Abstract 926: Extrinsic and intrinsic activation of RAS/MAPK signaling enables resistance to FLT3 inhibitor, gilteritinib, in acute myeloid leukemia
Authors: Joshi Sunil K. , Christy Stephen , Ribeiro Renata Scopim , McWeeney Shannon , Tyner Jeffrey W. , Tognon Cristina E. , Druker Brian J. , Traer Elie 
Published: August 2019

Abstract
Acute Myeloid Leukemia (AML) remains a highly fatal disease due to the development of drug resistance. Mutational activation of kinases is a frequent event in leukemia, driving malignant cell growth. The most commonly mutated kinase in AML is the FMS-like tyrosine kinase 3 (FLT3) receptor. Internal tandem duplication (ITD) events in the juxtamembrane domain of FLT3 have been reported in ~ 20% of patients and lead to constitutive activation of FLT3. Previous work from our laboratory has shown that fibroblast growth factor 2 (FGF2) secreted by mesenchymal stromal cells protects FLT3-ITD AML cells from quizartinib, a FLT3 inhibitor (Cancer Research, 2016). FGF2 binds the FGFR1 receptor and activates MAPK signaling, enabling leukemia cells to become drug resistant. Over months of treatment, FGF2-protected leukemia cells acquired FLT3 resistance mutations. This two-step mechanism of resistance in vitro mirrors what happens in patients treated with quizartinib. Newer FLT3 inhibitors, such as gilteritinib, have been developed that have activity against both ITD and kinase domain mutations that impart resistance to quizartinib. We used a similar approach and treated the FLT3-ITD+ AML cell line, MOLM-14, with 100 nM of gilteritinib in media alone (n=4), or supplemented with 10 ng/ml FGF2 (n=4) or FLT3 ligand (FL, n=4). After 7 weeks, all cultures supplemented with FGF2 or FL eventually resumed growth. In contrast, no MOLM-14 cells cultured in gilteritinib alone resumed growth even after 16 weeks, suggesting the importance of extrinsic factors in facilitating early drug resistance. Selective pressure was then put on the ligand-dependent cultures by removal of FGF2 and FL. This transiently restored sensitivity to gilteritinib, however within a month the cultures resumed exponential growth. To further explore the mechanism of resistance, we performed immunoblots of kinase signaling pathways on MOLM-14 resistant cultures pre- and post-withdrawal of ligands. We saw robust activation of FLT3 receptor in resistant cultures with FL. FGF2 activated the MAPK pathway, circumventing inhibition by gilteritinib. After removal of extrinsic FGF2 and FL, there was re-activation of MAPK signaling but FLT3 itself remained inhibited. Whole Exome Sequencing of resistant cells identified NRAS mutations as the source of MAPK signaling after ligand withdrawal. Two of the four FGF2-supplemented cultures also developed novel FLT3 point mutations, although there was no clear re-activation of FLT3 signaling in these cultures. Our in vitro results suggest that extrinsic signals from the microenvironment can re-activate MAPK pathways enough to keep cells alive but continued selective pressure eventually leads to direct activation of MAPK through RAS mutations. As such, there is a need to consider rational inhibitor combinations early in disease therapy to prevent the emergence of resistance.
Citation Format: Sunil K. Joshi, Stephen Christy, Renata Scopim Ribeiro, Shannon McWeeney, Jeffrey W. Tyner, Cristina E. Tognon, Brian J. Druker, Elie Traer. Extrinsic and intrinsic activation of RAS/MAPK signaling enables resistance to FLT3 inhibitor, gilteritinib, in acute myeloid leukemia [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 926.