Old Meets New: Hydroxyurea Synergistically Enhances the Antileukemic Activity of Gilteritinibagainst FLT3-ITD AML

About 25% of acute myeloid leukemia (AML) cases harbor FMS-like tyrosine kinase 3 (FLT3) internal tandem duplication (ITD) mutations which is associated with poor prognosis. Gilteritinib is a second generation FLT3 inhibitor approved for treating FLT3-mutated relapsed/refractory (R/R) AML patients. Monotherapy, while efficacious, shows short-lived responses, highlighting the need for combination therapies. Results from our preliminary studies show that ribonucleotide reductase (RNR), the rate-limiting enzyme in the biosynthesis of deoxynucleotides, is increased in a cytarabine (AraC)-resistant FLT3-ITD AML cell line (designated MV4-11/AraC-R) compared to the parental cells, accompanied by significantly increased levels of dNDPs. These findings prompted us to hypothesize that targeting RNR with hydroxyurea, an off-patent RNR inhibitor currently used to treat AML patients with hyperleukocytosis, will enhance the antileukemic activity of gilteritinib against R/R FLT3-ITD AML.

Increased level of RNR was also detected in another AraC-resistant FLT3-ITD AML cell line, MOLM-13/AraC-R, compared to the parental cells. The levels of RNR in patient-derived xenograft (PDX) cells derived from a relapsed FLT3-ITD AML patient after chemotherapy and bone marrow transplant (J000106565; Jackson Laboratory) were comparable to that in the cell lines, demonstrating consistent increase of RNR in AraC-resistant FLT3-ITD AML cells. Apoptosis levels induced by HU were negatively associated with RRM2 levels in these cells, indicating RRM2 downregulation would enhance the response to HU. Indeed, shRNA RRM2 knockdown significantly enhanced apoptosis induced by HU in MV4-11/AraC-R cells. It has been reported that RRM2 is a transcriptional target of c-Myc and gilteritinib treatment which downregulates c-Myc. Thus, gilteritinib could downregulate RRM2 to enhance the antileukemic activity of HU against AraC-resistant FLT3-ITD AML. Treatment of MV4-11/AraC-R and the J000106565 PDX cells with variable concentrations of gilteritinib (25-1000 nM) almost completely abolished c-Myc and RRM2 in the cells even at the lowest concentration. Treatment of these cells with a c-Myc inhibitor, 10058-F4, resulted in substantial downregulation of RRM2, confirming that downregulation of RRM2 by gilteritinib was at least partially through c-Myc. There was a plateau of apoptosis induced by gilteritinib under the same experimental conditions, indicating that low concentrations (e.g. 25-100 nM) of gilteritinib would be sufficient to enhance the antileukemic activity of HU. To test if combining gilteritinib with HU results in synergistic antileukemic effect, MV4-11/AraC-R cells were treated with HU (125 - 500 µM) and gilteritinib (25 - 100 nM), either alone or in combination simultaneously or sequentially (pretreatment with HU for 48 hours followed by gilteritinib for 24 hours or pretreatment with gilteritinib for 24 hours followed by HU for 48 hours). Simultaneous treatment with both agents resulted in significantly increased levels of apoptosis compared to the individual drug treatment. Calculation of combination indices revealed that the simultaneous combination was moderately synergistic. However, pretreatment with HU for 48 hours followed by gilteritinib for another 24 hours had a strong synergistic effect (CI < 0.5). In contrast, pretreatment with gilteritinib for 24 hours followed by HU for another 48 hours had an antagonistic effect. These results demonstrate that HU pretreatment followed by gilteritinib yields the best synergy between the two agents. An increase in gilteritinib concentrations by 10-fold did not result in further increase of apoptosis induced by the combination. Very strong synergy (CI < 0.17) between the two agents was also detected in the PDX and MOLM-13/AraC-R cells. HU treatment substantially induced RRM2 which was attenuated by gilteritinib. Additional studies are ongoing to determine the molecular mechanisms underlying the synergistic effect and in vivo efficacy of the combination in the PDX xenograft model.

This study highlights that gilteritinib synergistically enhances the antileukemic activity of HU against Ara-resistant FLT3-ITD AML at low concentrations. Our results suggest that the clinical dose of gilteritinib could be reduced to reduce toxicities and maintain efficacy at the same time.

No relevant conflicts of interest to declare.