Loss of the functional Adenomatous Polyposis Coli (APC-LOF) tumor suppressor gene represents the disease-initiating event in most colorectal cancer (CRC) cases. A newly identified dependency between PRMT5 and APC-LOF suggests that inhibiting PRMT5 may help intercept CRC. To circumvent hematological toxicities associated with orally bioavailable first-generation PRMT5 inhibitors, we aimed to limit systemic exposure after oral administration. We describe our efforts, challenges, and compound evaluation workflow resulting in gut-restricted PRMT5 inhibitors. A two-pronged approach was envisioned, consisting of (1) minimizing passive absorption, and (2) maximizing systemic clearance by incorporation of a metabolic "soft spot". This resulted in 9 and 18, displaying low absorption in preclinical species and high first-pass extraction mediated by aldehyde oxidase. 9 and 18 demonstrated in vivo colon pharmacodynamics without signs of systemic on-target toxicity, confirming gut-restriction. Administering 9 to dextran sodium sulfate (DSS)-treated polyp-bearing ApcMin/+ mice significantly reduced polyp number, indicating local treatment efficacy.

The RecQ helicase WRN is a synthetic lethal dependency in microsatellite instability-high (MSI-H) cancer and a therapeutic target of interest in that context. The two clinical-stage WRN inhibitors, HRO761 and RO7589831, both bind in an allosteric pocket near the interface of the two helicase domains (D1 and D2). To discover novel WRN inhibitors, we conducted a high-throughput phenotypic screen using γH2AX immunofluorescence imaging in MSI-H HCT116 colorectal cancer cells and identified a singleton hit, Compound A. Compound A phenocopied WRN siRNA knockdown in multiparametric analysis and was confirmed to engage and inhibit purified WRN protein. X-ray co-crystal structures with and without ATP revealed that Compound A binds to a novel allosteric site in the D1 domain which is absent in apo-WRN. The allosteric site was induced by localized rearrangement, including displacement of the DEAH-box upon ligand binding. Electron density was indicative of covalent bond formation via addition of Cys672 to the imine C=N bond of Compound A. Mechanistically, Compound A was ATP-noncompetitive, exhibited time-dependent inhibition of WRN ATPase and helicase activity, and did not inhibit the paralogous RecQ helicases BLM, RECL and RECQL4. Exploration of structure-activity relationships resulted in Compound B and Compound C that were characterized for therapeutic pharmacology. Both Compounds B and C demonstrated high potency and selectively induced γH2AX phosphorylation, cell cycle arrest, apoptosis, and clonogenic killing in MSI-H cells. The cytotoxicity of Compound C in HCT116 cells was unaffected by isogenic, CRISPR-mediated knockout of BLM, RECQL, RECQL4, RECQL5 or BRIP1 (FANCJ). In global proteomics and cell signaling studies, Compound C caused concentration-dependent degradation of WRN and upregulation of DNA damage response pathways in HCT116 but not microsatellite stable (MSS) SW620 colorectal cancer cells. Compound B was highly selective for MSI-H models and recapitulated the profile of sensitivity to WRN perturbation in a PRISM screen of 906 cell lines. Co-administration of Compound C with a CYP inhibitor to mice bearing HCT116 xenografts produced dose-dependent γH2AX phosphorylation in tumor tissue. Whole-genome CRISPR modifier screens with Compound C and a close analog of HRO761 in HCT116 and KM12 cells showed generally concordant functional genomic profiles, with perturbations in SMARCAL1, HELLS, and WDR76 enriched and POLQ and RBM6 depleted by WRN inhibition. In conclusion, we report a series of WRN inhibitors that engage a novel cryptic binding site and show a distinct mechanism of inhibition to WRN inhibitors currently in clinical development. Francis W. Hunter, Lindsey DeRatt, Kenneth Maksimchuk, Tatsuya Maehigashi, Seong Joo Koo, Liesbeth Micholt, Ranjeet P. Dash, Faraz Kazmi, Zhengyu Jiang, Albi Francis, Neetu Shukla, Aaron Patrick, Trpta Bains, Romy Perez-Abraham, Nikit Kumar, Zhijie Liu, Brian Reiss, Caitlin Morgan, Inha Heo, Werner Eyckmans, Gerald Chu, Anna Kuhn, Lorraine Angelillo, Lin-Chien Huang, Yu Sun, Ashley M. Rouleau, Yongkang Kim, John J. Shin, Harris Bell-Temin, Victoria Wong, Ruth Steele, Paul Shaffer, Kurtis E. Bachman, David Pocalyko, Scott Kuduk. Discovery of WRN helicase inhibitors that covalently engage a novel, induced allosteric site [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 4207.

Colorectal cancer (CRC) is one of the most common causes of cancer-related deaths in the world and loss of the Adenomatous Polyposis Coli (APC) gene represents the disease-initiating event in the majority of CRC cases. Strategies for targeting APC-loss of function (APC-LOF) may provide an opportunity to intercept tumorigenesis in high-risk CRC patients, such as those with Familial Adenomatous Polyposis (FAP). To discover targets for therapeutic intervention in APC-LOF-driven disease, we conducted a high-throughput screen in isogenic APC knockout (APCKO) colorectal organoids of >6000 compounds with known clinical relevance. From this screen, we uncovered a novel synthetic lethal dependency of APC-LOF on protein arginine methyltransferase 5 (PRMT5). JNJ-64619178 (JNJ-9178), a potent clinical PRMT5 inhibitor, demonstrated an >800-fold window in growth inhibition of engineered APCKO organoids compared to wild-type (WT). Pharmacodynamic assessments showed a dose-dependent decrease in SmD3, an established biomarker for PRMT5 target engagement, with JNJ-9178 treatment, while no decrease was observed with a weakly active compound, providing evidence that the antiproliferative activity observed in the APCKO organoids is a direct consequence of on-target PRMT5 inhibition. We further validated this PRMT5/APC dependency by testing other S-adenosylmethionine (SAM)-competitive PRMT5 inhibitors which show a similar trend in selectively inhibiting growth of the APCKO organoids vs WT. Furthermore, we assessed the impact of JNJ-9178 on colon polyp burden in the Apcmin/+-DSS mouse model, an in vivo model of FAP, where we started treatment either before (early interception) or after (late interception) significant polyp burden had been established. A 72% and 66% reduction in polyp burden was observed, respectively, compared to vehicle-treated mice after 3 weeks of dosing (BID). Taken together, these findings showcase the impact of PRMT5 inhibition in both human and mouse models of FAP disease. Orally bioavailable PRMT5 inhibitors, including JNJ-9178, present challenges as therapeutics for CRC interception due to on-target cytopenias observed with systemic exposure. To address these dose-limiting toxicities, we have developed a GI-restricted approach and discovered new compounds that only inhibit PRMT5 activity locally in the intestinal tract. This strategy is detailed in a second abstract. Dana S. Gaffney, Jan Willem Thuring, Fabian Hulpia, Eileen Vesely, Edward Retzbach, Zienab Etwebi, Zhengyu Jiang, Albi Francis, Bethany Mattson, Victoria Wong, Mariana Silva, Gerben ten Hag, Rene Overmeer, Carla Verissimo, Sylvia F. Boj, Johan Nicolai, Mark Hixon, Gerry Chu, Andrea DiSandro, Kathleen Clancy, Alexandra Liddane, Kurtis Bachman, David Pocalyko. PRMT5 as a target in CRC interception: Synthetic lethal dependency identified in APC-LOF preclinical models of FAP disease [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 2363.

To identify causal links between gut microbes and tumorigenesis, we colonized groups of germ-free colon tumor-susceptible mice ( Apc Min/+ ; Il10 −/−) with 15 cultured human fecal microbiotas from healthy individuals, as well as patients with inflammatory bowel disease and colorectal cancer. The number of colonic tumors in Apc Min/+ ; Il10 −/− mice varied by donor microbiota but not by the health status of the donor. In vitro screens of host cell proliferation, genotoxicity, and inflammation in bacteria-mammalian cell cocultures revealed that genotoxicity best predicted tumorigenic microbes in vivo with genotoxic microbes present in all tested individuals. The genotoxic subset of strains from each donor induced more tumors than the complete community – even when the complete community was not tumorigenic. Combining genotoxic microbes from multiple sources increased tumor number and decreased the time to tumor onset. Together these results suggest that most individuals harbor genotoxic bacterial strains and the balance of genotoxic to protective strains determines the timing and severity of tumorigenesis in vivo. ### Competing Interest Statement Jeremiah Faith is on the scientific advisory board of Vedanta Biosciences, reports receiving research grants from Janssen Pharmaceuticals, and reports receiving consulting fees from Genfit, Janssen Pharmaceuticals, BiomX, and Vedanta Biosciences. A. Nicole Desch and Dirk Gevers were employees of Janssen Research and Development LLC at the time of this work. Gerald Chu, Zhengyu Jiang, Jianyao Wang, David Pocalyko, Kurtis E. Bachman, and Lani R. San Mateo are current employees of Janssen Research and Development LLC.

Identifying genetic dependencies in human colon cancer could help identify effective treatment strategies. Genome-wide CRISPR-Cas9 dropout screens have the potential to reveal genetic dependencies, some of which could be exploited as therapeutic targets using existing drugs. In this study, we comprehensively characterized genetic dependencies present in a colon cancer organoid avatar, and validated tumor-specific selectivity of select pharmacologic agents. We conducted a genome-wide CRISPR dropout screen to elucidate the genetic dependencies that interacted with select driver somatic mutations. We found distinct genetic dependencies that interacted with WNT, MAPK, PI3K, TP53, and mismatch repair pathways and validated targets that could be exploited as treatments for this specific subtype of colon cancer. These findings demonstrate the utility of functional genomic screening in the context of personalized medicine.

Abstract HUB patient derived organoids, (HUB-OrganoidsTM or PDOs) are self-organized epithelial cell structures with near-physiological features, extensively used to model aspects of cancer initiation and progression. Microinjection of colibactin-producing pks+ E. coli into the lumen of PDOs resulted in the appearance of two co-occurring mutational signatures identified in a subset of colorectal cancer (CRC) patients, demonstrating that pks+ E. coli plays a causative role in CRC development. The scalability of PDO bacteria microinjection is, however, limited and represent a bottleneck in the screening of preventive therapies for these patients. Here we develop a bacteria-PDO co-culture system (PDO-fragment exposure model), alternative to PDO microinjection, that is compatible with medium-to-high throughput screening methods. We validated the genotoxicity of colibactin-producing bacteria and showed the potential of the PDO fragment exposure model for the screening of drugs targeting colibactin-dependent genotoxicity. Citation Format: Eider Valle-Encinas, Mayke Doorn, Farzin Pourfarzad, Lani San Mateo, Prashanth Gokare, David Pocalyko, Sylvia F. Boj, Carla S. Verissimo. Development of organoids-bacteria co-cultures for medium-to-high throughput screening [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 242.

Identifying genetic dependencies in human colon cancer could help identify effective treatment strategies. Genome-wide CRISPR-Cas9 dropout screens have the potential to reveal genetic dependencies, some of which could be exploited as therapeutic targets using existing drugs. In this study, we comprehensively characterized genetic dependencies present in a colon cancer organoid avatar, and validated tumor-specific selectivity of select pharmacologic agents. We conducted a genome-wide CRISPR dropout screen to elucidate the genetic dependencies that interacted with select driver somatic mutations. We found distinct genetic dependencies that interacted with WNT, MAPK, PI3K, TP53, and mismatch repair pathways and validated targets that could be exploited as treatments for this specific subtype of colon cancer. These findings demonstrate the utility of functional genomic screening in the context of personalized medicine.

Pathogens such as colibactin-producing bacteria strains of the Enterobacteriaceae family are highly prevalent in the gut microbiota of colorectal cancer (CRC) patients. HUB patient derived Organoids, (HUB-OrganoidsTM or PDOs) are self-organized epithelial cell structures with near-physiological features, extensively used to model aspects of cancer initiation and progression. Studies conducted by Pleguezuelos-Manzanos et al. (2020) showed that repetitive cycles of colibactin-producing bacteria into the organoid lumen via microinjection, led to two co-occurring mutational signatures appearance, a single-base substitution signature (SBS-pks) and an indel signature (ID-pks), identified in a subset of CRC patients (~2% and 7%, respectively). These results highlight the relevance of organoids for modelling host-pathogen interactions and the application of these systems as in vitro platforms for investigating long-standing cause-correlation relationship between the presence of genotoxic pathogens and CRC development. The limited scalability of sophisticated co-culture strategies with pathogens such as microinjection has represented a bottleneck in the use of organoids as throughput screening tools in the development of preventive therapies. Thus, there is an unmet need to develop alternative co-culture systems compatible with the process of compound screening. An organoid-bacteria co-culture system compatible with medium-to-high throughput screening readouts has been developed. This co-culture system is currently tailored for the modeling of colibactin genotoxic effects in the gut epithelium, but theoretically could be extended as a discovery platform to identify targetable, novel and complex interactions between host and pathogen. Key words: colibactin, organoids, CRC, screening Citation Format: Eider Valle-Encinas, Roshni Nair, Katerina Pisa, Mayke Doorn, Farzin Pourfarzad, Lani San Mateo, Nicole Desch, Prashanth Gokare, David Pocalyko, Sylvia F. Boj, Carla Verissimo. Development of a medium-to-high throughput organoid-bacteria co-culture platform for the assessment of host pathogen interaction [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 5917.

Colorectal cancer (CRC) is the second most common cause of cancer-related deaths worldwide. The disease-initiating event in most CRC cases is the loss of function (LOF) of the adenomatous polyposis coli (APC) gene, accounting for 70-80% of CRC. Strategies to target cells that harbor the truncation mutation in APC, which leads to its LOF, may provide the opportunity to intercept tumorigenesis in CRC patients. High-risk CRC populations include patients with familial adenomatous polyposis (FAP), the inherited disorder caused by germline APC mutations. Aurora Kinase (AK), a family of serine/threonine kinases, is essential for proper cell division, and overexpression of AK has been associated with multiple heme and solid tumor malignancies, including CRC. Aurora kinases have been well-studied, both preclinically and clinically, as a target for therapeutic intervention across multiple cancers; however, the role of AK in CRC disease progression has not been fully explored. Here we report a synthetic lethal dependency between APC and AK which was identified in a human isogenic colon organoid model containing a genetically engineered APC LOF (APCLOF). Aurora A-specific, Aurora B-specific, and Pan-AK inhibitors were evaluated in isogenic APCLOF or APCWT colon organoids derived from multiple donors. All AK inhibitors demonstrated selective growth inhibition of the APCLOF organoid. The strongest synthetic lethal effect was observed with a pan-AK inhibitor showing greater than 1000-fold selectivity in the APCLOF colon organoids compared to APCWT. Interestingly, this activity diminished as oncogenic driver mutations in KRAS and/or p53 genes were introduced into the APCLOF organoids. These findings suggest that therapeutic benefit of AK inhibitors would be greater in early APCLOF driven adenomas as opposed to advanced CRC. Despite these encouraging results indicating therapeutic opportunity in early adenoma disease, on-target genotoxicity was observed in vitro through increases in micronuclei at pharmacologic doses. Therefore, although there is a high unmet need in FAP for therapeutic options, potential genotoxicity associated with AK inhibitor treatments may limit utility as an intervention strategy. Citation Format: Dana S. Gaffney, Eileen Vesely, David Pocalyko, Kurtis Bachman, Karla Wiehagen, Glenn Cowley, Victoria Wong, Prashanth Gokare, Peggy Guzzie-Peck, Sandy Weiner, Ann De Smedt, Jacqueline Kinyamu-Akunda, Tammy Bush, Zhuming Zhang, Robert Schulingkamp, Sylvia F. Boj, Rene Overmeer, Carla Verissimo, Roshni Nair. Aurora kinase is synthetic lethal with APC loss of function in engineered human colon organoid models [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 1416.

Fibroblast-like synoviocytes (FLS), one of the main cell types of the rheumatoid arthritis (RA) synovium, possess phenotypic and molecular characteristics of transformed cells. JQ1, an inhibitor of the bromodomain and extra terminal domain family that includes BRD2, BRD3, BRD4, and BRDt, has shown efficacy in models of arthritis. We demonstrate that the active isomer of JQ1 but not its inactive isomer inhibits IL-1b-induced RA-FLS activation and proliferation. To understand the mechanism of JQ1 action, we subjected JQ1-treated RA-FLS to transcriptional profiling and determined BRD2 and BRD4 cistromes by identifying their global chromatin binding sites. In addition, assay for transposable accessible chromatin by high throughput sequencing was employed to identify open and closed regions of chromatin in JQ1-treated RA-FLS. Through an integrated analysis of expression profiling, Brd2/Brd4 cistrome data, and changes in chromatin accessibility, we found that JQ1 inhibited key BRD2/BRD4 super-enhancer genes, downregulated multiple crucial inflammatory pathways, and altered the genome-wide occupancy of critical transcription factors involved in inflammatory signaling. Our results suggest a pleiotropic effect of JQ1 on pathways that have shown to be individually efficacious in RA (in vitro, in vivo, and/or in humans) and provide a strong rationale for targeting BRD2/BRD4 for disease treatment and interception.

Transcriptomics technologies such as next-generation sequencing and microarray platforms provide exciting opportunities for improving diagnosis and treatment of complex diseases. Transcriptomics studies often share similar hypotheses, but are carried out on different platforms, in different conditions, and with different analysis approaches. These factors, in addition to small sample sizes, can result in a lack of reproducibility. A clear understanding and unified picture of many complex diseases are still elusive, highlighting an urgent need to effectively integrate multiple transcriptomic studies for disease signatures. We have integrated more than 3,000 high-quality transcriptomic datasets in oncology, immunology, neuroscience, cardiovascular and metabolic disease, and from both public and internal sources (DiseaseLand database). We established a systematic data integration and meta-analysis approach, which can be applied in multiple disease areas to create a unified picture of the disease signature and prioritize drug targets, pathways, and compounds. In this bipolar case study, we provided an illustrative example using our approach to combine a total of 30 genome-wide gene expression studies using postmortem human brain samples. First, the studies were integrated by extracting raw FASTQ or CEL files, then undergoing the same procedures for preprocessing, normalization, and statistical inference. Second, both p-value and effect size based meta-analysis algorithms were used to identify a total of 204 differentially expressed (DE) genes (FDR < 0.05) genes in the prefrontal cortex. Among these were BDNF, VGF, WFS1, DUSP6, CRHBP, MAOA, and RELN, which have previously been implicated in bipolar disorder. Finally, pathway enrichment analysis revealed a role for GPCR, MAPK, immune, and Reelin pathways. Compound profiling analysis revealed MAPK and other inhibitors may modulate the DE genes. The ability to robustly combine and synthesize the information from multiple studies enables a more powerful understanding of this complex disease.

DNA methylation is an epigenetic mechanism that regulates gene expression during many stages of development, including genomic imprinting, stem cell regulation, and X-chromosome inactivation. Moreover, aberrant DNA methylation patterns, characterized by genome-wide hypomethylation and promoter-specific hypermethylation, are a prominent feature of cancer. Methylation of DNA at the 5-position of cytosines is mediated by the DNA methyltransferase (DNMT) protein family, which regulates both maintenance methylation (DNMT1) and de novo methylation (DNMT3A and DNMT3B). Loss-of-function mutations of DNMT3A have been identified in hematological malignancies including acute myeloid leukemia (AML), where DNMT3A is mutated in approximately 25% of known cases. Published reports suggest the existence of a synthetic lethal interaction between DNMT3A and DNMT1/3B. To further study this potential genetic interaction, we are performing a CRISPR-Cas9 tiling screen to identify functional domains within DNMT1 and/or DNMT3B that are synthetic lethal with DNMT3A. We generated a lentiviral library containing 777 and 421 single guide RNAs (sgRNAs) that tile the coding region of DNMT1 and DNMT3B, respectively and performed viability screens in AML cell lines that are either wild-type or mutant for DNMT3A. This screen was designed to identify in-frame alterations within functional domains that lead to effects on cell viability. Next generation sequencing of sgRNAs identified three functional domains of DNMT1 which, when mutated, leads to decreases in cell viability. Current efforts are focused on verifying the essentiality of these functional domains using CRISPR-Cas9-based approaches as well as mutagenesis by integrated tiles (MITE)-seq analyses. Citation Format: Balpreet Bhogal, Barbara Weir, Ramona Crescenzo, Min Chul Kwon, Ulrike Philippar, Ricardo Attar, Glenn Cowley, David Pocalyko. A CRISPR-Cas9 tiling screen to identify functional domains within DNMT1 and/or DNMT3B that can be targeted for therapeutic intervention in AML [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 1383.

Epigenetics contributes to the pathogenesis of immune-mediated diseases like rheumatoid arthritis (RA). Here we show the first comprehensive epigenomic characterization of RA fibroblast-like synoviocytes (FLS), including histone modifications (H3K27ac, H3K4me1, H3K4me3, H3K36me3, H3K27me3, and H3K9me3), open chromatin, RNA expression and whole-genome DNA methylation. To address complex multidimensional relationship and reveal epigenetic regulation of RA, we perform integrative analyses using a novel unbiased method to identify genomic regions with similar profiles. Epigenomically similar regions exist in RA cells and are associated with active enhancers and promoters and specific transcription factor binding motifs. Differentially marked genes are enriched for immunological and unexpected pathways, with "Huntington's Disease Signaling" identified as particularly prominent. We validate the relevance of this pathway to RA by showing that Huntingtin-interacting protein-1 regulates FLS invasion into matrix. This work establishes a high-resolution epigenomic landscape of RA and demonstrates the potential for integrative analyses to identify unanticipated therapeutic targets.

BackgroundRheumatoid arthritis (RA) is a chronic autoimmune disease characterised by infiltration of immune cells into the synovium and hyperplasia of the synovial lining, resulting in the formation of pannus that degrades cartilage and bone. Fibroblast-like synoviocytes (FLS) are the main cell types of the rheumatoid synovium and possess phenotypic and molecular characteristics of transformed cells. JQ1, an inhibitor of the bromodomain and extra terminal domain (BET) family that includes BRD2, BRD3, BRD4 and BRDt has shown efficacy in vitro on RA-FLS proliferation and in vivo in a murine model of arthritis.ObjectivesWe sought to elucidate the mechanism of action of BET proteins in FLS biology and determine the potential therapeutic utility of targeting BRD2/BRD4 for RA disease treatment and interception.MethodsTo understand the mechanism of JQ1 action, we subjected JQ1-treated RA-FLS to transcriptional profiling by RNA-Seq and determined BRD2 and BRD4 cistromes by identifying global BRD2/BRD4 chromatin binding sites by ChIP-Seq. In addition, Assay for Transposable Accessible Chromatin by high throughput Sequencing (ATAC-Seq) was employed to identify open and closed regions of chromatin in JQ1-treated RA-FLS.ResultsWe demonstrate that the active isomer of JQ1 but not its inactive isomer inhibits IL-1b-induced RA-FLS activation and proliferation. Through an integrated analysis of RNA-Seq, ATAC-Seq to profile changes in chromatin accessibility and ChIP-Seq of BRD2/4 and Pol2 proteins, we found that JQ1 inhibited multiple key inflammatory pathways, and altered the genome wide occupancy of crucial transcription factors involved in inflammatory signalling. Specifically, JQ1 treatment resulted in reduced occupancy of both BRD2 and BRD4 in approximately 2000 regions genome-wide, and a loss of Pol2 occupancy in approximately 600 genomic regions. Collectively we found that 105 genes had altered occupancy in all three proteins (BRD2, BRD4 and Pol2) and were also differentially expressed. Most prominently, JQ1 resulted in down regulation of IL6, IL8, p38 MAP kinase and HMGB1/TLR4 signalling pathways. In addition, we have identified BRD2/BRD4 super-enhancer genes and demonstrate that JQ1 altered BRD2/BRD4 occupancy in the IL6 and IL8 super-enhancer regions, and significantly down-regulated IL6, IL8, TLR4 and IL1b expression.ConclusionsOur results suggest pleiotropic effects of JQ1 on pathways that have been individually targeted and shown to be efficacious for the treatment of RA. These studies provide a strong rationale for targeting of BRD2/BRD4 in RA.Disclosure of InterestS. Nagpal Shareholder of: Johnson and Johnson, Grant/research support from: Johnson and Johnson, Employee of: Johnson and Johnson, V. Krishna Shareholder of: Johnson and Johnson, Grant/research support from: Johnson and Johnson, Employee of: Johnson and Johnson, X. Yin Shareholder of: Johnson and Johnson, Grant/research support from: Johnson and Johnson, Employee of: Johnson and Johnson, D. Pocalyko Shareholder of: Johnson and Johnson, Grant/research support from: Johnson and Johnson, Employee of: Johnson and Johnson, A. Walsh Shareholder of: Johnson and Johnson, Grant/research support from: Johnson and Johnson, Employee of: Johnson and Johnson, K. Bachman Shareholder of: Johnson and Johnson, Grant/research support from: Johnson and Johnson, Employee of: Johnson and Johnson, I. Anderson Shareholder of: Johnson and Johnson, Grant/research support from: Johnson and Johnson, Employee of: Johnson and Johnson, L. Madakamutil Shareholder of: Johnson and Johnson, Grant/research support from: Johnson and Johnson, Employee of: Johnson and Johnson

Oxygen deprivation induces a range of cellular adaptive responses that enable to drive cancer progression. Here, we report that lysine‐specific demethylase 1 (LSD1) upregulates hypoxia responses by demethylating RACK1 protein, a component of hypoxia‐inducible factor (HIF) ubiquitination machinery, and consequently suppressing the oxygen‐independent degradation of HIF‐1α. This ability of LSD1 is attenuated during prolonged hypoxia, with a decrease in the cellular level of flavin adenine dinucleotide (FAD), a metabolic cofactor of LSD1, causing HIF‐1α downregulation in later stages of hypoxia. Exogenously provided FAD restores HIF‐1α stability, indicating a rate‐limiting role for FAD in LSD1‐mediated HIF‐1α regulation. Transcriptomic analyses of patient tissues show that the HIF‐1 signature is highly correlated with the expression of LSD1 target genes as well as the enzymes of FAD biosynthetic pathway in triple‐negative breast cancers, reflecting the significance of FAD‐dependent LSD1 activity in cancer progression. Together, our findings provide a new insight into HIF‐mediated hypoxia response regulation by coupling the FAD dependence of LSD1 activity to the regulation of HIF‐1α stability.

Abstract Therapies targeting epigenetic modifiers such as the DNA methyltransferase (DNMT) inhibitor, 5-azacytidine (Aza), are used in treating hematologic malignancies and also show promising results in subsets of solid tumors. The molecular mechanisms for these results, however, are not fully understood. Nonetheless, a number of clinical studies using combinations of epigenetic therapies plus chemo or immunotherapy to enhance the tumor response are ongoing. HCT-116, a colon cancer cell line genetically defined by microsatellite instability (MSI), resulting in a highly mutated, yet primarily diploid genome, has been routinely used to study epigenetic mechanisms and response to DNMT inhibition. In a previous study, we integrated a combination of data types including DNA methylation, DNA accessibility, histone modifications and transcriptomic data that revealed strong correlations between epigenomic changes and gene expression following Aza treatment. However, for some genes, we could not predict expression changes using these unidimensional, region-specific analysis methods. As others have demonstrated, genes do not work as single, isolated units, but rather interact with distal regulatory elements. These regulatory elements, often several Mb away can control gene expression through physical interactions such as bending and looping. We reason that in addition to direct demethylation of the immediate regulatory elements within the gene, Aza treatment can affect long-range chromosomal communications. In order to further understand the chromatin remodeling effect of Aza treatment, we performed high resolution genome-wide chromosome conformation capture (Hi-C) followed by NGS. HCT-116 cells were treated with DMSO or 1 μM Aza for 42 and 96 hours. Hi-C and RNA-Seq data was analyzed and integrated with other datatypes. The results showed that AZA treatment alters topologically associating domains (TAD), with the formation of new TAD boundaries and disappearance of others. The genome was then separated into 25K base pair consecutive bins, with each bin marked as type A (active) or type B (inactive). We observed bin switching following AZA treatment and differential loop formations (e.g. promoter-enhancer). Bins that switched from B to A at both time points were found enriched in genes that are related to acute phase response, interferon pathway, and cancer including PI3KCB, DDX58, CD274 and CDKN2A. Our findings using Hi-C were found to be in agreement with previous results using alternative experimental methods, which identified Aza and CTCF as the top upstream regulators of these genes. Our data also suggests that overall changes in 3D chromatin activities measured by Hi-C could be a better predictor of transcriptional regulation compared to H2K27me3 and H3K4me3 alone, particularly in situations where there are no significant changes in those marks but where changes in gene expression exist. Citation Format: Yuchen Vincent Bai, John Whitaker, Emanuele Palescandolo, Vinod Krishna, Vipul Bhargava, Satya Saxena, Xiang Yao, David Pocalyko, Kurt Bachman. Effect of DNA methyltransferase inhibitor 5-azacitidine on 3D chromatin structure measured by Hi-C [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 5371. doi:10.1158/1538-7445.AM2017-5371

Abstract DNA methyltransferase inhibitors (DNMTi) such as 5-azacytidine(Aza) are clinically used in treating hematologic malignancies. Promising effects were also observed in solid tumors, although the exact mechanism underlining the efficacy was unclear. There has been accumulating evidence suggesting an enhanced immune response to the tumor cells following such epigenetic therapies. Cancer immunotherapies targeting checkpoint inhibition in combination with epigenetic agents such as a DNMTi could hold promise for those patients that do not respond to immunotherapies alone. Aza was also found to stimulate an interferon response in tumor cells via inducing expression of endogenous retroviruses. In this study, we were interested to further explore the mechanisms of AZA on transcription regulation across the whole regulome. HCT-116 is a colon cancer cell line genetically defined by microsatellite instability (MSI) and mutations in KRAS(G13D) and PIK3CA(H1047R). In this study, HCT-116 cells were treated with DMSO (control) or 1μM Aza for 42 and 96 hours. RNA-seq, Mass spectrometry proteomics, RRBS, Chip-Seq, ATAC-Seq and Hi-C data was collected. Data was analyzed by integrative computational methods. Treatment with Aza resulted in global demethylation at CpG islands(CGI), which was moderately correlated with increased expression of the corresponding genes. DNA methylation, DNA accessibility, H3K4me3 (active promoter histone mark), H3K27me3 (repressive histone mark) and RNA expression revealed and cross-verified different states of genomic regions. Pathway and gene set enrichment analysis found activation of anti-viral defense and interferon pathways, as well as increased expression of cancer-testis antigens. IRF7 expression was increased by Aza, and IRF binding motifs were the most enriched TF motifs at the immediate promoter regions of the up-regulated genes. The data also suggested that Aza may remove gene repression by disrupting the scaffolding function of DNMTs. The effects of Aza on promoter-enhancer interactions were also examined by Hi-C. In conclusion, we found that DNMTi such as Aza regulates gene transcription through multiple modes of actions. Citation Format: Yuchen Bai, Emanuele Palescandolo, John Whitaker, Vinod Krishna, Vipul Bhargava, Satya Saxena, Xiang Yao, David Pocalyko, Kurtis Bachman. Integrated genomics and epigenomics analysis reveals genome-wide effect of the DNA methyltransferase inhibitor 5-azacitidine (Aza) on regulation of transcription. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 2783.

Cancer is a genetic disease with frequent somatic DNA alterations. Studying recurrent copy number aberrations (CNAs) in human cancers would enable the elucidation of disease mechanisms and the prioritization of candidate oncogenic drivers with causal roles in oncogenesis. We have comprehensively and systematically characterized CNAs and the accompanying gene expression changes in tumors and matched nontumor liver tissues from 286 hepatocellular carcinoma (HCC) patients. Our analysis identified 29 recurrently amplified and 22 recurrently deleted regions with a high level of copy number changes. These regions harbor established oncogenes and tumor suppressors, including CCND1 (cyclin D1), MET (hepatocyte growth factor receptor), CDKN2A (cyclin-dependent kinase inhibitor 2A) and CDKN2B (cyclin-dependent kinase inhibitor 2B), as well as many other genes not previously reported to be involved in liver carcinogenesis. Pathway analysis of cis-acting genes in the amplification and deletion peaks implicates alterations of core cancer pathways, including cell-cycle, p53 signaling, phosphoinositide 3-kinase signaling, mitogen-activated protein kinase signaling, Wnt signaling, and transforming growth factor beta signaling, in a large proportion of HCC patients. We further credentialed two candidate driver genes (BCL9 and MTDH) from the recurrent focal amplification peaks and showed that they play a significant role in HCC growth and survival. Conclusion: We have demonstrated that characterizing the CNA landscape in HCC will facilitate the understanding of disease mechanisms and the identification of oncogenic drivers that may serve as potential therapeutic targets for the treatment of this devastating disease. (Hepatology 2013;58:706–717)

PKM2 is an isoenzyme of the glycolytic enzyme pyruvate kinase that promotes aerobic glycolysis. Here, we describe an important role for PKM2 in regulating the survival of gastric cancer (GC) cells. We showed that PKM2 was overexpressed in gastric tumor tissues compared to normal tissues and its expression level was associated with poor survival of gastric cancer patients. We also showed that PKM2 affected cell survival by regulating Bcl-xL at the transcriptional level. PKM2 knockdown partially affected the stability of NF-kB subunit p65, suggesting that post-translational regulation of p65 by PKM2 is one of plausible mechanisms for the increased cell growth. Therefore, PKM2 may function as an upstream molecule that regulates p65 function and thus enhances the growth of tumor cells.