TET1 is a tumor suppressor of hematopoietic malignancy

The methylcytosine dioxygenase TET1 (‘ten-eleven translocation 1’) is an important regulator of 5-hydroxymethylcytosine (5hmC) in embryonic stem cells. The diminished expression of TET proteins and loss of 5hmC in many tumors suggests a critical role for the maintenance of this epigenetic modification. Here we found that deletion of Tet1 promoted the development of B cell lymphoma in mice. TET1 was required for maintenance of the normal abundance and distribution of 5hmC, which prevented hypermethylation of DNA, and for regulation of the B cell lineage and of genes encoding molecules involved in chromosome maintenance and DNA repair. Whole-exome sequencing of TET1-deficient tumors revealed mutations frequently found in non-Hodgkin B cell lymphoma (B-NHL), in which TET1 was hypermethylated and transcriptionally silenced. Our findings provide in vivo evidence of a function for TET1 as a tumor suppressor of hematopoietic malignancy.National Institutes of Health (U.S.) (5RO1HD045022)National Institutes of Health (U.S.) (5R37CA084198

Pharmacological evidence for transactivation within melatonin MT2 and serotonin 5-HT2C receptor heteromers in mouse brain

Association of G protein-coupled receptors into heterodimeric complexes has been reported for over 50 receptor pairs in vitro but functional in vivo validation remains a challenge. Our recent in vitro studies defined the functional fingerprint of heteromers composed of Gi-coupled melatonin MT2 receptors and Gq-coupled serotonin 5-HT2C receptors, in which melatonin transactivates phospholipase C (PLC) through 5-HT2C. Here, we identified this functional fingerprint in the mouse brain. Gq protein activation was probed by [35S]GTPγS incorporation followed by Gq immunoprecipitation, and PLC activation by determining the inositol phosphate levels in brain lysates of animals previously treated with melatonin. Melatonin concentration-dependently activated Gq proteins and PLC in the hypothalamus and cerebellum but not in cortex. These effects were inhibited by the 5-HT2C receptor-specific inverse agonist SB-243213, and were absent in MT2 and 5-HT2C knockout mice, fully recapitulating previous in vitro data and indicating the involvement of MT2/5-HT2C heteromers. The antidepressant agomelatine had a similar effect than melatonin when applied alone but blocked the melatonin-promoted Gq activation due to its 5-HT2C antagonistic component. Collectively, we provide strong functional evidence for the existence of MT2/5-HT2C heteromeric complexes in mouse brain. These heteromers might participate in the in vivo effects of agomelatine

Generation of a Novel, Multi-Stage, Progressive, and Transplantable Model of Multiple Myeloma

Abstract Abstract 327 Multiple myeloma is characterized by the progressive expansion of monoclonal plasma cells in the bone marrow, which leads to the production of serum and/or urine monoclonal proteins and systemic complications including lytic bone lesions, renal abnormalities hypercalcemia, and infections. Although the treatment of multiple myeloma has vastly improved, multiple myeloma remains a generally incurable disease. Transgenic mouse models have been generated that develop plasma cell accumulations or myeloma, however these models are quite imperfect in mimicking the human disease. Quite serendipitously, we have generated a multi-stage, progressive, and transplantable mouse model of multiple myeloma, crossing a genetically modified mouse with aberrant class switch recombination with another modified mouse that has elevated DNA damage response signaling. We have reported that cells expressing the hypermorphic Rad50s allele show constitutive ATM activation, leading to cancer predisposition and aggressive hematopoietic failure in Rad50s/s mice. While deficiency of the transcription factor Mef/Elf4, which regulates the quiescence of hematopoietic stem/progenitor cells, can mitigate hematopoietic failure observed in Rad50s/s mice, we found that 70% of Mef−/−Rad50s/s mice more than 200 days old died from multiple myeloma, plasmacytoma, or plasma cell leukemia, confirmed by pathology, immunohistochemistry, flowcytometry (CD138/B220 profiles), and PCR analysis for VDJ recombination. Prior to the onset of the plasma cell neoplasms, the Mef−/−Rad50s/s mice show abnormal plasma cell accumulation in the peripheral blood and bone marrow, which worsens with age. As the mice age, they also develop progressive increases in g-globulin levels and decreases in serum albumin levels. Monoclonal protein peaks were frequently observed in the serum of mice older than 200 days, and in step with the progressive nature of these manifestations, anemia and lower bone mineral density becomes apparent as the mice further age. Overall, the median survival of the Mef−/−Rad50s/s mice is approximately 470 days. The plasma cell neoplasms derived from Mef−/−Rad50s/s mice can be transplanted into recipient mice and the onset of the transplanted disease is markedly accelerated, to approximately 4 weeks post transplantation. Thus, the transplanted neoplastic Mef−/−Rad50s/s plasma cells appear to be more aggressive than the original ones. Taken together, our findings suggest that the Mef−/−Rad50s/s animal model can recapitulate the spectrum and pace of human plasma cell neoplasms, including the progression from monoclonal gammopathy to multiple myeloma. Class switch recombination is facilitated in Mef−/−Rad50s/s B cells in vitro, compared with control, Mef−/−, and Rad50s/s B cells, thus the plasma cell neoplasms found in Mef−/−Rad50s/s mice may result from Rad50s-driven oncogenesis. This novel Mef−/−Rad50s/s myeloma animal model should be useful for the drug screening of novel anti-myeloma compounds, as well as defining the pathogenesis of multiple myeloma/plasma cell neoplasms. Disclosures: No relevant conflicts of interest to declare

Notch pathway activation targets AML-initiating cell homeostasis and differentiation

Notch signaling pathway activation is known to contribute to the pathogenesis of a spectrum of human malignancies, including T cell leukemia. However, recent studies have implicated the Notch pathway as a tumor suppressor in myeloproliferative neoplasms and several solid tumors. Here we report a novel tumor suppressor role for Notch signaling in acute myeloid leukemia (AML) and demonstrate that Notch pathway activation could represent a therapeutic strategy in this disease. We show that Notch signaling is silenced in human AML samples, as well as in AML-initiating cells in an animal model of the disease. In vivo activation of Notch signaling using genetic Notch gain of function models or in vitro using synthetic Notch ligand induces rapid cell cycle arrest, differentiation, and apoptosis of AML-initiating cells. Moreover, we demonstrate that Notch inactivation cooperates in vivo with loss of the myeloid tumor suppressor Tet2 to induce AML-like disease. These data demonstrate a novel tumor suppressor role for Notch signaling in AML and elucidate the potential therapeutic use of Notch receptor agonists in the treatment of this devastating leukemia

The PcG Protein L3MBTL1 Transcriptionally Represses Human Embryonic and Fetal Globin Genes: a Novel Prospect for HbF Activation

Abstract Abstract 1014 L3MBTL1 is the human homolog of the Drosophila Polycomb Group tumor suppressor gene, lethal(3)malignant brain tumor. We demonstrated that human L3MBTL1 functions as a transcriptional repressor and after crystallizing the MBT repeat domain determined that L3MBTL1 compacts chromatin by binding mono- and di-methylated lysine residues in histones H1 (H1K26) and H4 (H4K20). Despite the known role of L3MBTL1 in affecting chromatin structure, the function of L3MBTL1 in human hematopoiesis has remained largely unknown. We recently demonstrated that L3MBTL1 enforces cell fate decision toward the erythroid lineage and that knockdown of L3MBTL1 accelerates the erythroid differentiation of human hematopoietic stem/progenitor cells, suggesting that its deletion contributes to the pathogenesis of 20q- erythroid malignancies. Consistently with its role in erythropoiesis, here we reveal that L3MBTL1 is a novel transcriptional repressor of fetal globin genes and it may work in concert with BCL11A and EKLF to control globin gene expression. By utilizing RNA interference to reduce L3MBTL1 expression, we have found that knockdown of L3MBTL1 in human cord blood hematopoietic stem/progenitor cells consistently upregulates the expression of the epsilon, gamma, and zeta globin genes, but not the beta globin gene. Similar effects were seen following knockdown of L3MBTL1 in the human erythroleukemia cell line K562, and knockdown of L3MBTL1 in human embryonic stem cells (ESCs) led to the inappropriate expression of fetal and embryonic globin genes (which increases more than 50-fold after the L3MBTL1-KD). These data suggest a role for L3MBTL1 in regulating the globin switch. To investigate the mechanism by which L3MBTL1 silences embryonic and fetal globin gene expression, we used chromatin immunoprecipitation (ChIP) assays to show that L3MBTL1 directly associates with the human β-globin locus. L3MBTL1 occupies several discrete regions within the human β-globin cluster and colocalizes with H4K20me within the Locus Control Region (LCR), a primary attachment site for chromatin modifiers. As confirmation, we found that treatment of K562 cells with hemin, which broadly increases H3K9 acetylation over the β-globin locus and activates the transcription of globin genes, leads to decreases in expression of the repressive H4K20me2 methylmark and L3MBTL1 to the beta-globin cluster. Given the recent identification of the repressor of gamma globin gene expression, BCL11A, we investigated a potential relationship between L3MBTL1 and BCL11A. We found that knockdown of L3MBTL1 led to downregulation of BCL11A mRNA. Accordingly, we have also found that overexpression of L3MBTL1 is associated with an upregulation of BCL11A mRNA, suggesting that L3MBTL1 and BCL11A may function cooperatively to silence globin gene expression. Knockdown of L3MBTL1 also upregulated EKLF mRNA levels which could relate to the decreased BCL11A expression. In summary our data demonstrate that knock-down of L3MBTL1 upregulates embryonic and fetal globin genes in cell contexts where they are usually silenced, indicating the functional importance of this Polycomb protein for repressing the globin gene locus. The clearance of L3MBTL1 and its associated histone mark (H4K20me2) during treatments that induce potent transcriptional activation of globin genes suggest that repression induced by L3MBTL1 is dynamic and may be involved in the fetal-to-adult globin switch. L3MBTL1 therefore emerges as a novel transcriptional repressor of fetal globin genes whose expression may be coordinated with that of BCL11A and EKLF. Understanding the role of L3MBTL1 and the H4K20 methylmark in globin gene switching offers the prospect of the targeted activation of HbF in erythroid cells of patients with hemoglobin disorders. Disclosures: No relevant conflicts of interest to declare

Tet2 Loss Leads to Increased Hematopoietic Stem Cell Self-Renewal and Myeloid Transformation

Somatic loss-of-function mutations in the ten-eleven translocation 2 ( TET2) gene occur in a significant proportion of patients with myeloid malignancies. Although there are extensive genetic data implicating TET2 mutations in myeloid transformation, the consequences of Tet2 loss in hematopoietic development have not been delineated. We report here an animal model of conditional Tet2 loss in the hematopoietic compartment that leads to increased stem cell self-renewal in vivo as assessed by competitive transplant assays. Tet2 loss leads to a progressive enlargement of the hematopoietic stem cell compartment and eventual myeloproliferation in vivo, including splenomegaly, monocytosis, and extramedullary hematopoiesis. In addition, Tet2 +/− mice also displayed increased stem cell self-renewal and extramedullary hematopoiesis, suggesting that Tet2 haploinsufficiency contributes to hematopoietic transformation in vivo. ► Tet2-expression silencing leads to increased self-renewal ability ► Tet2 deletion leads to progressive defects in hematopoiesis ► Tet2-deficient hematopoietic stem cells show increased repopulating ability ► Tet2-deficient animals develop CMML-like diseas

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