Targeting ribonucleotide reductase to enhance the antileukemic activity of gilteritinib against chemoresistant FLT3- internal tandem duplication acute myeloid leukemia

Acute myeloid leukemia (AML) cases harboring FMS-like tyrosine kinase 3 ( FLT3 ) internal tandem duplication ( FLT3 -ITD) mutations have poor clinical outcomes. Gilteritinib is a United States Food and Drug Administration–approved FLT3 inhibitor for treating relapsed/refractory (R/R) FLT3 -mutated AML; however, monotherapy shows short-lived responses, highlighting the need for combination therapies. Increased ribonucleotide reductase regulatory subunit M2 (RRM2) was detected in cytarabine-resistant (AraC-R) FLT3 -ITD AML cell lines and patient-derived xenograft (PDX) cells, accompanied by increased dNDPs determined by proteomics, western blotting, and metabolomics studies. shRNA knockdown of RRM2 significantly enhanced cell death induced by the ribonucleotide reductase inhibitor, hydroxyurea (HU). Treatment of MV4-11/AraC-R and the PDX cells with variable concentrations of gilteritinib (25–1000 nM) almost completely abolished RRM2 in the cells even at the lowest concentration and was accompanied by a plateau of cell death. Pretreatment with HU (12.5–500 μ M) for 48 hours followed by gilteritinib (25–100 nM) for another 24 hours had a strong synergistic effect on the AraC-R FLT3 -ITD AML cell lines and the PDX cells. Increasing gilteritinib concentrations by 10-fold did not result in further increased cell death. HU treatment induced RRM2 which was abolished by gilteritinib treatment, whereas gilteritinib treatment induced FLT3 which was canceled by HU, demonstrating reciprocal overcoming of drug resistance. Given that both drugs are Food and Drug Administration approved, and HU is well tolerated in both pediatric and adult populations as well as being cost-effective, establishing in vivo models could pave the path to clinical trials, ultimately providing a bridge to transplantation for R/R FLT3 -ITD AML while minimizing toxicity. This study demonstrates the reciprocal overcoming of drug resistance and supports further development of the combination of hydroxyurea and gilteritinib for the treatment of relapsed/refractory FMS-like tyrosine kinase 3 internal tandem duplication acute myeloid leukemia. Moreover, this study highlights the importance of drug repurposing.