Abstract 2371: Entospletinib, a potent SYK inhibitor, blocks constitutive and FCgRI activated signaling in FLT3-ITD cell lines
Authors: Tannheimer Stacey L. , Kashishian Adam , Sorensen Rick , Keegan Kathleen S. 
Published: August 2018

Spleen tyrosine kinase (SYK), a non-receptor protein tyrosine kinase, is an important mediator of immune receptor signaling in B cells, mast cells, neutrophils, and macrophages. SYK is expressed in 90% of AML blasts and has been reported to be is constitutively activated (Hahn et al., Cancer Cell 2009). High expression of phosphorylated SYK (pSYK) is associated with unfavorable outcomes, independent of other AML prognostic predictors such as age, cytogenetics, and white blood cell count (Boros et al., Oncotarget 2015). Entospletinib (ENTO) is a potent and selective inhibitor of SYK currently in clinical trials for the treatment of AML as monotherapy and in combination with standard-of-care therapies. We investigated the effects of ENTO on SYK phosphorylation and signaling in FLT3-ITD mutant AML cells. We found that ENTO blocked constitutive phosphorylation of pSYKY352, pSTAT5Y694, and MYC protein expression in FLT3-ITD-driven AML cell lines, MV4-11 and MOLM14. The concentration of ENTO required to inhibit pSYKY352 and pSTAT5Y694 correlated with inhibition of cell viability. Phosphorylation at these sites was inhibited by the selective FLT3 inhibitor, quizartinib; but not inhibited by Src family kinase inhibitor (dasatinib) or JAK inhibitors (ruxolitinib or tofacitinib). Further, a second-generation, highly selective SYK inhibitor, GS-9876, failed to inhibit the constitutive phosphorylation of these sites. Comparison of the biochemical and cellular kinase activities of the SYK and FLT3 inhibitors suggests that the observed inhibition of constitutive phosphorylation of pSYKY352 and pSTAT5Y694 and MYC expression was mediated by the direct activity of ENTO against FLT3-ITD, rather than SYK. Activation of FcγRI has been previously shown to activate SYK (Oellerich et al., Blood 2013). We demonstrate SYK activation at pSYKY348 and pSYKY323 after stimulation of the FCγRI with human IgG in the FLT3-ITD mutant cell line, MV4-11. Downstream signaling molecules pERK1/2T202/Y204, pVAV1Y160, and pLYNY507 were also activated by FCγR stimulation. ENTO inhibited phosphorylation of pSYKY348, pSYKY323 and pVAV1 Y160. The second-generation SYK inhibitor, GS-9876, also inhibited FCγRI-mediated activation of pSYKY348,pSYKY323 and pVAV1 Y160, suggesting that these phosphorylation events were mediated by SYK activity. In conclusion, ENTO inhibited constitutive and FCγRI-activated SYK phosphorylation and signaling pathways in FLT3-ITD mutant cell lines, resulting in inhibition of cell viability. Inhibition of constitutive SYK phosphorylation is mediated by direct activity of ENTO against FLT3-ITD; however, inhibition of FCγRI activated SYK results from direct inhibition of SYK.

Citation Format: Stacey L. Tannheimer, Adam Kashishian, Rick Sorensen, Kathleen S. Keegan. Entospletinib, a potent SYK inhibitor, blocks constitutive and FCgRI activated signaling in FLT3-ITD cell lines [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 2371.