Academic literature on the topic 'MEL cells; Epigenetic marks'

Abstract CD19 is expressed broadly on the surface of B-cells during normal development and malignant growth, making it a good target for immunotherapy. While immunotherapies targeting CD19 have had great success against pediatric B-cell acute lymphoblastic leukemia (B-ALL), relapses lacking the CD19 epitope still occur (Maude et al., 2014). We have discovered that alternative splicing of CD19, in particular the skipping of exon 2, is responsible for the loss of CD19 extracellular domains, causing resistance to therapy (Sotillo et al., 2015). Here we investigate the molecular mechanism of CD19 exon 2 skipping. The sequence-based algorithm AVISPA (Barash et al., 2013) predicts several splicing factors (SF) to bind near exon 2. We used RNA crosslink immunoprecipitation (CLIP) in nuclear lysates from Nalm-6 B-ALL cells to test the direct binding to exon 2 of 9 AVISPA-predicted SFs and 6 SFs commonly involved in exon skipping. This allowed us to identify SRSF3, hnRNP-A, and hnRNP-C as CD19 exon 2-bound proteins. Subsequent siRNA knockdown experiments reveled that downregulation of SRSF3, but not hnRNP-C, increases the frequency of exon 2 skipping in a dose dependent manner, suggesting that SRSF3 promotes the inclusion of exon 2. To further validate the role of SRSF3 in CD19 splicing we mined the publicly available GSE52834 dataset where 22 RNA binding proteins were knocked down in the GM19238 lymphoblastoid cell line. Of all siRNAs tested, only the anti-SRSF3 siRNA caused an increase in exon 2 skipping, suggesting that SRSF3 is indeed the key regulator of CD19 splicing. Interestingly, SRSF3 has been shown to interact with PSIP1, a cofactor known to "read" modified histone H3K36me3 (Pradeepa et al., 2012), suggesting a convergence of splicing-based and epigenetics mechanisms. Indeed, exonic regions in genomic DNA are enriched for H3K36me3, and knockdown of Setd2, the H3K36 methyltransferase, results in changes in exon inclusion (Luco et al., 2010; Brown et al., 2012; Hnilicova and Stanek, 2011). Thus, we are currently investigating the connection between the H3K36me3 marks in the CD19 locus and alternative splicing of CD19. Our data could suggest a method of restoring full-length CD19 expression in immunotherapy-resistant cancers using epigenetic drugs. Maude, S L, Noelle, F, Shaw, PA, Aplenc, R, Barrett, DM, Bunin, NJ, Chew, A, Gonzalez, VE, Zheng, Z, Lacey, SF, Mahnke, YD, Melenhorst, JJ, Rheingold, SR, Shen, A, Teachey, DT, Levine, BL, June CH, Porter, DL, and Grupp, SA. Chimeric antigen receptor T cells for sustained remissions in leukemia. N Engl J Med 2014; 371: 1507-1517. Sotillo, E, Barrett, D, Bagashev, A, Black, K, Lanauze, C, Oldridge, D, Sussman, R, Harrington, C, Chung, EY, Hofmann, TJ, Maude, SL, Martinez, NM, Raman, P, Ruella, M, Allman, D, Jacoby, E, Fry, T, Barash, Y, Lynch, KW, Mackall, C, Maris, J, Grupp, SA, and Thomas-Tikhonenko, A. Alternative splicing of CD19 mRNA in leukemias escaping CART-19 immunotherapy eliminates the cognate epitope andcontributes to treatment failure. 2015AACR Annual Meeting, Philadelphia. Barash Y, Vaquero-Garcia J, González-Vallinas J, Xiong HY, Gao W, Lee LJ, and Frey BJ. AVISPA: a web tool for the prediction and analysis of alternative splicing. Genome Biol 2013; 14(10):R114. Pradeepa, MM, Sutherland, HG, Ule, J, Grimes, GR, and Bickmore, WA. Psip1/Ledgf p52 binds methylated histone H3K36 and splicing factors and contributes to the regulation of alternative splicing. PLOS Genets 2012; 8:e1002717. Luco, RF, Pan, Q, Tominaga, K, Blencowe, BJ, Pereira-Smith, OM, Misteli, T. Regulation of alternative splicing by histone modifications. Science 2010; 327: 996-1000. Brown, SJ, Stoilov, P, and Xing, Y. Chromatin and epigenetic regulation of pre-mRNA processing. Human Mol Genets 2012; 21:R90-R96. Hnilicova, J, and Stanek, D. Where splicing joins chromatin. Nucleus 2011; 2:182-188. Disclosures No relevant conflicts of interest to declare.

Abstract Protein arginine methyltransferase-5 (PRMT5), a major type II arginine methyltransferase, is an important epigenetic modifier with oncogene-like properties due to its transcriptional repressive activity. When over-expressed, PRMT5 has been shown to target and silence the expression of multiple regulatory and tumor suppressor genes. Global symmetric dimethylation of arginine residues within the N-terminal of histones (H2A(Me2)R3, H3(Me2)R8, H4(Me2)R3) plays a critical role in B cell transformation, correlates with increased tumor cell proliferation and survival. PRMT5 expression is enhanced in aggressive B-cell non-Hodgkin's lymphomas, including mantle cell lymphoma (MCL) and supports constitutive CYCLIN D1/CDK4/6 activity leading to inactivation of the RBL2/E2F tumor suppressor pathway. Other work has identified PRMT5 as a vital contributor to MYC-driven oncogenesis. While PRMT5 has been characterized as a transcriptional repressor, few micro-RNAs (miRs) have been identified as direct targets. Here we utilize next generation sequencing and whole genome mapping to identify miRs targeted by PRMT5 in mantle cell lymphomas. ChIP-seq analysis revealed genome-wide recruitment of the PRMT5-specific epigenetic mark H3(Me2)R8 in B-cell lymphoma cell lines (Jeko, Pfeiffer, SUDHL2) with minimal enrichment on chromatin from normal B cells. PRMT5 was found to target as many as 8593 genes in each lymphoma cell line examined with 4662 genes that are common targets between three different lymphoma cell lines. Comparing Chip-Seq to RNA-Seq data of each lymphoma cell line treated with PRMT5 shRNA identified miR-33b, miR-96 and miR-503 as direct targets. Predicted 3' untranslated region (UTR) targets of these miRs included CYCLIN D1 (miR-33b, miR-96, miR-503), MYC (miR-33b) and PRMT5 (miR-96), three gene products that have been shown to be highly relevant to the malignant phenotype of aggressive MCL. Validation studies with ChIP-real-time polymerase chain reaction (RT-PCR) and quantitative-RT-PCR demonstrated that CRISPR-CAS9-mediated PRMT5 deletion led to transcriptional derepression of miR-33b, miR-96 and miR-503 in MCL lines (CC-MCL, Jeko, and SP53) and in primary MCL patient samples. Inhibition of PRMT5 led to loss of recruitment of an epigenetic repressor complex at the miR-96 promoter containing PRMT5 and HDAC3, gain of p65 and enrichment of hyperacetylated lysine epigenetic marks H4K8, H3K14 and H2BK12, changes consistent with restored transcriptional activity of miR96. Promoter regions of miR33b and miR503 showed loss of recruitment of a repressive complex consisting of SP1, HDAC2 and gain of p300, CBP and hyperacetylated lysine epigenetic marks H3K9 and H3K14 following PRMT5 inhibition. Restored expression of miR-33b led to simultaneous down-modulation of CYCLIN D1 and c-MYC, whereas miR-96 re-expression led to loss of CYCLIN D1 and PRMT5. Re-expression of miR-503 on the contrary only impacted CYCLIN D1 expression. Furthermore, luciferase reporter assays with wild-type and mutant 3' UTRs showed that binding sites of miR-33b, miR-96 and miR-503 were critical for translational regulation of CYCLIN D1 and C-MYC. Inducible re-expression of miR-33b, miR-96 and miR-503 inhibited proliferation of MCL cells as determined by MTS assay and promoted cellular apoptosis as measured by staining with Annexin V/PI staining and flow cytometry. In vivo studies with the MCL cell line CC-MCL1 designed to conditionally express miR-96 and or miR-33b are currently underway. These results link dysregulated PRMT5 expression to transcriptional silencing of tumor suppressor miRs capable of regulating critical drivers of aggressive histologic subtypes of MCL and indicate that multiple mechanisms are involved in the regulation of CYCLIN D1 expression in MCL. Furthermore, this data supports PRMT5 overexpression as a critical factor involved with maintenance of the malignant phenotype of MCL, and supports strategies to selectively inhibit this promising therapeutic target in this disease. Disclosures Baiocchi: Essanex: Research Funding.

Abstract Genetic correction of hematologic defects is currently impeded by inefficient vector technology. We find that vectors that insulate the correcting transgene from position effects and genotoxicity compromise viral titers. Here we present an improved vector system which utilizes a modified insulator element, without sacrificing viral titers. Specifically, our genetic and epigenetic analysis of the 1.2kb chicken β-globin hypersensitive site-4 (cHS4) insulator reveal heretofore unknown activities in regions of the chicken β-globin insulator element outside the canonical and well studied 250bp 5′ “core” element. Previously, the core insulator activity was mapped to CTCF and USF-1/2 binding sites, located only in the 5′ 250bp core. However, we find that the 5′ 250bp core alone is ineffective at shielding from position effects when it flanks transgenes, and gammaretrovirus/lentivirus vectors. In contrast, the entire 1.2kb cHS4 efficiently insulates, but significantly lowers titers of lentiviral vectors. To identify insulating activities which might be appended to the 5′ 250bp core to properly insulate transgene expression cassettes without sacrificing viral titers, we performed a structure-function analysis of the cHS4 insulator placed within the 3′LTR of a lentivirus containing the regulatory and coding sequences of human b-globin (Table 1). We compared single-copy clonal progeny of mouse erythroleukemia cells (MEL) and primary transduced and transplanted hematopoietic stem cells for position effects. Additionally, we studied repressive and activating histone marks over the transgene promoter and cHS4 in the different proviruses. Our data indicate that while all vectors containing the core reduced the coefficient of variation (CV) of human b-globin (HbA) expression, several constructs suggested that cHS4 sequences in the most 3′ 400bp (furthest from the core) may be critical to full length insulator activity. We next analyzed HbA expression in vector-corrected thalassemia mice, and generated single copy secondary CFU-S, the gold standard for studying chromatin position effects (Table 2). While all vectors containing the cHS4 core provided some ‘insulator’ activity when compared to the uninsulated vector control (conceivably by reducing the CV/clonal variegation) the full length 1.2kb insulator vector provided maximum shielding from position effects, with nearly 2.5-fold higher HbA expression compared to the uninsulated vector. These data were confirmed in secondary CFU-S. Epigenetic analyses of the vector b-globin promoter revealed that transcriptionally repressive histone modifications were decreased, and activating histone modifications increased when the last 400bp sequences of cHS4 were present. Notably, vectors carrying only the 3′ 400bp sequences of cHS4 reduced clonal variegation in MEL cells and secondary CFU-S, but did not increase HbA expressing cells both in vitro and in vivo (Table 2). However, full insulator activity was restored in MEL clones when both the 5′ 250bp core was combined with the 3′ 400bp element. The addition of the 3′ 400bp element to the core was accompanied with a significant enrichment of active histone marks and minimal repressive histone marks in the provirus as seen with the 1.2Kb insulator. These data consolidate the known insulating activity of the 5′ 250bp core element with a novel 3′ 400bp element which (together) constitutes a new insulated vector system with excellent insulating properties and viral titers. Our data have important implications in the design of gene therapy vectors, where optimal insulator activity can be achieved with a minimal reduction in viral titers. Table 1. Single copy MEL clones showing effect of insulator sequences on position effects in β-globin carrying lentiviruses MEL Clones Uninsulated 5′ 250bp Core 5′ 400bp 2 Cores 5′800bp 1.2Kb 3′ 400bp HbA+ cells (%) 59±5 49±6 51±3 52±3 49±6 84±3** 59±3 CV of HbA expression 53±4 37±3* 40±2* 42±1* 42±3 37±1* 38±1* Table 2. Effect of insulator sequences on the differentiated progeny of transduced and transplanted thalassemia hematopoietic stem cells Primary transplants (24 wks) Mock Uninsulated 5′250 bp 5′400bp 2 cores 1.2Kb 3′ 400bp *P<0.05; **P<0.01; ***P<0.001; HbA = human β and mouse α globin tetramers Statistical analysis was performed by ANOVA (Dunnell’s multiple comparison test to the control vector sBG) *Data has not been nomialized far vector copies; CFU-S represent those screened for carrying single integrants by qPCR Note: Copy number of vector with 1.2kb cHS4 insert is significantly lower compared to uninsulated vector ^RBC (M/μl) 6.6±0.4 8.6±0.5 8.5±0.3 7.9±0.2 7.5±0.2 8.9±0.2 7.9±0.2 ^Hematecrit (%) 24±2 32±3 36±1 36±1 35±2 38±1 34±1 ^MCHC (g/dL) 24±1 20±1 29±2 29±2 27±2 33±1* 28±1 ^Reticulocyte (%) 29±2 11±4 10±2 12±7 11±13 7±1 12±2 Vector Copy (VC)/Cell 0 1.2±0.18 0.90±0.12 0.97±0.13 1.2±0.26 0.63±0.10 0.76±0.16 HbA (%)/VC (HPLC) 19±6 26±4 30±3 22±3 43±3** 18±3 HbA+ RBC/VC (FACS) 40±8 56±5 62±7 51±9 100±5** 32±7 ^CV 618±171 375±36* 384±29* 294±29** 281±16** 270±44** Secondary CFU-S (single copy) ^HbA+cells/CFUS (%) 37±4 53±4 53±5 42±4 85±2** 8±1*** ^CV 98±15 70±5* 75±3* 91±5 57±2** 59±2**

Abstract Adult T-cell leukemia (ATL) is an aggressive T-cell malignancy caused by human T-cell leukemia virus type 1 (HTLV-1) that develops through a multistep carcinogenesis process involving 5 or more genetic events. We provide a comprehensive overview of recently uncovered information on the molecular basis of leukemogenesis in ATL. Broadly, the landscape of genetic abnormalities in ATL that include alterations highly enriched in genes for T-cell receptor–NF-κB signaling such as PLCG1, PRKCB, and CARD11 and gain-of function mutations in CCR4 and CCR7. Conversely, the epigenetic landscape of ATL can be summarized as polycomb repressive complex 2 hyperactivation with genome-wide H3K27 me3 accumulation as the basis of the unique transcriptome of ATL cells. Expression of H3K27 methyltransferase enhancer of zeste 2 was shown to be induced by HTLV-1 Tax and NF-κB. Furthermore, provirus integration site analysis with high-throughput sequencing enabled the analysis of clonal composition and cell number of each clone in vivo, whereas multicolor flow cytometric analysis with CD7 and cell adhesion molecule 1 enabled the identification of HTLV-1–infected CD4+ T cells in vivo. Sorted immortalized but untransformed cells displayed epigenetic changes closely overlapping those observed in terminally transformed ATL cells, suggesting that epigenetic abnormalities are likely earlier events in leukemogenesis. These new findings broaden the scope of conceptualization of the molecular mechanisms of leukemogenesis, dissecting them into immortalization and clonal progression. These recent findings also open a new direction of drug development for ATL prevention and treatment because epigenetic marks can be reprogrammed. Mechanisms underlying initial immortalization and progressive accumulation of these abnormalities remain to be elucidated.

Abstract Introduction: Aggressive histologic subtypes of lymphoma such as mantle cell (MCL) and activated B cell (ABC) are considered incurable and affected patients often have a short median survival despite multimodal therapy. It is well established that altered expression of oncogenes and epigenetic dysregulation of tumor suppressor and regulatory genes promote cellular transformation of normal B cells into malignant lymphoma. Hypermethylation of histone proteins (H3R8 and H4R3) by the protein arginine methyltransferase 5 (PRMT5) enzyme has been documented in multiple cancer types and has been shown to promote tumor cell growth and survival. Importantly, PRMT5 over expression does not occur in normal B cells (resting or activated) and is only detected in malignant lymphoma cells. We have previously shown that PRMT5 regulates the Polycomb-repressive complex 2 (PRC2) complex including EZH2, a core histone-lysine N methyl transferase. EZH2 has tumor suppressor functions and has been shown to regulate WNT antagonist’s gene expression. WNT/β-CATENIN signaling pathway has been associated with increased cell proliferation and survival in various forms of cancers including lymphoma. Until recently, the role of PRMT5 in controlling WNT/β-CATENIN signaling has been unclear. We hypothesized that PRMT5, through its ability to repress EZH2 expression, would control WNT/β-CATENIN signaling and orchestrate downstream pathways that are relevant to lymphomagenesis. Methods: PRMT5 inhibition of patient-derived lymphoma cell lines, primary lymphoma tumor cells and mouse primary Eμ-BRD2 transgenic lymphoma cells by infecting with sh-PRMT5 lentivirus (or sh-GFP control) or a selective small molecule PRMT5 inhibitor (tool compound CMP5). Gene expression was monitored by immunoblotting and reverse transcription (RT) real time PCR. Recruitment of target proteins to promoter regions was examined by ChIP assays. To evaluate PRMT5 and WNT antagonist expression in NHL patient samples, primary tumor samples were collected from 4 patients with MCL. Cellular growth and apoptosis was assessed by proliferation assay and FACS analysis. Results: PRMT5 supports WNT/β-CATENIN activity by direct transcriptional repression of AXIN2 and WIF1 via a PRMT5-EZH2 repressor complex. PRMT5 indirectly supports EZH2 expression via inactivation of the RB-E2F pathway. AXIN2 and WIF1 are two proteins that negatively regulate WNT/bCATENIN. Additionally, PRMT5 inhibition with shRNA or CMP5 leads to repression of the WNT/β-CATENIN signaling pathway by allowing de-repression of AXIN2 and WIF1, leading to decreased nuclear phospho-b-CATENIN and decreased transcription of the target genes CYCLIN D1, c-MYC and SURVIVIN. Reduced nuclear localization of phospho-β-catenin (S675) led to differential enhanced recruitment of co-repressors LSD and HDAC2 (and loss of epigenetic marks H3K4Me3 and H3K9Me3) and loss of activating epigenetic marks H3K9Ac and H3K14Ac on CYCLIN D1, c-MYC and SURVIVIN promoters. We also found that PRMT5 regulates target gene repression in primary blastic variant MCL patient samples and mouse primary lymphoma tumor cells. Significance: Our observations show that PRMT5 is an important epigenetic regulator that governs the expression of its own target genes, the PRC2 program as well as regulating the WNT/β-CATENIN-driven pro-growth and survival genes c-MYC, CYCLIND D1 and SURVIVIN. These results, together with the prevalence of PRMT5 and EZH2 over expression and activation of WNT targets in multiple lymphoma histologic subtypes, suggests that inhibiting PRMT5 is likely to result in removal of repressive histone arginine and lysine marks and promote restoration of normal growth and survival checkpoints in malignant lymphomas. Disclosures No relevant conflicts of interest to declare.

Abstract Correct reprogramming of epigenetic marks in the donor nucleus is a prerequisite for successful cloning by somatic cell transfer (SCT). In several mammalian species, repressive histone (H) lysine (K) trimethylation (me3) marks, in particular H3K9me3, form a major barrier to somatic cell reprogramming into pluripotency and totipotency. We engineered bovine embryonic fibroblasts (BEFs) for the doxycycline-inducible expression of a biologically active, truncated form of murine Kdm4b, a demethylase that removes H3K9me3 and H3K36me3 marks. Upon inducing Kdm4b, H3K9me3 and H3K36me3 levels were reduced about 3-fold and 5-fold, respectively, compared with noninduced controls. Donor cell quiescence has been previously associated with reduced somatic trimethylation levels and increased cloning efficiency in cattle. Simultaneously inducing Kdm4b expression (via doxycycline) and quiescence (via serum starvation) further reduced global H3K9me3 and H3K36me3 levels by a total of 18-fold and 35-fold, respectively, compared with noninduced, nonstarved control fibroblasts. Following SCT, Kdm4b-BEFs reprogrammed significantly better into cloned blastocysts than noninduced donor cells. However, detrimethylated donors and sustained Kdm4b-induction during embryo culture did not increase the rates of postblastocyst development from implantation to survival into adulthood. In summary, overexpressing Kdm4b in donor cells only improved their reprogramming into early preimplantation stages, highlighting the need for alternative experimental approaches to reliably improve somatic cloning efficiency in cattle.

Introduction: Post-translational histone modifications directly modify chromatin structure to influence a wide variety of cellular events including gene expression, DNA replication and repair, and cell cycle control. While histone lysine methylation can confer either transcriptionally active or repressive states, the symmetric dimethylation of arginine residues on histone tails is generally associated with transcriptional repression. Overexpression and dysregulation of PRMT5, the major type II protein arginine methyltransferase, has been shown to drive cellular proliferation and survival of multiple cancer types including mantle cell lymphoma (MCL), a subtype of non-Hodgkin lymphoma associated with poor prognosis. Our work has shown that PRMT5 drives the symmetric dimethylation of arginine (histones H3R8 and H4R3) and attenuates RB/E2F regulatory pathways leading to the expression and activation of the PRC2 lysine methylation programs. We have previously shown that PRMT5 promotes constitutive expression of the WNT/β-CATENIN target genes (MYC, CCND1, and SURVIVIN) via the epigenetic repression of AXIN2 and WIF1 regulatory genes. This PRMT5 mediated epigenetic program allows for constitutive activation of WNT and PI3/AKT downstream pathways relevant to MCL growth and survival. The inhibition of PRMT5 triggers histone deacetylation and H3K4me3 demethylation on promoters of β-CATENIN target genes. H3K4 is modified by several enzymes containing SET domains including the SETD7 and MLL1 proteins. We hypothesized that PRMT5 inhibition, through its ability to repress AKT phosphorylation, would regulate SETD7 and MLL1 activity and regulate downstream pathways relevant to lymphomagenesis. Methods: PRMT5 inhibition of patient-derived MCL cell lines and primary lymphoma tumor cells was achieved with sh-PRMT5 lentivirus (or sh-GFP control) and utilization of a selective small molecule, SAM-competitive PRMT5 inhibitor (PRT382). Gene and protein expression was monitored by reverse transcription (RT) real time PCR and western immunoblotting, respectively. Recruitment of target proteins to promoter regions was examined by ChIP-PCR assays. Interactions within the transcriptional complex were examined by co-immunoprecipitation. Cellular growth and apoptosis was assessed by proliferation assays and FACS analysis. Results: Inhibition of PRMT5 by shRNA-mediated knock down or treatment with PRT382 (Prelude Therapeutics) reduced H3K4me2 in MCL cells via indirect epigenetic repression of SETD7. ChIP-PCR studies showed PRMT5 to be recruited to the SETD7 promoter, suggesting that the activity of PRMT5 promoted the transcription of this lysine methyltransferase. Our findings show that reduced phosphor-AKT by PRMT5 inhibition inhibits dimerization of the MLL1 complex leading to dissociation of MLL1 from the transcriptional activation complex and decreased H3K4me1 and H3K4me3. PRMT5 inhibition led to dissociation of the BCL9- Pygopus-MLL1 transcriptional activating complex and to assembly of repressive LSD1 (the histone demethylase affecting H3K4me3)-HDAC2 (the histone lysine deacetylase) containing complexes. Furthermore, PRMT5 inhibition led to differentially enhanced recruitment of this repressive LSD1/HDAC2 complex and decreased H3K9Ac and H3K14Ac epigenetic marks on promoters of β-CATENIN target genes. PRMT5 inhibition regulates lysine methylation at H3K4 through epigenetic silencing of SETD7 and MLL1 activity, as well as histone H3 acetylation driven by histone acetyltransferase KAT2A. ChIP-Seq studies examining SETD7 and PRC2 recruitment in context of PRMT5 inhibition are currently underway. Conclusions: Our observations show that dysregulated PRMT5 activity acts as a master epigenetic regulator affecting arginine and lysine histone marks. PRMT5 can act directly to repress target tumor suppressor genes while simultaneously indirectly activating SETD7 and MLL1 to drive transcriptional activation of key oncogenes that promote the proliferation and survival of MCL. These results support the notion that inhibiting PRMT5 leads to erasure of repressive histone arginine and lysine marks and promote the restoration of normal growth and survival checkpoints in this disease. Disclosures Scherle: Prelude Therapeutics: Employment. Vaddi:Prelude Therapeutics: Employment. Baiocchi:Prelude: Consultancy.

Abstract To date numerous datasets of gene expression and epigenetic profiles for mouse and human hematopoietic cells have been generated. While individual data sets for a particular cell type have been correlated, no approach exists to harness all expression and epigenetic profiles for the different types of hematopoietic cells. Our goal is to develop a systems biology platform to compare epigenetic profiles of hematopoietic cells towards a better understanding of epigenetic mechanisms governing hematopoiesis. To provide the necessary foundation to support systematic studies of hematopoiesis, we have developed the Systems Biology Repository (SBR, http://sbrblood.nhgri.nih.gov), a data "ranch" for organizing and analyzing transcriptome and epigenome data cells throughout differentiation. To populate SBR, we extracted, curated, annotated, and integrated all human and mouse hematopoietic datasets available through the Encyclopedia of DNA Elements (ENCODE), the Gene Expression Omnibus (GEO) and the Short Read Repository (SRR). These include genome-wide profiles of DNA methylation, histone methylation and acetylation, transcription factor occupancy (ChIPSeq), chromatin accessibility (DNaseISeq, ATACSeq, FAIRESeq), and coding as well as non-coding transcriptional profiles (RNASeq). To demonstrate the utility of SBR, we conducted three different analyses. The first was a vertical study of HistoneSeq (H3K4me1, H3K4me2, H3K4me3, and H3K27ac), DNA methylation and RNASeq profiles during mouse erythroid differentiation. We found a global decrease in DNA methylation from hematopoietic stem and progenitor cells (HSC) through common myeloid progenitors (CMP), erythroid progenitor cells (MEP) and erythroblasts (ERY; 92936 peaks in HSC to 14422 in ERY). The number of expressed genes (using a tags per million cutoff of 10) increased in erythroid progenitors (8901 in HSC to 10778 in CMP and 10670 in MEP) before decreasing in ERY (8654). 62% of histone marks delineating active enhancers (H3K27ac, H3K4me1) are present in both HSC and ERY, while 48% arise de novo during differentiation. In contrast, only 16% of active promoter specific histone marks (H3K4me2, H3K4me3) are present in both HSC and ERY. For a horizontal analysis we compared the DNA methylation, RNASeq, histone modification (H3K4me1, H3K4me2, H3K4me3, and H3K27ac) and transcription factor binding (GATA1 and NFE2) profiles of erythroblasts (ERY) and megakaryocytes (MEG). We found a similar relationship between gene expression and the histone and DNA methylation profiles in each cell type but differences between expression and in transcription factor occupancy. DNA methylation and H3K4me3 was enriched in the gene body of expressed genes (>36%) for both ERY (p ≤ 0.001) and MEG (p ≤ 0.01). In contrast DNA methylation was enriched in the upstream and downstream regions of non-coding RNA genes (p ≤ 0.001). Transcription factor occupancy was cell type specific: 79% of GATA1 sites are in ERY and 72% of NFE2 sites are in MEG. In erythroblasts, DNA methylation and GATA1 binding in the gene body are associated with gene silencing (4 fold difference, p ≤ 0.001), while in megakaryocytes, DNA methylation and NFE2 binding in the gene body are associated with gene activation (8 fold difference, p ≤ 0.001). We used the Mouse Genome Informatics homology map data to perform a cross-species comparison of the expression profiles of mouse and human multipotent progenitors (MPP), proerythroblasts and orthochromatic erythroblasts. We found a total of 5247 genes expressed at significantly different levels (p ≤ 0.001) between human and mouse MPP, while only 2010 genes were expressed at significantly similar levels (p ≤ 0.001). At the proerythroblast and orthochromatic erythroblast stages 7696 genes and 6571 genes were expressed at significantly different levels (p ≤ 0.001) between human and mouse respectively, while 2024 and 2560 genes were expressed at significantly similar levels (p ≤ 0.001). These data are consistent with previous studies showing differences in the transcriptional profiles of mouse and human hematopoietic cells. In summary, SBR provides a foundation to model the genetic and epigenetic landscape in both the mouse and human hematopoietic system, and enables functional correlations to be made between the species. As SBR is expanded to include data from patient cells, it will be possible to model epigenetic changes associated with disease. Disclosures No relevant conflicts of interest to declare.

Abstract Abstract 2488 Lysine specific histone methylation and deacetylation are chromatin modifications that, along with DNA methylation, are involved in the epigenetic silencing of tumor suppressor genes (TSGs). This silencing is mediated by multi-protein complexes PRC (polycomb repressive complexes) 1 and 2. Of the three core protein components of PRC2, i.e., EZH2, SUZ12 and EED, EZH2 has the SET domain with its intrinsic histone methyltransferase activity, which induces the trimethylation (Me3) of lysine (K) 27 on histone (H) 3-a repressive histone modification mediating gene repression. The PRC1 components include BMI1, MEL18, RING1 and RING2, and it serves to further compact the chromatin at PRC2 target genes. The RING1 and RING2 proteins are responsible for the ubiquitylation of K119 on H2A. We have previously reported that treatment with the pan-histone deacetylase inhibitor panobinostat (PS, Novartis Pharmaceutical Corp) depletes PRC2 complex proteins EZH2 and SUZ12 and the DNA methyltransferase (DNMT) 1. We also showed that co-treatment with the S-adenosylhomocysteine hydrolase and EZH2 inhibitor, DZNep, further depleted PRC2 complex proteins and, in combination with PS, induced synergistic apoptosis of cultured and primary AML cells (Blood 2009; 114: 2733–43). In the present studies we determined the effects of DZNep and/or PS on the expression of PRC1 and PRC2 proteins in human Mantle Cell Lymphoma (MCL) cells. Treatment with DZNep dose-dependently depleted EZH2, SUZ12 and BMI1 expression as well as inhibited K27Me3, while inducing K27 acetylation on H3. DZNep treatment also induced p21, p27 and FBXO32, while depleting the levels of cyclin D1 in the cultured MCL JeKo-1 and MO2058 cells. Similar induction of p21, p27 and FBXO32 were also observed, following siRNA knockdown of EZH2 in the cultured MCL cells. Notably, DZNep also induced similar perturbations in primary, patient-derived MCL cells. Treatment with PS alone attenuated EZH2, SUZ12 and DNMT1, as well as depleted the protein expression of BMI1, RING2 and MEL18 in the cultured MCL cells. This was associated with attenuation of H3K27Me3 and augmentation of H3K4Me3 chromatin marks. PS treatment also induced heat shock protein (hsp) 90 acetylation, and depleted the levels of hsp90 client proteins in the MCL cells, including CDK4, c-RAF and AKT. As compared to treatment with each agent alone, co-treatment with DZNep and PS caused greater depletion of EZH2, SUZ12 and BMI1, accompanied with greater induction of p21 and p27 but attenuation of cyclin D1 expression. Co-treatment with DZNep and PS also induced cell cycle growth arrest and synergistically induced apoptosis of JeKo-1 and MO2058, as well as of primary MCL cells derived from 3 patients with MCL (combination indices <1.0). Taken together these findings indicate that by targeted depletion of the PRC2 and PRC1 components and associated chromatin and other protein modifications (hsp90 acetylation), co-treatment with DZNep and PS represents a superior therapy of human MCL cells. These studies also support the in vivo testing of combined epigenetic therapies involving agents that target deregulated epigenetic mechanisms, e.g., histone deacetylases, methyl transferases and demethylases, as well as target DNMTs in the therapy of MCL. Disclosures: Atadja: Novartis: Employment.

Background It is becoming increasingly recognized that evasion from immune control represents one of the main drivers of acute myeloid leukemia (AML) relapse after allogeneic hematopoietic cell transplantation (allo-HCT). In particular, alterations in the antigen processing and presentation machinery represent one of the most effective strategies enacted by tumor cells to avoid recognition from T cells. Whereas it is now well recognized that genomic loss of HLA is frequently at the basis of post-transplantation relapse, it was only recently reported that up to 40% of AML relapses display transcriptional downregulation and complete loss of surface expression of HLA class II molecules without any genetic lesion explaining this phenotype (Christopher et al, N Engl J Med, 2018; Toffalori et al, Nat Med, 2019). This led us to investigate the links between epigenetic changes, immune evasion and post-transplantation relapse. Methods Starting from primary AML samples pairwise collected from patients at diagnosis and relapse with non-genomic loss of HLA class II expression, we generated Patients-Derived Xenografts (PDXs) into NOD-SCID γ-chain null mice. Leukemic cells expanded in the mice and their original human counterparts were analyzed for surface expression of selected immune-related markers (HLA class I and II, PD-L1, B7-H3), and characterized for changes in gene expression (by RNA-Seq), DNA methylation profile (by RRBS), histone modifications associated with active promoters (H3K4me3) or regulatory elements (H3K27ac) (by ChIP-seq) and chromatin accessibility (by ATAC-Seq). The results obtained by all these approaches were integrated by Multi-Omics Factor Analysis (MOFA), followed by Gene Set Enrichment Analysis (GSEA). Finally, the immunological effects of epigenetic drugs and recombinant immune-modulatory cytokines on primary and PDX-derived AML samples were tested in ex-vivo short-term cultures on a layer of mesenchymal stromal cells. Results We verified that PDXs faithfully recapitulate immune-related differences between diagnosis and post-transplantation relapse, including loss of expression of HLA class II molecules (Figure 1A). Integration of all the high-throughput technologies by MOFA evidenced that the differences between diagnosis and post-transplantation relapse samples were mostly explained by changes in chromatin accessibility and histone marks, and largely unrelated to the DNA methylation profile (Figure 1B). We documented that the gene sets that emerged upon integrating epigenetic analyses by MOFA matched our previously published immune-related relapse signature (Toffalori et al, Nat Med, 2019) but also and most intriguingly lists of genes known to be targeted by EZH2, the enzymatic subunit of the PRC2 chromatin repressor complex (Figure 1C). These computational analyses were supported by the evidence of a relapse-specific closed chromatin status of HLA class II genes and their regulators (Figure 1D). To revert these epigenetic changes, we inhibited EZH2 with tazemetostat (EPZ-6438), an epigenetic drug currently being tested in early-phase clinical trials for lymphomas, in two AML relapses with non-genomic loss of HLA class II expression. EZH2 inhibition reduced the levels of the repressor mark H3K27me3 (Figure 1E), increased the surface expression of HLA class II molecules on leukemia cells (Figure 1F) and ultimately improved leukemia recognition by CD4+ T cells (Figure 1G). Notably, these effects were even more pronounced when EZH2 inhibition was combined with IFN-g treatment (Figure 1F,G), suggesting synergism between this epigenetic compound and cytokines released by immune cells upon target recognition. Conclusions Our results provide mechanistic insights into epigenetic regulation of HLA class II downregulation in leukemia and a strong therapeutic rationale to test EZH2 inhibition as an innovative strategy for the treatment of AML post-transplantation relapses. Figure 1 Disclosures Vago: GenDx: Research Funding; Moderna Therapeutics: Research Funding.

H1 linker histone facilitates the formation of higher order chromatin structure and is essential for mammalian development. Mice have 11 H1 variants which are differentially regulated and conserved in human. Previous research indicates that H1 regulates the expression of specific genes in mouse embryonic stem cells (ESCs). However, whether individual variants have distinct functions and how H1 participates in gene regulation remain elusive. An investigation of the precise localization of individual H1 variants in vivo would facilitate the elucidation of mechanisms underlying chromatin compaction regulated gene expression, while it has been extremely difficult due to the lacking of specific antibodies toward H1 variants. In this dissertation, I have generated a knock-in system in ESCs and shown that the N-terminally tagged H1 proteins are functionally interchangeable to their endogenous counterparts in vivo. H1d and H1c are depleted from GC- and gene-rich regions and active promoters, inversely correlated with H3K4me3, but positively correlated with H3K9me3 and associated with characteristic sequence features. Surprisingly, both H1d and H1c are significantly enriched at major satellites, which display increased nucleosome spacing compared with bulk chromatin. While also depleted at active promoters and enriched at major satellites, overexpressed H10 displays differential binding patterns in specific repetitive sequences compared with H1d and H1c. Depletion of H1c, H1d ,and H1e causes pericentric chromocenter clustering and de-repression of major satellites. Collectively, these results integrate the localization of an understudied type of chromatin proteins, namely the H1 variants, into the epigenome map of mouse ESCs, and demonstrate significant changes at pericentric heterochromatin upon depletion of this epigenetic mark.

O câncer de mama representa problema mundial de saúde pública e a causa mais frequente de morte por câncer entre as mulheres. A identificação de agentes moduladores de marcas epigenéticas, tais como metilação global do DNA e modificações pós-tradução em histonas, compreende alternativa promissora para estabelecimento de estratégias de controle da carcinogênese mamária. Dentre os nutrientes, o elemento traço essencial selênio (Se) pode ser destacado como agente dietético com potencial anti-câncer de mama e que poderia atuar modulando processos epigenéticos. Entretanto seus mecanismos de ação são pouco elucidados. Este estudo objetivou, assim, identificar efeitos do tratamento com selênio no crescimento e marcas epigenéticas de células de adenocarcinoma mamário humano MCF-7. Células MCF-7, positivas para o receptor de estrógeno, foram tratadas com ácido metilselenínico (MSA) ou selenito de sódio (ST) por diferentes tempos e em diferentes concentrações. Foram avaliados: padrão de proliferação (ensaio cristal violeta) e viabilidade celular (método de exclusão azul de tripan); integridade de membrana plasmática (citometria de fluxo); níveis de fragmentação do DNA (citometria de fluxo), distribuição das fases do ciclo celular (citometria de fluxo); apoptose (citometria de fluxo/ marcação dupla com Anexina V - Iodeto de propídio); níveis de lisina 9 acetilada (H3K9ac) e trimetilada (H3K9me3) em histona H3; níveis de lisina 16 acetilada (H4K16ac) em histona H4 (Western blot); padrão de metilação global do DNA (HPLC-DAD); expressão de gene supressor de tumor (RASSF1a; qPCR) e padrão de metilação da região promotora (RASSF1a e RAR&#946;; MS-PCR); expressão da enzima DNA metilstransferase 1 (DNMT1) (Western Blotting). Comparado ao grupo controle de células não tratadas (GC), ambos os tratamentos com MSA ou ST inibiram a proliferação e viabilidade de células MCF-7 de forma dose e tempo dependente. Ambas as formas químicas de Se induziram a parada do ciclo celular, aumentando (p< 0,05) a proporção de células na fase G2/M e reduzindo (p< 0,05) a proporção daquelas nas fases G0/G1 e S. Os tratamentos com MSA favoreceram a morte celular por apoptose, que foi associada com nível de fragmentação de DNA aumentado (p< 0,05), e reduzida ruptura da membrana plasmática associada com a exposição aumentada (p< 0,05) de fostadilserina. Por outro lado, o ST aumentou (p< 0,05) a fragmentação do DNA e (p< 0,05) a positividade ao iodeto de propídio associado à indução de necrose (p< 0,05). Dentre os mecanismos epigenéticos investigados, 1,6&#181;M e 2&#181;M reduziram a acetilação de H3K9ac (72h; p< 0,05) e aumentaram a de H4K16ac (96h; p< 0,05). O tratamento por 96h com 2&#181;M de MSA reduziu (p< 0,05) a metilação de H3K9me3. Ambos MSA e ST não alteraram o padrão de metilação global do DNA, mas reduziram a expressão de DNMT1, após 96h com 2&#181;M de MSA (p< 0,001; 88%) e após 120h com 10&#181;M de ST (p< 0,001; 96%). ST, mas não o MSA, aumentou (p< 0,05; 45%) a expressão do gene RASSF1a. Em ambos os grupos tratados com MSA ou ST, bem como no GC, a região promotora dos genes RASSF1a e RAR estavam predominantemente metiladas. Estes resultados fornecem evidências de que as ações anti-câncer de mama de compostos do selênio dependem de sua forma química. Além disso, a modulação de processos epigenéticos parecem ser relevantes para as ações inibitórias do MSA em células de câncer de mama.<br>Breast cancer is a global public health problem and the most frequent cause of cancer death among women. The identification of agents able to modulate epigenetic marks, such as global DNA methylation and histone post-translational modifications, comprises promising alternative for establishing control strategies on mammary carcinogenesis. Among the nutrients, the essential trace element selenium (Se) can be highlighted as a dietary agent with potential anti-breast cancer and could act by modulating epigenetic processes. However its mechanisms of action are poorly understood. This study aimed, therefore, to identify the effects of selenium treatment on growth and epigenetic marks of MCF-7 human breast adenocarcinoma cells. MCF-7 cells, positive for estrogen receptor, were treated with methylseleninic acid (MSA) or sodium selenite (ST) for different times and in different concentrations. Evaluated parameters included: cell proliferation (crystal violet assay) and cell viability (trypan blue exclusion assay); plasma membrane integrity (flow cytometry); levels of DNA fragmentation (flow cytometry), apoptosis (flow cytometry - double labeling with Annexin V - propidium iodide); distribution of cell cycle phases (flow cytometry); acetylated (H3K9ac) and trimethylated (H3K9me3) lysine 9 levels on histone H3; acetylated (H4K16ac) lysine 16 level on histone H4 (Western blot); global DNA methylation (HPLC-DAD); tumor suppressor gene expression (RASSF1a; qPCR) and promoter methylation (RASSF1a, RAR&#946;; MS-PCR); DNA methyltransferase 1 (DNMT1) expression (Western blot). Compared to untreated cells (controls), both MSA and ST inhibited (p< 0.05) MCF-7 cell proliferation and viability in a dose- and time-dependent manner. Treatments with MSA favored cell death by apoptosis, that was associated with increased (p< 0.05) DNA fragmentation level, reduced plasma membrane rupture associated with high (p< 0.05) phosphatidylserine exposure. On the other hand, ST increased (p< 0.05) DNA fragmentation, enhanced (p< 0.05) propidium iodide positivity associated to necrosis induction (p< 0,05). Both chemical forms of Se induced nduced cell cycle arrest, increasing (p< 0.05) the proportion of cells in G2/M phase and reducing (p< 0.05) the proportion of those in G0/G1 and S phases. Among the epigenetic mechanisms investigated, 1.6&#181;M and 2&#181;M of MSA reduced acetylation of H3K9ac (72h, p< 0.05) and increased the H4K16ac (96h, p< 0.05). The treatment for 96h with 2&#181;M of MSA reduced (p< 0.05) the H3K9me3 methylation. Neither MSA nor ST altered (p> 0.05) global DNA methylation, while both compounds reduced (p< 0.05) DNMT1 protein expression, after 96h with 2&#181;M of MSA (p< 0.001; 88%) and after 120h with 10&#181;m of ST (p< 0.001; 94%). ST, but not MSA, increased (p< 0.05; 45%) RASSF1a gene expression. In control and Se-treated cells promoter regions of RASSF1a and RAR&#946; were predominantly methylated. These results provide evidence that the anti-breast cancer actions of selenium compounds depend on its chemical form. Additionally, modulation of epigenetic processes seems to represent a relevant feature of MSA inhibitory effects in breast cancer cells.