Abstract 984: FGF2 from the bone marrow promotes resistance to FLT3 inhibitors in AML
Authors: Martinez Jacqueline , Traer Elie , Javidi-Sharifi Nathalie , Agarwal Anupriya , Dunlap Jennifer , English Isabel , Kovacsovics Tibor , Tyner Jeffrey , Wong Melissa , Druker Brian 
Published: August 2015

Abstract
Introduction: Activating mutations in the FLT3 kinase are present in ∼25% of newly diagnosed acute myeloid leukemia (FLT3+ AML) patients and confer a poor prognosis. Treatment with FLT3 inhibitors is initially effective, but residual leukemia cells survive in the bone marrow microenvironment and clinical resistance develops within months. We tested proteins from the bone marrow microenvironment for their ability to protect FLT3-ITD+ MOLM14 cells from AC220, and found that FLT3 ligand (FL) and fibroblast growth factor 2 (FGF2) were among the most protective.
Results: Consistent with previous reports, FL restored downstream FLT3 phosphorylation and signaling in the presence of the FLT3 inhibitor quizartinib (AC220). In contrast, FGF2 activated FGFR1 and the MAPK pathway, circumventing the effects of AC220 on MOLM14 cells and increasing cell survival. To model the effect of prolonged contact with the bone marrow microenvironment, MOLM14 cells were cultured continuously in FGF2 and AC220. Under these conditions, 4/4 cultures developed AC220 resistance and resumed exponential growth after 7 weeks. Development of FGF2-mediated resistance was accelerated compared to MOLM14 cultured continuously in AC220 alone (only 2/4 resistant cultures,12 weeks). Long-term resistant cultures grown in FGF2 were protected from AC220 across a wide range of concentrations, but concurrent treatment with the FGFR inhibitor PD173074 synergistically overcame FGF2-mediated protection.
Serial bone marrow biopsies of patients on the AC220 phase II clinical trial were analyzed by immunohistochemistry. Quantification of FGF2 revealed a significant increase in AC220 during treatment (34% to 51% after one month, p&lt;0.01) and FGF2 expression peaked at 61% after 4 months (p&lt;0.01), indicating that residual leukemia cells exist in a microenvironment abundant in FGF2. To test if FGFR inhibition could overcome FGF2-mediated protection from bone marrow stroma, we grew MOLM14 cells in co-culture with an FGF2-expressing stromal cell line (HS-5). HS-5 co-culture increased viability in MOLM14 in AC220 treatment, however concurrent PD173074 treatment abrogated stromal cell protection suggesting that combination of FLT3 and FGFR inhibitors may overcome resistance mediated by the microenvironment.
Conclusions: FGF2 ligand-induced activation of the FGFR1/MAPK pathway leads to protection from AC220 in vitro and eventual development of resistance. FGF2-mediated resistance can be overcome by concurrent FLT3 and FGFR inhibition. Consistent with our in vitro model, patients treated with AC220 had increasing FGF2 in their bone marrow, which peaked prior to resistance and relapse, suggesting that combined FLT3 and FGFR inhibitors may improve the durability of response. Our results provide an approach to develop rational inhibitor combinations that cirucumvent ligand-mediated pathways of resistance.
Citation Format: Jacqueline Martinez, Elie Traer, Nathalie Javidi-Sharifi, Anupriya Agarwal, Jennifer Dunlap, Isabel English, Tibor Kovacsovics, Jeffrey Tyner, Melissa Wong, Brian Druker. FGF2 from the bone marrow promotes resistance to FLT3 inhibitors in AML. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 984. doi:10.1158/1538-7445.AM2015-984