Acute myeloid leukemia (AML) is an aggressive hematologic malignancy with poor clinical outcomes. One of the major drivers of adverse prognosis is the internal tandem duplication (ITD) mutation in the FLT3 tyrosine kinase gene, which occurs in approximately 20% of newly diagnosed patients. Although recently approved FLT3 inhibitors have markedly improved therapeutic outcomes for FLT3-ITD–mutated AML, primary resistance and transient responses remain substantial clinical challenges. The objective of this study was to elucidate mechanisms underlying primed resistance to FLT3 inhibitors driven by intrinsic transcriptional heterogeneity and to identify novel agents capable of pre-sensitizing AML cells to FLT3-targeted therapy.

To identify pre-existing resistant states to FLT3 inhibitors midostaurin and quizartinib, we applied our single-cell lineage-tracing platform, ReSisTrace, in an FLT3-ITD–positive MOLM-13 AML cell line. Differential gene expression analyses comparing pre-resistant and pre-sensitive populations were performed to derive pre-resistance signatures. Candidate synergistic compounds were selected among drugs that target individual genes within the pre-resistance signatures, as well as by querying the L1000 database for drugs predicted to induce transcriptional changes opposing these signatures. Drug combinations were validated in AML cell lines, primary patient samples, and xenograft models.

Pharmacological targeting of the pre-resistance signature gene G1 to S phase transition 1 (GSPT1) using the selective cereblon E3 ligase modulator CC-90009 demonstrated strong synergistic activity with FLT3 inhibitors in FLT3-ITD–mutated AML cell lines and primary patient cultures. Moreover, CC-90009 and quizartinib combination significantly prolonged antitumor responses and improved overall survival in an FLT3-ITD AML patient-derived xenograft model compared with either agent alone. In addition, vistusertib (mTOR inhibitor), linsitinib (insulin receptor inhibitor), and meisoindigo (Src family kinase inhibitor)—all targeting pathways parallel or downstream of oncogenic FLT3 signaling—were predicted and experimentally validated in AML cell lines and primary patient samples as effective pre-sensitizing agents to FLT3 inhibition.

Our lineage-tracing approach enables the identification of transcriptional states that precede primed resistance to FLT3 inhibitors in FLT3-ITD–mutated AML. Targeting the pre-resistance gene GSPT1 with the molecular degrader CC-90009 produces robust synergistic effects with FLT3 inhibitors both in vitro and in vivo, highlighting a promising strategy to enhance therapeutic efficacy in FLT3-ITD–positive AML.

The authors declare no conflict of interest.