Focal Adhesion Kinase As a Potential Target in AML and MDS
Authors: Carter Bing Z , Kornblau Steven M. , Mak Po Yee , Yang Hui , Qiu Yihua , Jiang Xuejie , Coombes Kevin , Zhang Nianxiang , Garcia-Manero Guillermo , Andreeff Michael 
Published: October 2019

Abstract
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.

Disclosures
Carter: PrismBiolab: Research Funding. Andreeff:Oncoceutics, Inc.: Membership on an entity's Board of Directors or advisory committees.