Characterization of FLT3-ITD-mutated leukemic stem cells: functional and clinical insights

Abstract Despite treatment advances, relapsed/refractory FLT3 -ITD-mutated acute myeloid leukemia ( FLT3 -ITD mut AML) remains an unmet medical need. We previously identified a CD34/CD123/CD25/CD99+ leukemic progenitor cell (LPC) population that persists after induction treatment and drives disease recurrence in FLT3 -ITD mut AML. Here, we performed an integrated functional and molecular characterization of FLT3 -ITD mut LPCs. Multiparametric flow cytometry prospectively demonstrated that CD34/CD123/CD25/CD99+ expression levels above 7.4% robustly predict FLT3 -ITD mutation with 88.2% sensitivity and 100% specificity. Sorted LPCs efficiently engrafted conditioned NSG mice and reinitiated leukemia, with sequential passages expanding the LPC compartment, consistent with their role as a disease reservoir. Transcriptomic profiling of 13 paired LPCs and blasts revealed upregulation of protein kinases, primarily lymphocyte-specific protein tyrosine kinase (LCK), a finding confirmed in 28 additional FLT3 -ITD mut samples. Gilteritinib reduced proliferation and FLT3-STAT5 signaling in FLT3 -ITD mut cells and LPCs. Additional LCK inhibition further attenuated FLT3/STAT5 activity, disrupted aberrant FLT3 localization, and downregulated ABC transporter-associated multidrug resistance. Notably, combining gilteritinib with LCK blockade enhanced its anti-leukemic activity, consistent with a cooperative effect of these pathways in sustaining FLT3 -ITD mut leukemic progenitors. These findings provide the first functional evidence that FLT3 -ITD mut LPCs maintain stem cell potential in vivo, refine clinically actionable leukemic immunophenotype for diagnosis and MRD monitoring, and uncover novel molecular vulnerabilities. Collectively, they support strategies to selectively eradicate residual FLT3 -ITD mut LPCs to improve long-term outcomes in AML.