Anthony Deblasio

Complete Remission after Treatment of Acute Promyelocytic Leukemia with Arsenic Trioxide

BACKGROUND Two reports from China have suggested that arsenic trioxide can induce complete remissions in patients with acute promyelocytic leukemia (APL). We evaluated this drug in patients with APL in an attempt to elucidate its mechanism of action. METHODS Twelve patients with APL who had relapsed after extensive prior therapy were treated with arsenic trioxide at doses ranging from 0.06 to 0.2 mg per kilogram of body weight per day until visible leukemic cells were eliminated from the bone marrow. Bone marrow mononuclear cells were serially monitored by flow cytometry for immunophenotype, fluorescence in situ hybridization, reverse-transcription-polymerase-chain-reaction (RT-PCR) assay for PML-RAR-alpha fusion transcripts, and Western blot analysis for expression of the apoptosis-associated proteins caspases 1, 2, and 3. RESULTS Of the 12 patients studied, 11 achieved complete remission after treatment that lasted from 12 to 39 days (range of cumulative doses, 160 to 495 mg). Adverse effects were relatively mild and included rash, lightheadedness, fatigue, and musculoskeletal pain. Cells that expressed both CD11b and CD33 (antigens characteristic of mature and immature cells, respectively), and which were found by fluorescence in situ hybridization to carry the t(15;17) translocation, increased progressively in number during treatment and persisted in the early phase of complete remission. Eight of 11 patients who initially tested positive for the PML-RAR-alpha fusion transcript by the RT-PCR assay later tested negative; 3 other patients, who persistently tested positive, relapsed early. Arsenic trioxide induced the expression of the proenzymes of caspase 2 and caspase 3 and activation of both caspase 1 and caspase 3. CONCLUSIONS Low doses of arsenic trioxide can induce complete remissions in patients with APL who have relapsed. The clinical response is associated with incomplete cytodifferentiation and the induction of apoptosis with caspase activation in leukemic 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.

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.

Akt Phosphorylates the Transcriptional Repressor Bmi1 to Block Its Effects on the Tumor-Suppressing Ink4a-Arf Locus

Akt counteracts growth-promoting signals by stimulating the transcription of tumor suppressor genes. Silencing the Silencer The Polycomb group protein Bmi1 transcriptionally silences the Ink4a-Arf locus and thus decreases the abundance of the tumor suppressor proteins p16 and p19. Liu et al. found that phosphorylation of Bmi1 by the kinase Akt causes it to dissociate from the Ink4a-Arf locus, which results in increased abundance of p16 and p19 that decreases cellular proliferation, tumor growth, and self-renewal of stem and progenitor cells. Thus, Akt, which is typically activated downstream of growth-promoting signals, can mediate a feedback loop that ultimately attenuates these growth signals. The Polycomb group protein Bmi1 is a transcriptional silencer of the Ink4a-Arf locus, which encodes the cell cycle regulator p16Ink4a and the tumor suppressor p19Arf. 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 p16Ink4a and p19Arf seen in cancer cells with an activated phosphoinositide 3-kinase to Akt signaling pathway and identifies crosstalk between phosphorylation events and chromatin structure.

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 Rad50s allele show hematopoietic failure, which can be mitigated by the lack of a transcription factor, Mef/Elf4. However, we find that 70% of Mef−/−Rad50s/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 Rad50s and Mef/Elf4 pathways in initiating myelomagenic mutations that promote plasma cell transformation.