Characterization of leukemic stem cells in FLT3-ITD-positive acute myeloid leukemia

Despite major treatment advances, FLT3-ITD mutated acute myeloid leukemia (FLT3-ITDmut AML) still remains an unmet clinical need. We previously showed that rare CD34/CD123/CD25/CD99+ FLT3-ITDmut leukemic progenitor cells (LPCs), may persist after induction treatment and be responsible for drug resistance and disease progression. We aimed at the functional characterization of FLT3-ITDmut LPCs, also using a NSG mouse model. The transcriptomic profile of paired blasts and LPCs, purified from 13 FLT3-ITDmut AML using RNAseq showed significant upregulation of receptor tyrosine kinases pathway, primarily lymphocyte-specific protein tyrosine kinase (LCK), which also cooperates with STAT5. Interestingly, LCK upregulation was confirmed in 28 additional FLT3-ITDmut samples, suggesting a specific therapeutic vulnerability. Indeed, a clear anti-proliferative effect of the LCK inhibitor Dasatinib was detected on the FLT3-ITDmut MV4-11 cell line and 3 primary AML LPCs, together with inhibition of FLT3 and STAT5 signalling, further potentiated by the addition of Gilteritinib. Dasatinib also induced significant downregulation of ABC transporters, a well-known cause of LSCs multi-drug resistance. Sorted LPCs were then injected into conditioned NSG mice, demonstrating their potential to engraft and induce leukemia. Interestingly, after sequential passages in mice, the LPCs compartment expanded, indicating unveil of the ancestral leukemic precursor cells. In conclusions, we deeply characterized the FLT3-ITDmut LSCs compartment in AML, providing the first evidence that FLT3-ITDmut progenitors may engraft NSG mice, while maintaining the stem cell potential. Dasatinib/Gilteritinib combinations are able to target FLT3-ITDmut LSCs and may support eradication of residual leukemic cells harbinger of relapse.