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. 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

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.

Agency
National Institute of Health (NIH)
Institute
National Cancer Institute (NCI)
Type
Research Project (R01)
Project #
5R01CA065910-17
Application #
8510585
Study Section
Developmental Therapeutics Study Section (DT)
Program Officer
Snyderwine, Elizabeth G
Project Start
1996-09-30
Project End
2015-07-31
Budget Start
2013-08-01
Budget End
2014-07-31
Support Year
17
Fiscal Year
2013
Total Cost
$289,079
Indirect Cost
$82,621
Name
Roswell Park Cancer Institute Corp
Department
Type
DUNS #
824771034
City
Buffalo
State
NY
Country
United States
Zip Code
14263
Zhang, Hao; Shao, Huanjie; Golubovskaya, Vita M et al. (2016) Efficacy of focal adhesion kinase inhibition in non-small cell lung cancer with oncogenically activated MAPK pathways. Br J Cancer 115:203-11
Wilton, John; Kurenova, Elena; Pitzonka, Laura et al. (2016) Pharmacokinetic analysis of the FAK scaffold inhibitor C4 in dogs. Eur J Drug Metab Pharmacokinet 41:55-67
Marlowe, Timothy A; Lenzo, Felicia L; Figel, Sheila A et al. (2016) Oncogenic Receptor Tyrosine Kinases Directly Phosphorylate Focal Adhesion Kinase (FAK) as a Resistance Mechanism to FAK-Kinase Inhibitors. Mol Cancer Ther 15:3028-3039
Stewart, Jerry E; Ma, Xiaojie; Megison, Michael et al. (2015) Inhibition of FAK and VEGFR-3 binding decreases tumorigenicity in neuroblastoma. Mol Carcinog 54:9-23
Golubovskaya, Vita; Curtin, Leslie; Groman, Adrienne et al. (2015) In vivo toxicity, metabolism and pharmacokinetic properties of FAK inhibitor 14 or Y15 (1, 2, 4, 5-benzenetetramine tetrahydrochloride). Arch Toxicol 89:1095-101
Gogate, Priyanka N; Kurenova, Elena V; Ethirajan, Manivannan et al. (2014) Targeting the C-terminal focal adhesion kinase scaffold in pancreatic cancer. Cancer Lett 353:281-9
Golubovskaya, Vita M; Ylagan, Lourdes; Miller, Austin et al. (2014) High focal adhesion kinase expression in breast carcinoma is associated with lymphovascular invasion and triple-negative phenotype. BMC Cancer 14:769
Golubovskaya, Vita M; Sumbler, Brittany; Ho, Baotran et al. (2014) MiR-138 and MiR-135 directly target focal adhesion kinase, inhibit cell invasion, and increase sensitivity to chemotherapy in cancer cells. Anticancer Agents Med Chem 14:18-28
Gogate, Priyanka N; Ethirajan, Manivannan; Kurenova, Elena V et al. (2014) Design, synthesis, and biological evaluation of novel FAK scaffold inhibitors targeting the FAK-VEGFR3 protein-protein interaction. Eur J Med Chem 80:154-166
Kurenova, Elena; Ucar, Deniz; Liao, Jianqun et al. (2014) A FAK scaffold inhibitor disrupts FAK and VEGFR-3 signaling and blocks melanoma growth by targeting both tumor and endothelial cells. Cell Cycle 13:2542-53

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