Focal Adhesion Kinase - Tumor Biology and Therapeutics

Abstract public domain (NIH RePORTER)

DESCRIPTION (provided by applicant): Focal Adhesion Kinase (FAK) is a 125kDa protein tyrosine kinase that is overexpressed in a number of different types of solid tumors and has emerged as a major cancer therapeutic target. We have demonstrated that FAK functions as a survival signal for the tumor cells to suppress apoptosis as they develop the capacity for invasion and metastasis. FAK provides these survival signals through kinase-dependent and kinase-independent scaffolding functions by interacting with other proteins along its large carboxy-C-terminal and amino-N-terminal scaffolding domains. During this period of support, we have identified three sites on FAK that are critical for its kinase activity and survival function, each of which has the potential for developing novel therapeutics that target FAK. These sites are as follows: the FAK-vascular endothelial growth factor receptor-3 (VEGFR-3) interaction in the C-terminal domain, the Y397 autophosphorylation site of FAK in the N- terminal domain, and the FAK-p53 interaction in the N-terminal domain. We have developed specific inhibitors for each of these interactions and will use them to test hypotheses about the survival and kinase functions of FAK. We hypothesize that disrupting the interaction of FAK and VEGFR-3 will increase apoptosis in cancer cells, inhibit angiogenesis, lymphangiogenesis, and metastasis (Aim 1). We also hypothesize that targeting the Y397 site will inhibit the autophosphorylation kinase function of FAK without disrupting the ATP-binding site (Aim 2). We hypothesize that FAK sequesters p53 from its normal function and that disrupting interactions between FAK and p53 will increase apoptotic signals in cells (Aim 3). The first specific aim will determine the biological efficacy of our small molecule FAK /VEGFR-3 inhibitor, C4 or chloropyramine, and its derivatives that target the FAK-VEGFR-3 interaction. We will study the binding of C4 to FAK by ITC and co-crystallization analyses. We will perform structure-activity relationship studies (SAR) of C4 for the specific FAK-VEGFR-3 binding site. We will test off-target effects of C4 and derivatives on adjacent binding partners and downstream FAK effectors. We will determine the effect of C4 and derivatives in vivo in orthotopic models of human breast and pancreatic cancer on angiogenesis, lymphangiogenesis, and metastasis and will develop a pharmacokinetic (PK) model. The second specific aim will target the autophosphorylation kinase activity of FAK by blocking its Y397 autophosphorylation site. We have identified another small molecule lead inhibitor, Y15, that specifically blocks the Y397 FAK autophosphorylation site without targeting the ATP-binding site of the kinase. We will determine if Y15 directly binds to FAK by ITC and co-crystallization analyses. We will determine if chemical modifications of Y15 (derivatives) have stronger inhibition of FAK autophosphorylation and directly bind to FAK. We will test the biological and off-target effects of Y15 and derivatives on SH-2 domain-containing proteins (Src and PI3-kinase) that bind to FAK, other kinases and other FAK downstream targets. We will test Y15 and derivatives efficacy in orthotopic models of human breast and pancreatic cancer in vivo. The third specific aim will test our hypothesis that FAK sequesters p53 from its normal pro-apoptotic function. We will define the p53 binding site on the N-terminal domain of FAK by co-crystallization with the N-terminal domain. We will test the downstream biological effects of the FAK-p53 sequestration model using site-directed mutagenesis and synthetic peptides that target this interaction in vitro. We will detect the FAK-p53 binding site in mice and determine the functional significance of the FAK-p53 binding in vivo by generating knock-in mice with mutation of the FAK-p53 binding site.

Abstract public domain (NIH RePORTER)

The research proposed in this application will define specific mechanisms of the survival function of FAK and develop novel small molecule inhibitors that target the survival and kinase functions of FAK as a prelude to novel anti-cancer therapeutics

Public health relevance public domain (NIH RePORTER)

PUBLIC HEALTH RELEVANCE: This research will help to develop novel anti-cancer therapeutics using focal adhesion kinase (FAK) as a target. The research proposed in this application will validate FAK protein-protein interactions (scaffolding function) as therapeutic targets. It will provide novel small molecule inhibitors that target the FAK-VEGFR-3 and FAK-p53 interactions. It also provides a novel approach for targeting FAK kinase function by developing small molecule inhibitors that target the major autophosphorylation site of FAK (Y397). Thus, this application will provide novel molecular cancer therapeutics that target the focal adhesion kinase. PROJECT NARRATIVE This research will help to develop novel anti-cancer therapeutics using focal adhesion kinase (FAK) as a target. The research proposed in this application will validate FAK protein-protein interactions (scaffolding function) as therapeutic targets. It will provide novel small molecule inhibitors that target the FAK-VEGFR-3 and FAK-p53 interactions. It also provides a novel approach for targeting FAK kinase function by developing small molecule inhibitors that target the major autophosphorylation site of FAK (Y397). Thus, this application will provide novel molecular cancer therapeutics that target the focal adhesion kinase. __SpecificAimsTextDelimiter__ A. SPECIFIC AIMS Focal Adhesion Kinase (FAK) is a protein tyrosine kinase that is overexpressed in a number of solid tumors and has emerged as a major cancer therapeutic target. FAK provides survival signals to tumors through kinase-dependent and kinase-independent scaffolding functions by interacting with other proteins along its large carboxy-C-terminal and amino-N-terminal scaffolding domains. During this period of support, we have identified three sites on FAK that are critical for its kinase activity and survival function, each of which has the potential for developing novel therapeutics that target FAK: the FAK-vascular endothelial growth factor receptor-3 (VEGFR-3) interaction in the C-terminal domain (Aim 1), the Y397 autophosphorylation site of FAK in the N-terminal domain (Aim 2), and the FAK-p53 interaction in the N- terminal domain (Aim 3). We have developed specific inhibitors for each of these interactions and will use them to test hypotheses about the survival and kinase functions of FAK, and in Aims 1 and 2, to develop further preclinical data on their efficacy in breast and pancreatic cancer. We hypothesize that disrupting the interaction of FAK and VEGFR-3 will increase apoptosis in cancer cells and decrease tumorigenesis, lymphangiogenesis, and metastasis (Aim 1). We also hypothesize that targeting the Y397 site will inhibit the kinase function of FAK without disrupting the ATP-binding site and will prevent SH-2 proteins from binding to FAK (Aim 2). We have proposed a model for this scaffolding function whereby FAK also sequesters pro- apoptotic proteins, such as p53 (Science Signaling, 2008) to prevent their normal function. We hypothesize that FAK sequesters p53 from its normal function and that disrupting interactions between FAK and p53 will increase apoptotic signals in cells (Aim 3). We will test these hypotheses in our in vitro and in vivo model systems by performing the following specific aims: Aim 1. We will determine the biological efficacy of our small molecule FAK/VEGFR-3 inhibitor. VEGFR-3 is a critical mediator of lymphangiogenesis and directly binds to the C-terminus of FAK. We have identified small molecule C4 (chloropyramine) through in silico screening of a chemical library of small molecules and determined that C4 is an inhibitor of VEGFR-3 and FAK. Preliminary data have shown that C4 caused apoptosis in cancer cells in vitro, and inhibited tumor growth in subcutaneous mouse tumor xenograft models. We hypothesize that C4 directly binds to FAK, disrupts the FAK-VEGFR-3 interaction, and will inhibit

Public health relevance public domain (NIH RePORTER)

lymphangiogenesis and metastasis in tumor model systems. To test this, we will: A. Analyze the binding of small molecule C4 to FAK by isothermal titration calorimetry (ITC) and co- crystallization of C4 with the C-terminal domain of FAK B. Perform structure-activity relationship studies (SAR) of the C4 moiety for the specific FAK-VEGFR-3 binding site with a goal to find more potent agents with improved target-specificity and antitumor activity. C. Test off-target effects of C4 and derivatives on adjacent binding partners and downstream FAK effectors. D. Analyze the effect of C4 in vivo in orthotopic models of human breast and pancreatic cancer to test the effect on angiogenesis, lymphangiogenesis, and metastasis and develop a pharmacokinetic (PK) model. Aim 2. We will target the Y397 autophosphorylation site of FAK to affect its kinase-dependent function. Through our similar in silico screening approach, we have identified another small molecule lead compound (Y15) that effectively abrogated FAK kinase autophosphorylation activity at its Y397 site, without specifically targeting the ATP binding site. We hypothesize that this approach will provide more specific inhibition of kinase activity than other approaches that target the ATP binding site. We will use compound Y15 to: A. Determine if it directly binds to FAK at the Y397 site using ITC and co-crystallization analyses. B. Determine if chemical modifications of Y15 (derivatives) have stronger inhibition of FAK autophosphorylation and directly bind to FAK. C. Determine the biological and off-target effects of Y15 and derivatives on: SH-2 domain-containing proteins (Src and PI3-kinase) that bind to FAK, other kinases, and FAK downstream targets. D. Determine if Y15 and derivatives have efficacy in orthotopic models of human breast and pancreatic cancer with less toxicity than other FAK kinase inhibitors. Aim 3. We will determine whether FAK sequesters p53 from its normal pro-apoptotic function. We have demonstrated a direct interaction of the FAK-N-terminal domain and p53 and have identified the 7 amino-acid binding site inside p53 that binds to FAK. In this Aim, we will determine the biological significance of this interaction in vitro and in transgenic in vivo animal models. We hypothesize that targeting the FAK-p53 interaction and disrupting FAK-p53 binding will release the sequestration of p53, and activate p53 pro- apoptotic pathways. We will use our inhibitors of this binding site to test this hypothesis as follows: A. Define the p53 binding site on the N-terminus of FAK by co-crystallization with the N-terminal domain. B. Test the downstream biological effects of the FAK-p53 sequestration model using site-directed mutagenesis and synthetic peptides that target this interaction in vitro. C. Detect the FAK-p53 binding site in mice and determine the functional significance of the FAK-p53 binding in vivo by generating knock-in mice with mutation of the FAK-p53 binding site.