Reciprocal, Temporal Expression of SpeA and SpeB by Invasive M1T1 Group A Streptococcal Isolates In Vivo

ABSTRACT The streptococcal pyrogenic exotoxins (Spes) play a central role in the pathogenesis of invasive group A streptococcal (GAS) infections. The majority of recent invasive GAS infections have been caused by an M1T1 strain that harbors the genes for several streptococcal superantigens, including speA, speB,speF, speG, and smeZ. However, considerable variation in the expression of Spe proteins among clonal M1 isolates has been found, and many of thespeA-positive M1 strains do not produce detectable amounts of SpeA in vitro. This study was designed to test the hypothesis that speA gene expression can be induced in vivo. A mouse infection chamber model that allows sequential sampling of GAS isolates at various time points postinfection was developed and used to monitor the kinetics of Spe production in vivo. Micropore Teflon diffusion chambers were implanted subcutaneously in BALB/c mice, and after 3 weeks the pores became sealed with connective tissue and sterile fluid containing a white blood cell infiltrate accumulated inside the infection chambers. Representative clonal M1T1 isolates expressing no detectable SpeA were inoculated into the implanted chambers, and the expression of SpeA in the aspirated aliquots of the chamber fluid was analyzed on successive days postinfection. Expression of SpeA was detected in the chamber fluid as early as days 3 to 5 postinfection in most animals, with a significant increase in expression by day 7 in all infected mice. Isolates recovered from the chamber and grown in vitro continued to produce SpeA even after 21 passages in vitro, suggesting stable switch on of thespeA gene. A temporal relation between the upregulation of SpeA expression and the downregulation of SpeB expression was observed in vivo. These data suggest that in vivo host and/or environmental signals induced speA gene expression and suppressed speB gene expression. This underscores the role of the host-pathogen interaction in regulating the expression of streptococcal virulence factors in vivo. The model described here should facilitate such studies.

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Downregulation of S100A4 Alleviates Cardiac Fibrosis via Wnt/β -Catenin Pathway in Mice

Cellular Physiology and Biochemistry ◽

10.1159/000489683 ◽

2018 ◽

Vol 46 (6) ◽

pp. 2551-2560 ◽

Cited By ~ 12

Author(s):

LiJun Qian ◽

Jian Hong ◽

YanMei Zhang ◽

MengLin Zhu ◽

XinChun Wang ◽

...

Keyword(s):

Calcium Binding ◽

Cardiac Fibrosis ◽

Cardiac Fibroblasts ◽

Group A ◽

Ischemic Diseases ◽

Fibrotic Diseases ◽

Signaling Activation

Background/Aims: Cardiac fibrosis is a pathological change leading to cardiac remodeling during the progression of myocardial ischemic diseases, and its therapeutic strategy remains to be explored. S100A4, a calcium-binding protein, participates in fibrotic diseases with an unclear mechanism. This study aimed to investigate the role of S100A4 in cardiac fibrosis. Methods: Cardiac fibroblasts from neonatal C57BL/6 mouse hearts were isolated and cultured. Myocardial infarction was induced by ligating the left anterior descending coronary artery (LAD). The ligation was not performed in the sham group. A volume of 5×105pfu/g adenovirus or 5 µM/g ICG-001 was intramyocardially injected into five parts bordering the infarction zone or normal region. We used Western blotting, quantitative RT-PCR, immunofluorescence, immunohistochemistry and Masson’s trichrome staining to explore the function of S100A4. Results: We found significant increases of S100A4 level and cardiac fibrosis markers, and β-catenin signaling activation in vitro and in vivo. In addition, knockdown of S100A4 significantly reduced cardiac fibrosis and β-catenin levels. Moreover, the expression of S100A4 decreased after ICG-001 inhibited β-catenin signal pathway. Conclusion: Downregulation of S100A4 alleviates cardiac fibrosis via Wnt/β -catenin pathway in mice. S100A4 may be a therapeutic target of cardiac fibrosis.

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PRMT5 Is Required for Bovine Leukemia Virus Infection In Vivo and Regulates BLV Gene Expression, Syncytium Formation, and Glycosylation In Vitro

Viruses ◽

10.3390/v12060650 ◽

2020 ◽

Vol 12 (6) ◽

pp. 650 ◽

Cited By ~ 1

Author(s):

Wlaa Assi ◽

Tomoya Hirose ◽

Satoshi Wada ◽

Ryosuke Matsuura ◽

Shin-nosuke Takeshima ◽

...

Keyword(s):

Gene Expression ◽

Small Molecule ◽

Bovine Leukemia Virus ◽

Proviral Load ◽

Leukemia Virus ◽

Syncytium Formation ◽

High Proviral Load

Bovine leukemia virus (BLV) is the causative agent of enzootic bovine leukosis, which is the most common neoplastic disease of cattle and is closely related to human T-cell leukemia viruses. We investigated the role of a new host protein, PRMT5, in BLV infection. We found that PRMT5 is overexpressed only in BLV-infected cattle with a high proviral load, but not in those with a low proviral load. Furthermore, this upregulation continued to the lymphoma stage. PRMT5 expression was upregulated in response to experimental BLV infection; moreover, PRMT5 upregulation began in an early stage of BLV infection rather than after a long period of proviral latency. Second, siRNA-mediated PRMT5 knockdown enhanced BLV gene expression at the transcript and protein levels. Additionally, a selective small-molecule inhibitor of PRMT5 (CMP5) enhanced BLV gene expression. Interestingly, CMP5 treatment, but not siRNA knockdown, altered the gp51 glycosylation pattern and increased the molecular weight of gp51, thereby decreasing BLV-induced syncytium formation. This was supported by the observation that CMP5 treatment enhanced the formation of the complex type of N-glycan more than the high mannose type. In conclusion, PRMT5 overexpression is related to the development of BLV infection with a high proviral load and lymphoma stage and PRMT5 inhibition enhances BLV gene expression. This is the first study to investigate the role of PRMT5 in BLV infection in vivo and in vitro and to reveal a novel function for a small-molecule compound in BLV-gp51 glycosylation processing.

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Biologic consequences of androgen receptor (AR) activity in MDV3100-resistant LNCaP cells and dependence on AR full length.

Journal of Clinical Oncology ◽

10.1200/jco.2014.32.4_suppl.74 ◽

2014 ◽

Vol 32 (4_suppl) ◽

pp. 74-74

Author(s):

Yoshiaki Yamamoto ◽

Yohann Loriot ◽

Eliana Beraldi ◽

Tianyuan Zhou ◽

Youngsoo Kim ◽

...

Keyword(s):

Gene Expression ◽

Androgen Receptor ◽

Cell Growth ◽

Functional Role ◽

Full Length ◽

Lncap Cells ◽

Exon 1

74 Background: While recent reports link androgen receptor (AR) variants (AR-Vs) to castration resistant prostate cancer (CRPC), the biological significance of AR-Vs in AR-regulated cell survival and proliferation, independent of AR full length (AR-FL), remains controversial. To define the functional role of AR-FL and AR-Vs in MDV3100-resistant (MDV-R), we designed antisense oligonucleotide (ASO) targeting exon 1 and exon 8 in AR to knockdown AR-FL alone or in combination with AR-Vs and examined these effects in MDV-R LNCaP-derived cells in vitro and in vivo. Methods: We generated by selection MDV-R LNCaP-derived sub-lines that uniformly expressed high levels of both AR-FL and AR-V7 compared to CRPC LNCaP xenografts. Cell growth rates, protein and gene expression were analyzed using crystal violet assay, western blotting and real-time PCR, respectively. Exon 1 and 8 AR-ASO were evaluated in MDV-R49F CRPC LNCaP xenografts. Results: AR-V7 was transiently transfected in MDV-R49F cells and differential knockdown of AR-V7 and/or AR-FL by exon 1 versus exon 8 AR-ASO was used to evaluate relative biologic contributions of AR-FL versus AR-V7 in MDV-R LNCaP AR-V7 overexpressing cells. Exon 1 and 8 AR-ASO treatment in these cells similarly decreased prostate-specific antigen (PSA) expression and induced apoptosis as measured by caspase-3 and PARP cleavage and cell growth inhibition. To further define the functional role of AR-Vs in MDV-R LNCaP cells, we used a CE3 siRNA that specifically silenced AR-V7, but not AR-FL in MDV-R LNCaP cells. AR-V7 knockdown did not decrease PSA levels, did not induce apoptosis, and did not inhibit cell growth. In MDV-R LNCaP cells, exon 1 and 8 ASO similarly suppressed cell growth and AR-regulated gene expression in vitro and in vivo. Conclusions: These results indicate that the AR remains an important driver of MDV3100 resistance and, the biologic consequences mainly driven by AR-FL in MDV-R LNCaP models.

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Role of the snake venom toxin jararhagin in proinflammatory pathogenesis: In vitro and in vivo gene expression analysis of the effects of the toxin

Archives of Biochemistry and Biophysics ◽

10.1016/j.abb.2005.06.007 ◽

2005 ◽

Vol 441 (1) ◽

pp. 1-15 ◽

Cited By ~ 38

Author(s):

Paul Gallagher ◽

Yongde Bao ◽

Solange M.T. Serrano ◽

Gavin D. Laing ◽

R. David G. Theakston ◽

...

Keyword(s):

Gene Expression ◽

Expression Analysis ◽

Snake Venom ◽

Gene Expression Analysis ◽

Venom Toxin ◽

Snake Venom Toxin

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HEPATOCYTE TOLL-LIKE RECEPTOR 2 EXPRESSION IN VIVO AND IN VITRO: ROLE OF CYTOKINES IN INDUCTION OF RAT TLR2 GENE EXPRESSION BY LIPOPOLYSACCHARIDE

Shock ◽

10.1097/00024382-200014030-00021 ◽

2000 ◽

Vol 14 (3) ◽

pp. 361-365 ◽

Cited By ~ 46

Author(s):

Shubing Liu ◽

Neil A. Salyapongse ◽

David A. Geller ◽

Yoram Vodovotz ◽

Timothy R. Billiar

Keyword(s):

Gene Expression ◽

Toll Like Receptor ◽

Tlr2 Gene ◽

Toll Like Receptor 2

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Epigenetic factors in atherogenesis: microRNA

Biomeditsinskaya Khimiya ◽

10.18097/pbmc20166202134 ◽

2016 ◽

Vol 62 (2) ◽

pp. 134-140

Author(s):

A.V. Smirnova ◽

V.N. Sukhorukov ◽

V.P. Karagodin ◽

A.N. Orekhov

Keyword(s):

Gene Expression ◽

Antisense Oligonucleotides ◽

Noncoding Rna ◽

Cell Cycle Progression ◽

Rna Sequences ◽

Cycle Progression ◽

Atherosclerosis Progression

MicroRNAs (miRNAs) are small (~22 nucleotides in length) noncoding RNA sequences regulating gene expression at posttranscriptional level. MicroRNAs bind complementarily to certain mRNA and cause gene silencing. The involvement of miRNAs in the regulation of lipid metabolism, inflammatory response, cell cycle progression and proliferation, oxidative stress, platelet activation, endothelial and vascular smooth muscle cells (VSMC) function, angiogenesis and plaque formation and rapture indicates important roles in the initiation and progression of atherosclerosis. The key role of microRNAs in pathophysiology of cardiovascular diseases (CVDs), including atherosclerosis, was demonstrated in recent studies. Creating antisense oligonucleotides is a novel technique for selective changes in gene expression both in vitro and in vivo. In this review, we draw attention to the role of miRNAs in atherosclerosis progression, using miRNA as the potential biomarkers and targets in the CVDs, as well as possible application of antisense oligonucleotides

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ASXL2 Is Recurrently Mutated in t(8;21) AML and Regulates Hematopoietic Development

Blood ◽

10.1182/blood.v128.22.1051.1051 ◽

2016 ◽

Vol 128 (22) ◽

pp. 1051-1051

Author(s):

Vikas Madan ◽

Lin Han ◽

Norimichi Hattori ◽

Anand Mayakonda ◽

Qiao-Yang Sun ◽

...

Keyword(s):

Gene Expression ◽

Bone Marrow ◽

Research Funding ◽

Hematopoietic Differentiation ◽

Wild Type ◽

Flow Cytometric ◽

Deficient Mice

Abstract Chromosomal translocation t(8;21) (q22;q22) leading to generation of oncogenic RUNX1-RUNX1T1 fusion is a cytogenetic abnormality observed in about 10% of acute myelogenous leukemia (AML). Studies in animal models and recent next generation sequencing approaches have suggested cooperativity of secondary genetic lesions with t(8;21) in inducing leukemogenesis. In this study, we used targeted and whole exome sequencing of 93 cases (including 30 with matched relapse samples) to profile the mutational landscape of t(8;21) AML at initial diagnosis and post-therapy relapse. We identified recurrent mutations of KIT, TET2, MGA, FLT3, NRAS, DHX15, ASXL1 and KMT2Dgenes in this subtype of AML. In addition, high frequency of truncating alterations in ASXL2 gene (19%) also occurred in our cohort. ASXL2 is a member of mammalian ASXL family involved in epigenetic regulation through recruitment of polycomb or trithorax complexes. Unlike its closely related homolog ASXL1, which is mutated in several hematological malignancies including AML, MDS, MPN and others; mutations of ASXL2 occur specifically in t(8;21) AML. We observed that lentiviral shRNA-mediated silencing of ASXL2 impaired in vitro differentiation of t(8;21) AML cell line, Kasumi-1, and enhanced its colony forming ability. Gene expression analysis uncovered dysregulated expression of several key hematopoiesis genes such as IKZF2, JAG1, TAL1 and ARID5B in ASXL2 knockdown Kasumi-1 cells. Further, to investigate implications of loss of ASXL2 in vivo, we examined hematopoiesis in Asxl2 deficient mice. We observed an age-dependent increase in white blood cell count in the peripheral blood of Asxl2 KO mice. Myeloid progenitors from Asxl2 deficient mice possessed higher re-plating ability and displayed altered differentiation potential in vitro. Flow cytometric analysis of >1 year old mice revealed increased proportion of Lin-Sca1+Kit+ (LSK) cells in the bone marrow of Asxl2 deficient mice, while the overall bone marrow cellularity was significantly reduced. In vivo 5-bromo-2'-deoxyuridine incorporation assay showed increased cycling of LSK cells in mice lacking Asxl2. Asxl2 deficiency also led to perturbed maturation of myeloid and erythroid precursors in the bone marrow, which resulted in altered proportions of mature myeloid populations in spleen and peripheral blood. Further, splenomegaly was observed in old ASXL2 KO mice and histological and flow cytometric examination of ASXL2 deficient spleens demonstrated increased extramedullary hematopoiesis and myeloproliferation compared with the wild-type controls. Surprisingly, loss of ASXL2 also led to impaired T cell development as indicated by severe block in maturation of CD4-CD8- double negative (DN) population in mice >1 year old. These findings established a critical role of Asxl2 in maintaining steady state hematopoiesis. To gain mechanistic insights into its role during hematopoietic differentiation, we investigated changes in histone marks and gene expression affected by loss of Asxl2. Whole transcriptome sequencing of LSK population revealed dysregulated expression of key myeloid-specific genes including Mpo, Ltf, Ngp Ctsg, Camp and Csf1rin cells lacking Asxl2 compared to wild-type control. Asxl2 deficiency also caused changes in histone modifications, specifically H3K27 trimethylation levels were decreased and H2AK119 ubiquitination levels were increased in Asxl2 KO bone marrow cells. Global changes in histone marks in control and Asxl2 deficient mice are being investigated using ChIP-Sequencing. Finally, to examine cooperativity between the loss of Asxl2 and RUNX1-RUNX1T1 in leukemogenesis, KO and wild-type fetal liver cells were transduced with retrovirus expressing AML1-ETO 9a oncogene and transplanted into irradiated recipient mice, the results of this ongoing study will be discussed. Overall, our sequencing studies have identified ASXL2 as a gene frequently altered in t(8;21) AML. Functional studies in mouse model reveal that loss of ASXL2 causes defects in hematopoietic differentiation and leads to myeloproliferation, suggesting an essential role of ASXL2 in normal and malignant hematopoiesis. *LH and NH contributed equally Disclosures Ogawa: Takeda Pharmaceuticals: Consultancy, Research Funding; Sumitomo Dainippon Pharma: Research Funding; Kan research institute: Consultancy, Research Funding.

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Streptolysin-induced endoplasmic reticulum stress promotes group A streptococcal in vivo biofilm formation and necrotizing fasciitis

10.1101/183012 ◽

2017 ◽

Author(s):

Anuradha Vajjala ◽

Debabrata Biswas ◽

Kelvin Kian Long Chong ◽

Wei Hong Tay ◽

Emanuel Hanski ◽

...

Keyword(s):

Soft Tissue ◽

Er Stress ◽

Necrotizing Fasciitis ◽

Biofilm Formation ◽

Mammalian Cells ◽

Chronic Infections ◽

Group A

AbstractGroup A Streptococcus (GAS) is a human pathogen that causes infections ranging from mild to fulminant and life-threatening. Biofilms have been implicated in acute GAS soft-tissue infections such as necrotizing fasciitis (NF). However, most in vitro models used to study GAS biofilms have been designed to mimic chronic infections and insufficiently recapitulate in vivo conditions and the host-pathogen interactions that might influence biofilm formation. Here we establish and characterize an in vitro model of GAS biofilm development on mammalian cells that simulates microcolony formation observed in a murine model of human NF. We show that on mammalian cells, GAS forms dense aggregates that display hallmark biofilm characteristics including a three-dimensional architecture and enhanced tolerance to antibiotics. In contrast to abiotic-grown biofilms, host-associated biofilms require the expression of secreted GAS streptolysins O and S (SLO, SLS) resulting in the release of a host-associated biofilm promoting-factor(s). Supernatants from GAS-infected mammalian cells or from cells treated with endoplasmic reticulum (ER) stressors restore biofilm formation to an SLO and SLS null mutant that is otherwise attenuated in biofilm formation on cells, together suggesting a role for streptolysin-induced ER stress in this process. In an in vivo mouse model, the streptolysin-null mutant is attenuated in both microcolony formation and bacterial spread, but pre-treatment of softtissue with an ER-stressor restores the ability of the mutant to form wild type like microcolonies that disseminate throughout the soft tissue. Taken together, we have identified a new role of streptolysin-driven ER stress in GAS biofilm formation and NF disease progression.Significance StatementAlthough it is well-accepted that bacterial biofilms are associated with many chronic infections, little is known about the mechanisms by which group A Streptococcus (GAS) biofilms contribute to acute soft tissue-invasive diseases like necrotizing fasciitis (NF). In this study, we establish a physiologically relevant in vitro model to study GAS biofilm formation on mammalian cells and validate our findings in a mouse model that mimics human NF. This study demonstrates a novel role of GAS streptolysin-mediated ER stress in the development and spread of GAS biofilms in acute softtissue infections. We also show that biofilm formation depends on the release of a host-associated factor that promotes microcolony formation and GAS dissemination in vivo.

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The Potential Role of the Six1/Eya1 Pathway in the Establishment of Leukemia Stem Cells in MLL-ENL – Induced Leukemia

Blood ◽

10.1182/blood.v118.21.1362.1362 ◽

2011 ◽

Vol 118 (21) ◽

pp. 1362-1362 ◽

Cited By ~ 1

Author(s):

Sylvia Takacova ◽

Pavla Luzna ◽

Viktor Stranecky ◽

Vladimir Divoky

Keyword(s):

Bone Marrow ◽

Cellular Transformation ◽

Leukemia Cells ◽

Leukemia Stem Cells ◽

Transcription Profile ◽

Self Renewal ◽

Kinetics Of

Abstract Abstract 1362 During the multistep pathogenesis of acute leukemia (AL), a pool of leukemia stem cells (LSCs) emerges that is capable of limitless self-renewal and ensuring disease maintenance. The molecular mechanism that controls the kinetics of cellular transformation and development of LSCs is largely unknown. Using our MLL-ENL-ERtm mouse model, we have previously shown (Takacova et al., Blood 2009, 114 (22): 947–947, ASH abstract) activation of the ATR/ATM-Chk1/Chk2-p53/p21 checkpoint leading to senescence at early stages of cellular transformation (myeloproliferation), thereby preventing AL development in vivo. Experimental ATM/ATR inhibition accelerated the transition to immature cell states, acquisition of LSC properties and AL development in these mice. The MLL-ENL-ERtm mouse model allows us to study the kinetics of MLL-ENL-ERtm LSC development. We raised the questions how the transformation process progresses from the pre-LSC to the LSC state, and how DNA damage response (DDR) - mediated senescence affects the transition in gene expression. Given that the threshold of DDR signaling events is rate-limiting, we determined the transcription profile of the pre- LSC–enriched cell states derived from bone marrow and spleen of the MLL-ENL-ERtm mice at the early disease stage, and we correlated this transcription profile with the level of DDR, proliferation rate and induction of senescence. Pair-wise comparisons revealed up-regulation of the Six1 transcription factor gene and its cofactor Eya1 in the MLL-ENL-ERtm pre-LSCs in association with aberrant proliferation in both tissues. The notable difference between the two tissues concerning the barrier induction was the higher threshold of DDR and senescence in the bone marrow due to cooperation with inflammatory cytokines that fine-tune the DDR level. Interestingly, the expression of Six1 and Eya1 genes was down-regulated in senescence exclusively in the bone marrow. Consistent with these in vivo data, we found Six1 expression decreased in response to inflammation/DDR-induced senescence in the MLL-ENL-ERtm bone marrow cells cultured in vitro and correlated with SA-beta-gal positivity and p16 up-regulation. Six1 mRNA level was decreased only transiently after ionizing radiation (4 Gy)-induced DDR in the same cell line. These data suggest that Six1 expression is down-regulated in response to high DDR and permanent cell-cycle arrest in the MLL-ENL-ERtm pre-LSCs. Furthermore, we identified the transcription profile of the LSC-enriched cell state after inhibition of DDR in caffeine-treated MLL-ENL-ERtm mice in vivo. Interestingly, the expression level of Six1 and Eya1 was significantly increased in the bone marrow and spleen of the MLL-ENL-ERtm AML mice compared to the early (preleukemia) stage. High expression of Six1 and Eya1 and higher cell number expressing these genes was further confirmed by immunohistochemical staining on tissue sections. The MLL-ENL-ERtm LSC-enriched spleen cells showed increased colony forming ability in vitro and leukemia-initiating potential in serial transplantation experiments compared to pre-LSCs. Moreover, we detected Six1 and Eya1 expression in the infiltrating leukemia cells in tissues of the caffeine-treated MLL-ENL-ERtm AML mice and in a subset of leukemia cells in transplanted mice. Based on these findings and correlations, we hypothesized that the Six1/Eya1 pathway might be involved in regulation of some of the aspects of LSC development as well as invasion and maintenance of leukemia in our MLL-ENL-ERtm mice. Notably, our data indicate that senescence represses a subset of the MLL-ENL-downstream transcription response and prevents full activation of self-renewal. Experiments leading to more detailed understanding of the role of the Six1/Eya1 pathway in the MLL-ENL-induced cellular transformation are ongoing. Disclosures: No relevant conflicts of interest to declare.

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Context Dependent Role of Polycomb Repressive Complex 2 in Acute Leukemia

Blood ◽

10.1182/blood.v124.21.610.610 ◽

2014 ◽

Vol 124 (21) ◽

pp. 610-610

Author(s):

Etienne Danis ◽

Taylor Yamauchi ◽

Kristen Echanique ◽

Jessica Haladyna ◽

Huafeng Xie ◽

...

Keyword(s):

Gene Expression ◽

T Cell ◽

Acute Leukemia ◽

Murine Models ◽

Polycomb Repressive Complex ◽

Cell Precursor ◽

Polycomb Repressive Complex 2

Abstract Polycomb Repressive Complex 2 (PRC2) is a multi-protein complex with important roles in development and cancer. Both hyper- and hypoactivity of PRC2 are associated with blood-related malignancies. Activating mutations of the PRC2 methyltransferase EZH2 have been found in human B-lineage lymphomas. Inactivating mutations of PRC2 components EZH2, EED and SUZ12 have been described in early T-cell Precursor ALL (ETP-ALL) and inactivating PRC2-alterations are found in Myelodysplastic and Myeloproliferative Syndromes. The mechanisms underlying this paradox are incompletely understood. We here investigate the context dependent role of PRC2 in murine models. We initially studied PRC2 in normal hematopoiesis: Chip-seq analysis of the PRC2-mediated H3K27me3 chromatin mark demonstrates that many genes highly expressed in immature hematopoiesis gain H3K27me3 in the developmental transition from more immature Lin-Sca1+Kit+ (LSK) cells to lineage committed Granulocyte Macrophage Progenitors (GMPs). Transcription of these genes is enriched in EZH2ko GMP compared to EZH2ffGMP by Gene Set Enrichment Analysis (GSEA). These data suggest that PRC2 is important for the silencing of immature gene expression programs in the developmental transition from LSK to GMP. We next analyzed the role of PRC2 in two murine models of acute leukemia: MLL-AF9 driven leukemia, and a model of early T-cell precursor T-ALL (ETP-ALL). In MLL-AF9 leukemia, we previously found that inactivation of Eed completely abrogate leukemogenesis in vitro and in vivo. We now report that genetic inactivation of the tumor suppressor Cdkn2a (a canonical PRC2 target) partially rescued MLL-AF9 mediated leukemia in vitro and in vivo. However, Cdkn2akoEEDko MLL-AF9 leukemia remained compromised. In vitro growth was reduced to approximately 10% of Eedff controls. While control MLL-AF9 leukemia developed in vivo in 100% of the recipients, Cdkn2akoEEDkoMLL-AF9 leukemia developed with significantly prolonged latency and incomplete penetrance (25%). RNAseq analysis revealed that high level expression of genes with established roles in MLL-AF9 leukemia such as HoxA9, Cdk6 and Jmjd1c unexpectedly depends on Eed. These data are in keeping with the absence of alterations in PRC2-components in human MLL-rearranged leukemia. In contrast, PRC2 core components (EZH2/EED/SUZ12) are deleted or mutated in > 40% of ETP-ALL. ETP-ALL also often has direct or indirect activation of the RAS-pathway, and carries frequent deletions of the CDKN2A locus. To model the effects of EED and EZH2-inactivation in ETP-ALL, we established Cdkn2akoEedff vs Cdkn2akoEedko, and Cdkn2akoEzh2ff vs Cdkn2akoEZH2koleukemias by transduction with NRASQ61K followed by expansion on OP9DL1 stroma cells to activate T-lineage differentiation via Notch-signaling. Cdkn2ako NRASQ61K leukemia showed an immunophenotype similar to human ETP-ALL (positive for c-Kit, CD5 and myeloid markers and mostly negative for CD4/8). Inactivation of Eed or Ezh2 in this model led to a shortening of latency (p=0.03 for Eed, p=0.0001 for Ezh2). RNAseq revealed enrichment of genes associated with murine DN1 thymocytes and with human ETP-ALL in Eedko vs Eedff Cdkn2ako NRASQ61K leukemia. These genesets showed even more pronounced enrichment in Ezh2kocompared to Ezh2ff Cdkn2ako NRASQ61K leukemia. Genes highly expressed in early hematopoiesis were enriched in Eedko and Ezh2ko cells in both, the MLL-AF9 and NRASQ61K leukemia models. However, there was an opposing effect on HoxA9 gene expression, with PRC2 inactivation leading to decreased HoxA9 expression in MLL-AF9, and increased HoxA9 expression in Cdkn2ako NRASQ61K leukemia. Decreased HoxA9 has been shown to impair MLL-AF9 leukemia growth. To test the functional significance of elevated HoxA9-levels in the Eedko and EZH2koNRASQ61K leukemias, we co-expressed HoxA9 and NRASQ61K in the presence of intact Eed and Ezh2 loci. Preliminary data suggest that HoxA9 accelerates leukemia development in this setting. Alterations in chromatin modifiers, including PRC2, are frequent in leukemia and lymphoma. Our data demonstrate that manipulation of PRC2 can have opposite effects on leukemia phenotype and expression of key PRC2-repressed genes such as HoxA9 in the context of different tumors. We are currently characterizing the mechanisms leading to divergent outcomes of PRC2 manipulation in MLL-AF9 leukemia compared to NRASQ61K ETP-like leukemia. Disclosures Armstrong: Epizyme : Consultancy.

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