Ribonucleotide Reductase Inhibition Overcomes FLT3 Inhibitor Resistance in Acute Myeloid Leukemia

Tian, Z.; Wei, X.; Chatla, S.; Liu, Y.; Kim, D.; Li, Y.; Wang, P.; Liao, Y.; Liu, X.; Yang, D.; Octaviani, S.; Ma, G.; Pompetti, A.; Calendo, G.; Keough, M. P.; Xu, W.; Zhang, J.; Zheng, H.; Stieglitz, E.; Smith, C. C.; Huang, J.

doi:10.64898/2026.07.21.736123

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bioRxiv · 10.64898/2026.07.21.736123

Ribonucleotide Reductase Inhibition Overcomes FLT3 Inhibitor Resistance in Acute Myeloid Leukemia

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Abstract

Internal tandem duplication mutations in FLT3 (FLT3ITD) occur in approximately 30% of patients with acute myeloid leukemia (AML) and are among the most common genetic alterations in this disease. FLT3ITD is a major driver of AML and is associated with poor clinical outcomes. Although FLT3 inhibitors (FLT3is) have significantly improved outcomes for patients with FLT3ITD+ AML, acquired resistance remains a major barrier to durable clinical benefit. Reactivation of RAS/MAPK signaling, often driven by activating NRAS mutations, is a major mechanism of FLT3i resistance in AML; however, effective strategies to overcome this resistance remain lacking. Here, we identify ribonucleotide reductase (RNR) as a critical therapeutic vulnerability in NRAS-driven FLT3i-resistant FLT3ITD+ AML. Activation of RAS signaling through SPRY3 loss or oncogenic NRAS mutations confers robust resistance to FLT3is, whereas pharmacologic inhibition of RNR with multiple inhibitors, as well as siRNA-mediated RNR suppression, reverses FLT3i resistance and restores FLT3i sensitivity across multiple FLT3ITD+ AML models in vitro. In vivo, clofarabine, an FDA-approved RNR inhibitor (RNRi), significantly overcomes NRAS mutation-driven FLT3i resistance. In combination with FLT3 inhibition, clofarabine markedly suppresses the progression of FLT3i-resistant AML and significantly prolongs survival in cell line-derived xenograft (CDX) models. Importantly, the therapeutic efficacy of the gilteritinib/clofarabine combination was independently validated in two genetically distinct patient-derived xenograft (PDX) models harboring different NRAS mutations, demonstrating robust reduction of leukemia burden and confirming the generalizability of RNR inhibition in primary FLT3i-resistant AML. Together, these findings identify a previously unrecognized therapeutic vulnerability in FLT3i-resistant FLT3mut+ AML and establish RNR inhibition as an effective strategy to overcome FLT3i resistance, providing a strong rationale for the clinical evaluation of RNRis in combination with FLT3is in patients with resistant AML. SignificanceAlthough FLT3 inhibitors (FLT3i) are an important therapeutic advance in FLT3ITD+ AML, resistance commonly develops. We identified ribonucleotide reductase (RNR) as a new key vulnerability in NRAS-driven FLT3i-resistant AML and demonstrated that multiple RNRis, including the FDA-approved agent clofarabine, restore FLT3i sensitivity and enhance antileukemic activity, supporting a clinically actionable combination strategy.

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Tian, Z., Wei, X., Chatla, S., Liu, Y., Kim, D., Li, Y., Wang, P., Liao, Y., Liu, X., Yang, D., Octaviani, S., Ma, G., Pompetti, A., Calendo, G., Keough, M. P., Xu, W., Zhang, J., Zheng, H., Stieglitz, E., Smith, C. C., Huang, J.. 2026-07-22. Ribonucleotide Reductase Inhibition Overcomes FLT3 Inhibitor Resistance in Acute Myeloid Leukemia. https://doi.org/10.64898/2026.07.21.736123

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