Lethal Myelofibrosis Induced by Bmi1-Deficient Hematopoietic Cells Unveils a Tumor Suppressor Function of the Polycomb Group Genes

Abstract 390 Polycomb-group (PcG) proteins form polycomb repressive complex (PRC) 1 and PRC2, and function as transcriptional repressors through histone modifications. They maintain proliferative capacity of hematopoietic stem and progenitor cells by repressing transcription of tumor suppressor genes, thus have been characterized as oncogenes. However, identification of inactivating mutations of a PcG gene, EZH2, unveiled its tumor suppressor function in myeloid malignancies including primary myelofibrosis (PMF). Here, we show that loss of another PcG gene, Bmi1, causes pathological hematopoiesis reminiscent of PMF in mice. We previously reported that deletion of both Ink4a and Arf in Bmi1-deficient mice substantially restores the defective self-renewal capacity of HSCs. To evaluate the repopulating capacity of Bmi1−/−Ink4a-Arf−/− BM cells precisely, we transplanted wild-type, Ink4a-Arf−/−, and Bmi1−/−Ink4a-Arf−/− BM cells into lethally irradiated mice without competitor cells. The recipients repopulated with Bmi1−/−Ink4a-Arf−/− donor cells had 2-fold more megakaryocyte/erythroid progenitors (MEPs) and extramedullary hematopoiesis as evident from a significant increase in the number of LSK HSCs/MPPs in spleen. All the control recipient mice repopulated with Ink4a-Arf−/− BM cells eventually developed sarcoma or lymphoma and succumbed to die by 11 months post-transplant as previously reported with the Ink4a-Arf−/− mice. On the other hand, the recipient mice repopulated with Bmi1−/−Ink4a-Arf−/− BM cells died much earlier than the Ink4a-Arf−/− controls, displayed more progressive pancytopenia, and showed marked hepatosplenomegaly and hypoplastic BM with massive fibrosis at their terminal stage. Although abnormal megakaryocytosis was not obvious in BM and spleen because of severe fibrosis at the terminal stage of the disease, the mice at earlier time point after transplantation showed marked megakaryocytosis in both BM and spleen, implicating pathological megakaryocytosis in the development of lethal myelofibrosis. Together, lethal myelofibrosis induced by Bmi1−/−Ink4a-Arf−/− hematopoietic cells follows the natural course of human PMF. To identify the responsible genes for PMF-like disease in the absence of Bmi1, we compared gene expression profiles of LSK HSCs/MPPs and common myeloid progenitors (CMPs) from wild-type, Ink4a-Arf −/−, and Bmi1−/−Ink4a-Arf −/− BM cells. Absence of Bmi1 caused de-repression of a cohort of genes. We then compared the list of de-repressed genes with that of PMF-associated genes identified by a gene expression profiling of CD34+ cells from human PMF patients (Guglielmelli et al., Stem Cells 25:165–173, 2007). Hmga2, a well-known oncogene, appeared to be commonly upregulated in Bmi1−/−Ink4a-Arf −/− CMPs and PMF CD34+ cells. Chromatin immunoprecipitation assays demonstrated that Bmi1 directly represses the expression of Hmga2 by marking its promoter with a repressive histone mark. To test contribution of Hmga2 to the development of PMF-like disease in mice, we examined the effects of Hmga2 overexpression on hematopoiesis. Forced expression of Hmga2 in HSCs promoted expansion of progenitor cells and significantly facilitated megakaryocytopoiesis in vitro. Hmga2-overexpressing HSCs also induced a mild myeloproliferative state with enhanced megakaryocytopoiesis in recipient mice, although fibrosis was not obviously induced. These results implicated Hmga2 in the development of pathological hematopoiesis in the absence of Bmi1. Collectively, our findings unveiled the tumor suppressor function of Bmi1. Corresponding to our findings, mice with hypomorphic mutations of Eed and Suz12 reportedly showed enhanced hematopoiesis. All these findings might be in line with tumor suppressor function of EZH2 observed in MDS and MPN patients, and suggestive of a broad range of target genes of the PcG proteins, which include both oncogenes and tumor suppressor genes. Although the tumor suppressor genes have been stresses as PcG targets, our findings shed a light on the role of PcG proteins in gene silencing of oncogenes. Thus, the PcG proteins fine-tune the growth of hematopoietic cells in both a positive and a negative manner to maintain hematopoietic homeostasis. Disclosures: No relevant conflicts of interest to declare.