scholarly journals NPM1 Mutation Contributes to Hematological Dysfunction By Disrupting H3K79 Methylation

Blood ◽  
2016 ◽  
Vol 128 (22) ◽  
pp. 2702-2702
Author(s):  
Jie Xu ◽  
Wu Zhang ◽  
Xiaojing Yan ◽  
Chen Zhao ◽  
Jiang Zhu ◽  
...  
Keyword(s):  
Target Genes ◽  
Conflicts Of Interest ◽  
Leukemia Cell ◽  
Gene Profiling ◽  
In Vitro Assays ◽  
Hoxa Cluster ◽  
Hematopoietic Stem ◽  
Npm1 Mutation ◽  
H3k79 Methylation

Abstract NPM1 is one of the most frequent acquired mutated genes in acute myeloid leukemia (AML). Previous studies have shown that NPM1 mutation (NPMc+) established the distinctive gene expression signatures, which were associated with mixed lineage leukemia (MLL)-target genes, like MEIS1 and HOXA cluster. In AML carrying MLL fusion-oncoproteins, DOT1L-mediated histone 3 lysine 79 (H3K79) methylation is implicated in the regulation of MLL-target genes. Compared with MLL abnormalities, NPM1 variants preserve the similar transcriptional characteristics. However, whether NPM1 mutation could affect the histone modification of H3K79 methylation is unknown. In this study, we showed that NPM1 mutation dysregulated the homeostasis of hematopoietic stem and progenitor cells and resulted in ageing-related myeloproliferation in NPMc+ transgenic mouse model. Interestingly, through scanning the chromatin modification related gene profiling, di- and tri- methylated H3K79 were significantly elevated in bone marrow (BM) Lin-Sca-1+c-Kit+ cells (LSKs) of NPMc+ mice comparing to wild type (WT). Meanwhile, in the leukemia cell lines and AML primary BM samples, we confirmed that NPM1 mutated cells expressed the higher level of H3K79 methylation. In vitro assays also indicated that the decrease or increase of methylated H3K79 could be regulated respectively by knockdown or overexpression of NPM1 mutant but not WT. Importantly, with DOT1L inhibitor treatment, reduced di- and tri- methylated H3K79 was observed in OCI-AML3 (NPMc+) strains but not OCI-AML2 (NPM1 WT) cells. In contrast with OCI-AML2, DOT1L inhibitor significantly promoted the cell apoptosis and restrained the cell cycle of OCI-AML3. Moreover, by the means of murine BM colony formation assay, DOT1L inhibitor obviously weakened myeloid cell proliferation in NPMc+ mice, while colony number in WT group did not change. Also, leukemia development was repressed in OCI-AML3-xenografted NOD/SCID mice with the treatment of DOT1L inhibitor. Taken together, NPM1 mutation contributes to hematological dysfunction by disrupting H3K79 methylation, which could be largely attenuated by DOT1L inhibitor. Disclosures No relevant conflicts of interest to declare.

MiR-150 Suppresses MLL-AF9 Associated Leukemia Through Simultaneously Targeting Multiple Oncogenes,

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 3461-3461
Author(s):  
Beiyan Zhou
Keyword(s):  
Stem Cells ◽  
Stem Cell ◽  
Target Genes ◽  
Conflicts Of Interest ◽  
Expression Patterns ◽  
Gene Profiling ◽  
Fusion Genes ◽  
Leukemic Stem Cells ◽  
Hematopoietic Stem ◽  
Mll Gene

Abstract Abstract 3461 The mixed lineage leukemia (MLL) gene codes for an evolutionarily conserved histone methyltransferase that is crucial for early hematopoiesis. As a result of a chromosomal translocation involving locus 11q23 results in formation of chimeras composed of the 5' part of the MLL gene fused with more than 60 partner genes lead to disruption of normal function of MLL as a histone methytransferase and acquisition of transcriptional properties conferred by the partner genes. MLL fusion genes (MLL-FG) are often the causal mutations for aggressive acute myeloid and lymphoid leukemias (AML and ALL) that correlated with poor prognosis. In order to treat or even eliminate MLL-associated leukemias, extensive studies on the regulatory mechanism underlying MLL associated transformation and progression have been carried out. Leukemic stem cells (LSC) can derive from either hematopoietic stem or progenitor cells with the recruitment of MLL-fusion genes (MLL-FG) and wild type MLL protein. We report that miR-150, a key hematopoietic regulatory microRNA (miRNA) and one of the most downregulated miRNAs in MLL-associated leukemias, acts as a tumor suppressor to block the leukemogenic potency of leukemic stem cells. When expression of miR-150 was restored, a significantly suppressed leukemic stem cell potency of MLL-AF9 cells was observed both in vivo and in vitro. Gene profiling analysis demonstrated that elevated miR-150 altered various aspects of gene expression patterns in MLL-AF9 cells, including stem cell signatures, cancer pathways, and cell survival. By screening more than 30 predicted target genes, we identified multiple leukemia-associated oncogenes as bona fide miR-150 targets, and knockdown of these genes by shRNAs recapitulated the tumor suppressive effects observed after ectopically expression of miR-150 in MLL-AF9 cells. Disclosures: No relevant conflicts of interest to declare.

DNMT3A Mutation Leads to Hematopoietic Dysregulation.

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 2382-2382
Author(s):  
Jie Xu ◽  
Wei-na Zhang ◽  
Tao Zhen ◽  
Yang Li ◽  
Jing-yi Shi ◽  
...  
Keyword(s):  
Transcriptional Activation ◽  
Dna Methyltransferase ◽  
De Novo ◽  
Conflicts Of Interest ◽  
Epigenetic Modification ◽  
Hematopoietic Cells ◽  
In Vitro Assays ◽  
Clinical Samples ◽  
Hematopoietic Stem

Abstract Abstract 2382 Epigenetic modification process is required for the development of hematopoietic cells. DNA methyltransferase DNMT3A, responsible for de novo DNA methylation, was newly reported to have a high frequency of mutations in hematopoietic malignancies. Conditional knock-out of DNMT3A promoted self-renewal activity of murine hematopoietic stem cells (HSCs). However, the role of mutated DNMT3A in hematopoiesis and its regulative mechanism of epigenetic network mostly remain unknown. Here we showed that the Arg882His (R882H) hotspot locus on DNMT3A impaired the normal function of this enzyme and resulted in an abnormal increase of primitive hematopoietic cells. In both controlled in vivo and in vitro assays, we found that the cells transfected by R882H mutant promoted cell proliferation, while decreased the differentiation of myeloid lineage compared to those with wild type. Analysis of bone marrow (BM) cells from mice transduced by R882H reveals an expansion of Lin−Sca-1+C-kit+ populations and a reduction of mature myeloid cells. Meanwhile, a cluster of upregulated genes and downregulated lineage-specific differentiation genes associated with hematopoiesis were discovered in mice BM cells with R882H mutation. We further evaluated the association of mutated DNMT3A and HOXB4 which was previously detected to be highly expressed in clinical samples carrying R882 mutation. Compared with wildtype DNMT3A, R882H mutation disrupted the repression of HOXB4 by largely recruiting tri-methylated histone 3 lysine 4 (H3K4). Taken together, our results showed that R882H mutation disturbed HSC activity through H3K4 tri-methylation, and transcriptional activation of HSC-related genes. Disclosures: No relevant conflicts of interest to declare.

MiR-29a Maintains Hematopoietic Stem Cell Self-Renewal and Is Required For Myeloid Leukemogenesis

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 1190-1190
Author(s):  
Wenhuo Hu ◽  
James Dooley ◽  
Stephen S. Chung ◽  
Safak Yalcin ◽  
Yu Sup Shin ◽  
...  
Keyword(s):  
Stem Cell ◽  
Colony Formation ◽  
Target Genes ◽  
Conflicts Of Interest ◽  
Ectopic Expression ◽  
Null Mouse ◽  
Cell Cycle Regulators ◽  
Self Renewal ◽  
Hematopoietic Stem

Abstract microRNAs (miRNAs) are important regulators of both embryonic and adult tissue stem cell self-renewal. We previously showed that ectopic expression of miR-29a, a miRNA highly expressed in HSCs as well as in human acute myeloid leukemia (AML) stem cells, in immature mouse hematopoietic cells is sufficient to induce a myeloproliferative disorder that progresses to AML. During the early phase of this disease, miR-29a induces aberrant self-renewal of committed myeloid progenitors, strongly suggesting a role for miR-29a in regulating HSC self-renewal. In order to determine the role of miR-29a in HSC function, we have evaluated our recently described miR-29a/b1 null mouse. Homozygous deletion of miR-29a/b1 resulted in reduced bone marrow cellularity and reduced colony forming capacity of hematopoietic stem and progenitor cells (HSPCs). The phenotype was mediated specifically by miR-29a since miR-29b expression was not significantly altered in HSCs and reconstitution of miR-29a/b1 null HSPCs with miR-29a, but not miR-29b, rescued in vitro colony formation defects. Self-renewal defects were observed in miR-29a deficient HSCs in both competitive and non-competitive transplantation assays, and these deficits were associated with increased HSC cell cycling and apoptosis. Gene expression studies of miR-29a deficient HSCs demonstrated widespread gene dysregulation including a number of up-regulated miR-29a target genes including DNA methylation enzymes (Dnmt3a, -3b) and cell cycle regulators (e.g. Cdk6, Tcl1, Hbp1, Pten). Knockdown of one of these targets, Dnmt3a, in miR-29a deficient HSCs resulted in partial restoration of colony formation, providing functional validation that Dnmt3a mediates part of miR-29a null HSPCs functional defects. miR-29a loss also abrogated leukemogenesis in the MLL-AF9 retroviral AML model. Together, our results demonstrate that miR-29a positively regulates HSC self-renewal and is required for myeloid leukemogenesis. Disclosures: No relevant conflicts of interest to declare.

A Subset of MicroRNAs and Genes Involved in AML Has a Pivotal Role in the in Vitro differentiation of Hematopoietic Stem Cell Precursors

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 1290-1290
Author(s):  
Julian Pulecio ◽  
Leopoldo Laricchia-Robbio ◽  
Juan Carlos Izpisua ◽  
Montserrat Barragan ◽  
Marianna Vitaloni ◽  
...  
Keyword(s):  
Stem Cells ◽  
Conflicts Of Interest ◽  
Embryonic Stem ◽  
In Vitro Assays ◽  
Main Role ◽  
In Vitro Differentiation ◽  
Related Factors ◽  
Hematopoietic Stem

Abstract Abstract 1290 After the finding of a set of transcription factors capable of reprogram any somatic cell into an embryonic stem-like cell by Yamanaka's group a lot of effort has been put to differentiate and produce in-vitro engraftable cells that could replace and fix damaged tissues. One of the most attractive and promising fields is the differentiation towards blood, considering it is a tissue without a complex tridimensional structure and that the phenotypes of the different sublineages are already well characterized. Nonetheless, so far there are no reports of successful differentiation into blood progenitors which are able to completely recover functionally in vivo blood-depleted mice. We previously reported the differentiation from induced pluripotent stem cells (iPS) towards hematopoietic cells capable of distinguish into sub lineages in in vitro assays, while another group obtained blood precursors by transdifferentiation of fibroblasts; however a complete recovery of the hematopoietic lineages in vivo was not seen. Our hypothesis is that the gap missing in the current protocols to obtain repopulating blood stem cells can be filled by the microRNA profiling of Cord Blood (CB) progenitors, in order to find the key players in the maintenance of blood stemness. In particular, it has been shown that population with the highest capacity to be engrafted in mice is the CD34+/CD90+ from CB. Our preliminary results depict a set of miRNAs that are specifically overexpressed in the CD34+/CD90+ population from CB cells when compared against a less specific CD34+ population. These miRNAs are currently being tested as a tool to improve the efficiency of iPS differentiation and fibroblasts conversion towards blood progenitors by means of lentiviral infection of the miRNA precursors. Interestingly, we have found that these miRNAs have been previously reported to have a main role in the occurrence of Acute Myeloid Leukemia in humans and mice. These results led us to look for genes that are highly expressed in blood progenitors but also have been shown to be correlated with AML.As a safety study, we are currently evaluating the effect of overexpressing AML related factors (miRNAs and genes) when added to the established protocols to obtain blood progenitors from iPS and fibroblasts. Surprisingly, our initial results show that the overxpression of the above mentioned genes and miRNAs have an intrinsic potential to induce in vitro differentiation or conversion from iPS and fibroblasts towards blood progenitors. Disclosures: No relevant conflicts of interest to declare.

scholarly journals The stem cell–specific long noncoding RNA HOXA10-AS in the pathogenesis of KMT2A-rearranged leukemia

2019 ◽  
Vol 3 (24) ◽  
pp. 4252-4263
Author(s):  
Sina Al-Kershi ◽  
Raj Bhayadia ◽  
Michelle Ng ◽  
Lonneke Verboon ◽  
Stephan Emmrich ◽  
...  
Keyword(s):  
Noncoding Rna ◽  
Target Genes ◽  
Hox Genes ◽  
Locked Nucleic Acid ◽  
Monocytic Differentiation ◽  
Hoxa Cluster ◽  
Hematopoietic Stem ◽  
Human Cd34

Abstract HOX genes are highly conserved, and their precisely controlled expression is crucial for normal hematopoiesis. Accordingly, deregulation of HOX genes can cause leukemia. However, despite of intensive research on the coding HOX genes, the role of the numerous long noncoding RNAs (lncRNAs) within the HOX clusters during hematopoiesis and their contribution to leukemogenesis are incompletely understood. Here, we show that the lncRNA HOXA10-AS, located antisense to HOXA10 and mir-196b in the HOXA cluster, is highly expressed in hematopoietic stem cells (HSCs) as well as in KMT2A-rearranged and NPM1 mutated acute myeloid leukemias (AMLs). Using short hairpin RNA– and locked nucleic acid-conjugated chimeric antisense oligonucleotide (LNA-GapmeR)–mediated HOXA10-AS-knockdown and CRISPR/Cas9-mediated excision in vitro, we demonstrate that HOXA10-AS acts as an oncogene in KMT2A-rearranged AML. Moreover, HOXA10-AS knockdown severely impairs the leukemic growth of KMT2A-rearranged patient-derived xenografts in vivo, while high HOXA10-AS expression can serve as a marker of poor prognosis in AML patients. Lentiviral expression of HOXA10-AS blocks normal monocytic differentiation of human CD34+ hematopoietic stem and progenitor cells. Mechanistically, we show that HOXA10-AS localizes in the cytoplasm and acts in trans to induce NF-κB target genes. In total, our data imply that the normally HSC-specific HOXA10-AS is an oncogenic lncRNA in KMT2A-r AML. Thus, it may also represent a potential therapeutic target in KMT2A-rearranged AML.

scholarly journals Thrombopoietin (TPO) Induces Hematopoietic Differentiation of Mouse ES Cells Via HIF-1 Alpha-Dependent Activation of a BMP4 Autoregulatory Loop

Blood ◽  
2015 ◽  
Vol 126 (23) ◽  
pp. 1186-1186
Author(s):  
Azadeh Zahabi ◽  
Tatsuya Morishima ◽  
Andri Pramono ◽  
Dan Lan ◽  
Lothar Kanz ◽  
...  
Keyword(s):  
Molecular Mechanisms ◽  
Target Genes ◽  
Conflicts Of Interest ◽  
Es Cells ◽  
Embryonic Stem ◽  
Efficient Production ◽  
Hematopoietic Differentiation ◽  
Hematopoietic Stem

Abstract Understanding the molecular mechanisms underlying hematopoietic differentiation of embryonic stem (ES) cells may help to ascertain the optimal conditions for the production of hematopoietic cells as a source for transplantation or experimental use. Previously, we found that patients with congenital amegakaryocytic thrombocytopenia (CAMT), who develop pancytopenia early after birth, harbor mutations within the thrombopoietin (TPO) receptor, c-mpl. This knowledge, together with observations in vitro and in animal models in vivo, suggests that TPO/c-mpl signaling promotes early hematopoiesis. However, the downstream mechanisms underlying TPO signaling are not fully elucidated. Here, we describe for the first time a direct connection between the TPO and bone morphogenetic protein 4 (BMP4) signaling pathways in the hematopoietic fate decision of ES cells. BMP4 is a classical morphogen and a well-known inducer of early hematopoietic differentiation of ES cells. Treatment of ES cells with TPO induced the autocrine production of BMP4 by ES cells with concomitant upregulation of the BMP receptor, BMPR1A, phosphorylation of Smad1, 5, and 8 and activation of the specific target genes, Id1, 2, and 3, and Msx1 and 2. This was mediated by TPO-dependent binding of the HIF-1α transcription factor to the BMP4 gene promoter, resulting in further activation of the BMP4-autoregulatory loop. Treatment of ES cells with the BMP antagonist noggin substantially reduced TPO-dependent hematopoietic differentiation of ES cell. Taken together, our findings contribute to the understanding the mechanisms of hematopoietic differentiaiton of ES cells and might help to establish new methods for the efficient production of hematopoietic stem cells in vitro. Disclosures No relevant conflicts of interest to declare.

scholarly journals Comparative Studies of B Cell Precursor ALL Pathogenesis and Translational Biology Using a Conditional E2a-PBX1 Transgenic Mouse Model

Blood ◽  
2014 ◽  
Vol 124 (21) ◽  
pp. 283-283
Author(s):  
Jesus Duque-Afonso ◽  
Jue Feng ◽  
Florian Scherer ◽  
Zhong Wang ◽  
Michael L. Cleary
Keyword(s):  
B Cell ◽  
Conflicts Of Interest ◽  
Lymphoblastic Leukemia ◽  
Leukemia Cell ◽  
Disease Free Survival ◽  
Leukemia Cells ◽  
Mouse Strains ◽  
Driver Gene ◽  
Hematopoietic Stem

Abstract Pediatric acute lymphoblastic leukemia (ALL) represents a collection of orphan diseases that are defined by their genomic abnormalities. Their genetic diversity and low prevalence serve as major barriers to investigations of their molecular pathogenesis and translational biology. To address this, we have engineered novel mouse strains that conditionally activate and express E2a-PBX1, the fusion oncogene derived from chromosomal translocation t(1;19), present in 5-7% of pediatric ALL. Somatic activation of the oncogene is accomplished by Cre-recombinase expressed under the control of specific B-lineage promoters CD19 or Mb1, or in hematopoietic stem cells using the Mx1 interferon-inducible promoter. The three mouse strains show similar pre-leukemic and leukemic phenotypes. At the time of disease, mice exhibit leukocytosis, anemia and thrombocytopenia as well as lymphadenopathy and hepatosplenomegaly and infiltration of several organs including kidney, lung and central nervous system. Leukemia cell phenotypes (CD117+, CD19+, CD43+, CD45+, CD25+, sIgM-, Bp-1+, CD24+, CD127+, CD79a+ and TdT+) correspond to the phenotypic fraction B-C’ (or pro-B/large pre-B II stage, Basel nomenclature) that is very similar to human E2a-PBX1+ pre-B-ALL. Hence, we detected productive VDJ rearrangements and cytoplasmic heavy chain in 12.5 % of cases, a characteristic of human E2a-PBX1 leukemias. Leukemia incidence varies from 5-50% depending on the Cre driver gene and the median latency is about 10 months in E2a.PBX1/Mb1.Cre and Mx1.Cre lines, suggesting the need for secondary mutations. Whole exome sequencing detected secondary genetic aberrations, which were validated in a larger cohort of leukemias. Spontaneous deletions of Pax5, which are present in ~45% of pediatric ALLs with E2a-PBX1 gene fusions, were found in about 30 % of mouse E2a-PBX1 leukemias. Conditional deletion of Pax5 and E2a-PBX1 expression expanded progenitor B cell subpopulations in healthy 3-months old preleukemic mice. Consequently, Pax5 haplo-insufficiency in mice cooperates with E2a-PBX1 increasing the penetrance and shortening the latency of leukemia, providing the first evidence for cooperative oncogenic effects of Pax5 haplo-insufficiency. Tumor suppressor genes as Trp53 and Cdkn2a/b were inactivated by secondary mutations and deletions, respectively. Additionally, secondary recurrent activating mutations were detected in key signaling pathways such as Ras/Mapk and Jak/Stat on which the leukemia cells are strongly dependent. Furthermore, leukemia cells displayed higher basal levels of phosphorylated pSTAT5 and pAKT, pERK1/2, and were hyper-sensitive to stimulation with IL-7 and thymic stromal lymphopoietin (TSLP) as seen by induction of pSTAT5 and supported growth in colony-forming assays. The JAK1/2 inhibitor ruxolitinib blocked the induction of pSTAT5 by IL-7 and TSLP, inhibited colony formation in vitro, and increased disease-free survival after in vivo treatment. Human E2a-PBX1 primary cells and cell lines showed hypersensitivity to IL-7/pSTAT5 activation compared to other ALL karyotypes and pre-treatment with ruxolitinib blocked induction of pSTAT5 by IL-7. In summary, we have developed conditional transgenic E2a-PBX1 mouse models that consistently develop leukemias that resemble human pre-B-ALL carrying the t(1;19) and identified key cooperating oncogenic pathways. This model provides experimental validation of the multistep pathogenesis for a subset of ALL previously inferred from genomic analyses and provides a platform for comparative mechanistic and preclinical studies. Disclosures No relevant conflicts of interest to declare.

Myeloid Leukemia Cells With MLL partial Tandem Duplication Are Sensitive To Pharmacological Inhibition Of The H3K79 Methyltransferase DOT1L

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 1256-1256 ◽  
Author(s):  
Michael W.M. Kühn ◽  
Michael Hadler ◽  
Scott R. Daigle ◽  
Chun-Wei Chen ◽  
Amit U. Sinha ◽  
...  
Keyword(s):  
Cell Lines ◽  
Target Genes ◽  
Leukemia Cell ◽  
Leukemia Cell Line ◽  
Hoxa Cluster ◽  
Histone 3 ◽  
Leukemia Cell Lines ◽  
Equity Ownership

Abstract Genetic alterations of the mixed-lineage leukemia (MLL) gene are commonly identified in acute leukemias. In acute myeloid leukemia (AML), a partial tandem duplication (PTD) of MLL occurs in about 5-10% of AML patients and is associated with adverse prognosis. The mutation leads to an in-frame duplication of exons 5 to 11 resulting in the production of an aberrant MLL protein. Unlike chromosomal rearrangements of MLL, this mutation does not affect the functional histone 3 lysine 4 (H3K4) methyltransferase domain. However, AMLs carrying a MLL-PTD and MLL-rearranged leukemias share some common characteristics, such as overexpression of HOXA-cluster genes and dysregulated epigenetic functions. Recently, leukemias with various MLL-translocations have been shown to be dependent on the histone 3 lysine 79 (H3K79) methyltransferase, DOT1L, and are sensitive to EPZ004777, a recently described selective small-molecule DOT1L inhibitor. EPZ-5676, a DOT1L-inhibitor with improved potency and drug-like properties, has recently been identified and is currently under clinical investigation. To evaluate the therapeutic potential of DOT1L-inhibition in MLL-PTD positive leukemia cells, we assessed the effect of EPZ004777 on the MLL-PTD containing leukemia cell lines MUTZ-11 and EOL-1. In vitro treatment with EPZ004777 over a 14-day period resulted in dramatic reduction of cell proliferation compared to DMSO vehicle control in both cell lines beginning 7 days after the start of treatment. Similar results were obtained for MOLM-13, a leukemia cell line harboring a MLL-translocation, but not for HL-60, a non-MLL-rearranged leukemia cell line. To further investigate whether these findings reflect a selective response to EPZ004777 or non-specific drug toxicity, we first explored the genome-wide H3K79 dimethylation (H3K79me2) profile using chromatin immunoprecipitation (ChIP) followed by next generation sequencing in untreated MUTZ-11 cells. Across the HOXA-cluster locus, we detected a similar H3K79me2 distribution pattern as previously reported in MLL-rearranged leukemias. Further analysis of H3K79me2 in MUTZ-11 and EOL-1 cells after treatment with the inhibitor showed profound suppression of those marks as assessed by western blot and ChIP-PCR. Subsequent global gene expression analysis revealed concurrent downregulation of HOXA-cluster and other MLL-target genes after 7 days of DOT1L inhibition. To investigate the effect of EPZ004777 on the MLL-PTD positive cells in more detail, we analyzed cell differentiation and apoptosis upon a 10-day exposure to the compound. As previously described for EPZ004777-sensitive MLL-rearranged leukemias, drug treatment resulted in increased expression of CD11b and morphological changes consistent with myeloid cell differentiation. We further observed apoptotic cell death after EPZ004777 treatment as measured by an increase in the percentage of Annexin V positive cells and cleaved Caspase 3 protein compared to vehicle controls. In order to determine the effect of DOT1L inhibition in vivo, we tested the recently identified DOT1L-inhibitor, EPZ-5676, for its ability to inhibit leukemia growth in a subcutaneous EOL-1 xenograft model in immunocompromised rats. Similar to what we observed in vitro, continuous intravenous administration over 21 days led to substantial dose-dependent inhibition of tumor growth, abrogation of H3K79me2, and concurrent downregulation of selected MLL-target genes. Thus, we demonstrate unexpected sensitivity of MLL-PTD containing leukemia cell lines to the DOT1L inhibitors EPZ004777 in vitro and EPZ-5676 in vivo. These data suggest that patients with myeloid malignancies carrying this particular mutation might benefit from treatment with therapeutic approaches that target DOT1L. Disclosures: Daigle: Epizyme, Inc: Employment, Equity Ownership. Olhava:Epizyme Inc.: Employment. Pollock:Epizyme Inc.: Employment, Equity Ownership, Patents & Royalties, Stock Options Other. Armstrong:Epizyme Inc.: Has consulted for Epizyme Inc. Other.

scholarly journals Organoarsenic Compounds with In Vitro Activity against the Malaria Parasite Plasmodium falciparum

Biomedicines ◽  
2020 ◽  
Vol 8 (8) ◽  
pp. 260
Author(s):  
Sofia Basova ◽  
Nathalie Wilke ◽  
Jan Christoph Koch ◽  
Aram Prokop ◽  
Albrecht Berkessel ◽  
...  
Keyword(s):  
Plasmodium Falciparum ◽  
Malaria Parasite ◽  
Rapid Development ◽  
Sexual Transmission ◽  
Leukemia Cell ◽  
Leukemia Cell Line ◽  
In Vitro Assays ◽  
Parasite Plasmodium ◽  
Parasite Plasmodium Falciparum

The rapid development of parasite drug resistance as well as the lack of medications targeting both the asexual and the sexual blood stages of the malaria parasite necessitate the search for novel antimalarial compounds. Eleven organoarsenic compounds were synthesized and tested for their effect on the asexual blood stages and sexual transmission stages of the malaria parasite Plasmodium falciparum using in vitro assays. The inhibitory potential of the compounds on blood stage viability was tested on the chloroquine (CQ)-sensitive 3D7 and the CQ-resistant Dd2 strain using the Malstat assay. The most effective compounds were subsequently investigated for their effect on impairing gametocyte development and gametogenesis, using the gametocyte-producing NF54 strain in respective cell-based assays. Their potential toxicity was investigated on leukemia cell line Nalm-6 and non-infected erythrocytes. Five out of the 11 compounds showed antiplasmodial activities against 3D7, with half-maximal inhibitory concentration (IC50) values ranging between 1.52 and 8.64 µM. Three of the compounds also acted against Dd2, with the most active compound As-8 exhibiting an IC50 of 0.35 µM. The five compounds also showed significant inhibitory effects on the parasite sexual stages at both IC50 and IC90 concentrations with As-8 displaying the best gametocytocidal activity. No hemolytic and cytotoxic effect was observed for any of the compounds. The organoarsenic compound As-8 may represent a good lead for the design of novel organoarsenic drugs with combined antimalarial and transmission blocking activities.

scholarly journals Specific patterns of H3K79 methylation influence genetic interaction of oncogenes in AML

2020 ◽  
Vol 4 (13) ◽  
pp. 3109-3122
Author(s):  
Molly C. Kingsley ◽  
Hongbo M. Xie ◽  
Bo-Rui Chen ◽  
Simone S. Riedel ◽  
Taylor Pastuer ◽  
...  
Keyword(s):  
Myeloid Leukemia ◽  
Target Genes ◽  
Genetic Interaction ◽  
Leukemia Cell ◽  
Leukemic Cell ◽  
Partial Explanation ◽  
Histone 3 ◽  
Idh Mutations ◽  
H3k79 Methylation ◽  
Tandem Duplications

Abstract Understanding mechanisms of cooperation between oncogenes is critical for the development of novel therapies and rational combinations. Acute myeloid leukemia (AML) cells with KMT2A-fusions and KMT2A partial tandem duplications (KMT2APTD) are known to depend on the histone methyltransferase DOT1L, which methylates histone 3 lysine 79 (H3K79). About 30% of KMT2APTD AMLs carry mutations in IDH1/2 (mIDH1/2). Previous studies showed that 2-hydroxyglutarate produced by mIDH1/2 increases H3K79 methylation, and mIDH1/2 patient samples are sensitive to DOT1L inhibition. Together, these findings suggested that stabilization or increases in H3K79 methylation associated with IDH mutations support the proliferation of leukemias dependent on this mark. However, we found that mIDH1/2 and KMT2A alterations failed to cooperate in an experimental model. Instead, mIDH1/2 and 2-hydroxyglutarate exert toxic effects, specifically on KMT2A-rearranged AML cells (fusions/partial tandem duplications). Mechanistically, we uncover an epigenetic barrier to efficient cooperation; mIDH1/2 expression is associated with high global histone 3 lysine 79 dimethylation (H3K79me2) levels, whereas global H3K79me2 is obligate low in KMT2A-rearranged AML. Increasing H3K79me2 levels, specifically in KMT2A-rearrangement leukemias, resulted in transcriptional downregulation of KMT2A target genes and impaired leukemia cell growth. Our study details a complex genetic and epigenetic interaction of 2 classes of oncogenes, IDH1/2 mutations and KMT2A rearrangements, that is unexpected based on the high percentage of IDH mutations in KMT2APTD AML. KMT2A rearrangements are associated with a trend toward lower response rates to mIDH1/2 inhibitors. The substantial adaptation that has to occur for 2 initially counteracting mutations to be tolerated within the same leukemic cell may provide at least a partial explanation for this observation.

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