FLT3-ITD mutation promotes leukemic differentiation toward an immature cytotoxic stage

Abstract FLT3 mutations contribute to leukemogenesis and poor prognosis in acute myeloid leukemia (AML). Despite FLT3-targeted therapies, relapse rates remain high, suggesting other mechanisms of persistence. FLT3-ITD promotes leukemic stem cell survival and may alter the bone marrow microenvironment, leading to immune evasion. We hypothesized that FLT3-ITD allelic burden and clonal dominance affect immune status and treatment response. We analyzed NK and T cell immunophenotypes in a Flt3-ITD mouse model and in human AML samples to investigate this dysfunction. In the murine model, the Flt3 mutation was associated with reduction in NK cell (CD45hiCD19-CD3-NK1.1+) content characterized by predominance of immature phenotypes (CD27+CD11b-) and a reduction in cytotoxic subpopulations (CD27-CD11b+). Although these alterations were observed in both heterozygous and homozygous animals as compared to wild-type controls, they were more pronounced in the homozygous group. These results were corroborated by an extended phenotypic analysis using CD122, NK1.1, CD49b, and NKp46 expression to identify four NK cell more primitive maturation stages. Among these, a significant reduction in the frequency of more mature subsets (NKII to NKIV) was observed in Flt3-mutated homozygous mice, whose NK cells exhibited an increased expression of DNAM-1, a key activating receptor, and TIM-3, an inhibitory checkpoint receptor involved in immune regulation. In the T cell compartment, a significant increase in short-lived effector cells (CD127-KLRG1+) was observed among CD4⁺ T cells specifically in homozygous animals. In the CD8⁺ T cell population, homozygous animals showed an increased frequency of central memory (CD62L+CD44+) and memory precursor effector cells (CD127+KLRG1-), accompanied by elevated expression of activation and inhibitory receptors, including NKG2D, TIM-3, and CTLA-4, highlighting altered immune regulation within this subset. Furthermore, γδ T cells were expanded in homozygous mutant animals compared to wild-type mice. These results suggested that the Flt3 mutation content influenced the immune profile and inspired us to study NK cells in AML samples of different stages of leukemia arrest (HSC-L, MPP-L, CMP-L, GMP-L, MP-L, and GP-L) driven by their FLT3-ITD/NPM1 mutational status. Multiparametric flow cytometry analysis of bone marrow samples from 148 de novo AML patients with normal karyotype and known FLT3/NPM1 status revealed that FLT3mut/NPM1wtpatients (n=36) were predominantly associated with CMP-L (CD34+/-CD117+CD13+CD33+/-HLA-DR+MPO+) and GMP-L (CD34+/-CD117+/-CD13+/-CD33+HLA-DR+MPO+) leukemias, whereas FLT3wt/NPM1mut patients (n=80) predominantly presented GP-L (CD34-CD117+/-CD13+CD33+HLA-DR-MPO+) leukemias. Double-mutant patients (FLT3mut/NPM1mut, n=32) exhibited a similar profile to FLT3wt/NPM1mut patients, with a predominance of MP-L and GP-L stages. As compared to other maturation stages of leukemia arrest, GMP-L leukemias were associated with disease persistence after the first induction therapy and a trend toward earlier relapse, although impact on overall or relapse-free survival was not observed. In agreement, in silico analysis using the Beat AML 2.0 dataset indicated higher resistance to cytarabine, midostaurin, and azacitidine in patients with GMP-L FLT3mut/NPM1wt cells. No differences were observed in the frequencies of CD56+ NK cells, CD56bright, or CD56dim subsets among the FLT3wt/NPM1mut, FLT3mut/NPM1mut, and FLT3mut/NPM1wt groups. However, in FLT3-mutated patients, high AR showed a trend toward fewer CD56dim and more CD56bright NK cells compared to low AR. Regarding leukemic maturation, a trend toward increased CD56bright and decreased CD56dim populations was observed in more differentiated leukemias (GMP-L, MP-L, and GP-L), suggesting functional dysregulation linked to stage of leukemia arrest. Collectively, our findings show that FLT3-ITD promotes dysregulation in maturation and activation of cytotoxic immune cells, enabling immune evasion. Immature leukemic subsets are linked to impaired cytotoxic differentiation and altered receptor expression, possibly contributing to resistance and poor outcomes. Immunophenotypic alterations in mouse and human models support the potential of targeted immunotherapy and emphasize the need to consider leukemic maturation and immune status in therapeutic strategies.