Potent Focal Adhesion Kinase Research Articles

Focal Adhesion Kinase As a Potential Target in AML and MDS

Focal adhesion kinase (FAK), a non-receptor tyrosine kinase is overexpressed/activated in several solid cancers. It controls cell growth, survival, invasion, metastasis, cell movement, gene expression, as well as stem cell self-renewal. Integrin ligation and growth factor/receptor interaction activate FAK leading to SRC phosphorylation and subsequent FAK/SRC phosphorylation at multiple sites which relays the external signal into cells by activating multiple cell proliferating/survival pathways. Although it is extensively studies in solid tumors, the expression and function of FAK in myeloid leukemia are not well investigated. We determined FAK expression by reversed-phase protein array in samples from a large cohort of newly diagnosed AML (n = 511) and MDS (n = 279). We found that in AML, FAK expression is associated with unfavorable cytogenetic group (P = 2x10-4). The lowest expression was seen in patients with inv16 (n = 19), t8;21 (n = 15) and t15;17 (n = 20) chromosome translocations. Patients with -5, -7, +8 (n = 100) expressed relatively higher FAK. FAK expression is higher in relapsed compared to paired newly diagnosed samples (n = 47, P = 0.02). FAK expression was significantly lower in patients with FLT-ITD (n=83, P = 0.0024) or RAS mutation (n = 64, P = 0.05) suggesting functional redundancy of these signaling pathways and therefore mutually exclusive expression patterns. FAK expression is highly correlated with SRC/p-SRC and ITGb3/ITGa2 levels among over 200 proteins analyzed suggesting a role of intergrin/FAK/SRC signal in AML cells. We also observed overexpression of FAK in MDS. FAK expression levels were significantly higher in both CD34+ (P = 5.42e-20) and CD34+ CD38- MDS cells (P = 7.62e-9) than in normal CD34+ cells (n = 16). Patients with higher expression of FAK in CD34+ cells had a trend towards better overall survival (P = 0.05) in newly diagnosed MDS. Furthermore, we determined the FAK mRNA expression levels in bone marrow CD34+ cells from 63 patients (57 MDS and 6 AML). In this cohort of patients, fifty (79%) were previously untreated with chemotherapy (46 MDS and 4 AML). No significant difference in FAK gene expression was observed when comparing patients with or without prior chemotherapy in either MDS or AML patients. Aberrant up-regulation (≥ 2 fold) was observed in 23% and 50% of the patients respectively compared to normal CD34+ cells (n = 5). In patients who received no prior chemotherapy (n = 50), aberrant up-regulation (≥2 fold) was observed in 22% and 50% of the patients respectively. FAK protein levels by immunohistochemical analysis were correlated with mRNA levels.Growth factor GM-CSF and co-culture with mesenchymal stem cells (MSCs) increased the expression of FAK in leukemia cells suggesting that the microenvironment modulates leukemia cell function in part via activating FAK signaling. Inhibition of FAK with VS-4718, a potent selective FAK inhibitor decreased viable cells and induced apoptosis in various AML cell lines and in co-cultures with MSCs. Inhibition of FAK decreased adhesion and migration of OCI-AML3 to MSCs suggesting FAK signaling promotes leukemia/stromal interactions. Furthermore, VS-4718 induced cell death in bulk, CD34+, and CD34+ CD38- cells from patients with AML even in co-cultures with MSCs.In conclusion, our results demonstrate that FAK is expressed in AML patient samples and that high expressions associate with unfavorable patient characteristics. Overexpression of FAK in MDS suggests that FAK signaling may be involved in pathogenesis of the disease. FAK, activated by tumor microenvironment, supports the survival of leukemia cells and in turn promotes leukemia/stromal interaction. FAK inhibition can induce apoptosis in leukemia cells. FAK may hence be a potential therapeutic target in AML and MDS. DisclosuresCarter:PrismBiolab: Research Funding. Andreeff:Oncoceutics, Inc.: Membership on an entity's Board of Directors or advisory committees.

VS-4718, a Potent Focal Adhesion Kinase (FAK) Inhibitor, Exhibits Anticancer Activity in Leukemia Models in Vitro and in Vivo

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

Vs-6063 (Defactinib) Targets Mesothelioma Cancer Stem Cells Directly and Through Inhibition of Tumor-Associated Macrophages and Cytokine Production

ABSTRACT Aim: Focal adhesion kinase (FAK) is a non-receptor tyrosine kinase required for survival of cancer stem cells (CSCs). PYK2, the other FAK family member, shares 60% kinase domain homology. Based on the observation of reduced macrophage infiltration in PYK2 knockout mice, we investigated effects of PYK2 inhibition on tumor-associated macrophages (TAMs). Abundance of TAMs has been correlated with poor prognosis in multiple cancer types including mesothelioma. Defactinib is a potent, orally active FAK kinase inhibitor that has completed phase I and is currently in clinical development in several indications including mesothelioma. Methods: Cytokine release from human macrophages was measured in response to small molecule FAK kinase inhibitors. Serum IL-6 and F4/80-positive macrophages were assessed by ELISA and immunofluorescence respectively. Results: In addition to potent FAK inhibition, defactinib also inhibits PYK2 kinase activity with nanomolar potency. Using both FAK inhibitors and FAK siRNA, we demonstrated that FAK inhibition effectively reduces CSCs. Human monocyte-derived macrophages release IL-6 and IL-8, and these cytokines increased the proportion of ALDH-positive CSCs in mesothelioma and breast cancer cell lines. Defactinib dose-dependently inhibited production of IL-6 and IL-8, whereas a FAK-only reference inhibitor had no effect on these cytokines. Accordingly, PYK2 siRNA decreased IL-6 and IL-8 release from human macrophages. In a xenograft model in vivo, defactinib reduced serum IL-6 while the FAK-only reference compound did not. Furthermore, defactinib substantially reduced the number of F4/80-positive macrophages in the tumors. Conclusions: These data suggest that dual targeting of FAK and PYK2 by defactinib may more effectively suppress CSCs than inhibition of FAK alone. Given the sensitivity of mesothelioma CSCs to defactinib, the use of this agent in a maintenance setting after first line chemotherapy may be an attractive strategy to extend survival of patients with mesothelioma. These data support the ongoing COMMAND study with defactinib in mesothelioma (Clinical trial NCT01870609). Disclosure: M. Keegan: Employee of Verastem, Inc. Stock Ownership in Verastem, Inc.; J.E. Ring: Employee of Verastem, Inc. Stock Ownership in Verastem, Inc.; V.N. Kolev: Employee of Verastem, Inc. Stock Ownership in Verastem, Inc.; I.M. Shapiro: Employee of Verastem, Inc. Stock Ownership in Verastem, Inc.; M.V. Padval: Employee of Verastem, Inc. Stock Ownership in Verastem, Inc.; Q. Xu: Employee of Verastem, Inc. Stock Ownership in Verastem, Inc.; J.A. Pachter: Employee of Verastem, Inc. Stock Ownership in Verastem, Inc.

Design, synthesis, and biological evaluation of novel FAK scaffold inhibitors targeting the FAK–VEGFR3 protein–protein interaction

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. Previously, we reported that small molecule chlorpyramine hydrochloride (C4) specifically targets the interaction between FAK and VEGFR3 and exhibits anti-tumor efficacy. In this study, we designed and synthesized a series of 1 (C4) analogs on the basis of structure activity relationship and molecular modeling. The resulting new compounds were evaluated for their binding to the FAT domain of FAK and anti-cancer activity. Amongst all tested analogs, compound 29 augmented anti-proliferative activity in multiple cancer cell lines with stronger binding to the FAT domain of FAK and disrupted the FAK–VEGFR3 interaction. In conclusion, we hope that this work will contribute to further studies of more potent and selective FAK–VEGFR3 protein–protein interaction inhibitors.

Abstract A31: Merlin loss as a biomarker for defactinib (VS-6063) sensitivity: High frequency in malignant mesothelioma tumors.

Abstract Malignant pleural mesothelioma (MPM) is an aggressive tumor in the lining of the lung often caused by asbestos exposure. With a median survival on standard of care chemotherapy of only 12 months from diagnosis, new therapeutic modalities and predictive biomarkers are urgently needed for MPM patients. The Neurofibromatosis 2 (NF2) tumor suppressor gene that encodes the protein merlin is disrupted in approximately 50% of MPMs. The role of merlin in cell adhesion, invasion and cell motility may be partially mediated through the focal adhesion kinase (FAK), an important signaling node downstream of integrin cell adhesion proteins. An examination of 15 mesothelioma cell lines grown in 3-dimensional Matrigel culture demonstrates that low merlin by immunoblotting correlated with increased sensitivity to the FAK inhibitor, defactinib (VS-6063). An immunohistochemistry (IHC) test for merlin may be an optimal clinical tumor specimen measurement for merlin loss, provided that merlin reduction or loss is correlated with other indicators of NF2 change. The presence or absence of merlin and NF2 was evaluated using MPM patient-derived xenograft (PDX) tumor models using merlin IHC and an NF2 Fluorescence In situ Hybridization (FISH), as well as Western blotting to determine merlin levels in the tumor lysates. The PDX MPM-0235 tumor had low merlin by Western blotting, merlin IHC, and NF2 FISH, whereas PDX MPM-0237 showed high merlin levels by Western blotting, merlin IHC, and diploid chromosome 22 by FISH. In addition to PDX analysis, Merlin IHC and NF2 FISH optimized by these conditions was evaluated with in patient MPM Tissue Microarrays (TMAs) of 64 primary tumors. Chromosome 22 monosomy was significantly more frequent than NF2 regional deletion of chromosome 22. We observed that 42% of the tumors had a low merlin IHC score (merlin-low) that correlated with loss of NF2 (unpaired t-test, p&amp;lt;0.029). In a second cohort of archived tumor specimens from 300 MPM patients treated at one clinical site, we observed a similar association between NF2 loss/deletion by FISH and merlin-low IHC (unpaired t-test, p=0.0001). While phosphoFAK Y397 IHC (an indicator of FAK activation) have high IHC values in some tumors that are merlin-low, these two marker trends were not statistically associated for all the tumors in the cohort. These data support the clinical development of a merlin IHC test for evaluation of NF2-mutated malignant mesothelioma. Merlin IHC will be used as a predictive biomarker for responsiveness to defactinib, a selective and potent FAK inhibitor under investigation in a Phase 2 registration-directed clinical trial for patients with MPM. Citation Information: Mol Cancer Ther 2013;12(11 Suppl):A31. Citation Format: David T. Weaver, Irina Shapiro, Christian Vidal, Qunli Xu, Mahesh Padval, Jonathan Pachter, Daniel Paterson, Jullianne Barlow, William Richards, Sabina Signoretti, Raphael Bueno. Merlin loss as a biomarker for defactinib (VS-6063) sensitivity: High frequency in malignant mesothelioma tumors. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2013 Oct 19-23; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2013;12(11 Suppl):Abstract nr A31.

Synthesis, biological evaluation, and molecular docking studies of novel 1,3,4-oxadiazole derivatives possessing benzotriazole moiety as FAK inhibitors with anticancer activity

1,3,4-Oxadiazole derivatives have drawn continuing interest over the years because of their varied biological activities. In order to search for novel anticancer agents, we designed and synthesized a series of new 1,3,4-oxadiazole derivatives containing benzotriazole moiety as potential focal adhesion kinase (FAK) inhibitors. All the synthesized compounds were firstly reported. Among the compounds, compound 4 shows the most potent inhibitory activity against MCF-7 and HT29 cell lines with IC50 values of 5.68μg/ml and 10.21μg/ml, respectively. Besides, all the compounds were assayed for FAK inhibitory activity using the TRAP–PCR–ELISA assay. The results showed compound 4 exhibited the most potent FAK inhibitory activity with IC50 values of 1.2±0.3μM. Docking simulation by positioning compound 4 into the FAK structure active site was performed to explore the possible binding mode. Apoptosis which was analyzed by flow cytometry, demonstrated that compound 4 induced apoptosis against MCF-7 cells. Therefore, compound 4 may be a potential anticancer agent against MCF-7 cancer cell.

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

Abstract LB-192: The FAK inhibitors VS-4718 and VS-5095 attenuate breast cancer stem cell function in vitro and tumor growth in vivo

Abstract As a key mediator of integrin signaling, focal adhesion kinase (FAK) regulates cellular responses to extracellular matrix interactions. Amplification and overexpression of FAK have been observed in aggressive human cancers including breast cancer. FAK has been implicated in multiple steps in carcinogenesis including tumor growth, metastasis and angiogenesis. We now demonstrate the importance of FAK in breast cancer stem cell function, and the reduction of cancer stem cell function by the selective FAK inhibitors VS-4718 and VS-5095. VS-4718 and VS-5095 are potent and selective FAK inhibitors which were optimized following high throughput screening. Both VS-4718 and VS-5095 block fibronectin-stimulated FAK autophosphorylation of Tyr397 with low nanomolar cellular potency and are highly selective for FAK among a panel of protein kinases. Consistent with their mechanism of action, VS-4718 and VS-5095 showed greater inhibitory potency on the growth of multiple cancer cell lines in 3D matrigel culture as compared to conventional 2D culture. To determine if FAK plays a role in the biology of breast cancer stem cells in addition to its reported function in normal mammary stem cell biology, the effects of these FAK inhibitors were characterized using two different in vitro assays. It was previously demonstrated that immortalized mammary epithelial cells (HMLEs) driven to undergo epithelial to mesenchymal transition (EMT) by knockdown of E-cadherin (HMLE-shECad) exhibit many of the characteristics of cancer stem cells and can be used to identify agents that selectively target cancer stem cells. VS-4718 exhibited 25-fold greater potency against proliferation of mesenchymal HMLE-shECad cells as compared to epithelial HMLE-shGFP control cells, suggesting preferential effects on breast cancer stem cells. Furthermore, pre-treatment of SUM159 triple negative breast cancer cells with VS-5095 in matrigel attenuated secondary tumorsphere formation, suggesting that FAK is important for the self-renewal function of breast cancer stem cells. The role of FAK in breast cancer stem cell renewal was further corroborated by the observation that FAK shRNA inhibited tumorsphere formation by SUM159 cells. The in vivo efficacy of the FAK inhibitor VS-5095 was evaluated in the MDA-MB-231 human triple negative breast cancer xenograft model. By oral administration, VS-5095 induced significant dose-dependent tumor growth inhibition. In summary, these results demonstrate the importance of FAK in the self-renewal of breast cancer stem cells, and support the clinical development of the selective FAK inhibitors VS-4718 and VS-5095 to target breast cancer stem cells for the treatment of triple negative breast cancer. 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 LB-192. doi:1538-7445.AM2012-LB-192

Pharmacokinetics (PK) of PF-562271, a focal adhesion kinase (FAK) inhibitor, and its effect on CYP3A in patients with advanced nonhematologic malignancies.

2553 Background: FAK transduces signaling from integrins and growth factors to modulate tumor cell invasion, proliferation, and survival. PF-562271, a potent FAK inhibitor, is under development as an anticancer agent. Preclinical studies demonstrated that PF-562271 is an inhibitor of its primary metabolizing enzyme, CYP3A. PF-562271 PK and its effect on CYP3A were investigated in a dose-escalation trial in patients with nonhematologic malignancies. Methods: Escalating doses of PF-562271 were administered orally to a total of 67 patients twice a day (BID) or once a day (QD), with or without food. Serial serum samples were collected after the first dose and at steady state to determine PK of PF-562271. To evaluate the effect of PF-562271 on CYP3A activity, plasma PK of orally administered midazolam were determined prior to PF-562271 administration and after repeated PF-562271 dosing to steady state in 8 patients who received the recommended phase 2 dose of 125 mg BID. Serum PF-562271 and plasma midazolam concentrations were determined using validated HPLC-MS/MS methods. The concentration-time data for PF-562271 and midazolam were analyzed by noncompartmental methods. Results: After a single oral dose, PF-562271 was readily absorbed with the maximum serum concentration achieved in 0.5 to 6 hours. The increases in systemic exposure (AUCinf) were dose proportional over the 5 to 25 mg range, but greater than dose proportional from 35 to 225 mg. After multiple oral doses, PF-562271 demonstrated nonlinear accumulation in exposure; the mean ratio of steady state AUC during a dose interval to AUCinf after the first dose ranged from 1.3 to 5.3, indicating time-dependent PK. Similar PK parameters for PF-562271 were observed at comparable doses given with or without food. When co-administered, PF-562271 (125 mg BID) increased total midazolam plasma exposure approximately 5.9-fold. Conclusions: PF-562271 displayed time- and dose-dependent nonlinear PK, and is a potent CYP3A inhibitor. The nonlinear PK of PF-562271 likely resulted from auto-inhibition of CYP3A. In the limited number of subjects evaluated, food did not substantially alter the PK of PF-562271. Author Disclosure Employment or Leadership Position Consultant or Advisory Role Stock Ownership Honoraria Research Funding Expert Testimony Other Remuneration Pfizer Pfizer

Abstract A86: Design, synthesis, and SAR of focal adhesion kinase (FAK) inhibitors

Abstract Focal adhesion kinase (FAK) is a non-tyrosine kinase that localizes to focal adhesion plaques. It is activated in response to intergin binding to cellular ligands and when phosphorylated inhibits anoikis allowing for anchorage independent cell growth. Recent studies have shown increased FAK expression and phosphorylation status in many types of invasive and aggressive human tumors strongly suggesting FAK is a possible target for anticancer chemotherapy. Literature, in house HTS and de novo studies identified 2, 4-diaminopyrimidines as potent FAK inhibitors. Early SAR efforts quickly determined that smaller substituents, particularly CF3, were optimal in the C5 position. Parallel medicinal chemistry strategies were executed for the C2 and C4 positions. These studies suggested that substituted aryl and fused heteroaryl groups at the C2 position in conjunction with substituted phenyl and heterocycles at the C4 position imparted optimum activity and metabolic stability. Inhibitor-FAK co-crystal structures were utilized to guide in the SAR strategy around the 2, 4-diaminopyrimidine template which afforded several lead compounds. The team's effort culminated in the advancement of PF-562,271 as a potent and reversible inhibitor of FAK (kinase IC50 of 2 nM and cell IC50 of 5 nM) that is &amp;gt; 100x selective against a long list of non-target kinases. In summary, detailed SAR studies were executed on the 2, 4-diaminopyrimidine templates that produced potent inhibitors of FAK with improved ADME properties, and identified a novel and potent series of FAK inhibitors that are selective against most other kinases and have shown activity in clinical trials. This poster will present design, synthesis, challenging chemistry, optimization, and complete inhibitor chemical structures of lead analogs. Citation Information: Mol Cancer Ther 2009;8(12 Suppl):A86.

3D cell cultures of human head and neck squamous cell carcinoma cells are radiosensitized by the focal adhesion kinase inhibitor TAE226

Background and purpose Focal adhesion kinase (FAK), a main player in integrin signaling and survival, is frequently overexpressed in human cancers and therefore postulated as potential target in cancer therapy. The aim of this study was to evaluate the radiosensitizing potential of the FAK inhibitor TAE226 in three-dimensional (3D) tumor cell cultures. Materials and methods Head and neck squamous cell carcinoma (HNSCC) cells (FaDu, UT-SCC15, UT-SCC45), lung cancer cells (A549), colorectal carcinoma cells (DLD-1, HCT-116) and pancreatic tumor cells (MiaPaCa2, Panc1) were treated with different concentrations of TAE226 (0–1 μm; 1 or 24 h) without or in combination with irradiation (0–6 Gy, X-ray, single dose). Subsequently, 3D clonogenic survival assays (laminin-rich extracellular matrix) and Western blotting (expression/phosphorylation, e.g. FAK, Akt, ERK1/2) were performed. Results All investigated 3D cell cultures showed a dose-dependent reduction in clonogenic survival by TAE226. Intriguingly, TAE226 only significantly radiosensitized 3D HNSCC cell cultures accompanied by a pronounced dephosphorylation of FAK, Akt and ERK1/2. Conclusions Our data demonstrate TAE226 as potent FAK inhibitor that enhances the cellular radiosensitivity particularly of HNSCC cells grown in a 3D cell culture model. Future in vitro and in vivo investigations will clarify, to which extent this approach might be clinically relevant for radiotherapy of HNSCC.

3′,4′‐Dihydroxyflavonol down‐regulates monocyte chemoattractant protein‐1 in smooth muscle: role of focal adhesion kinase and PDGF receptor signalling

We investigated the effects of a synthetic flavonol, 3',4'-dihydroxyflavonol (DiOHF) on the expression of monocyte chemoattractant protein-1 (MCP-1) in rat vascular smooth muscle cells. MCP-1 expression was assessed by quantitative real-time PCR and protein phosphorylation by immunoprecipitation and Western blots. DiOHF (1-30 micromol x L(-1)) concentration-dependently reduced MCP-1 expression in both quiescent cells and cells stimulated with platelet-derived growth factor (PDGF) or interleukin 1-beta. The effect of DiOHF was associated with a suppression of focal adhesion kinase (FAK)-mediated signalling. In vitro kinase assays demonstrated that DiOHF is a potent inhibitor of FAK kinase activity (EC(50)= 2.4 micromol x L(-1)). Expression of FAK-related non-kinase reduced basal MCP-1 expression, but not that induced by PDGF or interleukin 1-beta. DiOHF also inhibited autophosphorylation of PDGF receptors. The PDGF receptor inhibitor AG-1296 potently suppressed basal and PDGF-induced MCP-1 expression. Inhibition of extracellular signal-regulated kinase activation by DiOHF, either directly or indirectly, may also be involved in its effects on MCP-1 expression. DiOHF had no inhibitory effect on either p38 or nuclear factor-kappaB activation. Moreover, DiOHF inhibited smooth muscle cell spreading (a FAK-mediated response) and proliferation. This is the first report on a flavonoid compound (DiOHF) that is a potent FAK inhibitor. DiOHF also inhibits PDGF receptor autophosphorylation. These effects underlie the inhibitory action of DiOHF on MCP-1 expression in smooth muscle cells. Our results suggest that DiOHF might be a useful tool for dissection of the (patho)physiological roles of FAK signalling.

Anti-cancer activity of NVP-TAE226, a potent dual FAK/IGF-IR kinase inhibitor, against pancreatic carcinoma

13162 Background: Focal adhesion kinase (FAK) is a non-receptor cytoplasmic tyrosine kinase that regulates multiple cell functions. Elevated expression levels of FAK have been detected in various tumor samples and are closely correlated with invasive potential. Activation of integrins and the growth factor receptors result in FAK autophosphorylation at Y397 and the presentation of suitable binding sites for proteins containing either SH2 or phosphotyrosine binding domains. Recent evidences suggest that FAK plays important roles in cancer cell proliferation and survival. IGF-IR function is required for tumor cell survival, but dispensable for survival of normal cells. Therefore, a dual inhibitor of both kinases may selectively block the growth, migration, and survival of FAK- and IGF-IR- expressing tumor cells compared to proliferating and migrating normal cells. Methods: In this study, anti-cancer activity of NVP-TAE226 that is identified as a potent and selective FAK inhibitor was evaluated in cancer cell lines panel and MIA PaCa-2 pancreatic carcinoma in vivo model. Results: Mean GI50 value of NVP-TAE226 against 37 cancer cell lines was 0.76 μmole/L. Inhibition of cancer cell proliferation was not affected by expression of P-glycoprotein, suggesting that NVP-TAE226 is not served as a substrate of P-glycoprotein. Oral administration of NVP-TAE226 efficiently inhibited MIA PaCa-2 human pancreatic tumor growth at all doses tested. Tumor stasis was observed at a dose of 30 mg/kg, qd for 7×/week and tumor regression was observed at a dose of 100 mg/kg, qd for 5×/week. All animals tolerated NVP-TAE226 treatment up to 100 mg/kg, 5×/wk, qd, po for 2 weeks with no body weight loss. Inhibition of downstream signaling such as phosphorylation of Akt at Serine473 was accompanied by inhibition of FAK phosphorylation in human pancreatic carcinoma cell lines. Conclusions: NVP-TAE226 is a novel class of selective and small molecule kinase inhibitors with a potent in vivo activity and potential therapeutic application. No significant financial relationships to disclose.

Design and synthesis of 7H-pyrrolo[2,3-d]pyrimidines as focal adhesion kinase inhibitors. Part 2

A series of 2-amino-9-aryl-7H-pyrrolo[2,3-d]pyrimidines were designed and synthesized as focal adhesion kinase (FAK) inhibitors using molecular modeling in conjunction with a co-crystal structure. Chemistry was developed to introduce functionality onto the 9-aryl ring, which resulted in the identification of potent FAK inhibitors. In particular, compound 32 possessed single-digit nanomolar IC50 and represents one of the most potent FAK inhibitors discovered to date.