High-Dimensional Analysis Identifies Mechanisms of Gilteritinib Resistance in FLT3-Mutated AML

While clinical benefit has been observed with gilteritinib in patients with FLT3 mutated relapsed/refractory acute myeloid leukemia (AML), most patients relapse through mechanisms that are incompletely understood. In this study, to investigate mechanisms of gilteritinib sensitivity and resistance, we performed targeted sequencing (21 patients) and scRNASeq analysis (8 patients) of FLT3-ITD-positive AML samples obtained before and during treatment. Before treatment, co-occurring mutations were observed in 33 genes among 21 patients. Mutations in RAS pathway genes (PTPN11, KRAS, NRAS, CBL) were the most common and observed in 57% (12/21) of patients. Seven patients pretreatment already contained RAS pathway mutations, of which 6 of these mutations were maintained over the course of treatment. During treatment, 9 patients showed emerging RAS mutations, 4 of which initially presented with a different RAS pathway mutation pre-treatment. Other mutations that arose during treatment were observed in CEBPA, IDH1, SF1 and WT1; as well as CSF3R, CUX1, PLEKHG5, and XPO1, not previously identified in gilteritinib-treated patients. Mutational clonality was generally maintained over treatment in both responders and non-responders. scRNASeq revealed global gene expression differences in myeloblast populations between gilteritinib-responsive and -unresponsive patients. Previous studies in vitro have shown that bone marrow-derived hematopoietic and inflammatory cytokines/chemokines confer resistance to FLT3 inhibitors. In the unresponsive group, we observed an increase in expression of CCL5, CXCL1, CXCL2, CXCL8, FLT3, IL6R, IL3RA, and CSF2RA during gilteritinib treatment, supporting the concept from preclinical studies that AML microenvironment-mediated factors play a critical role in drug resistance. Baseline expression of the Tec kinase BMX was significantly higher in unresponsive patients (Log2FoldChange, 6.65; adjusted P value, 0.00186), and this was maintained in the expanding myeloblast populations during treatment. Previously, upregulated BMX was shown to contribute to sorafenib resistance in patients with FLT3-ITD-positive AML, through cell-nonautonomous microenvironment hypoxia-dependent effects. Further in vitro investigation confirmed gilteritinib resistance could be reversed through genetic and pharmacological manipulation of BMX. Gene module analysis showed associations between gilteritinib responsive and upregulation of genes and pathways involved in lymphocyte differentiation and myeloid leukocyte activation, including TBX21, GATA3, CD33, and LYZ. By contrast, there was association between unresponsiveness to gilteritinib and upregulation of cell-cycle, DNA, and RNA metabolic processes, including pathways involving METTL1 and DNMT3A, as well as pre-treatment expression of pathways associated with protein translation. Together, these data provide support for microenvironment-dependent escape from targeted therapy and suggest that BMX may contribute to gilteritinib resistance. High-dimensional analysis with scRNA-seq provides a deeper understanding of targets and pathways for potential therapeutic intervention to restore gilteritinib sensitivity.