RUNX1 is generally considered a tumor suppressor in myeloid neoplasms. Inactivating RUNX1 mutations have frequently been found in patients with myelodysplastic syndrome (MDS) and cytogenetically normal acute myeloid leukemia (AML). However, no somatic RUNX1 alteration was found in AMLs with leukemogenic fusion proteins, such as core-binding factor (CBF) leukemia and MLL fusion leukemia, raising the possibility that RUNX1 could actually promote the growth of these leukemia cells. Using normal human cord blood cells and those expressing leukemogenic fusion proteins, we discovered a dual role of RUNX1 in myeloid leukemogenesis. RUNX1 overexpression inhibited the growth of normal cord blood cells by inducing myeloid differentiation, whereas a certain level of RUNX1 activity was required for the growth of AML1-ETO and MLL-AF9 cells. Using a mouse genetic model, we also showed that the combined loss of Runx1/Cbfb inhibited leukemia development induced by MLL-AF9. RUNX2 could compensate for the loss of RUNX1. The survival effect of RUNX1 was mediated by BCL2 in MLL fusion leukemia. Our study unveiled an unexpected prosurvival role for RUNX1 in myeloid leukemogenesis. Inhibiting RUNX1 activity rather than enhancing it could be a promising therapeutic strategy for AMLs with leukemogenic fusion proteins.

The accession numbers for the antibody nucleotide sequences were incorrectly noted in Methods. The correct sentence appears below.Antibody nucleotide sequences have been deposited in GenBank (JX458933-52); antibody X-ray structure has been deposited in the Protein Data Bank (4GSD).The authors regret the error.

The t(8;21) rearrangement, which creates the AML1-ETO fusion protein, represents the most common chromosomal translocation in acute myeloid leukemia (AML). Clinical data suggest that CBL mutations are a frequent event in t(8;21) AML, but the role of CBL in AML1-ETO-induced leukemia has not been investigated. In this study, we demonstrate that CBL mutations collaborate with AML1-ETO to expand human CD34+ cells both in vitro and in a xenograft model. CBL depletion by shRNA also promotes the growth of AML1-ETO cells, demonstrating the inhibitory function of endogenous CBL in t(8;21) AML. Mechanistically, loss of CBL function confers hyper-responsiveness to thrombopoietin and enhances STAT5/AKT/ERK/Src signaling in AML1-ETO cells. Interestingly, we found the protein tyrosine phosphatase UBASH3B/Sts-1, which is known to inhibit CBL function, is upregulated by AML1-ETO through transcriptional and miR-9-mediated regulation. UBASH3B/Sts-1 depletion induces an aberrant pattern of CBL phosphorylation and impairs proliferation in AML1-ETO cells. The growth-inhibition caused by UBASH3B/Sts-1 depletion can be rescued by ectopic expression of CBL mutants, suggesting that UBASH3B/Sts-1 supports the growth of AML1-ETO cells partly through modulation of CBL function. Our study reveals a role of CBL in restricting myeloid proliferation of human AML1-ETO-induced leukemia, and identifies UBASH3B/Sts-1 as a potential target for pharmaceutical intervention.

Eradication of leukemia stem cells (LSCs) is the ultimate goal of treating acute myeloid leukemia (AML). We recently showed that the combined loss of Runx1/Cbfb inhibited the development of MLL-AF9-induced AML. However, c-Kit+/Gr-1− cells remained viable in Runx1/Cbfb-deleted cells, indicating that suppressing RUNX activity may not eradicate the most immature LSCs. In this study, we found upregulation of several hemostasis-related genes, including the thrombin-activatable receptor PAR-1 (protease-activated receptor-1), in Runx1/Cbfb-deleted MLL-AF9 cells. Similar to the effect of Runx1/Cbfb deletion, PAR-1 overexpression induced CDKN1A/p21 expression and attenuated proliferation in MLL-AF9 cells. To our surprise, PAR-1 deficiency also prevented leukemia development induced by a small number of MLL-AF9 leukemia stem cells (LSCs) in vivo. PAR-1 deficiency also reduced leukemogenicity of AML1-ETO-induced leukemia. Re-expression of PAR-1 in PAR-1-deficient cells combined with a limiting-dilution transplantation assay demonstrated the cell-dose-dependent role of PAR-1 in MLL-AF9 leukemia: PAR-1 inhibited rapid leukemic proliferation when there were a large number of LSCs, while a small number of LSCs required PAR-1 for their efficient growth. Mechanistically, PAR-1 increased the adherence properties of MLL-AF9 cells and promoted their engraftment to bone marrow. Taken together, these data revealed a multifaceted role for PAR-1 in leukemogenesis, and highlight this receptor as a potential target to eradicate primitive LSCs in AML.

No abstract

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.

Leukemic stem cells (LSCs) are capable of limitless self-renewal and indefinitely propagating leukemia. Eradication of LSCs is the ultimate goal of treating acute myeloid leukemia (AML). Using a mouse model of AML induced by the MLL-fusion protein MLL-AF9, we recently showed that the combined loss of Runx1/Cbfb inhibited the development of leukemia in vivo (Goyama S…Mulloy JC. Transcription factor RUNX1 promotes survival of acute myeloid leukemia cells. Journal of Clinical Investigation 123(9): 3876-3888, 2013). However, LSC-enriched cells with immature surface phenotype (cKit+Gr1-) remained viable in Runx1/Cbfb-deleted MLL-AF9 cells, indicating that RUNX targeting may not eradicate the most immature LSCs. Gene expression analyses of Runx1/Cbfb-deleted MLL-AF9 cells revealed the upregulation of thrombin pathway genes including a thrombin-activatable receptor PAR-1. Interestingly, both overexpression and knockout of PAR-1 inhibit leukemogenesis but do so through distinct mechanisms. Similar to the effect of Runx1/Cbfb-depletion, PAR-1 overexpression induced p21 expression and attenuated proliferation in MLL-AF9 cells. To our surprise, PAR-1-deficiency also prevented leukemia development induced by a small number of MLL-AF9 LSCs in vivo. Re-expression of PAR-1 in PAR-1-deficient cells combined with a limiting-dilution transplantation assay demonstrated the cell-dose dependent role of PAR-1 in MLL-AF9 leukemia: PAR-1 inhibited rapid leukemic proliferation when there are a large number of LSCs, while a small numbers of LSCs required PAR-1 for their growth. Mechanistically, PAR-1 increased adhering properties of MLL-AF9 cells and promoted their engraftment to bone marrow. PAR-1-deficiency also reduced leukemogenicity of AML1-ETO-induced leukemia. Together, these data reveal a multifaceted role for PAR-1 in leukemogenesis, and highlight this receptor as a potential target to eradicate primitive LSCs in AML. Disclosures No relevant conflicts of interest to declare.

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. Disclosures Goyama: Fuji Film: Research Funding. Iwamura:FUJIFILM Corporation: Employment. Saito:Fuji Film: Employment.