Melissa A. Fischer

HDAC3 is essential for DNA replication in hematopoietic progenitor cells

Histone deacetylase 3 (HDAC3) contributes to the regulation of gene expression, chromatin structure, and genomic stability. Because HDAC3 associates with oncoproteins that drive leukemia and lymphoma, we engineered a conditional deletion allele in mice to explore the physiological roles of Hdac3 in hematopoiesis. We used the Vav-Cre transgenic allele to trigger recombination, which yielded a dramatic loss of lymphoid cells, hypocellular bone marrow, and mild anemia. Phenotypic and functional analysis suggested that Hdac3 was required for the formation of the earliest lymphoid progenitor cells in the marrow, but that the marrow contained 3-5 times more multipotent progenitor cells. Hdac3(-/-) stem cells were severely compromised in competitive bone marrow transplantation. In vitro, Hdac3(-/-) stem and progenitor cells failed to proliferate, and most cells remained undifferentiated. Moreover, one-third of the Hdac3(-/-) stem and progenitor cells were in S phase 2 hours after BrdU labeling in vivo, suggesting that these cells were impaired in transit through the S phase. DNA fiber-labeling experiments indicated that Hdac3 was required for efficient DNA replication in hematopoietic stem and progenitor cells. Thus, Hdac3 is required for the passage of hematopoietic stem/progenitor cells through the S phase, for stem cell functions, and for lymphopoiesis.

MTGR1 is required for tumorigenesis in the murine AOM/DSS colitis-associated carcinoma model.

Myeloid Translocation Gene, Related-1 (MTGR1) CBFA2T2 is a member of the Myeloid Translocation Gene (MTG) family of transcriptional corepressors. The remaining two family members, MTG8 (RUNX1T1) and MTG16 (CBFA2T3) are identified as targets of chromosomal translocations in acute myeloid leukemia (AML). Mtgr1(-/-) mice have defects in intestinal lineage allocation and wound healing. Moreover, these mice show signs of impaired intestinal stem cell function. Based on these phenotypes, we hypothesized that MTGR1 may influence tumorigenesis arising in an inflammatory background. We report that Mtgr1(-/-) mice were protected from tumorigenesis when injected with azoxymethane (AOM) and then subjected to repeated cycles of dextran sodium sulfate (DSS). Tumor cell proliferation was comparable, but Mtgr1(-/-) tumors had significantly higher apoptosis rates. These phenotypes were dependent on epithelial injury, the resultant inflammation, or a combination of both as there was no difference in aberrant crypt foci (ACF) or tumor burden when animals were treated with AOM as the sole agent. Gene expression analysis indicated that Mtgr1(-/-) tumors had significant upregulation of inflammatory networks, and immunohistochemistry (IHC) for immune cell subsets revealed a marked multilineage increase in infiltrates, consisting predominately of CD3(+) and natural killer T (NKT) cells as well as macrophages. Transplantation of wild type (WT) bone marrow into Mtgr1(-/-) mice, and the reciprocal transplant, did not alter the phenotype, ruling out an MTGR1 hematopoietic cell-autonomous mechanism. Our findings indicate that MTGR1 is required for efficient inflammatory carcinogenesis in this model, and implicate its dysfunction in colitis-associated carcinoma. This represents the first report functionally linking MTGR1 to intestinal tumorigenesis.

Myeloid translocation gene 16 is required for maintenance of haematopoietic stem cell quiescence

The t(8;21) and t(16;21) that are associated with acute myeloid leukaemia disrupt two closely related genes termed Myeloid Translocation Genes 8 (MTG8) and 16 (MTG16), respectively. Many of the transcription factors that recruit Mtg16 regulate haematopoietic stem and progenitor cell functions and are required to maintain stem cell self‐renewal potential. Accordingly, we found that Mtg16‐null bone marrow (BM) failed in BM transplant assays. Moreover, when removed from the animal, Mtg16‐deficient stem cells continued to show defects in stem cell self‐renewal assays, suggesting a requirement for Mtg16 in this process. Gene expression analysis indicated that Mtg16 was required to suppress the expression of several key cell‐cycle regulators including E2F2, and chromatin immunoprecipitation assays detected Mtg16 near an E2A binding site within the first intron of E2F2. BrdU incorporation assays indicated that in the absence of Mtg16 more long‐term stem cells were in the S phase, even after competitive BM transplantation where normal stem and progenitor cells are present, suggesting that Mtg16 plays a role in the maintenance of stem cell quiescence.

Discovery and biological characterization of potent myeloid cell leukemia‐1 inhibitors

Myeloid cell leukemia 1 (Mcl‐1) is an antiapoptotic member of the Bcl‐2 family of proteins that when overexpressed is associated with high tumor grade, poor survival, and resistance to chemotherapy. Mcl‐1 is amplified in many human cancers, and knockdown of Mcl‐1 using RNAi can lead to apoptosis. Thus, Mcl‐1 is a promising cancer target. Here, we describe the discovery of picomolar Mcl‐1 inhibitors that cause caspase activation, mitochondrial depolarization, and selective growth inhibition. These compounds represent valuable tools to study the role of Mcl‐1 in cancer and serve as useful starting points for the discovery of clinically useful Mcl‐1 inhibitors.

Mtg16/Eto2 contributes to murine T-cell development

ABSTRACT Mtg16/Eto2 is a transcriptional corepressor that is disrupted by t(16;21) in acute myeloid leukemia. Using mice lacking Mtg16, we found that Mtg16 is a critical regulator of T-cell development. Deletion of Mtg16 led to reduced thymocyte development in vivo, and after competitive bone marrow transplantation, there was a nearly complete failure of Mtg16−/− cells to contribute to thymocyte development. This defect was recapitulated in vitro as Mtg16−/− Lineage−/Sca1+/c-Kit+ (LSK) cells of the bone marrow or DN1 cells of the thymus failed to produce CD4+/CD8+ cells in response to a Notch signal. Complementation of these defects by reexpressing Mtg16 showed that 3 highly conserved domains were somewhat dispensable for T-cell development but required the capacity of Mtg16 to suppress E2A-dependent transcriptional activation and to bind to the Notch intracellular domain. Thus, Mtg16 integrates the activities of signaling pathways and nuclear factors in the establishment of T-cell fate specification.

Defective replication stress response inhibits lymphomagenesis and impairs lymphocyte reconstitution

DNA replication stress promotes genome instability in cancer. However, the contribution of the replication stress response to the development of malignancies remains unresolved. The DNA replication stress response protein SMARCAL1 stabilizes DNA replication forks and prevents replication fork collapse, a cause of DNA breaks and apoptosis. While the fork regression/remodeling functions of SMARCAL1 have been investigated, its in vivo functions in replication stress and cancer are unclear. Using a gamma radiation (IR)-induced replication stress T-cell lymphoma mouse model, we observed a significant inhibition of lymphomagenesis in mice lacking one or both alleles of Smarcal1. Notably, a quarter of the Smarcal1-deficient mice did not develop tumors. Moreover, hematopoietic stem/progenitor cells (HSPCs) and developing thymocytes in Smarcal1-deficient mice showed increased DNA damage and apoptosis during the proliferation burst following IR and an impaired ability to repopulate the thymus after IR. Additionally, mice lacking Smarcal1 showed significant HSPC defects when challenged to respond to other replication stress stimuli. Thus, our data reveal the critical function of the DNA replication stress response and, specifically, Smarcal1 in hematopoietic cell survival and tumor development. Our results also provide important insight into the immunodeficiency observed in individuals with mutations in SMARCAL1 by suggesting that it is an HSPC defect.

Therapeutic approaches in myelofibrosis and myelodysplastic/myeloproliferative overlap syndromes

The discovery of JAK2V617F a decade ago led to optimism for a rapidly developing treatment revolution in Ph− myeloproliferative neoplasms. Unlike BCR–ABL, however, JAK2 was found to have a more heterogeneous role in carcinogenesis. Therefore, for years, development of new therapies was slow, despite standard treatment options that did not address the overwhelming symptom burden in patients with primary myelofibrosis (MF), post-essential thrombocythemia MF, post-polycythemia vera MF, and myelodysplastic syndrome (MDS)/myeloproliferative neoplasm (MPN) syndromes. JAK–STAT inhibitors have changed this, drastically ameliorating symptoms and ultimately beginning to show evidence of impact on survival. Now, the genetic foundations of myelofibrosis and MDS/MPN are rapidly being elucidated and contributing to targeted therapy development. This has been empowered through updated response criteria for MDS/MPN and refined prognostic scoring systems in these diseases. The aim of this article is to summarize concisely the current and rationally designed investigational therapeutics directed at JAK–STAT, hedgehog, PI3K–Akt, bone marrow fibrosis, telomerase, and rogue epigenetic signaling. The revolution in immunotherapy and novel treatments aimed at previously untargeted signaling pathways provides hope for considerable advancement in therapy options for those with chronic myeloid disease.

MTG16 is a tumor suppressor in colitis-associated carcinoma

MTG16 is a member of the myeloid translocation gene (MTG) family of transcriptional corepressors. While MTGs were originally identified in chromosomal translocations in acute myeloid leukemia, recent studies have uncovered a role in intestinal biology. For example, Mtg16-/- mice have increased intestinal proliferation and are more sensitive to intestinal injury in colitis models. MTG16 is also underexpressed in patients with moderate/severe ulcerative colitis. Based on these findings, we postulated that MTG16 might protect against colitis-associated carcinogenesis. MTG16 was downregulated at the protein and RNA levels in patients with inflammatory bowel disease and in those with colitis-associated carcinoma. Mtg16-/- mice subjected to inflammatory carcinogenesis modeling exhibited worse colitis and increased tumor multiplicity and size. Loss of MTG16 also increased severity of dysplasia, apoptosis, proliferation, DNA damage, and WNT signaling. Moreover, transplantation of WT marrow into Mtg16-/- mice failed to rescue the Mtg16-/- protumorigenic phenotypes, indicating an epithelium-specific role for MTG16. While MTG dysfunction is widely appreciated in hematopoietic malignancies, the role of this gene family in epithelial homeostasis, and in colon cancer, was unrealized. This report identifies MTG16 as an important modulator of colitis and tumor development in inflammatory carcinogenesis.

Abstract 4696: The LSD1 inhibitor INCB059872 is synergistic with ATRA in models of non-APL acute myelogenous leukemia

Acute myeloid leukemia (AML) is a disease characterized by the expansion of a hematopoietic stem cell like population caused in part by a block of myeloid differentiation. In AML, an altered epigenetic landscape, often arising from genetic lesions in epigenetic regulators, enforces an oncogenic expression profile and suppresses myeloid differentiation. Lysine specific demethylase 1 (LSD1) catalyzes the demethylation of lysine 4 and 9 of histone H3 through an FAD-dependent redox process. Aberrant LSD1 activity has been proposed to maintain oncogenic programs and prevent differentiation of multiple subtypes of AML. Intriguingly, recent studies indicate that LSD1 inhibition can reactivate an all-trans retinoic acid (ATRA)-dependent differentiation program in non-acute promyelocytic leukemia (APL) hematologic malignancies, a genetically heterogeneous group of blood cancers that normally respond poorly to ATRA therapy. In this study, we assessed the in vitro and in vivo effects of combining ATRA with INCB059872, a potent and selective FAD-directed LSD1 inhibitor, in non-APL AML models. As a single agent, INCB059872 induced differentiation of AML cells and when combined with ATRA, synergistically promoted differentiation as indicated by induction of CD86 and CD11b expression. In addition, the combination of INCB059872 and ATRA increased apoptosis in a panel of non-APL AML cell lines. Similarly, the combination significantly increased the fraction of CD86+CD11b+ cells and reduced cell viability in a panel of primary AML cells ex vivo. These effects in both human AML cell lines and human primary AML cells were observed across distinct FAB subtypes and genetic mutation profiles. Microarray profiling coupled with bioinformatic analysis of MV-4-11 cells demonstrated that the number of regulated genes related to differentiation and apoptotic pathways was markedly elevated in cells treated with the combination of LSD1 inhibition and ATRA relative to single agents. The synergistic increase in levels of myeloid lineage transcription factors GFI1, PU.1, CEBP and a decrease in levels of the oncogene c-MYC in the combination groups were validated by q-RT-PCR and western blot analyses. In vivo, the combination of INCB059872 and ATRA enhanced CD86 and CD11b induction and reduced tumor growth in the THP-1 xenograft model of AML compared with monotherapy. Similarly, oral administration of INCB059872 and ATRA in PDX mouse models markedly increased levels of CD11b+ cells in bone marrow. Collectively, these data underscore the synergy that can exist between LSD1 inhibition and retinoic acid receptor agonism, and provide a scientific rationale for the clinical evaluation of INCB059872 and ATRA in non-APL AML patients. Citation Format: Min Ye, Mike Liu, Jin Lu, Yvonne Lo, Margaret Favata, Gengjie Yang, Melody Diamond, Valerie Dostalik, Paul Waeltz, Melissa Ann Fischer, Chunhong He, Liangxing Wu, Xiaochuan Shan, Hong Chang, Maryanne Covington, Yanlong Li, Tim Burn, Richard Wynn, Wenqing Yao, Gregory Hollis, Reid Huber, Peggy Scherle, Michael Savona, Martin Carroll, Bruce Ruggeri, Sang Hyun Lee. The LSD1 inhibitor INCB059872 is synergistic with ATRA in models of non-APL acute myelogenous leukemia. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 4696.

112 MTG16 Functions as a Tumor Suppressor in Murine Inflammatory Carcinogenesis

median age was 60 years (range 45-72 and 45-77 for cases and controls respectively). Four cases and 5 controls had primary sclerosing cholangitis (PSC). Case neoplasms included 9 cancers [median size 2.3 (0.8-5) cm; 6/9 (66%) were proximal to splenic flexure; median AJCC stage was I (range I-IIIC)], and 10 dysplastic lesions [8 visible adenomas (dysplasia grade: 3 high, 5 low) with median size 2.3 (1.0-6.2) cm and two flat lesions (1 high grade, 1 low grade) detected by random biopsy]. Univariate and multivariate logistic regression tested the association of marker copy numbers, and potential confounders, to IBD-CRN and cancer. Results: All 3 markers individually showed high discrimination for IBD-CRN: areas under the receiver operating characteristics curves (AUC) with VIM, NDRG4 and EYA4 were 0.91, 0.84 and 0.85, respectively. For cancer the AUC with VIM, NDRG4 and EYA4 were 0.97, 0.94 and 0.95, respectively. Stool assay of VIM alone at 95% specificity yielded a sensitivity for IBD-CRN of 68% (95% CI 43-86%) and for cancer of 89% (95% CI 51-99%). Neither IBD disease duration nor comorbid PSC influencedmarker levels. Combiningmarkers did not improve discrimination. Conclusions: Stool assay of methylated VIM, NDRG4 or EYA4 highly discriminates IBD-CRN cases from IBD controls. These data corroborate our earlier proof-of-concept report of IBD-CRN detection by stool assay of methylated DNA markers. Further studies are indicated to evaluate this noninvasive approach as a complement to endoscopic strategies in IBD surveillance cohorts.