Novel Myelodysplastic Syndromes-like Mouse Models By Cooperating Genetic/Epigenetic Mutations Reveal the Critical Role of HIF-1α for Disease Development

Myelodysplasitc syndromes (MDS) are associated with genetic and epigenetic defects in transcription factors and/or chromatin-modifying enzymes that regulate self-renewal, survival, and differentiation. Mixed lineage leukemia (MLL) is a transcription factor which regulates its downstream targets by an epigenetic manner and plays a critical role for regulation of normal hematopoiesis. Mutations in MLL have been identified in variety of hematopoietic malignancies. Internal partial tandem duplication (MLL-PTD) is a major aberration of MLL gene. MLL-PTD is often found in the patients with MDS, secondary AML (sAML), and de novo AML. We have previously shown that MLL-PTD knock-in (MLL^PTD/WT) mice present increased self-renewal and apoptosis in hematopoietic stem/progenitor cells (HSPCs) and skewed myeloid differentiation, but never develop MDS or AML phenotypes. This suggests that additional genetic and/or epigenetic defects are necessary for the transformation. Interestingly, MLL-PTD is significantly associated with RUNX1 mutations in sAML and de novo AML. Thus, we hypothesized that combination of these commonly co-mutated genes might generate faithful mouse models for human diseases.

To understand the impact of RUNX1 mutations in the MLL-PTD background, we retrovirally introduced MDS patient-derived RUNX1 mutants (RUNX1-D171N and RUNX1-291sX300) into BM cells obtained from 5-FU treated MLL^PTD/WT mice. These transduced cells were transplanted into lethally irradiated recipient mice. Both MLL^PTD/WT/D171N and MLL^PTD/WT/291fsX300 BMT mice quickly developed macrocytic anemia and mild thrombocytopenia. Tri-lineage dysplasia was observed in BM from both groups. Anemia in MLL^PTD/WT/291fsX300 BMT mice was more severe than that in MLL^PTD/WT/D171N mice. MLL^PTD/WT/D171N BMT mice presented hypo-cellular marrow with excess blasts (blasts< 5%), while MLL^PTD/WT/291fsX300 BMT mice presented hyper-cellular marrow with higher percentage of blasts (blasts < 20%). Interestingly monocytes in PB from MLL^PTD/WT/291fsX300 BMT mice were significantly increased compared to control and MLL^PTD/WT/D171N BMT mice. MLL^PTD/WT/291fsX300 BMT mice also developed BM fibrosis with slight splenomegaly. Interestingly, 20-30% of MDS patients, especially high-risk MDS with BM fibrosis, are also accompanied with splenomegaly.

We have previously shown that MLL-PTD enhances self-renewal of HSPCs. To understand the mechanism of enhanced self-renewal, we performed gene expression array analysis and mRNA-sequencing, followed by KEGG pathway analysis. Interestingly, one of the top dysregulated pathways was Glycolysis/Gluconeogenesis pathway. HIF-1α is one well-known transcription factor for this pathway. Thus, we measured HIF-1α expression in HSPCs from MLL^PTD/WT mice at both mRNA and protein levels. HIF-1α protein and mRNA of HIF-1α target genes in HSPCs from MLL^PTD/WT mice were significantly increased compared with those from WT mice. To determine the direct interaction between MLL-PTD and HIF-1α, we introduced WT MLL or MLL-PTD together with HIF-1α cDNA into 293T cells and performed western blot (WB) and imuunoprecipitation-WB. We found the strong accumulation of HIF-1α in MLL-PTD co-transfectants and interaction between MLL-PTD and HIF-1α. To assess the significance of HIF-1α in MLL-PTD mediated pathogenesis, we performed CFU assay in the presence of a HIF-1α inhibitor, Echinomycin. MLL^PTD/WT cells formed significantly fewer colonies than WT cells. RUNX1 mutants transduced MLL^PTD/WT cells were hyper-sensitive to Echinomycin while WT HSPCs or AML cells derived from Cbfb/SMMHC knock-in mice showed less sensitivity. Echinomycin could induce myeloid differentiation and inhibit MDS-initiating cells. We further took a genetic approach and performed BMT assay. HIF-1α deletion significantly reduced reconstitution ability of MLL^PTD/WT cells and MDS development in our mouse models. These results strongly suggest that HIF-1α is essential for MLL^PTD/WT and MLL^PTD/WT/RUNX1 mutant mediated self-renewal and MDS-like disease development.

In conclusion, 1) we have established novel MDS-like mouse models which recapitulate human diseases by using genetic/epigenetic mutations (RUNX1/MLL-PTD); 2) we identified HIF-1α as a critical factor for MDS-like disease by using our robust MDS-like mouse models; 3) targeting HIF-1α could eradicate the MDS phenotypes and the MDS-initiation cells.