Michael Yemma

Pharmacologic Blockade of FAK Autophosphorylation Decreases Human Glioblastoma Tumor Growth and Synergizes with Temozolomide

Malignant gliomas are characterized by aggressive tumor growth with a mean survival of 15 to 18 months and frequently developed resistance to temozolomide. Therefore, strategies that sensitize glioma cells to temozolomide have a high translational impact. We have studied focal adhesion kinase (FAK), a tyrosine kinase and emerging therapeutic target that is known to be highly expressed and activated in glioma. In this report, we tested the FAK autophosphorylation inhibitor, Y15, in DBTRG and U87 glioblastoma cells. Y15 significantly decreased viability and clonogenicity in a dose-dependent manner, increased detachment in a dose- and time-dependent manner, caused apoptosis, and inhibited cell invasion in both cell lines. In addition, Y15 treatment decreased autophosphorylation of FAK in a dose-dependent manner and changed cell morphology by causing cell rounding in DBTRG and U87 cells. Administration of Y15 significantly decreased subcutaneous DBTRG tumor growth with decreased Y397-FAK autophosphorylation, activated caspase-3 and PARP. Y15 was administered in an orthotopic glioma model, leading to an increase in mouse survival. The combination of Y15 with temozolomide was more effective than either agent alone in decreasing viability and activating caspase-8 in DBTRG and U87 cells in vitro. In addition, the combination of Y15 and temozolomide synergistically blocked U87 brain tumor growth in vivo. Thus, pharmacologic blockade of FAK autophosphorylation with the oral administration of a small-molecule inhibitor Y15 has a potential to be an effective therapy approach for glioblastoma either alone or in combination with chemotherapy agents such as temozolomide. Mol Cancer Ther; 12(2); 162–72. ©2012 AACR.

A small molecule focal adhesion kinase (FAK) inhibitor, targeting Y397 site: 1-(2-hydroxyethyl) -3, 5, 7-triaza-1-azoniatricyclo [3.3.1.13,7]decane; bromide effectively inhibits FAK autophosphorylation activity and decreases cancer cell viability, clonogenicity and tumor growth in vivo

Focal adhesion kinase (FAK) is a protein tyrosine kinase that is overexpressed in most solid types of tumors and plays an important role in the survival signaling. Recently, we have developed a novel computer modeling combined with a functional assay approach to target the main autophosphorylation site of FAK (Y397). Using these approaches, we identified 1-(2-hydroxyethyl)-3, 5, 7-triaza-1-azoniatricyclo [3.3.1.1(3,7)]decane; bromide, called Y11, a small molecule inhibitor targeting Y397 site of FAK. Y11 significantly and specifically decreased FAK autophosphorylation, directly bound to the N-terminal domain of FAK. In addition, Y11 decreased Y397-FAK autophosphorylation, inhibited viability and clonogenicity of colon SW620 and breast BT474 cancer cells and increased detachment and apoptosis in vitro. Moreover, Y11 significantly decreased tumor growth in the colon cancer cell mouse xenograft model. Finally, tumors from the Y11-treated mice demonstrated decreased Y397-FAK autophosphorylation and activation of poly (ADP ribose) polymerase and caspase-3. Thus, targeting the major autophosphorylation site of FAK with Y11 inhibitor is critical for development of cancer therapeutics and carcinogenesis field.

A FAK scaffold inhibitor disrupts FAK and VEGFR-3 signaling and blocks melanoma growth by targeting both tumor and endothelial cells

Melanoma has the highest mortality rate of all skin cancers and a major cause of treatment failure is drug resistance. Tumors heterogeneity requires novel therapeutic strategies and new drugs targeting multiple pathways. One of the new approaches is targeting the scaffolding function of tumor related proteins such as focal adhesion kinase (FAK). FAK is overexpressed in most solid tumors and is involved in multiple protein-protein interactions critical for tumor cell survival, tumor neovascularization, progression and metastasis. In this study, we investigated the anticancer activity of the FAK scaffold inhibitor C4, targeted to the FAK-VEGFR-3 complex, against melanomas. We compared C4 inhibitory effects in BRAF mutant vs BRAF wild type melanomas. C4 effectively caused melanoma tumor regression in vivo, when administered alone and sensitized tumors to chemotherapy. The most dramatic effect of C4 was related to reduction of vasculature of both BRAF wild type and V600E mutant xenograft tumors. The in vivo effects of C4 were assessed in xenograft models using non-invasive multimodality imaging in conjunction with histologic and molecular biology methods. C4 inhibited cell viability, adhesion and motility of melanoma and endothelial cells, specifically blocked phosphorylation of VEGFR-3 and FAK and disrupted their complexes. Specificity of in vivo effects for C4 were confirmed by a decrease in tumor FAK and VEGFR-3 phosphorylation, reduction of vasculogenesis and reduced blood flow. Our collective observations provide evidence that a small molecule inhibitor targeted to the FAK protein-protein interaction site successfully inhibits melanoma growth through dual targeting of tumor and endothelial cells and is effective against both BRAF wild type and mutant melanomas.

Targeting the C-terminal focal adhesion kinase scaffold in pancreatic cancer

Preliminary studies in our laboratory have demonstrated the importance of both the NH2 and COOH terminus scaffolding functions of focal adhesion kinase (FAK). Here, we describe a new small molecule inhibitor, C10, that targets the FAK C-terminus scaffold. C10 showed marked selectivity for cells overexpressing VEGFR3 when tested in isogenic cell lines, MCF7 and MCF7-VEGFR3. C10 preferentially inhibited pancreatic tumor growth in vivo in cells with high FAK-Y925 and VEGFR3 expression. Treatment with C10 led to a significant inhibition in endothelial cell proliferation and tumor endothelial and lymphatic vessel density and decrease in interstitial fluid pressure. These results highlight the underlying importance of targeting the FAK scaffold to treat human cancers.

Abstract C211: Demonstration of the efficacy of compound CFAK-C4, Targeting FAK-VEGFR-3 protein-protein interaction in animal models of gastric cancer.

While the emerging data strongly suggests that FAK is an excellent target for developmental therapeutics of cancer, small molecule kinase inhibitors of FAK have shown crossreactivity with some other protein kinases. We have shown that FAK acts pleiotropically, as a kinase and as a scaffolding protein, to regulate the functions of multiple downstream survival/metastasis/vasculogenesis-related proteins. Therefore, our objective is to target the scaffolding function of FAK to inhibit protein-protein interactions and to yield promising new drug leads. Previously, we have shown that FAK physically interact with VEGFR-3 and both are overexpressed in cancer cells to provide important survival signals. We subsequently identified a novel small molecule inhibitor C4 that targeted this VEGFR-3-FAK site of interaction. Both of these kinases are overexpressed in gastric cancer and were found to be an independent poor prognostic factors. The prognosis of patients with gastric cancer remains unfavorable and molecular based treatments are necessary for a potential breakthrough in the therapy of this disease. We hypothesize that FAK-VEGFR-3 interaction provides essential survival signals for gastric tumor growth and that simultaneous inhibition of these signals will inhibit tumor progression. Using in silico high throughput methods, we screened a chemical library of 240,000 compounds against the known protein-protein interaction site between FAK and VEGFR-3. The ability of one of the lead compounds CFAK-C4 on human gastric cancer cell lines AGS and NCI-N87 were examined by MTT assay (viability), colony formation assay and Western blotting (phosphorylation, apoptosis). Subcutaneous and orthotopic mouse models of gastric cancer were used to demonstrate effect of C4 in vivo. FAK and VEGFR-3 are overexpressed in human gastric cancer cells as determined by Western blotting. C4 specifically blocked phosphorylation of VEGFR-3 and FAK. It directly and significantly (p<0.05) inhibited cell viability, increased cell detachment and inhibited colony formation in a dose-dependent manner (range 1-100μM). C4 (50mg/kg, intraperitoneal injection 5 days a week) effectively caused tumor regression in vivo and effects of C4 were confirmed by a decrease in tumor FAK and VEGFR3 phosphorylation, and disruption of their complexes. In this study, we have shown that CFAK-C4 inhibits FAK-VEGFR3 signaling in gastric cancer cells and affects tumor grows. These results demonstrate that targeting the FAK-VEGFR-3 interaction with a small molecules inhibited the survival function of these two tyrosine kinases, representing a unique approach for molecular-targeted highly-specific cancer therapeutics. Targeting the scaffolding function of FAK with the small-molecule inhibitors can be effectively used to develop potential oral-based gastric cancer therapeutics. Citation Information: Mol Cancer Ther 2013;12(11 Suppl):C211. Citation Format: Elena Kurenova, Sartaj Sanghera, Jian Liao, Michael Yemma, William Cance. Demonstration of the efficacy of compound CFAK-C4, Targeting FAK-VEGFR-3 protein-protein interaction in animal models of gastric cancer. [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 C211.

Abstract C191: Preclinical investigation of the antitumor efficacy in lung cancer of Y15, a novel focal adhesion kinase inhibitor.

Introduction: Focal adhesion kinase (FAK) is a nonreceptor tyrosine kinase that associates with a number of cell adhesion components, such as integrins, paxillin and talin, as well as with various signaling proteins such as phosphatidylinositol 3-kinase (PI3K) and the Src family of protein kinases. It thus plays an important role in cell motility, invasion, growth and cell survival. Y15 (1,2,4,5-benzentetraamine tetra hydrochloride) is a small molecule that inhibits FAK autophosphorylation by selectively targeting the Y397 phosphorylation site of FAK. It has shown activity against breast, colon and pancreatic cancers. We herein present data in lung cancer. Methods: Various lung cancer cell lines as well as minimally passaged patient-derived tumor (containing PIK3CA mutation E545K) propagated in vitro were treated with escalating doses of Y15 or DMSO for 72 hours to evaluate single-agent activity. Cell lines were also treated with cytotoxic agents (cisplatin, gemcitabine, pemetrexed, paclitaxel) in combination with escalating doses of Y15. Cellular viability was assessed by trypan blue exclusion assay and colony formation assay. For in vivo experiments, selected cell lines were implanted into nude mice and vehicle versus Y15 treatment, via either intraperitoneal or oral gavage administration, once daily was started when tumors reached a mean volume of 50–60 mm3. Western blot will be performed to investigate alterations in the activity of various signaling proteins. Results: Y15 exhibited time and dose-dependent cell death in 6 out of 9 lung cancer cell lines tested. Activity was demonstrated beginning at 4 uM dose, with almost 100% cell kill demonstrated at 10 uM dose. There was also corresponding dose-dependent loss of clonogenic ability starting at 1 uM dose in sensitive cell lines. In addition, Y15 enhanced paclitaxel-induced cell death. Mice treated with Y15 at 100 mg/kg oral gavage administration showed reduced tumor growth compared to control mice. Conclusion: Y15 demonstrates promising antitumor efficacy against various non-small cell lung cancer cell lines. Further development of this agent in lung cancer is warranted. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2011 Nov 12-16; San Francisco, CA. Philadelphia (PA): AACR; Mol Cancer Ther 2011;10(11 Suppl):Abstract nr C191.

Abstract B3: Anti-angiogenic properties of FAK-VEGFR3 scaffold inhibitors, C9 and C10.

Introduction: Focal adhesion kinase (FAK) and vascular endothelial growth factor receptor 3 (VEGFR3) are tyrosine kinases, which function as key modulators of survival and metastasis signals in cancer cells. We have previously shown that VEGFR3 binds to the FAT domain of FAK and this interaction promotes the survival of tumor cells. We developed novel small molecule compounds C9 and C10 which specifically bind and inhibit the FAK-VEGFR3 interaction. Both these compounds inhibit cancer cell proliferation, induce apoptosis, disrupt the FAK-VEGFR3 signaling axis, and significantly inhibit tumor growth in xenograft mouse models of breast and pancreatic cancer. Since FAK and VEGFR3 have been implicated in tumor angiogenesis, we investigated the potential anti-angiogenic effects of C9 and C10. Methods: Binding of C9 and C10 to the FAT domain of FAK was tested using fluorescence polarization. Effects of C9 and C10 were tested in vitro by MTS assay (viability) and tube formation assay using HUVEC cells. Directed in vivo angiogenesis assay (DIVAA) kit was used to determine anti-angiogenic effects of both compounds. Single agent efficacy of C9 and C10 was tested in a pancreatic xenograft mouse model for 30 days. Mouse tumor tissues were analyzed for markers of proliferation (Ki67), endothelial vessels (CD31), and lymphangiogenic vessels (LYVE1) using immunohistochemistry. Results: Fluorescence polarization confirmed that C9 and C10 effectively bind to the FAT domain of FAK. C9 and C10 reduced cell viability of HUVEC cells and dramatically decreased tube formation by reducing total branching points, total loops and total tube length in a dose and time dependent manner. DIVAA assay confirmed that C9 and C10 significantly reduced the formation of endothelial blood vessels as compared to untreated groups. In vivo, 30 days treatment with low doses of C9 and C10 as single agents led to 40% to 45% reduction in tumor volume (P Conclusions: We have shown that targeting the FAK-VEGFR3 protein protein interaction site allows for dual inhibition of tumor proliferation as well as tumor angiogenesis. This study highlights the importance of oral-based small molecule inhibitors that might impact the therapeutic management of solid tumors. Citation Information: Mol Cancer Ther 2013;12(11 Suppl):B3. Citation Format: Priyanka A. Agharkar, Manivannan Ethirajan, Jianqun Liao, Michael Yemma, Brian Buckley, Mikhail Chernov, William Cance, Ravindra Pandey, Elena Kurenova. Anti-angiogenic properties of FAK-VEGFR3 scaffold inhibitors, C9 and C10. [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 B3.

Abstract A293: Novel allosteric small molecule FAK inhibitors effectively inhibit cancer cell viability and clonogenicity.

Introduction: Focal Adhesion Kinase is overexpressed and activated in many types of tumors and plays an important role in survival. Recently, we developed FAK inhibitor, 1,2,4,5-benzenetetraamine tetrahydrochloride, called Y15, which inhibited FAK autophosphorylation and blocked breast, neuroblastoma, pancreatic, brain and colon cancer tumor growth. Experimental procedures: In this study, eight novel chemical derivatives of Y15, called Y15-I1-I8 were synthesized and tested in a panel of different cancer cell lines. To test the effect on FAK autophosphorylation we performed in vitro ADP-Glo kinase assay with recombinant FAK protein and Western blotting with Y397-FAK antibody. To test the in vivo effect of FAK inhibitors we performed MTT assay, AnnexinV apoptosis and clonogenicity assays in different cancer cell lines. As a control, we used Novartis NVP-TAE-226 FAK inhibitor. Results: By in vitro kinase assay Y15-I1-8 derivatives effectively inhibited FAK autophosphorylation with recombinant purified full length FAK protein. Western blotting demonstrated decreased Y397-FAK and Y418-Src phosphorylation in many cancer cell lines. In addition, these novel FAK inhibitors demonstrated decreased viability, increased apoptosis and inhibition of clonogenicity in lung, melanoma, glioblastoma, pancreatic, breast, and colon cancer cell lines. Some of the novel derivatives were better than parental Y15 inhibitor. Conclusion: Targeting FAK autophosphorylation with novel allosteric derivatives of Y15 is an effective therapy approach. Citation Information: Mol Cancer Ther 2013;12(11 Suppl):A293. Citation Format: Vita M. Golubovskaya, Iryna Lebedyeva, Baotran Ho, Michael Yemma, Neha Singh, Makayla Arcara, David Ostrov, Alan Katritzky, William Cance. Novel allosteric small molecule FAK inhibitors effectively inhibit cancer cell viability and clonogenicity. [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 A293.

Abstract 3247: Novel small molecule inhibitors C9 and C10 specifically disrupt the FAK-VEGFR3 signaling axis in cancer cells.

Introduction: FAK functions as both, a critical signaling kinase and a scaffolding protein to collectively mediate proliferation, survival, cell adhesion and migration signals. VEGFR3 is a receptor tyrosine kinase that acts as a key modulator of survival signals and lymphangiogenesis. Upregulation of FAK and VEGFR3 in multiple tumor types, with low levels in normal tissues make them attractive anti-cancer targets. Previously we have shown that VEGFR3 binds to FAK and the physical interaction between the two proteins confers a significant survival advantage to tumor cells. Subsequently we discovered small molecule C4 that specifically targets the VEGFR3 binding site on the FAT domain of FAK and exerts anti-tumor effects at moderate concentrations. In this study we designed, synthesized and characterized novel C4 analogs which show enhanced potency and specificity for the target site. Methods: Modifications were made on the C4 moiety and the resulting analogs were screened for anti-cancer inhibitory effects in different cancer cell lines by MTS assay. Effects of C4 analogs on cancer cell lines were determined by MTS assay (viability), Clonogenecity assay, Western blot (phosphorylation, apoptosis), Immunofluorescence staining (apoptosis) and Flow cytometry (apoptosis). Computer modeling, target specificity (immunoprecipitation) and binding confirmation (Forte-Bio9s Octet Red assay) of all analogs were tested. In vitro wound healing and cell invasion assays were also performed. In vivo anti-tumor activity of lead analogs was done using a subcutaneous xenograft mouse model of pancreatic cancer. Results: Based on the preliminary screening results in several cancer cell lines, we selected analogs C9 and C10 for further analyses. Both analogs inhibit colony formation of pancreatic cancer cells and show target specificity by disrupting FAK-VEGFR3 interaction at ≤10μM. Binding of C9 and C10 to the FAT domain of FAK was confirmed with Octet method. Induction of apoptosis after treatment with C9 and C10 was confirmed via caspase 3/7 activation, caspase-3 and PARP cleavage. Down-regulation of phosphorylated forms of Akt, Erk1/2, Jnk, Grb2 and paxillin was seen in a dose and time dependent manner after 24h treatment with C9 and C10. These drugs also inhibited migration and invasion of pancreatic cancer cells at ≤10μM. In vivo, 30 days treatment with low doses of C9 and C10 led to massive reduction in tumor growth when treated as single agents (P Conclusions: FAK-VEGFR3 targeted therapeutics represents a novel approach towards anti-cancer treatment options. Our promising results thereby form the basis for future clinical applications of FAK-VEGFR3 small molecule inhibitors for cancer therapy. Citation Format: Priyanka A. Agharkar, Manivannan Ethirajan, Andrew Magis, Jian Liao, Michael Yemma, William Cance, Ravindra Pandey, Elena Kurenova. Novel small molecule inhibitors C9 and C10 specifically disrupt the FAK-VEGFR3 signaling axis in cancer 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 3247. doi:10.1158/1538-7445.AM2013-3247

Abstract 3756: Structure activity relationship studies of novel FAK inhibitor C4

Introduction: FAK and VEGFR3 have stirred considerable interest in recent years as attractive anti-cancer targets, owing to their high expression across several tumor types and relatively low levels in normal tissues. Our lab has shown that VEGFR3 binds to FAK and the physical interaction between the two proteins confers a significant survival advantage to tumor cells. We discovered small molecule C4 that specifically targets and disrupts FAK-VEGFR3 interaction to exert anti-tumor effects, but does so at moderate concentrations. This prompted us to design C4 analogs that display enhanced potency, minimum toxicity and high specificity for the target site. Here, we have shown that some C4 analogs disrupt FAK-VEGFR3 interaction and dramatically inhibit pancreatic tumor growth at low concentrations as single agents as well as in combination with Gemcitabine. Methods: The structure of C4 was modified at various positions by substituting alkyl and aryl moieties. The resulting analogs were screened for anti-cancer inhibitory effects in different cancer cell lines by MTS assay. Effects of C4 analogs on pancreatic cancer cell lines were determined by MTS assay (viability), Clonogenecity assay, Western blot (phosphorylation, apoptosis) and TUNEL assay. Target specificity (immunoprecipitation) and binding confirmation (Forte-Bio9s Octet Red assay) of all analogs were performed. In vivo anti-tumor activity of lead analogs was done using a subcutaneous xenograft mouse model of pancreatic cancer. Results: Twenty C4 analogs were screened and four were selected for further analyses. These four analogs strikingly decreased survival of six pancreatic cancer cell lines. Analog C9 showed highest cytotoxic effects, inhibited colony formation of Panc-1 cells at 1µM, induced dose dependent cleavage of caspase-3 and PARP and decreased phosphorylated forms of Akt and Erk within 24h treatment. Disruption of FAK-VEGFR3 interaction was seen with 1µM C9. Octet method revealed that 20µM C9, KD (M) = 1.29×10-7. In vivo, 30 days treatment with low doses of the four analogs led to massive reduction in tumor growth when treated as single agents (60%) and in combination with Gemcitabine (80%)(P Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 3756. doi:1538-7445.AM2012-3756