Uncovering pre-sensitizing agents to FLT3 inhibitors in acute myeloid leukemia with ReSisTrace lineage tracing

FLT3 inhibitors have significantly advanced the treatment of aggressive FLT3-mutated acute myeloid leukemia (AML), yet the development of resistance remains a major therapeutic challenge. Using the single-cell lineage-tracing method ReSisTrace, we identified transcriptional signatures distinguishing pre-resistant from pre-sensitive AML cells exposed to midostaurin or quizartinib. Targeting the pre-resistance gene GSPT1 with a CC-90009 degrader synergized with FLT3 inhibitors in AML cell lines, patient samples, and a PDX model, significantly improving survival. W further identified small molecules, including vistusertib (mTOR inhibitor), linsitinib (IGF1R/INSR inhibitor), and meisoindigo (IGF1R/Src inhibitor), capable of reversing pre-resistance transcriptional programs and shifting cells toward a FLT3 inhibitor-sensitive state. In summary, ReSisTrace unveils pre-existing transcriptional features of treatment vulnerability in hematological cancers, and elucidates novel strategies for enhancing FLT3 inhibitor treatment efficacy in FLT3-ITD-positive AML. Overall design: We applied ReSisTrace to reveal cell states that are primed to FLT3 inhibitor resistance in an FLT3-ITD-mutated MOLM-13 AML cell line. Uniquely labeled, synchronized cells were allowed to divide once, after which half of the cells were analyzed by scRNA-seq (pre-treatment samples), while the other half underwent DMSO (control), midostaurin, or quizartinib treatment. The lineage labels of cells that survived the treatment were determined by scRNA-sequencing (post-treatment sample) and were used to identify pre-resistant cells in the pre-treatment sample.