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Despite the remarkable clinical responses achieved with immune checkpoint blockade therapy, the response rate is relatively low and only a subset of patients can benefit from the treatment. Aberrant RNA accumulation can mediate IFN signaling and stimulate an immune response, suggesting that targeting RNA decay machinery might sensitize tumor cells to immunotherapy. With this in mind, we identified an RNA exoribonuclease, XRN1, as a potential therapeutic target to suppress RNA decay and stimulate antitumor immunity. Silencing of XRN1 suppressed tumor growth in syngeneic immunocompetent mice and potentiated immunotherapy efficacy, while silencing of XRN1 alone did not affect tumor growth in immunodeficient mice. Mechanistically, XRN1 depletion activated IFN signaling and the viral defense pathway; both pathways play determinant roles in regulating immune evasion. Aberrant RNA-sensing signaling proteins (RIG-I/MAVS) mediated the expression of IFN genes, as depletion of each of them blunted the elevation of antiviral/IFN signaling in XRN1-silenced cells. Analysis of pan-cancer CRISPR-screening data indicated that IFN signaling triggered by XRN1 silencing is a common phenomenon, suggesting that the effect of XRN1 silencing may be extended to multiple types of cancers. Overall, XRN1 depletion triggers aberrant RNA-mediated IFN signaling, highlighting the importance of the aberrant RNA-sensing pathway in regulating immune responses. These findings provide the molecular rationale for developing XRN1 inhibitors and exploring their potential clinical application in combination with cancer immunotherapy.Significance:Targeting XRN1 activates an intracellular innate immune response mediated by RNA-sensing signaling and potentiates cancer immunotherapy efficacy, suggesting inhibition of RNA decay machinery as a novel strategy for cancer treatment.

Despite the remarkable clinical responses achieved with immune checkpoint blockade therapy, the response rate is relatively low and only a subset of patients can benefit from the treatment. Aberrant RNA accumulation can mediate IFN signaling and stimulate an immune response, suggesting that targeting RNA decay machinery might sensitize tumor cells to immunotherapy. With this in mind, we identified an RNA exoribonuclease, XRN1, as a potential therapeutic target to suppress RNA decay and stimulate antitumor immunity. Silencing of XRN1 suppressed tumor growth in syngeneic immunocompetent mice and potentiated immunotherapy efficacy, while silencing of XRN1 alone did not affect tumor growth in immunodeficient mice. Mechanistically, XRN1 depletion activated IFN signaling and the viral defense pathway; both pathways play determinant roles in regulating immune evasion. Aberrant RNA-sensing signaling proteins (RIG-I/MAVS) mediated the expression of IFN genes, as depletion of each of them blunted the elevation of antiviral/IFN signaling in XRN1-silenced cells. Analysis of pan-cancer CRISPR-screening data indicated that IFN signaling triggered by XRN1 silencing is a common phenomenon, suggesting that the effect of XRN1 silencing may be extended to multiple types of cancers. Overall, XRN1 depletion triggers aberrant RNA-mediated IFN signaling, highlighting the importance of the aberrant RNA-sensing pathway in regulating immune responses. These findings provide the molecular rationale for developing XRN1 inhibitors and exploring their potential clinical application in combination with cancer immunotherapy.Significance:Targeting XRN1 activates an intracellular innate immune response mediated by RNA-sensing signaling and potentiates cancer immunotherapy efficacy, suggesting inhibition of RNA decay machinery as a novel strategy for cancer treatment.

ZFP36L1 is a tandem zinc-finger RNA-binding protein that recognizes conserved adenylate-uridylate–rich elements (ARE) located in 3′untranslated regions (UTR) to mediate mRNA decay. We hypothesized that ZFP36L1 is a negative regulator of a posttranscriptional hub involved in mRNA half-life regulation of cancer-related transcripts. Analysis of in silico data revealed that ZFP36L1 was significantly mutated, epigenetically silenced, and downregulated in a variety of cancers. Forced expression of ZFP36L1 in cancer cells markedly reduced cell proliferation in vitro and in vivo, whereas silencing of ZFP36L1 enhanced tumor cell growth. To identify direct downstream targets of ZFP36L1, systematic screening using RNA pull-down of wild-type and mutant ZFP36L1 as well as whole transcriptome sequencing of bladder cancer cells {plus minus} tet-on ZFP36L1 was performed. A network of 1,410 genes was identified as potential direct targets of ZFP36L1. These targets included a number of key oncogenic transcripts such as HIF1A, CCND1, and E2F1. ZFP36L1 specifically bound to the 3′UTRs of these targets for mRNA degradation, thus suppressing their expression. Dual luciferase reporter assays and RNA electrophoretic mobility shift assays showed that wild-type, but not zinc-finger mutant ZFP36L1, bound to HIF1A 3′UTR and mediated HIF1A mRNA degradation, leading to reduced expression of HIF1A and its downstream targets. Collectively, our findings reveal an indispensable role of ZFP36L1 as a posttranscriptional safeguard against aberrant hypoxic signaling and abnormal cell-cycle progression.Significance:RNA-binding protein ZFP36L1 functions as a tumor suppressor by regulating the mRNA stability of a number of mRNAs involved in hypoxia and cell-cycle signaling.

This file contains supplementary methods, Figs. S1 to S6 and Tables S1 to S5.

ZFP36L1 is significantly downregulated and methylated in breast and bladder cancer, and inhibits cell cycle progression and hypoxia/HIF1A signaling.

Liposarcoma, the most common soft tissue sarcoma, is a group of fat cell mesenchymal tumors with different histological subtypes. The dysregulation of long non-coding RNAs (lncRNAs) has been observed in human cancers including a few studies in sarcoma. However, the global transcriptome analysis and potential role of lncRNAs remain unexplored in liposarcoma. The present investigation uncovers the transcriptomic profile of liposarcoma by RNA sequencing to gain insight into the global transcriptional changes in liposarcoma. Our RNA sequencing analysis has identified that many oncogenic lncRNAs are differentially expressed in different subtypes of liposarcoma including MALAT1, PVT1, SNHG15, LINC00152, and MIR210HG. Importantly, we identified a highly overexpressed, unannotated, and novel lncRNA in dedifferentiated liposarcomas. We have named it TODL, transcript overexpressed in dedifferentiated liposarcoma. TODL lncRNA displayed significantly higher expression in dedifferentiated liposarcoma cell lines and patient samples. Interestingly, functional studies revealed that TODL lncRNA has an oncogenic function in liposarcoma cells by regulating proliferation, cell cycle, apoptosis, differentiation, and tumorigenesis in the murine model. Silencing of TODL lncRNA highlighted the enrichment of several key oncogenic signaling pathways including cell cycle, transcriptional misregulation, FOXM1 network, p53 signaling, PLK1 signaling, FoxO, and signaling Aurora signaling pathways. RNA pull-down assay revealed the binding of TODL lncRNA with FOXM1, an oncogenic transcription factor, and the key regulator of the cell cycle. Silencing of TODL lncRNA also induces adipogenesis in dedifferentiated liposarcomas. Altogether, our finding indicates that TODL could be utilized as a novel, specific diagnostic biomarker, and a pharmacological target for therapeutic development in controlling aggressive and metastatic dedifferentiated liposarcomas.

Proteasome inhibitors, such as bortezomib and carfilzomib, have shown efficacy in anti-cancer therapy in hematological diseases but not in solid cancers. Here, we found that liposarcomas (LPS) are susceptible to proteasome inhibition, and identified drugs that synergize with carfilzomib, such as selinexor, an inhibitor of XPO1-mediated nuclear export. Through quantitative nuclear protein profiling and phospho-kinase arrays, we identified potential mode of actions of this combination, including interference with ribosome biogenesis and inhibition of pro-survival kinase PRAS40. Furthermore, by assessing global protein levels changes, FADS2, a key enzyme regulating fatty acids synthesis, was found down-regulated after proteasome inhibition. Interestingly, SC26196, an inhibitor of FADS2, synergized with carfilzomib. Finally, to identify further combinational options, we performed high-throughput drug screening and uncovered novel drug interactions with carfilzomib. For instance, cyclosporin A, a known immunosuppressive agent, enhanced carfilzomib’s efficacy in vitro and in vivo. Altogether, these results demonstrate that carfilzomib and its combinations could be repurposed for LPS clinical management. Electronic supplementary material The online version of this article (10.1007/s00018-020-03620-w) contains supplementary material, which is available to authorized users.

Precision medicine aims to move from traditional reactive medicine to a system where risk groups can be identified before the disease occurs. However, phenotypic heterogeneity amongst the diseased and healthy poses a major challenge for identification markers for risk stratification and early actionable interventions. In Ayurveda, individuals are phenotypically stratified into seven constitution types based on multisystem phenotypes termed “Prakriti”. It enables the prediction of health and disease trajectories and the selection of health interventions. We hypothesize that exome sequencing in healthy individuals of phenotypically homogeneous Prakriti types might enable the identification of functional variations associated with the constitution types. Exomes of 144 healthy Prakriti stratified individuals and controls from two genetically homogeneous cohorts (north and western India) revealed differential risk for diseases/traits like metabolic disorders, liver diseases, and body and hematological measurements amongst healthy individuals. These SNPs differ significantly from the Indo-European background control as well. Amongst these we highlight novel SNPs rs304447 (IFIT5) and rs941590 (SERPINA10) that could explain differential trajectories for immune response, bleeding or thrombosis. Our method demonstrates the requirement of a relatively smaller sample size for a well powered study. This study highlights the potential of integrating a unique phenotyping approach for the identification of predictive markers and the at-risk population amongst the healthy.

Recurrent loss-of-function mutations of spliceosome gene, ZRSR2, occur in myelodysplastic syndromes (MDS). Mutation/loss of ZRSR2 in human myeloid cells primarily causes impaired splicing of the U12-type introns. In order to further investigate the role of this splice factor in RNA splicing and hematopoietic development, we generated mice lacking ZRSR2. Unexpectedly, Zrsr2-deficient mice developed normal hematopoiesis with no abnormalities in myeloid differentiation evident in either young or ≥1-year old knockout mice. Repopulation ability of Zrsr2-deficient hematopoietic stem cells was also unaffected in both competitive and non-competitive reconstitution assays. Myeloid progenitors lacking ZRSR2 exhibited mis-splicing of U12-type introns, however, this phenotype was moderate compared to the ZRSR2-deficient human cells. Our investigations revealed that a closely related homolog, Zrsr1, expressed in the murine hematopoietic cells, but not in human cells contributes to splicing of U12-type introns. Depletion of Zrsr1 in Zrsr2 KO myeloid cells exacerbated retention of the U12-type introns, thus highlighting a collective role of ZRSR1 and ZRSR2 in murine U12-spliceosome. We also demonstrate that aberrant retention of U12-type introns of MAPK9 and MAPK14 leads to their reduced protein expression. Overall, our findings highlight that both ZRSR1 and ZRSR2 are functional components of the murine U12-spliceosome, and depletion of both proteins is required to accurately model ZRSR2-mutant MDS in mice. Ministry of Education (MOE) Ministry of Health (MOH) National Medical Research Council (NMRC) National Research Foundation (NRF) Published version This work was funded by the Leukemia and Lymphoma Society, the Singapore Ministry of Health’s National Medical Research Council (NMRC) under its Singapore Translational Research (STaR) Investigator Award to HPK (NMRC/STaR/0021/2014), the NMRC Center Grant awarded to the National University Cancer Institute of Singapore (NMRC/CG/012/2013) and the National Research Foundation Singapore and the Singapore Ministry of Education under its Research Centers of Excellence initiatives. This research is also supported by the RNA Biology Center at the Cancer Science Institute of Singapore, NUS, as part of funding under the Singapore Ministry of Education’s Tier 3 grants, grant number MOE2014-T3-1-006. We thank the Melamed Family for their generous support.

N6-methyladenosine (m6A) is the most predominant internal mRNA modification in eukaryotes, recognised by its reader proteins (so-called m6A-readers) for regulating subsequent mRNA fates — splicing, export, localisation, decay, stability, and translation — to control several biological processes. Although a few m6A-readers have been identified, yet the list is incomplete. Here, we identify a new m6A-reader protein, Moloney leukaemia virus 10 homologue (MOV10), in the m6A pathway. MOV10 recognises m6A-containing mRNAs with a conserved GGm6ACU motif. Mechanistic studies uncover that MOV10 facilitates mRNA decay of its bound m6A-containing mRNAs in an m6A-dependent manner within the cytoplasmic processing bodies (P-bodies). Furthermore, MOV10 decays the Gsk-3ß mRNA through m6A that stabilises the ß-CATENIN expression of a WNT/ß-CATENIN signalling pathway to regulate downstream NANOG expression for maintaining the mouse embryonic stem cells (mESCs) state. Thus, our findings reveal how a newly identified m6A-reader, MOV10 mediates mRNA decay via m6A that impact embryonic stem cell biology.

ZFP36L1 is a tandem zinc-finger RNA-binding protein that recognizes conserved adenylate-uridylate–rich elements (ARE) located in 3′untranslated regions (UTR) to mediate mRNA decay. We hypothesized that ZFP36L1 is a negative regulator of a posttranscriptional hub involved in mRNA half-life regulation of cancer-related transcripts. Analysis of in silico data revealed that ZFP36L1 was significantly mutated, epigenetically silenced, and downregulated in a variety of cancers. Forced expression of ZFP36L1 in cancer cells markedly reduced cell proliferation in vitro and in vivo, whereas silencing of ZFP36L1 enhanced tumor cell growth. To identify direct downstream targets of ZFP36L1, systematic screening using RNA pull-down of wild-type and mutant ZFP36L1 as well as whole transcriptome sequencing of bladder cancer cells {plus minus} tet-on ZFP36L1 was performed. A network of 1,410 genes was identified as potential direct targets of ZFP36L1. These targets included a number of key oncogenic transcripts such as HIF1A, CCND1, and E2F1. ZFP36L1 specifically bound to the 3′UTRs of these targets for mRNA degradation, thus suppressing their expression. Dual luciferase reporter assays and RNA electrophoretic mobility shift assays showed that wild-type, but not zinc-finger mutant ZFP36L1, bound to HIF1A 3′UTR and mediated HIF1A mRNA degradation, leading to reduced expression of HIF1A and its downstream targets. Collectively, our findings reveal an indispensable role of ZFP36L1 as a posttranscriptional safeguard against aberrant hypoxic signaling and abnormal cell-cycle progression. Significance: RNA-binding protein ZFP36L1 functions as a tumor suppressor by regulating the mRNA stability of a number of mRNAs involved in hypoxia and cell-cycle signaling.

Precise regulation of chromatin architecture is vital to physiological processes including hematopoiesis. ARID1A is a core component of the mammalian SWI/SNF complex, which is one of the ATP-dependent chromatin remodeling complexes. To uncover the role of ARID1A in hematopoietic development, we utilized hematopoietic cell-specific deletion of Arid1a in mice. We demonstrate that ARID1A is essential for maintaining the frequency and function of hematopoietic stem cells and its loss impairs the differentiation of both myeloid and lymphoid lineages. ARID1A deficiency led to a global reduction in open chromatin and ensuing transcriptional changes affected key genes involved in hematopoietic development. We also observed that silencing of ARID1A affected ATRA-induced differentiation of NB4 cells, suggesting its role in granulocytic differentiation of human leukemic cells. Overall, our study provides a comprehensive elucidation of the function of ARID1A in hematopoiesis and highlights the central role of ARID1A-containing SWI/SNF complex in maintaining chromatin dynamics in hematopoietic cells.

Profiling of active gene-regulatory DNA elements in brain cancer tissues illuminates disease biology and therapeutic targets., Molecular profiling of the most aggressive brain tumor glioblastoma (GBM) on the basis of gene expression, DNA methylation, and genomic variations advances both cancer research and clinical diagnosis. The enhancer architectures and regulatory circuitries governing tumor-intrinsic transcriptional diversity and subtype identity are still elusive. Here, by mapping H3K27ac deposition, we analyze the active regulatory landscapes across 95 GBM biopsies, 12 normal brain tissues, and 38 cell line counterparts. Analyses of differentially regulated enhancers and super-enhancers uncovered previously unrecognized layers of intertumor heterogeneity. Integrative analysis of variant enhancer loci and transcriptome identified topographies of transcriptional enhancers and core regulatory circuitries in four molecular subtypes of primary tumors: AC1-mesenchymal, AC1-classical, AC2-proneural, and AC3-proneural. Moreover, this study reveals core oncogenic dependency on super-enhancer–driven transcriptional factors, long noncoding RNAs, and druggable targets in GBM. Through profiling of transcriptional enhancers, we provide clinically relevant insights into molecular classification, pathogenesis, and therapeutic intervention of GBM.

Personalized medicine relies on successful identification of genome-wide variations that governs inter-individual differences in phenotypes and system level outcomes. In Ayurveda, assessment of composite constitution types “Prakriti” forms the basis for risk stratification, predicting health and disease trajectories and personalized recommendations. Here, we report a novel method for identifying pleiotropic genes and variants that associate with healthy individuals of three extreme and contrasting “Prakriti” constitutions through exome sequencing and state-of-the-art computational methods. Exome Seq of three extreme Prakriti types from 108 healthy individuals 54 each from genetically homogeneous populations of North India (NI, Discovery cohort) and Western India (VADU, Replication cohort) were evaluated. Fisher’s Exact Test was applied between Prakriti types in both cohorts and further permutation based p-value was used for selection of exonic variants. To investigate the effect of sample size per genetic association test, we performed power analysis. Functional impact of differentiating genes and variations were inferred using diverse resources -Toppfun, GTEx, GWAS, PheWAS, UK Biobank and mouse knockdown/knockout phenotype (MGI). We also applied supervised machine learning approach to evaluate the association of exonic variants with multisystem phenotypes of Prakriti. Our targeted investigation into exome sequencing from NI (discovery) and VADU (validation) cohorts datasets provide ~7,000 differentiating SNPs. Closer inspection further identified a subset of SNPs (2407 (NI) and 2393 (VADU)), that mapped to an overlapping set of 1181 genes. This set can robustly stratify the Prakriti groups into three distinct clusters with distinct gene ontological (GO) enrichments. Functional analysis further strengthens the potential pleiotropic effects of these differentiating genes/variants and multisystem phenotypic consequences. Replicated SNPs map to some very prominent genes like FIG4, EDNRA, ANKLE1, BCKDHA, ATP5SL, EXOCS5, IFIT5, ZNF502, PNPLA3 and IL6R. Lastly, multivariate analysis using random forest uncovered rs7244213 within urea transporter SLC14A2, that associate with an ensemble of features linked to distinct constitutions. Our results reinforce the concept of integration of Prakriti based deep phenotypes for risk stratification of healthy individuals and provides markers for early actionable interventions.

Multiple myeloma (MM) is a hematological disease marked by abnormal growth of B cells in bone marrow. Inherent chromosomal instability and DNA damage are major hallmarks of MM, which implicates an aberrant DNA repair mechanism. Studies have implicated a role for CDK12 in the control of expression of DNA damage response genes. In this study, we examined the effect of a small molecule inhibitor of CDK12–THZ531 on MM cells. Treatment of MM cells with THZ531 led to heightened cell death accompanied by an extensive effect on gene expression changes. In particular, we observed downregulation of genes involved in DNA repair pathways. With this insight, we extended our study to identify synthetic lethal mechanisms that could be exploited for the treatment of MM cells. Combination of THZ531 with either DNA-PK inhibitor (KU-0060648) or PARP inhibitor (Olaparib) led to synergistic cell death. In addition, combination treatment of THZ531 with Olaparib significantly reduced tumor burden in animal models. Our findings suggest that using a CDK12 inhibitor in combination with other DNA repair inhibitors may establish an effective therapeutic regimen to benefit myeloma patients.

It is estimated from twin studies that heritable factors account for at-least half of asthma-risk, of which genetic variants identified through population studies explain only a small fraction. Multi-generation large families with high asthma prevalence can serve as a model to identify highly penetrant genetic variants in closely related individuals that are missed by population studies. To achieve this, a four-generation Indian family with asthma was identified and recruited for examination and genetic testing. Twenty subjects representing all generations were selected for whole genome genotyping, of which eight were subjected to exome sequencing. Non-synonymous and deleterious variants, segregating with the affected individuals, were identified by exome sequencing. A prioritized deleterious missense common variant in the olfactory receptor gene OR2AG2 that segregated with a risk haplotype in asthma, was validated in an asthma cohort of different ethnicity. Phenotypic tests were conducted to verify expected deficits in terms of reduced ability to sense odors. Pathway-level relevance to asthma biology was tested in model systems and unrelated human lung samples. Our study suggests that OR2AG2 and other olfactory receptors may contribute to asthma pathophysiology. Genetic studies on large families of interest can lead to efficient discovery.

SUMMARYTranscriptional network is controlled by master transcription factors (TFs) andcis-regulatory elements through interacting with target sequences and recruiting epigenetic regulators. By integration of enhancer profiling and chromatin accessibility, we establish super-enhancer (SE) mediated core regulatory circuitry (CRC) for esophageal squamous cell carcinoma (ESCC) and identify tumor cells-dependent CRC TFs-TP63, SOX2 and KLF5. They preferentially co-occupy SE loci and form a positive interconnected auto-regulatory loop through SEs to orchestrate chromatin and transcriptional programming. SE-associated oncogene-ALDH3A1is identified as a novel CRC target contributing to ESCC viability. Using circular chromosome conformation capture sequencing (4C-seq) and CRISPR/Cas9 genome editing, the direct interaction betweenTP63promoter and functional enhancers which is mediated by CRC TFs is identified. Deletion of each enhancer decreases expression of CRC TFs and impairs cell viability, phenocopying the knockdown of each CRC TF. Targeting epigenetic regulation by inhibition of either the BET bromodomain or HDAC disrupts the CRC program and its dependent global epigenetic modification, consequently suppressing ESCC tumor growth. Importantly, combination of both compounds result in synergistic anti-tumor effect.Graphical AbstractHIGHLIGHTSSuper-enhancers mediated transcriptional regulatory circuitry is established for ESCCTP63, SOX2 and KLF5 as CRC TFs co-localize super-enhancer loci to orchestrate chromatin accessibility and transcriptional dysregulationComplex interaction between functional enhancers andTP63promoter is mediated by CRC TFsALDH3A1is a key downstream target of ESCC CRC and is essential for ESCC cell survivalBET degrader-ARV-771 and HDAC inhibitor-Romidepsin synergistically inhibit ESCC tumor growth

Lipomas are benign fatty tumors with a high prevalence rate, mostly found in adults but have a good prognosis. Until now, reason for lipoma occurrence not been identified. We performed whole exome sequencing to define the mutational spectrum in ten lipoma patients along with their matching control samples. We presented genomic insight into the development of lipomas, the most common benign tumor of soft tissue. Our analysis identified 412 somatic variants including missense mutations, splice site variants, frameshift indels, and stop gain/lost. Copy number variation analysis highlighted minor aberrations in patients. Kinase genes and transcriptions factors were among the validated mutated genes critical for cell proliferation and survival. Pathway analysis revealed enrichment of calcium, Wnt and phospholipase D signaling in patients. In conclusion, whole exome sequencing in lipomas identified mutations in genes with a possible role in development and progression of lipomas.

Background & Aims We investigated the transcriptome of esophageal squamous cell carcinoma (ESCC) cells, activity of gene regulatory (enhancer and promoter regions), and the effects of blocking epigenetic regulatory proteins. Methods We performed chromatin immunoprecipitation sequencing with antibodies against H3K4me1, H3K4me3, and H3K27ac and an assay for transposase-accessible chromatin to map the enhancer regions and accessible chromatin in 8 ESCC cell lines. We used the CRC_Mapper algorithm to identify core regulatory circuitry transcription factors in ESCC cell lines, and determined genome occupancy profiles for 3 of these factors. In ESCC cell lines, expression of transcription factors was knocked down with small hairpin RNAs, promoter and enhancer regions were disrupted by CRISPR/Cas9 genome editing, or bromodomains and extraterminal (BET) family proteins and histone deacetylases (HDACs) were inhibited with ARV-771 and romidepsin, respectively. ESCC cell lines were then analyzed by whole-transcriptome sequencing, immunoprecipitation, immunoblots, immunohistochemistry, and viability assays. Interactions between distal enhancers and promoters were identified and verified with circular chromosome conformation capture sequencing. NOD-SCID mice were given injections of modified ESCC cells, some mice where given injections of HDAC or BET inhibitors, and growth of xenograft tumors was measured. Results We identified super-enhancer-regulated circuits and transcription factors TP63, SOX2, and KLF5 as core regulatory factors in ESCC cells. Super-enhancer regulation of ALDH3A1 mediated by core regulatory factors was required for ESCC viability. We observed direct interactions between the promoter region of TP63 and functional enhancers, mediated by the core regulatory circuitry transcription factors. Deletion of enhancer regions from ESCC cells decreased expression of the core regulatory circuitry transcription factors and reduced cell viability; these same results were observed with knockdown of each core regulatory circuitry transcription factor. Incubation of ESCC cells with BET and HDAC disrupted the core regulatory circuitry program and the epigenetic modifications observed in these cells; mice given injections of HDAC or BET inhibitors developed smaller xenograft tumors from the ESCC cell lines. Xenograft tumors grew more slowly in mice given the combination of ARV-771 and romidepsin than mice given either agent alone. Conclusions In epigenetic and transcriptional analyses of ESCC cell lines, we found the transcription factors TP63, SOX2, and KLF5 to be part of a core regulatory network that determines chromatin accessibility, epigenetic modifications, and gene expression patterns in these cells. A combination of epigenetic inhibitors slowed growth of xenograft tumors derived from ESCC cells in mice.

Lipomas are benign fatty tumors with a high prevalence rate, mostly found in adults but have a good prognosis. Until now, reason for lipoma occurrence not been identified. We performed whole exome sequencing to define the mutational spectrum in ten lipoma patients along with their matching control samples. We identified 412 somatic variants including missense mutations, splice site variants, frameshift indels, and stop gain/lost. Kinase genes and transcriptions factors were among the validated mutated genes critical for cell proliferation and survival. Pathway analysis revealed enrichment of calcium, Wnt and phospholipase D signaling in patients. Whole exome sequencing in lipomas identified mutations in genes with a possible role in development and progression of lipomas.Author SummaryWe presented genomic insight into the development of lipomas, the most common benign tumor of soft tissue. Until date, no one knows the cause of lipoma development and its progression. Our group for the first time profiled ten lipoma patients’ samples and their matching normal controls to delineate the somatic mutation pattern using whole exome sequencing. Interestingly, exome-profiling results highlighted the potential role of important kinase genes and transcription factors in lipoma development. In addition, calcium, Wnt and phospholipase D signaling pathway speculated to be involve in pathogenesis of this disease.

Background: Glioblastoma (GBM) is the most aggressive and therapy-refractory brain tumor in adults. Molecular profiling of GBM on the basis of gene expression, DNA methylation and genomic variations has advanced both cancer research and clinical diagnosis. However, the enhancer architectures and regulatory circuitries governing tumor-intrinsic transcriptional diversity and subtype identity are still elusive. Methods: Chromatin immunoprecipitation followed by sequencing analysis was applied to examine H3K27ac deposition and to map the active regulatory landscapes across 95 GBM biopsies and 12 normal brain tissues. RNA-sequencing analysis was performed to measure transcriptome and to classify transcriptional subtypes. Super-enhancer associated genes and master transcriptional factors were identified. Function of novel cancer associated genes was explored using both patient-derived GBM propagating cells and orthotopic xenograft models. Results: Analyses of differentially regulated enhancers and super-enhancers uncovered previously unrecognized layers of inter-tumor heterogeneity. Integrative analysis of variant enhancer loci and transcriptome identified topographies of transcriptional enhancers and core regulatory circuitries in four molecular subtypes of primary tumors: AC1-mesenchymal, AC1-classical, AC2-proneural and AC3-proneural. Subtype-specific enhancer domains contributed to transcriptional diversity and shaped subtype identity. Moreover, this study reveals novel oncogenic dependency of GBM on various super-enhancer-driven transcriptional factors and druggable targets (e.g., BRD4). Conclusion: Through profiling of transcriptional enhancers, we provide clinically relevant insights into the molecular classification, pathogenesis and therapeutic intervention of GBM. Citation Format: Liang Xu, Ye Chen, Yulun Huang, Edwin Sandanaraj, John S. Yu, Ruby Yu-Tong Lin, Pushkar Dakle, Xin-Yu Ke, Yuk Kien Chong, Lynnette Koh, Anand Mayakonda, Kassoum Nacro, Jeffrey Hill, Mo-Li Huang, Sigal Gery, See Wee Lim, Zhengyun Huang, Ying Xu, Jianxiang Chen, Longchuan Bai, Shaomeng Wang, Hiroaki Wakimoto, Tseng Tsai Yeo, Beng Ti Ang, Markus Müschen, Carol Tang, Tuan Zea Tan, Phillip H. Koeffler. Chromatin profiling of glioblastoma tissues identifies core oncogenic dependency and therapeutic opportunities [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 2123.

Background: As a high-grade neoplasm of connective tissues, de-differentiated liposarcoma (DDLPS) is featured by genomic amplification of MDM2, CDK4 and JUN. Super-enhancer-driven master transcription factors (RUNX1, FOSL2 and MYC) establish a core feed-forward transcriptional circuit underpinning the DDLPS malignancy. However, an important question remains unanswered: how do genomic insults interact with epigenetic dysregulation in this disease? Methods: We performed ChIP-seq analysis to map loading of transcription factors and histone marks on chromatin in DDLPS cells. Functional assays following genetic manipulation of target genes were conducted to explore their disease relevance. Results: Genomic activation of JUN represents a promising prognostic marker for DDLPS. Meanwhile, JUN locus engaged excessive H3K27ac signals in both DDLPS tissues and cells, while a markedly lower level of H3K27ac deposition was observed in normal adipose tissues, adipocytes and MSCs. JUN formed a complex with FOSL2 and other co-factors in active chromatin, especially super-enhancers. Silencing of either JUN or its co-factors disrupted the core regulatory circuitry, which was accompanied by strong deleterious impacts on both cell viability and tumorigenicity. As BET bromodomain proteins dictate transcriptionally active chromatin, targeted degradation of BET proteins by PROTAC compounds exerted potent inhibitory effects on both JUN cistrome and DDLPS development. Conclusion: Our study reveals that amplification and overexpression of JUN contribute to DDLPS development. DDLPS-associated genomic abnormalities collaborate with BET proteins to fuel disease-driving core regulatory circuitry. Citation Format: Ye Chen, Pushkar Dakle, Tuan Zea Tan, Ruby Yu-Tong Lin, Mark E. Puhaindran, Sigal Gery, Liang Xu, Phillip H. Koeffler. Genetic activation of JUN fuels the core regulatory circuitry of de-differentiated liposarcoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 2438.

Acute promyelocytic leukemia (APL) is a unique subtype of acute myeloid leukemia (AML). The disease is identified by distinctive morphology and is distinguished by a balanced reciprocal translocation between chromosomes 15 and 17. This aberration leads to the fusion between promyelocytic leukemia (PML) gene located on chromosome 15q21, and retinoic acid receptor α (RARA) gene from chromosome 17q21, leading to the resultant chimeric onco-fusion protein PML-RARA, which is detectable in more than 95% patients and disturbs proper promyelocytic differentiation. All-trans retinoic acid (ATRA) can induce granulocytic differentiation in APL and is used to treat APL patients. Genes containing PML-RARA-targeted promoters are transcriptionally suppressed in APL and most likely constitute a major mechanism of transcriptional repression occurring in APL. A growing body of evidence points to the role of distal regulatory elements, including enhancers, in the control of gene expression. In order to understand the unique sets of enhancers that might be under the control of PML-RAR and crucial for granulocytic differentiation of NB4 cells, we analysed the enhancer landscape of control and ATRA treated NB4 cells. H3K9Ac mapping identified a repertoire of enhancers that were gained in NB4 cells treated with ATRA. Closer investigation of these enhancer elements revealed enrichment of H3K9Ac signals around major drivers of myeloid differentiation. Of note, we identified a gain in enhancer signature for a region about 7kb downstream of the CEBPE gene. Our previous studies identified a novel enhancer for CEBPE in murine hematopoietic cells, which was 6 downstream of CEBPE core promoter. It appears that the +7kb region we identified in human APL cells may be analogous to the murine enhancer. We also observed that PML-RAR binds this +7kb region and ATRA treatment of NB4 cells displaced binding of PML-RAR from the + 7kb region, suggestive of a transcriptional repressive effect of PML-RAR at such enhancer elements. To test the transcription regulating potential of this +7kb region, we used catalytically inactive Cas9 fused to Krüppel associated box (KRAB) domain (dCas9-KRAB). We designed three guide RNAs covering this regulatory region. The sgRNAs effectively repressed expression of CEBPE accompanied by lowered granulocytic differentiation of these guide RNA targeted NB4 cells after ATRA treatment. To explore transcription factor (TF) occupancy at this +7 kb region, we analysed public available ChIP-seq datasets for hematopoiesis-specific factors. Analysis revealed that the +7kb region was marked by an open chromatin signature, accompanied by binding of a majority of hematopoietic TFs around this putative regulatory element with concurrent binding of EP300. Strikingly we noticed binding of CEBPA, CEBPB and CEBPE at this regulatory element. To assess whether binding of these members of the CEBP family of TFs is functionally relevant, luciferase reporter and electrophoretic mobility shift assays (EMSA) were performed. Co expression of the CEBP TFs led to significant induction of luciferase expression, and this data was further confirmed using EMSA assays. Based on these observations, we propose that PML-RAR blocks granulocytic differentiation by occupying this +7kb enhancer of CEBPE, hinders binding of other cell type/lineage specific TFs, and blocks CEBPE expression. When cells are stimulated with ATRA, PML-RAR is displaced from the CEBPE enhancer, allowing for efficient binding of myeloid-specific TFs. This results in increased CEBPE expression, which in turn promotes efficient granulocytic differentiation. The findings from our study expands our current understanding of the mechanism of differentiation therapy, the role of onco-fusion proteins in inhibiting myeloid differentiation, and may provide new therapeutic approaches to many acute myeloid leukemias. Disclosures Ong: National University of Singapore: Other: Royalties.

Mutations in genes encoding components of the RNA splicing machinery constitute the leading class of genetic alterations in myelodysplastic syndromes (MDS). Somatic inactivating alterations of splicing factor, ZRSR2, are observed in ~10% of MDS patients. Mutations/loss of ZRSR2 in human myeloid cells causes aberrant splicing, primarily impairing splicing of the U12-type introns. However, the precise role of ZRSR2 in splicing and hematopoietic development needs further exploration. To understand the function of ZRSR2 in hematopoietic development and splicing, we generated Zrsr2 knockout mice. RNA-Seq of sorted myeloid precursor populations (common myeloid progenitors, granulocyte monocyte progenitors and megakaryocyte erythrocyte progenitors) revealed aberrant retention of U12-type introns in ZRSR2-deficient murine cells, similar to our previous observation in ZRSR2 mutant MDS bone marrow cells (Madan et al, Nat Commun 2015). However, despite complete loss of ZRSR2 in our mouse model, the effect on splicing of U12-type introns was modest compared to the ZRSR2 mutant MDS bone marrow and ZRSR2 knockdown human AML cells (K562 and TF1). Moreover, our comprehensive analyses of hematopoietic compartment in ZRSR2-deficient (Zrsr2Δ/Y) compared to WT (Zrsr2+/Y) male mice, indicated that ZRSR2 was dispensable for hematopoietic differentiation. We did not observe any significant difference in peripheral blood counts, bone marrow cellularity, proportion of hematopoietic stem cells (HSCs) and myeloid precursors (CMP, GMP, MEP) in the bone marrow of both young (7-10 weeks) and old (>1 year) mice of either genotypes. Competitive and non-competitive reconstitution assays also demonstrated that loss of ZRSR2 does not affect repopulation potential of HSCs. Collectively, our data demonstrate that ZRSR2 is not essential for hematopoietic development in mice. Further, to understand the underlying determinant for our unexpected observation that deletion of Zrsr2 in mice did not impact hematopoietic development and affected modestly splicing of U12-type introns, we investigated the role of a closely-related homolog, ZRSR1, in splicing of U12-type introns. We utilized shRNA-mediated silencing of ZRSR1 in murine myeloid precursors (Lin−Kit+ bone marrow cells), and assessed splicing using RNA-Sequencing. Wildtype myeloid cells expressing Zrsr1 shRNA did not display significant mis-splicing of the U12-type introns. However, knockdown of Zrsr1 in ZRSR2-deficient myeloid cells exacerbated mis-splicing of the U12-introns. This suggested that ZRSR1 contributes to regulation of U12-spliceosome in murine hematopoietic cells. Increased mis-splicing of U12-type introns in ZRSR1/ZRSR2-deficient cells was validated using quantitative RT-PCR. Further to verify the compensatory role of murine ZRSR1 in splicing of U12-type introns, we used CRISPR/Cas9 technology to generate 32D cells lacking either one or both ZRSR proteins. Quantitative RT-PCR analysis showed notable aberrant retention of U12-type introns in Zrsr1/Zrsr2 double-deficient 32D cells compared to either WT or single KO cells. We also demonstrated that aberrant retention of U12-type introns of MAPK9 and MAPK14 caused by deficiency of ZRSR proteins led to their reduced protein expression in both human and murine myeloid cells. Taken together, our study highlights functional role of murine ZRSR1 in splicing of U12-type introns in murine hematopoietic cells where it can partially compensate for the deficiency of ZRSR2. Therefore, deficiency of ZRSR2 alone is insufficient to impact extensively RNA splicing in mice, and further studies with concurrent deficiency of ZRSR1 and ZRSR2 are warranted to replicate complete loss of ZRSR activity. Disclosures Chng: Novartis: Honoraria; Abbvie: Honoraria; Amgen: Honoraria, Research Funding; Celgene: Honoraria, Research Funding; Janssen: Honoraria, Research Funding.

CCAAT/enhancer binding protein ε (CEBPE) is an essential transcription factor for granulocytic differentiation. Mutations of CEBPE occur in individuals with neutrophil-specific granule deficiency (SGD), which is characterized by defects in neutrophil maturation. Cebpe-knockout mice also exhibit defects in terminal differentiation of granulocytes, a phenotype reminiscent of SGD. Analysis of DNase I hypersensitive sites sequencing data revealed an open chromatin region 6 kb downstream of the transcriptional start site of Cebpe in murine myeloid cells. We identified an interaction between this +6-kb region and the core promoter of Cebpe using circular chromosome conformation capture sequencing (4C-seq). To understand the role of this putative enhancer in transcriptional regulation of Cebpe, we targeted it using catalytically inactive Cas9 fused to Krüppel-associated box (KRAB) domain and observed a significant downregulation of transcript and protein levels of CEBPE in cells expressing guide RNA targeting the +6-kb region. To further investigate the role of this novel enhancer further in myelopoiesis, we generated mice with deletion of this region using CRISPR/Cas9 technology. Germline deletion of the +6-kb enhancer resulted in reduced levels of CEBPE and its target genes and caused a severe block in granulocytic differentiation. We also identified binding of CEBPA and CEBPE to the +6-kb enhancer, which suggests their role in regulating the expression of Cebpe. In summary, we have identified a novel enhancer crucial for regulating expression of Cebpe and required for normal granulocytic differentiation.

ZFP36L1 is a tandem zinc-finger RNA-binding protein that recognizes conserved adenylate-uridylate-rich elements (ARE) located in 3'untranslated regions (UTR) to mediate mRNA decay. We hypothesized that ZFP36L1 is a negative regulator of a posttranscriptional hub involved in mRNA half-life regulation of cancer-related transcripts. Analysis of

ARID1A is a key component of ATP-dependent SWI/SNF complex involved in chromatin remodeling. Chromatin remodeling mediated by SWI/SNF complex is crucial for gene expression and affects a broad range of biological processes including hematopoietic development. ARID1A is frequently mutated across several solid tumors as well as hematopoietic malignancies, including Burkitt's lymphoma, diffuse large B-cell lymphoma and acute promyelocytic leukemia. Nevertheless, function of ARID1A in adult hematopoiesis and implications of its deficiency in development and progression of hematopoietic diseases has not been explored. In this study, we used a murine model of ARID1A deficiency to establish its essential function in maintaining normal hematopoietic development. Germline loss of Arid1a is embryonic lethal; therefore, we generated mice with deletion of Arid1a specifically in the hematopoietic compartment using Vav-iCre and Mx1-Cre transgenic mice. Arid1afl/fl;Vav-iCre+ mice occurred at a lower than expected frequency, suggesting some perinatal mortality. For the Mx1-Cre model, Arid1a exon 9 was excised by administrating poly(I:C) to adult mice and hematopoiesis was evaluated using flow cytometry. An increase in both percentage and absolute number of long-term hematopoietic stem cells (LTHSCs) defined as Lin-Sca1+Kit+CD34-FLT3- or Lin-Sca1+Kit+CD48-CD150+ occurred in the bone marrow using both models of Arid1a deficiency. RNA-sequencing of sorted LTHSCs from Arid1a KO bone marrow revealed dysregulated expression of several genes involved in cell cycle, G2/M checkpoint and related pathways. In vivo BrdU incorporation assays showed a substantially lower proportion of quiescent hematopoietic stem cells in Arid1a deficient bone marrow. To assess the reconstitution ability of ARID1A deficient HSCs, sorted KO or WT LTHSCs were transplanted into irradiated congenic recipient mice in competitive repopulation assays. Proportion of donor-derived cells in recipients transplanted with KO cells was strikingly lower compared to wild-type cells, suggesting poor reconstitution ability of Arid1a KO LTHSCs. Also, differentiation of both myeloid and lymphoid lineages was impaired in Arid1a KO mice compared to WT controls. To investigate the mechanism of perturbed differentiation of the myeloid and erythroid lineages, RNA-Seq was performed on sorted CMPs, GMPs and MEPs from WT and Arid1a KO BM. Our analysis showed significant decrease in expression of several transcription factors (Runx1, Gata2, Cebpa), which play a crucial role in lineage differentiation. To determine how Arid1a deficiency alters chromatin accessibility in myeloid precursors, Assay for Transposase Accessible Chromatin with high-throughput sequencing (ATAC-Seq) was performed on sorted Lin-Kit+ BM cells from both Arid1a KO and WT mice. A global reduction in open chromatin in Arid1a KO cells was noted compared to WT cells. A substantial overlap occurred between down regulated genes (RNA-seq) and reduced chromatin accessibility in Arid1a KO myeloid progenitors. Motifs for PU.1, RUNX1, GATA and CEBPA were significantly enriched in loci with reduced ATAC-seq signals in Arid1a KO cells. Our findings demonstrate an indispensable function of Arid1a in hematopoietic development and underline the importance of precise chromatin dynamics maintained by ARID1A-containing SWI/SNF complex in hematopoiesis. Disclosures No relevant conflicts of interest to declare.

Background Coats plus syndrome is an autosomal recessive, pleiotropic, multisystem disorder characterized by retinal telangiectasia and exudates, intracranial calcification with leukoencephalopathy and brain cysts, osteopenia with predisposition to fractures, bone marrow suppression, gastrointestinal bleeding and portal hypertension. It is caused by compound heterozygous mutations in the CTC1 gene. Case presentation We encountered a case of an eight-year old boy from an Indian family with manifestations of Coats plus syndrome along with an unusual occurrence of dextrocardia and situs inversus. Targeted resequencing of the CTC1 gene as well as whole exome sequencing (WES) were conducted in this family to identify the causal variations. The identified candidate variations were screened in ethnicity matched healthy controls. The effect of CTC1 variation on telomere length was assessed using Southern blot. A novel homozygous missense mutation c.1451A > C (p.H484P) in exon 9 of the CTC1 gene and a rare 3′UTR known dbSNP variation (c.*556 T > C) in HES7 were identified as the plausible candidates associated with this complex phenotype of Coats plus and dextrocardia. This CTC1 variation was absent in the controls and we also observed a reduced telomere length in the affected individual’s DNA, suggesting its likely pathogenic nature. The reported p.H484P mutation is located in the N-terminal 700 amino acid regionthat is important for the binding of CTC1 to ssDNA through its two OB domains. WES data also showed a rare homozygous missense variation in the TEK gene in the affected individual. Both HES7 and TEK are targets of the Notch signaling pathway. Conclusions This is the first report of a genetically confirmed case of Coats plus syndrome from India. By means of WES, the genetic variations in this family with unique and rare complex phenotype could be traced effectively. We speculate the important role of Notch signaling in this complex phenotypic presentation of Coats plus syndrome and dextrocardia. The present finding will be useful for genetic diagnosis and carrier detection in the family and for other patients with similar disease manifestations. Electronic supplementary material The online version of this article (doi:10.1186/s12881-015-0151-8) contains supplementary material, which is available to authorized users.