An Impdh Inhibitor, FF-10501 Possesses Activating Effects on Myeloid Differentiation As Well As Anti-Proliferation

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.