Necdin, a p53 target gene, regulates the quiescence and response to genotoxic stress of hematopoietic stem/progenitor cells.

We recently defined a critical role for p53 in regulating the quiescence of adult hematopoietic stem cells (HSCs) and identified necdin as a candidate p53 target gene. Necdin is a growth-suppressing protein and the gene encoding it is one of several that are deleted in patients with Prader-Willi syndrome. To define the intrinsic role of necdin in adult hematopoiesis, in the present study, we transplanted necdin-null fetal liver cells into lethally irradiated recipients. We show that necdin-null adult HSCs are less quiescent and more proliferative than normal HSCs, demonstrating the similar role of necdin and p53 in promoting HSC quiescence during steady-state conditions. However, wild-type recipients repopulated with necdin-null hematopoietic stem/progenitor cells show enhanced sensitivity to irradiation and chemotherapy, with increased p53-dependent apoptosis, myelosuppression, and mortality. Necdin controls the HSC response to genotoxic stress via both cell-cycle-dependent and cell-cycle-independent mechanisms, with the latter occurring in a Gas2L3-dependent manner. We conclude that necdin functions as a molecular switch in adult hematopoiesis, acting in a p53-like manner to promote HSC quiescence in the steady state, but suppressing p53-dependent apoptosis in response to genotoxic stress.

Generation of a novel, multi-stage, progressive, and transplantable model of plasma cell neoplasms.

Multiple myeloma is a plasma cell neoplasm with an extremely variable clinical course. Animal models are needed to better understand its pathophysiology and for preclinical testing of potential therapeutic agents. Hematopoietic cells expressing the hypermorphic Rad50(s) allele show hematopoietic failure, which can be mitigated by the lack of a transcription factor, Mef/Elf4. However, we find that 70% of Mef(-/-)Rad50(s/s) mice die from multiple myeloma or other plasma cell neoplasms. These mice initially show an abnormal plasma cell proliferation and monoclonal protein production, and then develop anemia and a decreased bone mineral density. Tumor cells can be serially transplanted and according to array CGH and whole exome sequencing, the pathogenesis of plasma cell neoplasms in these mice is not linked to activation of a specific oncogene, or inactivation of a specific tumor suppressor. This model recapitulates the systemic manifestations of human plasma cell neoplasms, and implicates cooperativity between the Rad50(s) and Mef/Elf4 pathways in initiating myelomagenic mutations that promote plasma cell transformation.

Akt phosphorylates the transcriptional repressor bmi1 to block its effects on the tumor-suppressing ink4a-arf locus.

The Polycomb group protein Bmi1 is a transcriptional silencer of the Ink4a-Arf locus, which encodes the cell cycle regulator p16(Ink4a) and the tumor suppressor p19(Arf). Bmi1 plays a key role in oncogenesis and stem cell self-renewal. We report that phosphorylation of human Bmi1 at Ser³¹6 by Akt impaired its function by triggering its dissociation from the Ink4a-Arf locus, which resulted in decreased ubiquitylation of histone H2A and the inability of Bmi1 to promote cellular proliferation and tumor growth. Moreover, Akt-mediated phosphorylation of Bmi1 also inhibited its ability to promote self-renewal of hematopoietic stem and progenitor cells. Our study provides a mechanism for the increased abundance of p16(Ink4a) and p19(Arf) seen in cancer cells with an activated phosphoinositide 3-kinase to Akt signaling pathway and identifies crosstalk between phosphorylation events and chromatin structure.

JAK2V617F-mediated phosphorylation of PRMT5 downregulates its methyltransferase activity and promotes myeloproliferation.

The JAK2V617F constitutively activated tyrosine kinase is found in most patients with myeloproliferative neoplasms. While examining the interaction between JAK2 and PRMT5, an arginine methyltransferase originally identified as JAK-binding protein 1, we found that JAK2V617F (and JAK2K539L) bound PRMT5 more strongly than did wild-type JAK2. These oncogenic kinases also acquired the ability to phosphorylate PRMT5, greatly impairing its ability to methylate its histone substrates, and representing a specific gain-of-function that allows them to regulate chromatin modifications. We readily detected PRMT5 phosphorylation in JAK2V617F-positive patient samples, and when we knocked down PRMT5 in human CD34+ cells using shRNA, we observed increased colony formation and erythroid differentiation. These results indicate that phosphorylation of PRMT5 contributes to the mutant JAK2-induced myeloproliferative phenotype.

p53 regulates hematopoietic stem cell quiescence.

The importance of the p53 protein in the cellular response to DNA damage is well known, but its function during steady-state hematopoiesis has not been established. We have defined a critical role of p53 in regulating hematopoietic stem cell quiescence, especially in promoting the enhanced quiescence seen in HSCs that lack the MEF/ELF4 transcription factor. Transcription profiling of HSCs isolated from wild-type and p53 null mice identified Gfi-1 and Necdin as p53 target genes, and using lentiviral vectors to upregulate or knockdown the expression of these genes, we show their importance in regulating HSC quiescence. Establishing the role of p53 (and its target genes) in controlling the cell-cycle entry of HSCs may lead to therapeutic strategies capable of eliminating quiescent cancer (stem) cells.

The ETS protein MEF plays a critical role in perforin gene expression and the development of natural killer and NK-T cells.

We utilized gene targeting by homologous recombination to define the role that MEF, a transcriptional activating member of the ETS family of transcription factors, plays in lymphopoiesis. MEF-/- mice have a profound reduction in the number of NK-T and NK cells. Purified MEF-/- NK cells cannot lyse tumor cell targets and secrete only minimal amounts of IFNgamma. Perforin protein expression is severely impaired in MEF-deficient NK cells, likely accounting for the lack of tumor cell cytotoxicity. Promoter studies and chromatin immunoprecipitation analyses demonstrate that MEF and not ETS-1 directly regulates transcription of the perforin gene in NK cells. Our results uncover a specific role of MEF in the development and function of NK cells and in innate immunity.