Resistance to azacitidine is a major issue in the treatments of myelodysplastic syndrome and acute myeloid leukemia, and previous studies suggest that changes in drug metabolism are involved in the resistance. Therefore, drugs with mechanisms resistant or alternative to such metabolic changes have been desired for the treatment of resistant disease. We generated azacitidine‐resistant cells derived from SKM‐1 and MOLM‐13 leukemia cell lines in vitro, analyzed the mechanisms, and examined the impact on the efficacy of other antimetabolic drugs. It appeared that the cell growth‐inhibitory effect of azacitidine, expression levels of uridine–cytidine kinase 2, and the concentrations of azacitidine triphosphate were remarkably decreased in the resistant cells compared with those in parent cells. These results were consistent with previous observations that azacitidine resistance is derived from metabolic changes. Cross‐resistance of greater than 10‐fold (shift in IC50 value) was observed in azacitidine‐resistant cells for decitabine and for cytarabine, but not for gemcitabine or the inosine‐5′‐monophosphate dehydrogenase (IMPDH) inhibitors FF‐10501 and mycophenolate mofetil (cross‐resistance to 5‐fluorouracil was cell line dependent). The IMPDH inhibitors maintained their cell growth‐inhibitory activities in the azacitidine‐resistant cell lines, in which the levels of adenine phosphoribosyltransferase (which converts FF‐10501 to its active form, FF‐10501 ribosylmonophosphate [FF‐10501RMP]), FF‐10501RMP, and the target enzyme, IMPDH, were equivalent to those in the parent cell lines. These results suggest that an IMPDH inhibitor such as FF‐10501 could be an alternative therapeutic treatment for leukemia patients with acquired resistance to azacitidine.

Inosine monophosphate dehydrogenase (IMPDH) is a rate‐limiting enzyme in de novo guanine nucleotide synthesis pathway. Although IMPDH inhibitors are widely used as effective immunosuppressants, their antitumor effects have not been proven in the clinical setting. Here, we found that acute myeloid leukemias (AMLs) with MLL‐fusions are susceptible to IMPDH inhibitors in vitro. We also showed that alternate‐day administration of IMPDH inhibitors suppressed the development of MLL‐AF9‐driven AML in vivo without having a devastating effect on immune function. Mechanistically, IMPDH inhibition induced overactivation of Toll‐like receptor (TLR)‐TRAF6‐NF‐κB signaling and upregulation of an adhesion molecule VCAM1, which contribute to the antileukemia effect of IMPDH inhibitors. Consequently, combined treatment with IMPDH inhibitors and the TLR1/2 agonist effectively inhibited the development of MLL‐fusion AML. These findings provide a rational basis for clinical testing of IMPDH inhibitors against MLL‐fusion AMLs and potentially other aggressive tumors with active TLR signaling.

One of the major symptoms of myelodysplastic syndromes (MDS) is severe cytopenia. Despite cytokine therapies, such as erythropoiesis-stimulating agents, many patients still require blood transfusions, and the development of new therapeutic approaches is needed. In this work, we studied the effects of the inosine-5'-monophosphate (IMP) dehydrogenase (IMPDH) inhibitor FF-10501 on erythropoiesis of human hematopoietic cells. Differentiation of K562 chronic myeloid leukemia cells to an erythroid lineage was promoted by FF-10501 in a dose-dependent manner. Interestingly, we found that metabolic conversion of IMP to hypoxanthine leads to elevation of reactive oxygen species (ROS). The differentiative effects of FF-10501 were abolished by the ROS scavenger dimethylthiourea or the p38 MAPK inhibitor SB203580. Furthermore, FF-10501 promoted erythropoiesis from CD34 hematopoietic stem/progenitor cells, accompanied with ROS accumulation, while high-dose FF-10501 mainly showed cytotoxic effects. These findings denote the potential of IMPDH inhibition therapy with FF-10501 in amelioration of anemia in MDS patients.

Myelodysplastic syndromes (MDS) are clonal hematopoietic stem cell disorders, characterized by peripheral blood cytopenia and risk of leukemic progression. The treatment strategy is subdivided into anti-cancer therapy and the improvement of refractory cytopenia. The therapeutic options are still limited while the number of patients is increasing with growth of the aging population. Interestingly, some of the cytotoxic agents such as alkylating agents, antimetabolites, and antitumor antibiotics have been reported the potency of promoting the differentiation of leukemic cell-lines in cultures. FF-10501, the inhibitor of inosine-5-monophosphate dehydrogenase (IMPDH), which interferes with the synthesis of nucleic acids, is one of candidates for a novel agent for MDS treatment, and now phase I clinical trials for high-risk MDS patients are running in the United States (FF10501US101) and Japan (FF10501JP101). In this study, we evaluated the effects of this agent on myeloid differentiation using human hematopoietic cells. Human samples were collected after informed consent with protocols approved by the Investigational Review Board of Osaka University Hospital. First, FF-10501 inhibited the proliferation of 9 cell-lines of hematological malignancy, including K562, HL-60 and MOLM-13. The erythroid differentiation of K562 human leukemic cells and F36P MDS cells was promoted in the same condition. The induction of differentiation to either granulocytes or monocytes in acute myeloid leukemia cells, MOLM-13 and HL-60 was also observed. These effects were dose-dependent. To elucidate the detailed mechanisms of the FF-10501-induced differentiation, we studied the alternative pathway activated after the IMPDH inhibition. As expected, the production of GMP, GDP and GTP was suppressed while that of ADP was not altered. The accumulation of inosine-5-monophosphate (IMP) was recognized. Among the metabolic changes induced by FF-10501 treatment, we found the elevation in the concentration of inosine and hypoxanthine. Because it is known that reactive oxygen species (ROS) are generated during the degradation of IMP, we examined whether the imbalance of ROS would be associated with the differentiation effect. When ROS scavenger, dimethylthiourea was added into cultures with FF-10501, the erythroid differentiation of K562 and the granulocytic differentiation of MOLM-13 were attenuated. Moreover, the inhibition of p38 also cancelled the effects. These results indicate that overproduction of ROS and the consequent activation of p38 contribute to the promotion of myeloid differentiation of hematological tumor cells by FF-10501. To apply our findings for clinical settings, we examined the effects of FF-10501 using human mononuclear blood cells. When bone marrow cells derived from healthy donors and MDS patients were cultured in colony-forming unit assays, the number of recovered colonies decreased in a dose-dependent manner. Although high dose of FF-10501 (≥ 40 μM) showed strong cytotoxicity, we found that the intermediate dose of FF-10501 promoted generation of erythroid cells from CD34+ hematopoietic stem/progenitor cells. After 3 week-cultures with 4 μM of FF-10501 under erythroid condition (20 ng/ml IL-3, 10 ng/ml Flt3-ligand, 50 ng/ml thrombopoietin, 20 ng/ml stem cell factor and 10 ng/ml erythropoietin) on MS5 stromal cells, the development of glycophorin A+ cells were significantly promoted. Regarding ROS accumulation, we examined cultured cells under the erythroid condition with a fluorescent sensor, Red CC-1. As a result, FF-10501 increased the ROS production. In summary, FF-10501 showed enough anti-proliferative effects to consider the clinical use. Furthermore, in this study we showed that FF-10501 improves normal erythropoiesis. The effect was cancelled by the inhibition of ROS accumulation or MAPK signaling. Our results suggest that FF-10501 could ameliorate myelosuppression during MDS treatment by maintaining myelopoiesis, and the low-dose therapy for low-risk MDS patients might be effective for the improving refractory anemia. Disclosures Murase: FUJIFILM Corporation: Employment. Komatsu:FUJIFILM Corporation: Employment. Saito:FUJIFILM Corporation: Employment. Iwamura:FUJIFILM Corporation: Employment. Kanakura:Alexion Pharma: Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau.