Chromosomal Heterogeneity and Enrichment of Mitotic Regulatory Programs Reveal a Therapeutic Vulnerability in...

Cytarabine is a cornerstone of acute myeloid leukemia (AML) therapy; however, acquired resistance remains a major clinical challenge. FMS-like tyrosine kinase 3 internal tandem duplication (FLT3-ITD), a common genetic alteration in AML, is associated with high relapse rates and poor outcomes. Here, we investigated the cellular and molecular mechanisms of acquired cytarabine resistance in FLT3-ITD AML. Cytarabine-resistant MV4-11-CR and MOLM-13-CR cells were generated from parental MV4-11 and MOLM-13 cells, respectively, by stepwise drug selection. Both models exhibited markedly elevated cytarabine IC₅₀ values and enhanced proliferation. FLT3 expression and activation increased in MV4-11-CR cells but decreased in MOLM-13-CR cells. Midostaurin pretreatment failed to restore cytarabine sensitivity, indicating that altered FLT3 signaling is not a shared mechanism of resistance. Cytogenetic analyses and interphase FISH revealed greater numerical and structural chromosomal heterogeneity in the resistant cells than in their parental counterparts, including the presence of polyploid and near-tetraploid subpopulations. RNA sequencing identified common transcriptional alterations, with DCK among the most significantly downregulated genes. Reduced DCK expression was confirmed at the mRNA and protein levels. DCK knockdown in parental MV4-11 cells attenuated cytarabine-induced cytotoxicity, whereas its re-expression in MV4-11-CR cells partially restored cytarabine sensitivity, supporting the functional contribution of DCK downregulation to acquired resistance. Gene set enrichment analysis demonstrated shared enrichment of G2/M checkpoint and mitotic spindle pathways. Notably, combined TTK/Mps1 and FLT3 inhibition with luvixasertib and midostaurin synergistically suppressed cell viability in both resistant cell lines. These findings identify divergent FLT3 regulation, DCK downregulation, increased chromosomal heterogeneity, and enrichment of mitotic regulatory programs as key features associated with acquired cytarabine resistance. They also support the combined inhibition of TTK/Mps1 and FLT3 as a potential therapeutic strategy for cytarabine-resistant FLT3-ITD AML.