Focal Adhesion Kinase Inhibitor Treatment Research Articles

Mechanism of collagen type IV regulation by focal adhesion kinase during retained fetal membranes in dairy cows

Retained fetal membranes (RFM) is an important reproductive disease in dairy cows, caused by maternal and fetal placental tissue adhesion. The main collagen in maternal and fetal placenta tissues is collagen type IV (COL-IV) and its breakdown is the key to placental expulsion. Focal adhesion kinase (FAK) has been shown to regulate the hydrolysis of Col-IV by affecting the activity of MMP-2 and MMP-9 activity, but the regulation of the mechanisms involved in placenta expulsion in dairy cows after postpartum are still unclear. The aim of this study was to investigate the pathogenic mechanism of RFM by studying the relationship between the FAK signaling pathway and COL-IV regulation. Maternal placental tissues were collected from six healthy and six cows with RFM of similar age, parity, body condition and milk yield at 12 h postpartum. In vitro experiments were performed on bovine endometrial epithelial cells from three groups including a FAK inhibitor group, a FAK activator group and a control group without FAK inhibitor and activator. The abundance of molecules involved in the FAK signaling pathway and COL-IV was detected by immunohistochemistry, quantitative real-time polymerase chain reaction (qRT-PCR) and western blot. The immunohistochemical results showed that the key molecules of FAK signaling pathway FAK, Src, MMP-2 and MMP-9 and Col-IV were expressed in placental tissues. The expression level of FAK, Src, MMP-2, and MMP-9 were significantly down-regulated (P < 0.05) and the abundances of COL-IV were significantly up-regulated (P < 0.05) in maternal placental tissues of RFM cows compared with healthy cows. In the FAK inhibitor treatment group, the relative expression levels of FAK and other related proteins were significantly down-regulated (P < 0.05) and the relative expression levels of COL-IV were significantly up-regulated (P < 0.05) with the results of the FAK activation group the opposite. These results indicated that FAK in maternal endometrial epithelial cells could regulate the hydrolysis process of Col-IV through the expression of key factors of signaling pathways and promote collagen hydrolysis, which in turn facilitated the process of postpartum placenta expulsion in dairy cows.

Regulation of intracellular cAMP levels in osteocytes by mechano-sensitive focal adhesion kinase via PDE8A.

Osteocytes are the primary mechano-sensitive cell type in bone. Mechanical loading is sensed across the dendritic projections of osteocytes leading to transient reductions in focal adhesion kinase (FAK) activity. Knowledge regarding the signaling pathways downstream of FAK in osteocytes is incomplete. We performed tyrosine-focused phospho-proteomic profiling in osteocyte-like Ocy454 cells to identify FAK substrates. Gsα, parathyroid hormone receptor (PTH1R), and phosphodiesterase 8A (PDE8A), all proteins associated with cAMP signaling, were found as potential FAK targets based on their reduced tyrosine phosphorylation in both FAK- deficient or FAK inhibitor treated cells. Real time monitoring of intracellular cAMP levels revealed that FAK pharmacologic inhibition or gene deletion increased basal and GPCR ligand-stimulated cAMP levels and downstream phosphorylation of protein kinase A substrates. Mutating FAK phospho-acceptor sites in Gsα and PTH1R had no effect on PTH- or FAK inhibitor-stimulated cAMP levels. Since FAK inhibitor treatment augmented cAMP levels even in the presence of forskolin, we focused on potential FAK substrates downstream of cAMP generation. Indeed, PDE8A inhibition mimicked FAK inhibition at the level of increased cAMP, PKA activity, and expression of cAMP-regulated target genes. In vitro kinase assay showed that PDE8A is directly phosphorylated by FAK while immunoprecipitation assays revealed intracellular association between FAK and PDE8A. Thus, FAK inhibition in osteocytes acts synergistically with signals that activate adenylate cyclase to increase intracellular cAMP. Mechanically-regulated FAK can modulate intracellular cAMP levels via effects on PDE8A. These data suggest a novel signal transduction mechanism that mediates crosstalk between mechanical and cAMP-linked hormonal signaling in osteocytes.

Astrocyte focal adhesion kinase reduces passive stress coping by inhibiting ciliary neurotrophic factor only in female mice

Astrocytes have been implicated in stress responses and produce ciliary neurotrophic factor (CNTF), which we have shown in the mouse medial amygdala (MeA) to promote passive stress coping response only in females. Pharmacological inhibition of focal adhesion kinase (FAK) upregulates CNTF expression. Here, we found that inducible knockout of FAK in astrocytes or systemic treatment with an FAK inhibitor increased passive coping behavior, i.e., immobility, in an acute forced swim stress test in female, but not male, mice. Strikingly, four weeks of chronic unpredictable stress (CUS) did not further increase passive coping in female astrocytic FAK knockout mice, whereas it exacerbated it in female wildtype mice and male mice of both genotypes. These data suggest that astrocyte FAK inhibition is required for chronic stress-induced passive coping in females. Indeed, CUS reduced phospho-FAK and increased CNTF in the female MeA. Progesterone treatment after ovariectomy activated amygdala FAK and alleviated ovariectomy-induced passive coping in wildtype, but not astrocytic FAK knockout females. This suggests that progesterone-mediated activation of FAK in astrocytes reduces female stress responses. Finally, astrocytic FAK knockout or FAK inhibitor treatment increased CNTF expression in the MeA of both sexes, although not in the hippocampus. As mentioned, MeA CNTF promotes stress responses only in females, which may explain the female-specific role of astrocytic FAK inhibition. Together, this study reveals a novel female-specific progesterone-astrocytic FAK pathway that counteracts CNTF-mediated stress responses and points to opportunities for developing treatments for stress-related disorders in women.

Development of Combination Strategies for Focal Adhesion Kinase Inhibition in Diffuse Gastric Cancer.

Diffuse gastric cancer (DGC) is an aggressive and frequently lethal subtype of gastric cancer. Because DGC often lacks genomic aberrations that indicate clear candidate therapeutic targets, it has been challenging to develop targeted therapies for this gastric cancer subtype. Our previous study highlighted the contribution of focal adhesion kinase (FAK) in the tumorigenesis of DGC and the potential efficacy of small-molecule FAK inhibitors. However, drug resistance to monotherapy often hinders the efficacy of treatment. We generated a genome-scale library of open reading frames (ORF) in the DGC model of Cdh1-/-RHOAY42C/+ organoids to identify candidate mechanisms of resistance to FAK inhibition. Compensatory activated pathways were also detected following treatment with FAK inhibitors. Candidates were investigated by cotargeting in vitro and in vivo experiments using DGC. We found that cyclin-dependent kinase 6 (CDK6) promoted FAK inhibitor resistance in ORF screen. In addition, FAK inhibitor treatment in DGC models led to compensatory MAPK pathway activation. Small-molecule CDK4/6 inhibitors or MAPK inhibitors effectively enhanced FAK inhibitor efficacy in vitro and in vivo. Our data suggest that FAK inhibitors combined with MAPK inhibitors or CDK4/6 inhibitors warrant further testing in clinical trials for DGC.

Female-specific neuroprotection after ischemic stroke by vitronectin-focal adhesion kinase inhibition

We found that blood vitronectin (VTN) leaks into the brain and exacerbates tissue loss after stroke by increasing pro-inflammatory IL-6 expression in female, but not male, mice. VTN signals through integrins and downstream focal adhesion kinase (FAK). Here, a two day systemic treatment with a small molecule FAK inhibitor starting 6 h after middle cerebral artery occlusion reduced ipsilateral brain injury size by ∼40–45% at 7 and 14 d, as well as inflammation and motor dysfunction in wild-type female, but not male, mice. FAK inhibition also reduced IL-6 expression in the injured female striatum at 24 h by 62%. Inducible selective gene deletion of FAK in astrocytes also reduced acute IL-6 expression by 72% only in females, and mitigated infarct size by ∼80% and inflammation at 14 d after stroke. Lastly, VTN−/− females had better outcomes, but FAK inhibitor treatment had no additional protective or anti-inflammatory effects. Altogether, this suggests that VTN is detrimental in females primarily through FAK and that FAK inhibition provides neuroprotection (cerebroprotection) by reducing VTN-induced IL-6 expression in astrocytes. Thus, VTN signaling can be targeted to mitigate harmful inflammation with relevance to treatments for women with ischemic stroke, who often have worse outcomes than men.

Highlights from Recent Cancer Literature

Highlights from Recent Cancer Literature

FAK inhibition protects condylar cartilage under excessive mechanical stress.

Excessive mechanical stress is assumed to be a major cause of temporomandibular joint (TMJ) osteoarthritis (OA). +Focal adhesion kinase (FAK) is a cytoplasmic non-receptor tyrosine kinase involved in a variety of signaling pathways. Little has been reported on the function of FAK in TMJ-OA. In the present study, we investigated the effect of FAK inhibition on TMJ cartilage under excessive mechanical loading stress. Articular cartilage explants were harvested from the TMJ of rats and subjected to mechanical loading in the presence of an FAK inhibitor in organ culture. The gene expression of inflammatory cytokines was examined after the application of mechanical loading with or without FAK inhibitor. Paraffin-embedded sections of articular cartilage were stained with hematoxylin and eosin, safranin O and fast Green, toluidine blue, TUNEL staining, and immunohistochemical staining and was performed to investigate the protein expression of IL-1β and MMP-13. Treatment with FAK inhibitor reduced the gene expression of inflammatory cytokines and inhibited the degradation of articular cartilage, as determined histologically. FAK inhibitor treatment also suppressed the protein expression of IL-1β and MMP-13 in the hypertrophic zone, as determined immunohistologically. Treatment with FAK inhibitor suppresses inflammation and protects condylar cartilage under excessive mechanical loading.

Focal Adhesion Kinase-Mediated Sequences, Including Cell Adhesion, Inflammatory Response, and Fibrosis, as a Therapeutic Target in Endometriosis.

Endometriosis has several distinguishing features in the ectopic endometrium, including chronic inflammation and fibrosis. According to the retrograde menstruation theory, endometriotic cells are derived from eutopic endometrial cells, and adhesion of endometrial cells to the extracellular matrix can be the initial step in the development of endometriosis. Therefore, we hypothesized that cell adhesion, which mediates a sequence of events in the development of endometriosis triggering inflammatory responses and tissue fibrosis could be a possible therapeutic target for endometriosis. We found co-upregulation of focal adhesion kinase (FAK) and monocyte chemoattractant protein-1 (MCP-1) in the endometriotic tissues compared with that in the normal endometrium. MCP-1 secretion was significantly higher in the endometriotic stromal cells than in the eutopic endometrial stromal cells. Furthermore, co-culture of U937 cells and endometriotic stromal cells upregulated secretion of transforming growth factor-β1 (TGF-β1). A FAK inhibitor significantly inhibited the secretion of MCP-1 in the endometriotic stromal cells and TGF-β1 in the co-culture with macrophages. FAK inhibitor treatment in the murine endometriosis model demonstrated a decrease in the formation of endometriotic lesions as well as the expression of MCP-1 and TGF-β1. Our results suggest that the FAK-mediated sequential development of endometriosis, including inflammatory response and tissue fibrosis, can be a new therapeutic target in endometriosis.

Inhibition of cell migration by focal adhesion kinase: Time-dependent difference in integrin-induced signaling between endothelial and hepatoblastoma cells.

Angiogenesis plays an important role in the development and progression of tumors, and it involves a series of signaling pathways contributing to the migration of endothelial cells for vascularization and to the invasion of cancer cells for secondary tumor formation. Among these pathways, the focal adhesion kinase (FAK) signaling cascade has been implicated in a variety of human cancers in connection with cell adhesion and migration events leading to tumor angiogenesis, metastasis and invasion. Therefore, the inhibition of FAK in endothelial and/or cancer cells is a potential target for anti-angiogenic therapy. In the present study, a small-molecule FAK inhibitor, 1,2,4,5-benzenetetramine tetrahydrochloride (Y15), was used to study the effects of FAK inhibition on the adhesion and migration behaviors of vascular endothelial cells (VECs) and human hepatoblastoma cells. Furthermore, the time-dependent differences in proteins associated with the integrin-mediated FAK/Rho GTPases signaling pathway within 2 h were examined. The results indicated that the inhibition of FAK significantly decreased the migration ability of VECs and human hepatoblastoma cells in a dose-dependent manner. Inhibition of FAK promoted cell detachment by decreasing the expression of focal adhesion components, and blocked cell motility by reducing the level of Rho GTPases. However, the expression of crucial proteins involved in integrin-induced signaling in two cell lines exhibited a time-dependent difference with increased duration of FAK inhibitor treatment, suggesting different mechanisms of FAK-mediated cell migration behavior. These results suggest that the mechanism underlying FAK-mediated adhesion and migration behavior differs among various cells, which is expected to provide evidence for future FAK therapy targeted against tumor angiogenesis.

CKAP2 Promotes Ovarian Cancer Proliferation and Tumorigenesis Through the FAK-ERK Pathway.

Upregulation of cytoskeleton-associated protein 2 (CKAP2) has been observed in ovarian cancer. This study aimed to determine the effects of overexpression of CKAP2 on cell proliferation and tumorigenesis and to explore the potential signal pathway in the oncogenic process. Expression of CKAP2 in tissue of ovarian cancer patients was detected. Human cancer cell line SKOV3 was overexpressed with CKAP2 and measured for cell growth and mobility. Moreover, CKAP2 effect was estimated using mouse tumor xenografts. mRNA expression levels of related genes were assessed. Expression levels of CKAP2 and p-ERK2 in SKOV3 cells were detected before and after treatment of focal adhesion kinase (FAK) inhibitor. mRNA and protein levels of CKAP2 were significantly elevated in human ovarian tumor tissues. Overexpression of CKAP2 in SKOV3 cells promoted cell proliferation and migration. Implantation of overexpressed SKOV3 cells showed significant tumorigenesis ability. mRNA expression levels of proliferating cell nuclear antigen (PCNA), matrix metalloproteinase 2 (MMP2), matrix metalloproteinase 9 (MMP9), and SNAI1 were elevated in overexpressed SKOV3 cells. Cells with CKAP2 overexpression also had significantly increased p-ERK2 level. Moreover, FAK inhibitor treatment significantly decreased CKAP2 expression levels as well as the phosphorylation level of ERK2 in SKOV3 cells. This study suggests that overexpression of CKAP2 enhanced proliferation and mobility of carcinoma cells through the FAK-ERK2 signaling pathway.

Abstract 4236: FAK inhibitor VS-6063 (defactinib) targets mesothelioma cancer stem cells, which are enriched by standard of care chemotherapy

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

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.

Abstract 752: Genetic and pharmacological FAK inhibition disrupt a β5 integrin signaling axis controlling anchorage-independent ovarian carcinoma growth

Abstract Ovarian cancer spreads via cell shedding and growth within malignant ascites. Effective targeted therapies have not been developed for ovarian cancer. Ascites contains an abundance of matrix proteins, and spheroids maintain integrin receptor expression. Through databases analyses we find that elevated osteopontin (OPN), β5 integrin, and focal adhesion kinase (FAK) mRNA levels are associated with decreased overall survival of serous ovarian cancer patients treated with platinum and taxol. In ovarian tumor tissue arrays, increased FAK activation (FAK Y397 phosphorylation) correlated with elevated tumor grade in parallel with increased in β5 integin and OPN levels. FAK is a cytoplasmic tyrosine kinase that remains active in spheroids, and treatment of seven ovarian carcinoma cell lines with sub-micromolar levels of FAK inhibitor (PND-1186) identified sensitive (HEY and OVCAR8), intermediate (OVCAR3, ID8-IP, and IGROV1-IP), and resistant (SKOV3-IP and OVCAR10) cells to blockage of growth under anchorage-independent conditions. Genetic or pharmacological FAK inhibition within ID8-IP or HEY cells selectively prevents anchorage-independent growth in culture and tumor growth in mice with corresponding reductions in β5 integrin and OPN expression. β5 knockdown reduced HEY growth in soft agar, tumor growth in mice, FAK Y397 phosphorylation, and OPN expression in spheroids. Although FAK inhibitor resistant ovarian carcinoma cells (SKOV3-IP and OVCAR10) were associated with anchorage-independent Akt S473 phosphorylation, membrane-targeted and activated Akt expression in sensitive cells (HEY and OVCAR8) resulted in only a partial rescue of FAK inhibitor-associated growth block. These results support the hypothesis that OPN, αvβ5 integrins, and FAK may function as a signaling axis promoting ovarian tumor progression. Although Akt signaling pathway activation is a common event in serous ovarian cancer, our results suggest that this may not impart complete resistance to FAK inhibitor treatment. Supported by NIH CA102310 Citation Format: Isabelle Tancioni, Sean Uryu, Florian Sulzmaier, Nina Shah, Christine Lawson, Nichol L.G. Miller, Christine Jean, Xiao Lei Chen, Kristy K. Ward, David D. Schlaepfer. Genetic and pharmacological FAK inhibition disrupt a β5 integrin signaling axis controlling anchorage-independent ovarian carcinoma growth. [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 752. doi:10.1158/1538-7445.AM2014-752

Merlin deficiency predicts FAK inhibitor sensitivity: a synthetic lethal relationship.

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.

Abstract C262: Malignant mesothelioma lacking merlin shows enhanced sensitivity to the FAK inhibitor defactinib (VS-6063): Elucidation of the merlin-FAK relationship.

Abstract 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.

Abstract B13: Predictive biomarkers for FAK inhibitor sensitivity in ovarian cancer

Abstract Ovarian cancer is the fifth leading cause of cancer related death among women in the US and has the highest mortality rate among all gynecologic malignancies. Approximately 80% of patients diagnosed with ovarian epithelial cancer will relapse after first-line chemotherapy. Although targeted therapies tailored to specific genetic lesions have been developed for lung cancer, melanoma and CML, the molecular heterogeneity of the ovarian cancer genome has made it challenging to couple patient stratification markers with targeted agents. Focal adhesion kinase (FAK) is a non-receptor protein tyrosine kinase that plays important roles in proliferation, cell survival, migration, invasion and cancer stem cell (CSC) renewal. FAK amplification and overexpression in ovarian cancer have been correlated with poor clinical outcome. In addition, high levels of phosphorylated FAK (pFAK) have been linked to poor prognosis and high ovarian tumor grade. Anti-tumor activity of the FAK inhibitors VS-6063 and VS-4718 has been previously demonstrated in preclinical models including ovarian cancer xenograft models (1,2). We have aimed to identify molecular markers predictive of ovarian cancer sensitivity to FAK inhibitors. Human ovarian cancer cell lines were analyzed for FAK inhibitor sensitivity in 3D Matrigel culture that mimics the ovarian tumor microenvironment. We then performed gene expression analysis of ovarian cancer cell lines most sensitive or least sensitive to FAK inhibitor treatment and identified a 64 gene signature that unambiguously clustered cell lines into sensitive and resistant subgroups (fold change &amp;gt;2, AUC &amp;gt;0.85). The gene signature associated with increased sensitivity to VS-4718 contained 27 genes with elevated expression, such as SLIT2, and 37 genes with decreased expression, such as LPNH2. Gene expression data were validated using qPCR as well as Western blot analysis to assess corresponding protein expression levels. As these genes may be important predictive biomarkers for patient selection or stratification, the variability of gene expression across human ovarian tumors was investigated using RNA microarray data of 733 patient tumor specimens derived from 5 study groups. As desired, SLIT2, LPNH2, and other genes in the signature exhibited a broad range of expression in ovarian tumor samples. Seven genes encoded for secreted proteins which could enable development of detection assays in blood. To assess regional expression of some of the above biomarkers within the tumor, we used immunohistochemistry analysis of a human ovarian Tissue MicroArray (TMA). Potential biomarkers were then evaluated in an independent test set of ovarian cancer samples. We first analyzed expression of the 64-gene signature in RNA microarray datasets from 6 ovarian patient-derived xenograft (PDX) models to predict their response to FAK inhibitor treatment. Then, an ex vivo clonogenic 3D assay was used to assess cell viability of ovarian PDX samples treated with FAK inhibitor. Interestingly, expression of several markers correlated with sensitivity to FAK inhibitor. In a similar fashion, we analyzed cell viability of ovarian cancer surgical samples from patients following ex vivo treatment with the FAK inhibitor. Tumors that expressed high levels of a number of genes from the 64 gene signature were especially sensitive to FAK inhibitor ex vivo. Our data identify biomarkers predictive of response to FAK inhibition in ovarian cancer models and provide a rationale for validation of these markers in a clinical trial of a FAK inhibitor in patients with ovarian cancer.

Abstract 924: FAK inhibition preferentially attenuates growth of Merlin-negative malignant mesotheliomas: role of cancer stem cells.

Abstract Malignant pleural mesothelioma (MPM) is an aggressive tumor in the pleural lining of the lung often caused by asbestos exposure. MPM patients are usually diagnosed at an advanced stage of the disease and the prognosis is poor. Median survival after diagnosis is 9 to 12 months and standard-of-care agents such as pemetrexed are relatively ineffective in increasing median survival time for MPM patients. New therapeutic modalities are urgently needed for MPM patients. Neurofibromatosis 2 (NF2) is a tumor suppressor gene that encodes the protein Merlin. Biallelic inactivation of NF2 by mutation and/or deletion occurs in ∼40% of MPMs leading to inactive or absent Merlin. Merlin has been demonstrated to play roles in cell adhesion, invasion and cell motility in tumor cell lines partially through regulation of focal adhesion kinase (FAK) which in turn mediates signal transduction by integrins and growth factor receptors. Increased activation of FAK has been demonstrated in NF2-mutated mesothelioma cells, indicating that FAK may represent an important therapeutic target for MPM. A potent and selective FAK inhibitor VS-4718 was evaluated in a panel of MPM cell lines with wild-type or mutated NF2. Mutant NF2 MPM cells were found to be especially sensitive to the FAK inhibitor VS-4718 in a 3D Matrigel assay with EC50 values below 100 nM, in contrast to wild type NF2 MPM cell lines which were less sensitive with EC50 values above 1 μM. Decrease in cell growth in response to FAK inhibitor treatment was found to result from reduction of cell proliferation and induction of apoptosis. Ectopic expression of a non-phosphorylatable mutant form of NF2 (NF2-S518A) in NF2 mutant MPM cells abolished the enhanced sensitivity to VS-4718 in both proliferation and apoptosis assays, confirming the hypothesis that Merlin loss confers hypersensitivity to FAK inhibition. In addition, VS-4718 inhibited tumor cell growth in an orthotopic Merlin-negative mesothelioma xenograft model in a dose-dependent manner with corresponding inhibition of tumor FAK autophosphorylation. It has been reported that cancer stem cells (CSCs) are enriched in pemetrexed-resistant mesothelioma. Using Aldefluor activity as a measure of CSCs, we have found that the FAK inhibitor VS-4718 preferentially reduced the percentage of CSCs in Merlin-negative MPM in contrast to the standard-of-care agent pemetrexed which increased the percentage of CSCs. In summary, our results indicate that the FAK inhibitor VS-4718 is especially potent in Merlin-negative MPM tumor cells, and that NF2 status may be a valuable stratification marker for response to FAK inhibition. Furthermore, cancer stem cells in Merlin-negative mesotheliomas appear to be particularly resistant to pemetrexed, but sensitive to VS-4718. These data support the clinical development of a selective FAK inhibitor for treatment of Merlin-negative malignant mesothelioma. Citation Format: Irina M. Shapiro, Vihren N. Kolev, Christian M. Vidal, Jennifer E. Ring, Mitchell Keegan, Qunli Xu, Craig W. Menges, Joseph R. Testa, Jonathan A. Pachter. FAK inhibition preferentially attenuates growth of Merlin-negative malignant mesotheliomas: role of cancer stem cells. [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 924. doi:10.1158/1538-7445.AM2013-924

Role of Intercellular Junctions in Redistribution of Focal Adhesions and Orientation of Vascular Endothelial Cells Exposed to Cyclic Stretching

The redistribution of focal adhesions (FAs) containing integrin β1 and paxillin plays an important role in the cyclic stretching-induced morphological changes of endothelial cells (ECs). In addition to focal adhesion kinase (FAK), known to be a primary regulator for FA redistribution, intercellular junctions (IJs) have recently been reported to be involved in signaling upstream of FAs. Here, we addressed the role of IJs in the morphological changes and redistribution of FAs in ECs exposed to cyclic stretching. Both confluent and sparse ECs were oriented nearly perpendicularly to the stretch direction after 10 min of exposure. Orientation of sparse ECs, but not confluent ECs, was suppressed by treatment with a phospho-FAK inhibitor. FAK inhibitor blocked integrin β1 redistribution in ECs, which was observed in non-inhibited cells after 10-min stretch exposure. However, paxillin redistribution in confluent ECs was observed regardless of FAK inhibitor treatment after 2-min stretch exposure. When we blocked signals from IJs with an inhibitor of Src homology 2 domain-containing tyrosine phosphatase-2, the percentage of oriented ECs decreased and paxillin redistribution, but not integrin β1, was suppressed. These findings suggest that IJs are involved in the orientation of ECs subjected to cyclic stretching through signaling pathways other than FAK.