Effects Of Focal Adhesion Kinase Inhibition Research Articles

Focal adhesion kinase (FAK) inhibition induces aquaporin 2 (AQP2) plasma membrane accumulation in renal epithelial cells by remodeling the actin cytoskeleton

The actin cytoskeleton is involved in the cellular traffcking of the AQP2 water channel in kidney collecting duct (CD) principal cells (PC): this plays an important role in water homeostasis. Focal adhesion kinase (FAK) is known to modulate the actin cytoskeleton through small GTPase-mediated pathways and we, therefore, investigated the potential role of FAK in AQP2 traffcking. We and others have previously shown that AQP2 constitutively traffcs and recycles independently of vasopressin (VP) and that this involves actin depolymerization/polymerization; therefore, FAK could regulate AQP2 traffcking through actin remodeling, bypassing the VP-V2R (cAMP/PKA) signaling pathway. To examine this, we used small molecule FAK inhibitors, VS-4718, GSK2256098 (GSK), or Defactinib-Hydrochloride (DH). We used immunofluorescence staining, a transferrin endocytosis assay ± cold block treatment, a fluorometric exocytosis assay using cells expressing secreted yellow fluorescent protein (ssYFP), a RhoA activity assay, and western blotting. Inhibiting FAK with 1mM VS-4718 for 30 min increased AQP2 membrane accumulation in LLCPK1 and IMCD kidney epithelial cells expressing AQP2. VS-4718 resulted in reduced clathrin-mediated endocytosis, as well as increased vesicle exocytosis, both mechanisms contributing to increased AQP2 membrane accumulation. This effect of FAK inhibition was independent of vasopressin/cAMP signaling and did not require phosphorylation of the Serine 256 residue of AQP2, since AQP2 membrane accumulation also occurred in cells expressing the constitutive dephosphorylation AQP2 S256A mutant. The FAK inhibitor VS-4718 promoted actin depolymerization (phalloidin staining assay) which would facilitate AQP2 vesicle exocytosis into the plasma membrane. We then examined the activity of the small GTPase, RhoA, whose activity increases actin polymerization and vesicle traffcking in the exocytic and endocytic pathways. We found that FAK inhibition by VS-4718 downregulated RhoA activity using GST-RBD, a substrate that binds to active RhoA, as seen by western blotting using phospho-specific antibodies. Similar results were obtained with GSK and DH. In summary, our study revealed that the interaction between FAK and cytoskeletal architecture plays a crucial role in regulating AQP2 recycling and its subsequent entry into the endocytic pathway. Thus, targeting FAK could be a potential therapeutic strategy for water balance disorders involving defective AQP2 reabsorption, such as nephrogenic diabetes insipidus. This work was supported by NIDDK R01DK096015 (JHL) and DK096586 (DB). This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

FAK inhibitor PF-431396 suppresses IgE-mediated mast cell activation and allergic inflammation in mice

Mast cells (MCs) initiate and maintain allergic inflammation. Upon being stimulated with immunoglobulin (Ig)E and antigen (Ag), MCs exhibit FcεRI (high-affinity IgE) receptor-mediated degranulation, cytokine secretion, and increased focal adhesion kinase (FAK) activity. The aims of this study were to examine mechanisms of FAK regulation in IgE-mediated MC activation and the effects of FAK inhibition on MC-mediated allergic responses. FAK activity was manipulated with short hairpin RNA (shRNA) knockdown, FAK overexpression, and the FAK inhibitor PF-431396 (PF). Gene expression and kinase activation were analyzed with quantitative molecular biology assays. PF effects were tested in the passive cutaneous anaphylaxis (PCA), active systemic anaphylaxis (ASA), and allergic conjunctivitis (AC) mouse models. Our results showed that FAK overexpression increased IgE-mediated degranulation and reduced the dexamethasone inhibitory effect on MCs activation. The FAK inhibitor PF diminished MC release of β-hexosaminidase (β-hex), histamine, and inflammatory cytokines, via a mechanism that involves MAPK and NF-κB signaling pathways. CaMKII was identified as a robust FAK-associating protein. Inhibition of CaMKII activation by KN-93 suppressed FAK activity and its downstream pathway. PF attenuated inflammatory responses in our PCA and ASA models, and relieved signs of allergic disease in AC model mice. In conclusions, MC degranulation and production of inflammatory mediators in allergic disease may be consequent to FcεRI crosslinking inducing CaMKII-mediated activation of FAK activity. FAK inhibition may represent a new MC-suppressing treatment strategy for the treatment of allergic diseases.

Abstract P2-06-07: Investigating the role of focal adhesion kinase in regulating CSC activity in invasive ductal carcinoma

Abstract 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 = <0.0016.58 (1.87-23.10) p=0.0032.23 (1.08-4.58) p=0.03016.7 (3.7-73.9) p=<0.00112.8 (1.37-13.2,) p=0.012Hazard ratios calculated using cox-proportional hazard regression analysis. 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=<0.01, unpaired t-test) in MDA-MB-231 cells. VS4718 reduced primary mammosphere forming efficiency in all cell lines and reduced self-renewal in ER negative cell lines. FAK inhibition led to a reduction in mammosphere forming efficiency and self-renewal in 25 primary breast cancer specimens as outlined below: 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=<0.05, One way Anova used with post hoc Tukeys test. 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.

Abstract SY06-03: Reprogramming the tumor microenvironment to improve responses to therapy

Abstract Immunotherapeutics represent highly promising agents with the potential to improve patient outcomes in a variety of cancer types. Unfortunately, single-agent immunotherapy has achieved limited clinical benefit to date in patients suffering from pancreatic ductal adenocarcinoma (PDAC). This may be due to the presence of a uniquely immunosuppressive tumor microenvironment (TME) present in PDACs, which creates a barrier to effective immune surveillance. Critical obstacles to immunotherapy in PDAC tumors include the dense desmoplastic stroma that acts as a barrier to T-cell infiltration and the high numbers of tumor-associated immunosuppressive cells. We have identified hyperactivated focal adhesion kinase (FAK) activity in neoplastic PDAC cells as a significant regulator of the fibrotic and immunosuppressive TME. We found that FAK activity was elevated in human PDAC tissues and correlates with high levels of fibrosis and poor CD8+ cytotoxic T-cell infiltration. Single-agent FAK inhibition (VS-4718) significantly limited tumor progression, resulting in a doubling of survival in the p48-Cre/LSL-KrasG12D/p53Flox/+ (KPC) mouse model of human PDAC. This alteration in tumor progression was associated with dramatically reduced tumor fibrosis, decreased numbers of tumor-infiltrating immature myeloid cells, and immunosuppressive macrophages. We postulated that these desirable effects of FAK inhibition on the TME might render PDAC tumors more sensitive to immunotherapy. Accordingly, we found that FAK inhibition rendered the previously unresponsive KPC mouse model responsive to anti-PD1 and anti-CTLA4 antagonists, leading to a near tripling of survival times. These data suggest that FAK inhibition increases immune surveillance by overcoming the fibrotic and immunosuppressive PDAC TME and thus renders tumors responsive to immunotherapy. To test this hypothesis in human PDAC patients, we have opened a phase I clinical trial testing the safety and impact on the tumor microenvironment of the FAK inhibitor defactinib, in combination with pembrolizumab and gemcitabine (NCT02546531). These studies are ongoing in patients with advanced cancer, including metastatic PDAC. Early biomarker studies on PDAC tissue collected pre- and post-therapy suggest that this combination impacts the immunosuppressive TME. Citation Format: David G. Denardo. Reprogramming the tumor microenvironment to improve responses to therapy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr SY06-03. doi:10.1158/1538-7445.AM2017-SY06-03

Abstract 420: Focal Adhesion Kinase Disrupts Nuclear Factor-kB Signaling and Alleviates Atherogenesis

Introduction: While we have discovered that focal adhesion kinase (FAK) is critical for global pro-inflammatory gene expression, it remains unclear what role FAK plays in vascular inflammation. Our preliminary data suggested that FAK might regulate a major inflammatory signaling factor, NF-kB. The goals of this study are to investigate the mechanisms underlying FAK-mediated inflammatory gene expression in endothelial cells (ECs), and the effect of FAK inhibition on atherogenesis. Hypothesis: FAK activity is required for pro-inflammatory gene expression via NF-kB activation in ECs. Methods: Human aortic ECs (HAoECs) were treated with TNF-α and with/without a FAK inhibitor (FAK-I). Inflammatory gene expression and NF-kB signaling was evaluated via western blot, immunocytochemistry or qRT-PCR. To access vascular inflammation in vitro , primary mouse monocytes were used in an adhesion assay on TNF-α stimulated HAoECs. To evaluate the importance of FAK activity in atherogenesis, partial carotid artery ligation was performed in ApoE-/- mice. Mice were fed a high fat/high cholesterol diet and were given either FAK-I or vehicle by oral gavage for two weeks. Both carotid arteries were harvested and analyzed. Results: We found that FAK activity is required for expression of various pro-inflammatory molecules such as VCAM-1 and MCP-1, and FAK inhibition blocks sustained NF-kB activation. Mechanistically, while FAK inhibition does not block NF-kB activation after 1hr TNF-α stimulation, NF-kB activity significantly reduced after 3hr when compared to TNF-α only. This decrease in NF-kB activity was correlated with a decrease in NF-kB nuclear localization and an increase in IkB protein stability. FAK inhibition reduced monocyte attachment to TNF-α stimulated HAoECs (3.8 vs 22.1 fold). Mice treated with FAK-I showed decreased VCAM-1 expression, macrophage infiltration and lipid accumulation compared to vehicle group. Conclusion: These results suggest that FAK is critical for global inflammatory gene expression and plays a key role in maintaining sustained NF-kB activity during chronic inflammation in ECs. FAK inhibitors may prove useful as an “anti-inflammatory drug” in the treatment of cardiovascular disease.

Abstract IA22: Reprograming the tumor microenvironment to facilitate responses to immunotherapy

Abstract Immunotherapeutics represent highly promising agents with the potential to improve patient outcomes in a variety of cancer types. Unfortunately, single-agent immunotherapy has achieved limited clinical benefit to date in patients suffering from pancreatic ductal adenocarcinoma (PDAC). This may be due to the presence of a uniquely immunosuppressive tumor microenvironment (TME) present in PDACs, which creates a barrier to effective immune surveillance. Critical obstacles to immunotherapy in PDAC tumors include the dense desmoplastic stroma that acts as a barrier to T-cell infiltration and the high numbers of tumor-associated immunosuppressive cells. We have identified hyperactivated focal adhesion kinase (FAK) activity in neoplastic PDAC cells as a significant regulator of the fibrotic and immunosuppressive TME. We found that FAK activity was elevated in human PDAC tissues and correlates with high levels of fibrosis and poor CD8+ cytotoxic T-cell infiltration. Single-agent FAK inhibition (VS-4718) significantly limited tumor progression, resulting in a doubling of survival in the p48-Cre/LSL-KrasG12D/p53Flox/+ (KPC) mouse model of human PDAC. This alteration in tumor progression was associated with dramatically reduced tumor fibrosis, decreased numbers of tumor-infiltrating immature myeloid cells and immunosuppressive macrophages. We postulated that these desirable effects of FAK inhibition on the TME might render PDAC tumors more sensitive to immunotherapy. Accordingly, we found that FAK inhibition rendered the previously unresponsive KPC mouse model responsive to anti-PD1 and anti-CTLA4 antagonists leading to a nearly tripling of survival times. These data suggest that FAK inhibition increases immune surveillance by overcoming the fibrotic and immunosuppressive PDAC TME and thus renders tumors responsive to immunotherapy. The results of these pre-clinical studies have led to the development of a new clinical trial (NCT02546531) testing the combination of FAK inhibition (Defactinib) with Pembrolizumab and Gemcitabine in advanced PDAC Patients. Citation Format: David Glenn DeNardo.{Authors}. Reprograming the tumor microenvironment to facilitate responses to immunotherapy. [abstract]. In: Proceedings of the AACR Special Conference on Pancreatic Cancer: Advances in Science and Clinical Care; 2016 May 12-15; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2016;76(24 Suppl):Abstract nr IA22.

Abstract B031: FAK/PYK2 inhibition enhances antitumor efficacy of anti-PD-1 and anti-4-1BB antibodies

Abstract 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.

Abstract IA14: Targeting focal adhesion kinase to awaken responses to immunotherapy in pancreatic cancer

Abstract Immunotherapeutics represent highly promising agents with the potential to improve patient outcomes in a variety of cancer types. Unfortunately, single-agent immunotherapy has achieved limited clinical benefit to date in patients suffering from pancreatic ductal adenocarcinoma (PDAC). This may be due to the presence of a uniquely immunosuppressive tumor microenvironment (TME) present in PDACs, which creates a barrier to effective immune surveillance. Critical obstacles to immunotherapy in PDAC tumors include the dense desmoplastic stroma that acts as a barrier to T-cell infiltration and the high numbers of tumor-associated immunosuppressive cells. We have identified hyperactivated focal adhesion kinase (FAK) activity in neoplastic PDAC cells as a significant regulator of the fibrotic and immunosuppressive TME. We found that FAK activity was elevated in human PDAC tissues and correlates with high levels of fibrosis and poor CD8+ cytotoxic T-cell infiltration. Single-agent FAK inhibition (VS-4718) significantly limited tumor progression, resulting in a doubling of survival in the p48-Cre/LSL-KrasG12D/p53Flox/+ (KPC) mouse model of human PDAC. This alteration in tumor progression was associated with dramatically reduced tumor fibrosis, decreased numbers of tumor-infiltrating immature myeloid cells and immunosuppressive macrophages. We postulated that these desirable effects of FAK inhibition on the TME might render PDAC tumors more sensitive to immunotherapy. Accordingly, we found that FAK inhibition rendered the previously unresponsive KPC mouse model responsive to anti-PD1 and anti-CTLA4 antagonists leading to a nearly tripling of survival times. These data suggest that FAK inhibition increases immune surveillance by overcoming the fibrotic and immunosuppressive PDAC TME and thus renders tumors responsive to immunotherapy. Citation Format: David G. Denardo. Targeting focal adhesion kinase to awaken responses to immunotherapy in pancreatic cancer [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 IA14.

Abstract 568: FAK/PYK2 inhibition enhances immune checkpoint inhibitor efficacy

Abstract 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.

Effects of combining erlotinib and RNA-interfered downregulation of focal adhesion kinase expression on gastric cancer.

ObjectiveTo investigate the synergistic effects of combining erlotinib and RNA-interference downregulation of focal adhesion kinase (FAK) expression on the proliferation, apoptosis, invasion and migration of the human gastric adenocarcinoma cell line AGS.MethodsCells were divided into five experimental groups: Group A, nontransfected control; Group B, transfected with empty vector; Group C, transfected with FAK-shRNA; Group D, erlotinib treatment; Group E, combination erlotinib treatment and transfected with FAK-shRNA. FAK protein levels were confirmed via Western blotting. Cell proliferation (CCK-8 assay, apoptosis (flow cytometry), cell invasion (transwell assay) and migration (scratch assay) were evaluated.ResultsRNA interference significantly decreased FAK protein levels. Cell proliferation, invasion and migration were significantly lower in Groups C, D and E compared with Group A, and significantly lower in Group E than in Groups C and D.ConclusionsRNA interference effectively silences FAK expression and inhibits malignant cell proliferation and invasion in gastric cancer cells. The effect of FAK inhibition is increased by co-treatment with erlotinib.

Abstract B197: Focal adhesion kinase inhibition enables efficacy of checkpoint immunotherapy in pancreatic cancer

Abstract 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.

Targeting focal adhesion kinase reprograms the pancreatic tumor microenvironment and renders pancreas cancer responsive to checkpoint immunotherapy

Checkpoint immunotherapeutics are promising agents with potential to improve patient outcomes in several cancer types. 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 immunotherapy in PDAC tumors include the dense desmoplastic stroma that acts as a barrier to T cell infiltration and high numbers of tumor-associated immunosuppressive cells, such as 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 and that FAK activity correlates with highly fibrotic tumors with poor CD8+ T cell infiltration. The oral FAK kinase inhibitor VS-4718, currently in Phase I 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 FOXP3+ 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 nearly tripling survival times. We next assessed the mechanism of this potentiation. We found that FAK in tumor cells regulates pro-inflammatory and pro-fibrotic cytokine secretion. Furthermore, we found that shRNA knockdown of FAK in PDAC cells results 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. Taken together, 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.

Focal Adhesion Kinase Inhibitors Reverse the Stromal Adhesion Phenotype of Ikaros-Mutant B-ALL, Induce Apopotosis, and Synergize with ABL1 Tyrosine Kinase Inhibitors: A New Paradigm for Pathogenesis and Therapy of High-Risk B-ALL

B-cell acute lymphoblastic leukemia (B-ALL) is a malignancy of precursor B-lymphocytes affecting both children and adults. Deletions and dominant-negative mutations in IKZF1, the gene encoding the 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 imatinib have been added to chemotherapy regimens for Ph+ B-ALL, over half of these patients will still relapse, which correlates with residual disease burden in the bone marrow (BM) following induction therapy. Hence, 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) where the recombined allele is similar to the dominant-negative Ik6 mutant found in human B-ALL, we demonstrated recently that Ikaros DNA-binding function is required in the B-lymphoid lineage for transition from the large to small pre-B cell stage of differentiation, and that arrest at this stage of development can give rise to B-ALL (Joshi et al., Nat. Immunol. 2014;15:294). The 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, which plays important roles in cancer stem cell biology, the tumor microenvironment and tumorigenesis. VS-4718 and VS-6063 (defactinib) are potent, orally bioavailable FAK inhibitors that inhibit tumor growth and metastasis in preclinical models, and are currently under evaluation in clinical trials in patients with various solid tumors. VS-6063 has demonstrated tolerability and preliminary signs of clinical activity as a single agent and in combination with paclitaxel in phase I trials (ASCO, 2014). Here, we show that BCR-ABL1 cooperates with Ikzf1 mutation to accelerate B-leukemogenesis in mice. BCR-ABL1+ Ikaros-mutant B-ALLs exhibit stroma-mediated resistance to ABL1 TKIs, while the FAK inhibitors VS-4718 and VS-6063 are effective in blocking stromal adhesion and inducing apoptosis in both mouse and human Ikaros-mutant B-ALL samples.To test whether dysregulation of TK signaling cooperates with Ikzf1 mutation in the pathogenesis of high-risk B-ALL, we isolated BM B-lymphoid progenitor cells from wild-type (WT), IkE5fl/+CD2-Cre, and IkE5fl/flCD2-Cre donors, transduced them with BCR-ABL1 retrovirus and transplanted the cells into recipient mice. We observed a dramatic acceleration of precursor B-lymphoid leukemia induced by BCR-ABL1 in IkE5Δ/+ and particularly in IkE5Δ/Δ donor cells that correlated with a striking (~30-fold) increase in the frequency of engrafting leukemia-initiating or leukemic stem cells (LSCs). Relative to Ikzf1 WT BCR-ABL1+ leukemic cells, Ikzf1-mutant BCR-ABL1+ blasts showed significant resistance to imatinib and dasatinib that was dependent on the presence of OP9 stroma.The effect of FAK inhibition, using the FAK inhibitors VS-4718, VS-6062, and VS-6063 (Verastem), was first tested on murine B-ALL cells (genotypes Ikzf1 mutant, Ikzf1 mutant BCR-ABL1+, and Ikzf1 WT BCR-ABL1+) grown on OP9 stroma. FAK inhibitor treatment abolished stromal adhesion of Ikzf1-mutant B-ALL and induced apoptosis in non-adherent cells, but had little effect on Ikzf1 WT B-ALL cells. VS-4718 and VS-6063 were each synergistic with dasatinib in reducing the viability of Ikzf1-mutant BCR-ABL1+ B-ALL cells cultured on OP9 stroma. For primary human B-ALL samples grown on OP9 stroma, IKZF1-mutant cells were also more sensitive to FAK inhibitor treatment than WT IKZF1 WT B-ALL, with or without BCR-ABL1 expression.Collectively, these observations suggest a new model to explain the pathogenesis of high-risk B-ALL and its resistance to 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 LSCs. Inhibition of FAK signaling in Ph+ or Ph­–IKZF1-mutant B-ALL may reverse the stromal-mediated resistance to ABL1 TKIs and/or chemotherapy. Therefore, FAK inhibitors warrant further investigation for the treatment of high-risk IKZF1-mutant B-ALL patients. DisclosuresJoshi:Verastem: Research Funding. Yoshida:Verastem, Inc.: Research Funding. Paraskevopoulou:Verastem, Inc.: Research Funding. Zhang:Verastem, Inc.: Research Funding. Krause:Glycomimetics. Inc.: Research Funding. Shapiro:Verastem: Employment, Equity Ownership. Weaver:Verastem: Employment, Equity Ownership. Pachter:Verastem Inc.: Employment, Equity Ownership. Georgopoulos:Verastem, Inc.: Research Funding.

FAK Inhibition Abrogates the Malignant Phenotype in Aggressive Pediatric Renal Tumors

Despite the tremendous advances in the treatment of childhood kidney tumors, there remain subsets of pediatric renal tumors that continue to pose a therapeutic challenge, mainly malignant rhabdoid kidney tumors and nonosseous renal Ewing sarcoma. Children with advanced, metastatic, or relapsed disease have a poor disease-free survival rate. Focal adhesion kinase (FAK) is a nonreceptor tyrosine kinase that is important in many facets of tumor development and progression. FAK has been found in other pediatric solid tumors and in adult renal cellular carcinoma, leading to the hypothesis that FAK contributes to pediatric kidney tumors and would affect cellular survival. In the current study, FAK was present and phosphorylated in pediatric kidney tumor specimens. Moreover, the effects of FAK inhibition upon G401 and SK-NEP-1 cell lines were examined using a number of parallel approaches to block FAK, including RNA interference and small-molecule FAK inhibitors. FAK inhibition resulted in decreased cellular survival, invasion and migration, and increased apoptosis. Furthermore, small-molecule inhibition of FAK led to decreased SK-NEP-1 xenograft growth in vivo. These data deepen the knowledge of the tumorigenic process in pediatric renal tumors, and provide desperately needed therapeutic strategies and targets for these rare, but difficult to treat, malignancies. This study provides a fundamental understanding of tumorigenesis in difficult to treat renal tumors and provides an impetus for new avenues of research and potential for novel, targeted therapies.

Abstract P5-03-01: Focal adhesion kinase inhibition abrogates ductal carcinoma in situ cancer stem cell activity and self-renewal capacity via down regulation of WNT3a and potentiates effects of radiation

Abstract Background - Ductal carcinoma in situ (DCIS) recurs in 20% of patients after conserving surgery and radiotherapy. Research shows cancer stem cells (CSCs) are tumour initiating, resistant to current therapies and therefore may play a role in cancer recurrence. Focal Adhesion Kinase (FAK) signalling plays an important role in breast cancer, metastasis and radioresistance. These studies investigate FAK signalling in the regulation of human DCIS CSCs. Methods - Immunohistochemical (IHC) expression of active FAK was analysed in DCIS patients (n = 63) with known recurrence. DCIS cell lines (DCIS.com & SUM225) and human primary DCIS cells (n = 13) were cultured as primary and secondary mammospheres to assess CSC/progenitor activity and self-renewal after inhibition of FAK (PF573228 or siRNA) in combination with radiation (2Gy). Western Blotting and IHC staining were used to measure DNA-damage, FAK and Wnt signalling. Cell lines were cultured in 3D matrigel as DCIS acini and in vivo as DCIS xenografts to examine the effects of FAK inhibition. Results - Mammosphere initiating cells from DCIS cell lines and human primary DCIS have increased FAK activity and are more radioresistant than the total population (TP) (TP-6Gy vs AR-6Gy; DCIS.com-0.51±0.01 vs 0.05±0.01 Survival fraction (SF), P<0.001; SUM225-0.52±0.03 vs 0.03±0.01 SF, P<0.001; patient-derived DCIS–67.61±6.4% vs 13.8±7% mammosphere formation efficiency (MFE), P = 0.0005). FAK inhibition using PF573228 and FAK siRNA in mammosphere or 3D matrigel culture reduced mammosphere formation alone (mammosphere culture-control vs PF573228 0.5μM; DCIS.com-1.2±0.09% vs 0.7±0.06% MFE, P<0.001; SUM225-2.2±0.1% vs 1.4±0.06% MFE, P<0.001; patient derived DCIS-100±2.6% vs 72.1±6.2% MFE compared to control, P<0.001). Moreover, FAK inhibition in combination with 2Gy radiation abrogated mammosphere formation more than either treatment alone and increased DNA double strand breaks (2Gy vs 2Gy/0.5μM PF573228; DCIS.com-0.66±0.02% vs 0.39±0.01% MFE, P<0.05; SUM225-1.59±0.06% vs 1.22±0.05% MFE, P<0.01; patient derived DCIS-0.63±0.04 vs 0.39±0.03 fold change compared to control, P<0.01). Expression of active FAK in DCIS (n = 63) was associated with a shorter time to recurrence after surgery (Univariate analysis Cox's regression model, p = 0.049). Further investigation revealed FAK inhibition also decreased self-renewal capacity (SR) in vitro and in vivo (control vs PF573228 0.5μM; DCIS.com-0.85±0.03 vs 0.4±0.08 SR, P<0.001; SUM225-0.66±0.06 vs 0.28±0.03 SR, P<0.001; patient-derived DCIS-1 vs 0.54±0.19 SR compared to control, P = 0.001; DCIS.com xenograft-vehicle vs PF562271 25mg/kg; 1.3±0.12% vs 0.65±0.05% MFE, P<0.0001) and could be rescued through the addition of Wnt3a, showing for the first time FAK-Wnt signalling crosstalk in DCIS. Conclusion - Targeting FAK reduced self-renewal of DCIS stem/progenitor cells via down regulation of Wnt3a and improves sensitivity to radiotherapy. These data would support further investigations through a peri-operative trial of short term FAK inhibition before radiotherapy on the recurrence rates of DCIS patients after breast conserving surgery. Funding - Breast Cancer Campaign and The Royal College of Surgeons. Citation Information: Cancer Res 2013;73(24 Suppl): Abstract nr P5-03-01.

Focal adhesion kinase serves as a marker of cervical lymph node metastasis and is a potential therapeutic target in tongue cancer

The aims of the present study were to examine whether focal adhesion kinase (FAK) expression is correlated with cervical lymph node metastasis of tongue cancer and to investigate the roles of FAK in the process of cancer cell migration, invasion and anoikis resistance using the human tongue cancer cell line, Tca8113. FAK expression was evaluated in 5 normal oral mucosa, 10 premalignant lesions, 80 primary tongue cancers and 41 lymph node metastases using anti-FAK immunohistochemistry. The migration, invasion and anoikis resistance of tongue cancer cells were evaluated using wound healing assays, invasion assays and anoikis induction. The effect of FAK inhibition was evaluated using RNA interference (RNAi). In total, 55 of 80 primary tongue cancers (69%) showed high expression of FAK, and 25 of 80 tumors (31%) and all normal oral mucosa or premalignant lesions showed low expression of FAK. There was a significant correlation between FAK expression and the cervical lymph node metastasis of tongue cancer. Moreover, RNAi-mediated FAK reduction decreased tongue cancer cell migration, invasion and anoikis resistance. These results suggest that FAK may serve as a marker of cervical lymph node metastasis of tongue cancer and that RNAi targeting FAK could serve as a potential therapeutic for the treatment of tongue cancer.

Inhibition of Focal Adhesion Kinase Suppresses Adverse Features of Endocrine-Resistant Breast Cancer Cells and Improves Endocrine Response in ER+, Endocrine-Sensitive Cells.

Abstract Background: Focal adhesion kinase (FAK) regulates signalling pathways central to cell adhesion, migration and survival and its expression is frequently deregulated in cancer, including breast cancer. Acquired resistance to endocrine therapy in breast cancer is a major clinical problem: relapses present as local and/or regional recurrences, frequently with metastases and the outlook for these patients is poor. Interestingly, cell models of acquired endocrine resistance have altered cell-matrix adhesion and a highly migratory phenotype, features which may impact on tumour spread in vivo. We have used the novel FAK inhibitor, PF573228 ('PF228'), to address the role of FAK in the development of these characteristics.Material and Methods: FAK expression and activity was determined in endocrine-sensitive MCF7 cells and their acquired tamoxifen-resistant ('TamR') and fulvestrant-resistant ('FasR') counterparts in response to PF228 (0-5µM) by Western blotting using phospho-specific antibodies. Changes in cell adhesion were measured by seeding cells, pre-treated with PF228, onto matrix (fibronectin and laminin)-coated plates. After 20 minutes adherent cells were quantified by MTT assay. Cell migration was assessed by seeding cells onto matrix-coated porous membranes ± PF228. After 24hrs, migratory cells were stained and counted. Changes in cell morphology and focal adhesion structure were determined using interference contrast microscopy and immunofluorescence staining respectively. The effects of FAK inhibition on cell proliferation was determined by MTT assay.Results: Whilst FAK expression was similar between MCF7, TamR and FasR cells, phosphorylation of FAK (Y397 and Y861) was elevated in the resistant cells. PF573 promoted a dose-dependent inhibition of phosphorylated FAK (Y397) but did not affect other FAK activation sites (Y407, Y576 and Y861). Endocrine-resistant cells were significantly more sensitive to these inhibitory effects versus MCF7 (mean IC50 for FAK Y397 inhibition: 0.26µM, 0.077µM and 0.011µM for MCF7, TamR and FasR cells respectively). Inhibition of FAK Y397 was associated with a reduction in TamR and FasR adhesion to, and migration over, matrix components; accompanying this was a loss of focal adhesion integrity and a change in appearance, with cells reverting from an EMT-like morphology to one akin to their endocrine-sensitive MCF7 counterparts. Whilst PF228 modestly inhibited proliferation of all three cell types, with resistant cells appearing most sensitive, treatment of endocrine-sensitive MCF7 cells with endocrine agent and PF228 combined resulted in greater suppression of proliferation versus single agent treatment.Conclusion: These data suggest a role for FAK in mediating the aggressive behaviour of endocrine-resistant breast cancer cells. Combining FAK inhibitors and endocrine agent may provide additional benefit and circumvent the development of these adverse characteristics. Citation Information: Cancer Res 2009;69(24 Suppl):Abstract nr 3130.