Inhibition of Impdh As an Effective Treatment for MLL-Fusion Leukemia

Abstract

Uncontrolled cell proliferation is a hallmark of cancer and requires adequate nucleotide biosynthesis. Inosine monophosphate dehydrogenase (IMPDH) is a rate-limiting enzyme that catalyzes the conversion of IMP to XMP at the branch point of guanine nucleotide biosynthesis. Inhibition of IMPDH results in the depletion of guanine nucleotides, thereby suppressing the growth of cancers cells. Therefore, IMPDH inhibition constitutes a rational approach to treat cancers including acute myeloid leukemia (AML).We have developed an experimental system to model myeloid leukemogenesis using primary human cord blood (CB) cells. AML1-ETO is a leukemogenic fusion protein and promotes the self-renewal and long-term proliferation of CB cells in vitro. Another fusion protein MLL-AF9 immortalizes CB cells in vitro and produces human leukemia in immunodeficient mice. These engineered pre-leukemic and leukemic cells recapitulate many features of the clinical diseases and have been useful in testing potential anti-leukemic drugs. Using these human cell-based models, we assessed the effect of a specific IMPDH inhibitor, mycophenolic acid (MPA), on CB cells and those expressing AML1-ETO and MLL-AF9. MPA showed substantial growth-inhibitory effect against MLL-AF9-expressing CB cells, while it had only marginal effects on normal CB cells and AML1-ETO cells. Mechanistically, MPA treatment caused a cell cycle arrest at G0/G1-phase, triggered apoptosis, and induced upregulation of p53 and its downstream target genes including p21/CDKN1A in MLL-AF9 cells. The MPA-mediated changes in MLL-AF9 cells were almost fully reversed by the supplementation of guanosine, confirming that guanine nucleotide depletion underlies the effects of MPA. In contrast, knockdown of p53 or p21, or expression of a dominant-negative form of p53 (p53-DD), did not abrogate the growth-inhibitory effect of MPA on MLL-AF9 cells, indicating that MPA inhibits the leukemic growth largely through p53-independent mechanisms.To examine whether MPA has single-agent activity for MLL-fusion leukemia in vivo, we next established a mouse bone marrow transplant assay for MLL-AF9 leukemia. Bone marrow progenitors were transduced with MLL-AF9 and were transplanted into recipient mice. MLL-AF9-expressing bone marrow progenitors produced AML within 3 months. Leukemic cells were isolated from the spleens of moribund primary mice, and were transplanted into secondary recipient mice. These mice were treated either with injections of MPA (100 mg kg−1) every other day or with vehicle. MPA administration led to a delay in disease progression and significantly extended survival. We also tested the in vivo effect of another IMPDH inhibitor FF-10501-01 on MLL-AF9 leukemia. FF-10501-01 is a potent new competitive IMPDH inhibitor undergoing phase I clinical trials for patients with AML and high-risk myelodysplastic syndrome (MDS). As expected, FF-10501-01 also showed significant therapeutic value, providing survival benefit in a mouse MLL-AF9 leukemia model. Finally, we assessed the sensitivity of p53-deficient mouse MLL-AF9 leukemia cells to IMPDH inhibition. We generated p53-deficient leukemia cells by expressing MLL-AF9 into bone marrow progenitors derived from p53 knockout mice. Consistent with earlier results, the p53-deficient MLL-AF9 cells were still sensitive to MPA and FF-10501-01 both in vitro and in vivo, indicating that p53 activation is dispensable for the anti-leukemia effect of these IMPDH inhibitors.Taken together, these findings establish the inhibition of IMPDH as a promising therapy for MLL-fusion leukemia, including those with defective p53 signaling. DisclosuresGoyama:Fuji Film: Research Funding. Iwamura:FUJIFILM Corporation: Employment. Saito:Fuji Film: Employment.