Leukemia-Associated Mutations Of DNMT3A Inhibit Differentiation Of Hematopoietic Stem Cells and Promote Leukemic Transformation Through Abberant Recruitment Of Bmi1

Whole genome sequencing has revealed DNMT3A mutation is present in over 20% of cytogenetically normal acute myeloid leukemia (CN-AML) and R882 is the most frequent and recurrent mutated site. Cumulating clinical data have emphasized the importance of the mutation as a poor prognostic factor of AML. Since the functional role of DNMT3A mutation in leukemogenesis remains largely unknown, we aimed to elucidate the impact of DNMT3A mutation on the development and maintenance of AML. To investigate the effect of exogenous expression of DNMT3A R882 mutant (Mut) in hematopoiesis, we transplanted 5-FU primed mouse bone marrow cells transduced with empty vector (EV), DNMT3A wild type (WT), or DNMT3A Mut to lethally irradiated mice. Recipients transplanted with DNMT3A Mut-transduced cells exhibited hematopoietic stem cell (CD150+CD48-Lin-Sca1+c-Kit+) accumulation and enhanced repopulating capacity compared with EV and DNMT3A WT recipients. To identify the downstream target genes of DNMT3A Mut that evoked hematopoietic stem cell accumulation, we sorted vector-transduced LSK cells from transplanted mice and conducted quantitative PCR (Q-PCR) of various hematopoiesis-related genes. Q-PCR revealed that Hoxb cluster expression was up-regulated and differentiation-associated genes, such as PU.1 and C/ebpa, were down-regulated in DNMT3A Mut-transduced LSK cells. Targeted bisulfite sequencing showed hypomethylation of the Hoxb2 promoter-associated CpG island in DNMT3A Mut-transduced cells compared with EV-transduced cells, which suggests dominant-negative effect of DNMT3A R882 mutation. DNMT3A Mut caused no change in methylation status of PU.1 promoter-associated CpG island, indicating that DNA methylation-independent mechanism underlies PU.1 downregulation. Given that DNMT3A interacts with several histone modifiers to regulate target gene transcription, we performed co-immunoprecipitation to investigate whether these interactions are altered by DNMT3A mutation. We found that DNMT3A Mut has the emhanced capacity to interact with polycomb repressive complex 1 (PRC1), which is thought to be a potential mechanism of the DNMT3A Mut-induced differentiation defect. Co-immunoprecipitation experiments showed that DNMT3A R882H and R882C mutant exhibited augmented interaction with BMI1 and MEL18, respectively. In addition, RING1B, an essential component of PRC1, co-localized with DNMT3A Mut more frequently than WT, irrespective of the type of amino acid substitution. Furthermore, heterozygosity of Bmi1 restored the PU.1 mRNA to the normal level and canceled the effect of stem cell accumulation in mice transplanted with DNMT3A Mut bone marrow cells. Chromatin immunoprecipitation in AML cell lines showed that BMI1 and RING1B were more efficiently recruited to the upstream regulatory element of PU.1 upon expression of DNMT3A Mut than WT, while the amount of DNMT3A recruited were comparable between DNMT3A WT and Mut. In the murine transplantation model, we found that exogenous PU.1 expression impaired repopulating capacity in both EV and R882H-transduced cells to the similar level. Exogenous expression of DNMT3A WT inhibited proliferation and induced terminal myeloid differentiation, whereas DNMT3A Mut-transduced cells remained immature in AML cell lines. DNMT3A Mut-transduced cells were resistant to ATRA-induced differentiation compared to EV-transduced cells. Furthermore, R882 mutation promoted blastic transformation of murine c-Kit+ bone marrow cells in vitro in combination with HOXA9 which is highly expressed in clinical cases harboring DNMT3A mutation. Morphological and surface marker analysis revealed these cells were F4/80+ monocytic blasts, consistent with clinical observation that DNMT3A mutation is found frequently in FAB M4/M5 leukemia. These results indicate a distinct role for DNMT3A Mut as well as a potential collaboration between DNMT3A Mut and HOXA9 in malignant transformation of hematopoietic cells. Interestingly, Bmi1 heterozygosity impaired this monoblastic transformation of R882H and HOXA9 co-transduced progenitors. Taken together, our results highlight the functional role of DNMT3A mutation in differentiation block of hematopoietic stem cells and in promoting leukemic transformation via aberrant recruitment of Bmi1 and other PRC1 components.