bluuebunny/crossref_metadata_2025_split huggingface dataset:
Eradication of Chronic Myelogenous Leukemia By Inactivation of the Polycomb Group Protein EZH2
Authors: Xie Huafeng , Peng Cong , Qi Jun , Cheloni Giullia , Das Partha , Huang Jialiang , Nguyen Minh , Li Shaoguang , Bradner James E. , Orkin Stuart H. 
Published: October 2019

Abstract
Chronic myelogenous leukemia (CML) is a stem-cell driven malignancy caused by BCL/ABL, the oncoprotein derived from the Philadelphia chromosome 9/22 translocation. Although tyrosine kinase inhibitors (TKIs) are highly effective in managing chronic phase CML, challenges remain, including acquisition of TKI resistance, toxicities of long-term TKI administration and third-generation TKIs, and persistence of leukemia initiating cells (LICs). Reliable curative chemotherapy remains elusive. Alternative approaches to definitive treatment of CML would enhance current management.
We report here that the Polycomb group protein EZH2, a catalytic subunit of Polycomb repressive complex 2 (PRC2), is a potential target for curative chemotherapy for CML. EZH2 is overexpressed in LICs, compared to normal hematopoietic stem cells (HSCs), and is sustained by BCR/ABL signaling, as TKIs suppress EZH2 expression in CML cells. Removal of EZH2 in an engineered mouse model prevents CML initiation and development. Moreover, Ezh2 is also required for maintenance of established CML. In a tamoxifen-inducible EZH2 deletion mouse model, conditional inactivation of EZH2 eradicates existing disease induced by wild-type BCR/ABL or the gatekeeper T315I mutant, and leads to long-term survival. Importantly, EZH2 inactivation also eliminates phenotypic and functional LICs. Relevance of the genetic studies in mice is supported by shRNA knockdown and EZH2-inhibitor treatment of human CML cell lines and primary samples, which leads to cell cycle inhibition, apoptosis, and reduced colony formation. Effects of EZH2 loss in CML are context-specific, as EZH2 is dispensable for HSCs, in part due to decreased EZH1 expression in CML LICs compared to normal HSCs. EZH1 expression decreases even further in LICs upon the loss of EZH2. Thus, loss of EZH2 in CML LICs creates a scenario resembling complete inactivation of PRC2, which is essential for the maintenance of HSCs.
EZH2-dependence of CML LICs raises prospects for curative chemotherapy based on addition of EZH2 inhibitors to conventional TKI administration.

Disclosures
No relevant conflicts of interest to declare.
CREB Regulates Cell Cycle Progression through RFC3-PCNA Axis in Acute Myeloid Leukemia
Authors: Chae Hee-Don , Mitton Bryan , Sakamoto Kathleen 
Published: October 2019

Abstract
CREB (cAMP Response Element Binding protein) is a transcription factor overexpressed in normal and neoplastic myelopoiesis and regulates cell cycle progression, although its oncogenic mechanism has not been well characterized. Replication Factor C3 (RFC3), a 38 kDa subunit of the RFC complex, is required for chromatin loading of proliferating cell nuclear antigen (PCNA) which is a sliding clamp platform for recruiting numerous proteins in DNA replication and repair processes. CREB1 expression was coupled with RFC3 expression during the G1/S progression in the KG-1 acute myeloid leukemia (AML) cell line, suggesting that RFC3 and CREB1 might be target genes of E2F, a key transcriptional regulator of the G1/S progression. Though there were two potential E2F binding sites in the RFC3 promoter region, chromatin immunoprecipitation assays provided no evidence for E2F1 binding to the RFC3 promoter, whereas E2F1 could directly act on the CREB1 expression. Treatment with the cyclin-dependent kinase (CDK) inhibitor AT7519 decreased expression of CREB1 and RFC3 as well as well-known E2F target genes such as CCNE1, CCNA2 and CCNB1 in KG-1 cells. These results indicate that CREB1 overexpression, a potentially important prognostic marker in leukemia patients, may be associated with dysregulated CDK-E2F activity in leukemia. There was also a direct correlation between the expression of RFC3 and CREB1 in human AML cell lines as well as in AML cells from patients. CREB interacted directly with the CRE site in RFC3 promoter region. CREB knockdown primarily inhibited G1/S cell cycle transition, decreasing expression of RFC3 as well as PCNA loading onto chromatin. Exogenous expression of RFC3 was sufficient to rescue the impaired G1/S progression and PCNA chromatin loading [Chromatin-bound PCNA-positive cells (%), control vs. CREB-knockdown vs. CREB-knockdown with RFC3 overexpression, 8h after release from mitotic arrest: 66.87 +/– 0.90 vs. 24.77 +/– 0.99 vs. 79.17 +/– 0.12, n=3, p< 0.01, mean +/– SEM] caused by CREB knockdown. Taken together, our results suggest that RFC3 may play a role in neoplastic myelopoiesis by promoting the G1/S progression, and its expression is regulated by CREB. These data provide new insight into CREB-driven regulation of the cell cycle in AML cells, and may contribute to leukemogenesis associated with CREB overexpression.

Disclosures
No relevant conflicts of interest to declare.
Genome Engineering to Prospectively Investigate the Pathogenesis of MLL-AF9 Acute Leukemia
Authors: Buechele Corina , Breese Erin H. , Schneidawind Dominik , Porteus Matthew H. , Cleary Michael L. 
Published: October 2019

Abstract
Chromosomal rearrangements involving the MLL gene occur in both primary and treatment-related leukemias, and are associated with a poor prognosis. While animal models of MLL-AF9 translocations have improved our understanding of the role of MLL oncogenes in leukemia pathogenesis, in this study a more representative approach based on generation of endogenous activated oncogenes is used to more faithfully model the genomic events on human leukemia. This system allows for prospective investigation of the downstream effects of the initiating event on gene expression, epigenetic regulation, stem cell biology, and genome stability in order to understand the key steps critical for the pathogenesis of MLL-rearranged leukemias. We designed a set of TALENs that cut in intron 11 of the MLL gene (MLL-11 TALENs) to facilitate insertion of sequences encoding for AF9 and the fluorescent marker gene coding NeonGreen (knock-in approach). This resulted in MLL-AF9 expression controlled through the endogenous MLL promoter. Co-expression of MLL-11 TALENs with the AF9 knock-in template resulted in AF9 NeonGreen insertion both in K562 cells and in primary human hematopoietic stem cells isolated from umbilical cord blood. This approach showed a knock-in efficiency of ~20% (range 2-40%) in K562 and CD34+ cells based on NeonGreen expression detected by flow cytometry and confocal microscopy. Long-term culture of primary human hematopoietic cells showed increased frequency of knock-in cells suggesting a survival advantage. Remarkably, further enrichment of knock-in cells was promoted by colony-forming assays in semi-solid medium. Subsequent transplantation into NSG mice was performed to assess their leukemogenic potential. Strikingly, leukemia was induced within 8-14 weeks post transplantation. All mice presented with a similar disease profile that included peripheral blood blasts and splenomegaly. Histologic examination confirmed extensive replacement of bone marrow cells and splenic infiltration by leukemic blasts expressing MLL-AF9 detected by RT-PCR. The leukemic blasts showed increased levels of common MLL target genes (HoxA9, Meis1) compared to non-MLL leukemias and comparable levels to known MLL-AF9 cell lines (Mono Mac 6, THP-1). The blast cells displayed a pre-lymphoid phenotype with CD10+/CD19++/CD38++/CD34+ and were negative for mature B cell markers CD20 and IgM as measured by flow cytometry. Our studies establish a novel experimental model to generate MLL leukemia deriving from primary human hematopoietic stem cells expressing the fusion oncogene under control of the endogenous promoter. The model will allow for further prospective study of leukemia initiating and stem cell biology for a genetic subtype of poor prognosis leukemia.

Disclosures
No relevant conflicts of interest to declare.
Acquired Aplastic Anemia: New Genetics, New Genomics
Authors: Young Neal S. , Dumitriu Bogdan , Ogawa Seishi 
Published: October 2019

Clinical and laboratory data have established that aplastic anemia is an immune-mediated disease in which T lymphocytes destroy hematopoietic stem and progenitor cells. Immunosuppressive therapies are effective in the majority of patients. Addition of the thrombopoietin mimetic eltrombopag increases the overall and complete response rates to anti-thymocyte globulin and as a single agent can rescue patients refractory to immunosuppressive therapy (IST). Multi-lineage robust blood count improvements with increased marrow cellularity suggest activity of eltrombopag on the hematopoietic stem cell. Recently, genetic factors have been identified that increase the risk of bone marrow failure: adults may present in the clinic with late manifestations of a pediatric syndrome (dyskeratosis congenita), but more typically there are no physical anomalies, often no family history, and earlier blood counts may be normal. In the telomeropathies, which in later life are almost always due to mutations in TERT (which encodes the telomerase) or TERC (which encodes the RNA template), there may be personal or familial multi-organ involvement, especially of liver and lung; early hair greying is a useful clinical clue. Detection of extremely short telomeres of leukocytes, accompanied by gene sequencing, is used to establish the diagnosis. In the syndrome defined by GATA2 mutations, there may be a history of unusual or persistent mycobacterial and viral infections, and monocytopenia preceding pancytopenia. Diagnosis requires sophisticated interpretation of gene sequencing. Large genomic data sets are now available acquired (somatic) mutations in aplastic anemia. For almost 300 NIH aplastic anemia patients treated with IST, candidate gene sequencing of myeloid cells obtained six months after treatment revealed somatic mutations in about one-third of cases. PIGA was most frequently abnormal, followed by BCOR1, DNMT3A, and ASXL1. Initial variant allele frequency was usually low. Mutations in a subset of genes predicted poor survival and progression to myelodysplastic syndrome and acute myeloid leukemia, while mutations in BCOR and PIGA correlated favorably with outcomes. When we also examined a subset of patients at the time of progression to monosomy 7 with pancytopenia and/or incipient leukemia by whole exome sequencing.DNMT3A and ASXL1 were implicated in only two cases, RUNX1 in another, and there were no novel recurring mutations. Telomere attrition was markedly accelerated in these monosomy 7 cases. A good model of evolution from bone marrow failure to myeloid malignancy centers on haploinsufficiency of specific genes, by mutations or chromosome loss, both of which would be favored in a stressed, regenerative environment. The failed marrow environment may select for defective cells, as, for example, in differentiation capability. Malignant evolution should be predictable in the clinic and amenable to therapeutic intervention.

Disclosures
Off Label Use: Eltrombopag in bone marrow failure syndromes, research protocols.
Post-Transcriptional Defects and Erythroid Pathobiology
Authors: Sankaran Vijay G. 
Published: October 2019

A great body of work has focused on understanding the role that gene regulation at the transcriptional level plays in blood cell production and diseases that disrupt this process. However, until recently there has been limited insight on the role that post-transcriptional gene regulation has in both normal and pathological disorders of human hematopoiesis. Specifically, the regulation of messenger RNA translation can have a significant impact upon gene expression, and how this process affects hematopoiesis has only been explored in limited studies. In this talk, the role of ribosomal protein gene mutations in the specific disorder of red blood cell production, Diamond-Blackfan anemia, will be discussed. Recent findings from our laboratory show that mutations in the key hematopoietic transcription factor gene, GATA1, can result in Diamond-Blackfan anemia in rare cases. We have gone on to show that more common mutations in ribosomal protein genes can disrupt translation of GATA1 and thereby suggest a common underlying mechanism for the impaired erythropoiesis observed in Diamond-Blackfan anemia. We discuss both the mechanistic underpinnings of our observations and how these findings have important therapeutic implications. Other recent examples of how disordered translation can impair human hematopoiesis will also be examined. This talk will provide a cohesive framework to understand the implications of these recent findings for both normal and disordered human hematopoiesis.

Disclosures
No relevant conflicts of interest to declare.
Erythron to the Liver: Send Iron
Authors: Nemeth Elizabeta , Ganz Tomas , Kautz Léon 
Published: October 2019

For successful expansion of erythropoiesis, the activity of the hormone erythropoietin (EPO) must be coordinated with the supply of iron to erythroid precursors. Increased iron supply for erythropoiesis is ensured by the suppression of hepcidin, the iron-regulatory hormone produced by the liver. Low hepcidin levels allow greater absorption of dietary iron and greater mobilization of iron from the stores in the spleen and the liver. The mechanisms coordinating erythropoietic activity with iron delivery are not well understood. We recently identified erythroferrone as a new mediator of hepcidin suppression during stress erythropoiesis1. Erythroferrone (ERFE) is a member of the C1q/TNF-related protein (CTRP) family of metabolic mediators. ERFE is produced in response to EPO by erythroblasts of the bone marrow and spleen of mice. The induction of ERFE by EPO was dependent on Jak2/Stat5 signaling. Ex vivo treatment of human erythroblasts with EPO also resulted in a dramatic induction of ERFE expression. The essential role of ERFE in acute hepcidin suppression by erythropoiesis was demonstrated in ERFE-deficient mice. In contrast to wild-type mice which suppressed hepcidin ~10-fold within hours after hemorrhage or erythropoietin injection, no hepcidin suppression was observed in ERFE knockout mice within 24 h. As a consequence, ERFE-deficient mice exhibited delayed recovery of hemoglobin after hemorrhage or severe inflammation. Treatment of mice or hepatocytes with recombinant ERFE protein confirmed the hepcidin-suppressive activity of the protein. It remains to be seen whether administration of ERFE protein would be useful for the treatment of anemia of inflammation mediated by elevated hepcidin. In iron-loading anemias including β-thalassemia, hepcidin is chronically suppressed by the exuberant but ineffective erythropoietic activity. This is the cause of iron overload in untransfused thalassemia patients and may contribute to iron loading even in transfused patients. We found that ERFE expression is greatly increased in the bone marrow and spleen of mice with β-thalassemia intermedia (th3 model). Transgenic ablation of ERFE in th3 mice normalized hepcidin and partially corrected their iron overload. Although human studies of the role of ERFE in health and disease are clearly needed, ERFE is a promising candidate for the pathological suppressor of hepcidin in anemias with ineffective erythropoiesis.
References:
1. Kautz L, Jung G, Valore EV, et al. Identification of erythroferrone as an erythroid regulator of iron metabolism. Nat Genet. 2014; 46: 678-684.

Disclosures
Nemeth: Intrinsic LifeSciences: Equity Ownership, Membership on an entity's Board of Directors or advisory committees; Merganser Biotech: Equity Ownership. Ganz:Intrinsic LifeSciences: Consultancy, Equity Ownership, Membership on an entity's Board of Directors or advisory committees; Keryx Pharma: Consultancy; Merganser Biotech: Consultancy, Equity Ownership.
Maintenance of Pluripotent Stem Cells is Influenced by Ser/Thr Metabolism
Authors: Cantley Lewis C. 
Published: October 2019

Abstract
Recent studies have suggested not only that stem cells have different metabolic requirements than terminally differentiated cells, but also that metabolic intermediates may play a role in the maintenance of stem cells. It has long been clear that changes in acetylation and methylation of histones, as well as methylation of DNA play critical roles in deciding cell fate. The availability of critical intermediates in metabolism, especially S-adenosylmethionine (SAM), acetyl-CoA, nicotinamide adenine dinucleotide (NAD) and a-ketoglutarate play critical roles in modulating acetylation and methylation of histones and methylation of DNA. In the course of evaluating an unusual requirement of threonine (Thr) for the growth of murine embryonic stem cells, we found that metabolism of Thr to glycine (Gly) and the subsequent use of the methyl group of Gly for converting homocysteine to methionine is critical for maintaining high levels of SAM and low levels of S-adenosyl-homocysteine. Importantly, depletion of Thr from the media resulted in decreased tri-methylation of histone H3 lysine-4 (H3K4me3), leading to slowed growth and increased differentiation. Thus, abundance of SAM appears to influence H3K4me3, providing a possible mechanism by which modulation of a metabolic pathway might influence stem cell fate. Demethylation of histones and DNA can also be controlled by metabolic intermediates. Mutated forms of isocitrate dehydrogenase 1 (IDH1) and IDH2 that drive acute myeloid leukemia (AML) and other cancers, produce an oncometabolite (2-hydrogyglutarate) that can compete with the a-ketoglutarate requirement for enzymes involved in hydroxy-methylation and subsequent demethylation of DNA and histones. Recent studies indicate that 2-hydroxyglutarate plays a role in blocking differentiation of cancer cells. These and other observations linking intermediates in metabolism to stem cell maintenance will be discussed.

Disclosures
Cantley: Agios Pharmaceuticals: Consultancy, Equity Ownership, Membership on an entity's Board of Directors or advisory committees, Patents & Royalties.
IDH Mutations, 2-Oxoglutarate-dependent Dioxygenases, and Leukemia
Authors: Kaelin William G. 
Published: October 2019

Abstract
Somatic isocitrate dehydrogenase 1 (IDH1) and IDH2 mutations have been discovered in a variety of neoplasms, including glioblastomas and acute myelogenous leukemias. Cancer-associated IDH mutants produce large quantities (low mM) of the R enantiomer of 2-hydroxyglutarate (R-2HG). R-2HG resembles 2-oxoglutarate, which is a Krebs Cycle intermediate and an essential cofactor for a variety of enzymes including the ~70 2-oxoglutarate-dependent dioxygenases. Included among these enzymes are the EglN prolyl hydroxylases that earmark the hypoxia-inducible factor (HIF) for destruction under normoxic conditions, the JmjC histone demethylases, and the ten-eleven translocation (TET) enzymes that hydroxylate methylcytosine residues. In general, both R-2HG and the alternative enantiomer, S-2HG, inhibit 2-oxoglutarate-dependent dioxygenases, with the (S) enantiomer typically being more potent, at least as measured in vitro. We recently discovered that R-2HG, in contrast to S-2HG, potentiates, rather than inhibits, EglN activity; we linked this, and subsequent downregulation of HIF, to the ability of mutant IDH1 to transform human astrocytes. Moreover, we found that R-2HG, but not S-2HG, is sufficient to promote leukemic transformation and that its effects are reversible. In this setting, inactivation of EglN, as occurs with S-HG, appears to be antithetical to transformation. R-2HG promotes leukemic transformation by inhibiting targets such as TET2, while sparing EglN activity. Recent clinical data show that inhibiting R-2HG production can induce complete remissions in some patients with IDH mutant leukemias. We are currently trying to identify the relevant downstream targets of TET2 in IDH mutant leukemias and are also exploring whether loss of EglN activity has antileukemic effects in mouse models in vivo. Finally, we are exploring whether any other 2-oxoglutarate-dependent dioxygenases, such as the RBP2/JARID1A/KDM5A, might be viable drug targets in leukemia.

Disclosures
Kaelin: Agios: Consultancy, Membership on an entity's Board of Directors or advisory committees; FibroGen: Consultancy, Membership on an entity's Board of Directors or advisory committees; Lilly: Membership on an entity's Board of Directors or advisory committees; Nextech: Consultancy, Membership on an entity's Board of Directors or advisory committees; Peloton: Consultancy, Membership on an entity's Board of Directors or advisory committees; TRACON: Consultancy.
The Tissue Macrophage Lineage
Authors: Merad Miriam 
Published: October 2019

Despite accumulating evidence suggesting local self-maintenance of tissue macrophages in the steady state, the dogma remains that tissue macrophages derive from monocytes. In this presentation, I will discuss parabiosis, fate mapping, and adoptive transfer approaches that we developed in the laboratory to assess the contribution of monocytes to tissue resident macrophages in the steady state and after cell turnover. Using these approaches we found that monocytes contribute minimally to tissue resident macrophages in the steady state. Similarly, we found that after depletion of lung macrophages, the majority of repopulation occurs by local proliferation in a Csf-1 and Csf-2 dependent manner. This repopulation from a non-circulating population was stochastic and observed in both infection- and inflammatory-induced cytotoxicity of local macrophages. We also found that after bone marrow transplantation, host tissue macrophages retain the capacity to expand when the development of donor tissue macrophages is compromised. These results are consistent with recent results obtained in humans showing that macrophages can form in the absence of circulating monocytes. Collectively, these results indicate that tissue resident macrophages and circulating monocytes should be classified as independent mononuclear phagocyte lineages.

Disclosures
No relevant conflicts of interest to declare.