inhibitors target cancer stem cells both in vitro and in xenograft tumors in vivo and support the planned clinical development of VS-6063 and VS-4718 to achieve durable clinical responses for cancer patients.

The PI3 kinase/mTOR pathway plays a central role in cancer cell proliferation and survival. Depending on the mode of pathway activation, different PI3K isoforms and mTOR complexes have been shown to play essential roles in oncogenesis. VS-5584 potently and selectively inhibits PI3K/mTOR signaling with equipotency against all four human Class I PI3K isoforms and the mTOR kinase. VS-5584 also displays potent anticancer activities in a number of human cancer xenograft models in vivo either as a single agent or in combination. In a high-throughput screen designed to identify compounds that selectively target cancer stem cells (CSCs), the PI3K/mTOR pathway was found to be an important target. The effect of the PI3K/mTOR inhibitor VS-5584 was evaluated in multiple CSC assays. Using HMLE immortalized human mammary cells driven through epithelial-mesenchymal transition (EMT) by knock down of E-cadherin, the PI3K/mTOR inhibitor VS-5584 selectively inhibited proliferation of CSC-like mesenchymal HMLEs. Accordingly, VS-5584 preferentially decreased the proportion of CD44Hi/CD24Lo CSC-like cells in Ras-transformed HMLER cells in contrast to paclitaxel that increased the proportion of CD44Hi/CD24Lo CSCs in the same model. CSCs express higher levels of aldehyde dehydrogenase (ALDH), thus an ALDEFLUOR assay that measures the enzymatic activity of ALDH was used to assess CSCs. In SUM159 triple negative breast cancer cells, VS-5584 dose-dependently decreased the percentage of ALDEFLUOR-positive cells, in contrast to paclitaxel which again enriched CSCs in the same assay. Another characteristic of CSCs is the enhanced ability to exclude cytotoxic agents. This drug-resistant CSC population, termed the side population, can be identified by virtue of exclusion of Hoechst 33342 dye. In SUM159 cells, VS-5584 effectively eliminated the CSC side population, while paclitaxel increased this population. The effect of VS-5584 on cancer stem cells was further evaluated in the MDA-MB-231 triple negative breast cancer model in vivo. After one week of oral dosing, VS-5584 reduced the proportion of cancer stem cells in tumors as evidenced by decreases in the percentage of ALDEFLUOR -positive cells, tumorsphere-forming efficiency, and tumor-initiating capability in an in vivo limiting dilution re-implantation assay. Lastly, VS-5584 caused tumor regression in a primary triple negative breast cancer model, likely as a result of potent inhibitory activities against both cancer stem cells and cancer cells in general. These data taken together provide strong rationale for the clinical development of VS-5584 for breast and other cancers to achieve durable clinical responses. Citation Format: Qunli Xu, Vihren N. Kolev, Quentin G. Wright, David T. Weaver, Mahesh V. Padval, Jonathan A. Pachter. The dual PI3K/mTOR inhibitor VS-5584 preferentially targets cancer stem cells in vitro and in vivo. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 239. doi:10.1158/1538-7445.AM2013-239

Focal adhesion kinase (FAK) is a cytoplasmic tyrosine kinase that orchestrates cell signaling through integrins and 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 multiple aggressive human cancers including breast and ovarian. VS-4718 is a potent and selective FAK kinase inhibitor that was previously shown by us to exhibit preferential inhibitory activities on breast cancer stem cells. We have further extended our investigation of the role of FAK on cancer stem cells to other solid tumors and report here that pharmacological attenuation of FAK activity by VS-4718 or RNAi-mediated depletion of FAK exhibits preferential inhibitory effects on cancer stem cells. To determine if FAK plays a role in the biology of cancer stem cells, we depleted FAK expression in breast, ovarian and mesothelioma cancer cell lines by RNAi. Our results indicated that shRNA-mediated knock-down of FAK inhibits tumorsphere formation in vitro. In parallel, VS-4718 was evaluated in a multitude of cancer stem cell assays both in vitro and in vivo. Pre-treatment of SUM159 cells with VS-4718 in matrigel reduced the percentage of ALDEFLUOR+ cancer stem cells and side population (SP). Similar effects were observed in ovarian cancer cell lines OVCAR-8 and OVCAR-5 where VS-4718 inhibited cancer stem cells as measured by multiple CSC assays. In an analogous fashion, VS-4718 also reduced the proportion of the ALDEFLUOR+ cells in H2052 human mesothelioma cells. In direct contrast, standard-of-care agents paclitaxel, carboplatin or pemetrexed increased the percentage of cancer stem cells, suggesting these agents do not effectively target cancer stem cells. Importantly combination of VS-4718 with standard-of-care agents attenuated chemotherapy-induced increases in the percentage of cancer stem cells in vitro in all three cancer models. 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 significantly reduced the proportion of cancer stem cells in tumors as evidenced by decreases in the percentage of ALDEFLUOR+ cells and tumorsphere-forming efficiency relative to vehicle-treated tumors and significantly abrogated tumor-initiating capabilities of cancer cells in a limiting dilution re-implantation assay. In summary, our results indicate the importance of FAK in the self-renewal of cancer stem cells in vitro and in vivo, and support the clinical development of FAK inhibitors to target cancer stem cells for the treatment of multiple cancers. Citation Format: Vihren Kolev, Quentin Wright, Christian Vidal, Irina Shapiro, Mahesh Pavdal, Mitchell Keegan, Qunli Xu, Jonathan Pachter. Pharmacological and genetic inhibition of FAK attenuates cancer stem cell function in vitro and in vivo. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 236. doi:10.1158/1538-7445.AM2013-236