Abstract 2363: Targeting Src-family kinases to combat acquired inhibitor resistance in FLT3-ITD+ AML
Authors: Patel Ravi K. , Weir Mark , Hellwig Sabine , Dorman Heather , Smithgall Thomas E. 
Published: July 2017

Activating internal tandem duplication (ITD) mutations in the FMS-like receptor tyrosine kinase 3 (Flt3) are found in approximately 30% of acute myeloid leukemia (AML) patients. Flt3-ITD expression is associated with aggressive, chemotherapy-resistant disease and decreased overall survival. Multiple selective Flt3 kinase inhibitors have been developed as targeted therapy for Flt3-ITD+ AML. However, the clinical utility of these kinase inhibitors has been limited by the rapid recurrence of drug-resistant disease that often results from Flt3 kinase domain mutations that prevent inhibitor action. Recent studies revealed that multiple non-receptor tyrosine kinases are over-expressed in AML, including the myeloid Src-family kinases Hck, Lyn and Fgr, and may cooperate with Flt3 to drive disease. These observations suggest that kinase inhibitors with specificity profiles targeting Flt3 and these AML-associated cytoplasmic kinases may be less prone to acquired resistance. In the present study, we determined the contributions of Hck and Fgr kinase activities to Flt3-ITD+ AML cell growth using a chemical genetics approach. While RNAi-mediated knockdown of each of these kinases reduces proliferation and increases apoptosis in primary AML cells, the impact of pharmacologic inhibition of their kinase activities on AML cell growth is not known. Because no selective Hck or Fgr inhibitors exist, we developed kinase domain mutants that desensitize each kinase to the broad-spectrum SRC-family kinase inhibitor, A-419259. Examination of a previous crystal structure of Hck with A-419259 bound to the active site suggested a role for the ‘gatekeeper’ residue in compound binding. Substitution of the gatekeeper threonine of Hck and Fgr with methionine conferred 3-fold resistance to A-419259 in vitro without diminishing kinase activity. When expressed in human TF-1 myeloid cells transformed with Flt3-ITD, these inhibitor-resistant Hck and Fgr mutants conferred resistance to growth inhibition by A-419259 to a similar extent, while expression of the corresponding wild-type kinases was without effect. The sensitivity of the TF-1/Flt3-ITD cell lines to A-419259 correlated with inhibition of mutant Hck and Fgr kinase activity in the cells. Our findings suggest that suppression of AML cell growth by A-419259 is due in part to inhibition of Hck and Fgr, and identify these AML-associated kinases as therapeutic targets. Ongoing studies are addressing the role of Hck and Fgr as inhibitor targets in Flt3-ITD+ AML cell lines and patient samples as well as the propensity of A-419259 and other multi-targeted inhibitors of AML-associated kinases for acquired drug resistance.

Citation Format: Ravi K. Patel, Mark Weir, Sabine Hellwig, Heather Dorman, Thomas E. Smithgall. Targeting Src-family kinases to combat acquired inhibitor resistance in FLT3-ITD+ AML [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 2363. doi:10.1158/1538-7445.AM2017-2363