Single-Cell Lineage Tracing Uncovers Resistance Signatures and Sensitizing Strategies to FLT3 Inhibitors in Acute Myeloid Leukemia.

[UNLABELLED] Whereas FMS-like tyrosine kinase-3 (FLT3) inhibitors have significantly improved the treatment of aggressive FLT3-mutated acute myeloid leukemia (AML), the emergence of resistance remains as a major challenge. In this study, we applied our recently developed single-cell lineage-tracing method ReSisTrace to identify cells that are intrinsically resistant or sensitive to the FLT3 inhibitors midostaurin and quizartinib in AML with FLT3-ITD mutations. Comparison of the gene expression profiles of these cells revealed transcriptional resistance signatures, including upregulation of GSPT1. Depletion of GSPT1 with CRISPR-Cas9-mediated knockout resulted in increased sensitivity of AML cells to quizartinib treatment. Furthermore, targeting GSPT1 with the small molecule CC-90009 exhibited strong synergistic effects when combined with FLT3 inhibitors in the FLT3-ITD cell lines and primary AML patient samples. In addition, in an FLT3-ITD-positive AML patient-derived xenograft mouse model, the CC-90009 and quizartinib combination showed significantly higher antitumor efficacy and prolonged overall survival compared with either treatment alone. Furthermore, compounds that induced transcriptomic changes opposite to the resistance signatures prompted cells to acquire FLT3 inhibitor-sensitive states. Vistusertib (mTOR inhibitor), linsitinib (IGF1R and INSR inhibitor), and meisoindigo (IGF1R and SRC family kinase inhibitor), all inhibiting pathways parallel to or downstream of oncogenic FLT3 signaling, were predicted and validated to sensitize FLT3-mutated cell lines and primary cells to FLT3 inhibitors. Collectively, these findings demonstrate the ability of ReSisTrace to unveil preexisting transcriptional features of treatment vulnerability in hematologic cancers and elucidate strategies for enhancing FLT3 inhibitor treatment efficacy in FLT3-ITD-mutated AML.

[SIGNIFICANCE] Single-cell lineage tracing with ReSisTrace reveals cellular states that prime treatment resistance and identifies approaches to sensitize FLT3-ITD-positive acute myeloid leukemia to FLT3 inhibitors.