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Pancreatic ductal adenocarcinoma (PDAC) remains one of the most challenging cancer to treat. Due to the asymptomatic nature of the disease and ineffective drug treatment modalities, the survival rate of PDAC patients remains one of the lowest. The recurrent genetic alterations in PDAC are yet to be targeted; therefore, identifying effective therapeutic combinations is desperately needed. Here, we performed anin vivoCRISPR screening in a clinically relevant patient-derived xenograft (PDX) model system to identify synergistic drug combinations for PDAC treatment. Our approach revealed protein arginine methyltransferase gene 5 (PRMT5) as a promising druggable candidate whose inhibition creates synergistic vulnerability of PDAC cells to gemcitabine. Genetic and pharmacological inhibition results indicate that of PRMT5 depletion results in synergistic cytotoxicity with Gem due to depleted replication protein A (RPA) levels and an impaired non-homology end joining (NHEJ) DNA repair. Thus, the novel combination creates conditional lethality through the accumulation of excessive DNA damage and cell death, bothin vitroandin vivo. The findings demonstrate that unbiased genetic screenings combined with a clinically relevant model system is an effective approach in identifying synthetic lethal drug combinations for cancer treatment.STATEMENT of SIGNIFICANCEIdentify synergistic drug combinations for PDAC is a significant unmet need. Through CRISPR screening, we discovered and validated that PRMT5 depletion creates synergistic vulnerability of PDAC cells to gemcitabine. Mechanistically, the combination impairs DNA repair, synergistic accumulation of DNA damage and cell deathin vitroandin vivo.

Immune checkpoint blockade (ICB) therapy has improved long-term survival for patients with advanced melanoma. However, there is critical need to identify potential biomarkers of response and actionable strategies to improve response rates. Through generation and analysis of over 200 chromatin modification maps for ICB-treated melanoma patient samples, melanoma cells and T cells, we identified significant enrichment of active enhancer states in non-responders at baseline. Enhancer mapping by bulk ChIP-Seq or single cell ATAC-Seq methods in two independent cohorts of ICB-treated melanoma samples identified an enhancer signature that predicted response to anti-PD-1 therapy. The activated non-responder enhancers marked a group of key regulators of several pathways in melanoma cells (including c-MET, TGFβ, EMT and AKT) that are known to mediate resistance to ICB therapy. In addition, several checkpoint receptors were alternatively activated by aberrant enhancers in T cells. Unbiased CRISPRi screening in melanoma cells and T cells identified novel functional enhancers, such as one upstream of c-MET, that mediate ICB response or T cell mediated killing. Finally, inhibition of enhancers and repression of these pathways using bromodomain inhibitors along with anti-PD-1 therapy significantly decreased melanoma tumor burden and increased T-cell infiltration. Epigenomic experiments identified a signature of 107 genes that could be used as pharmacodynamic marker for BET inhibitor response. Together, these findings identify enhancer upregulation as a key mechanism of adaptive resistance to ICB response, a potential enhancer-based biomarker of resistance to anti-PD-1 and enhancer blockade in combination with ICB as a potential strategy to improve responses. Citation Format: Kunal Rai, Mayinuer Maitituoheti, Alvin Shi, Ming Tang, Li-Lun Ho, Firas Youssef Kreidieh, Christopher Terranova, Kyriakitsa Galani, Emily Z. Keung, Caitlin A. Creasy, Manrong Wu, Jiajia Chen, Nana Chen, Anand K. Singh, Apoorvi Chaudhri, Nazanin E. Anvar, Giuseppe Tarrantino, Jiekun Yang, Sharmistha Sarkar, Shan Jiang, Jared Malke, Lauren Haydu, Elizabeth Burton, Michael A. Davies, Jeffrey E. Gershenwald, Patrick Hwu, Alexander Lazar, Jaime H. Cheah, Christian K. Soule, Stuart S. Levine, Chantale Bernatchez, Srinivas V. Saladi, David Liu, Hussein Tawbi, Jennifer Wargo, Genevieve M. Boland, Manolis Kellis. Functional roles of enhancers in immune microenvironment & immunotherapy response. [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 6425.

Background: While CTLA-4 and PD-1 immune-checkpoint blockade (ICB) have revolutionized melanoma treatment, these treatments have different mechanisms, response rates, and toxicities. Differential predictors of response are poorly characterized. Combination ICB has the highest response rates but also much higher toxicity, and which patients to treat with single agent vs. combination ICB is controversial. In this study, we set out to understand the differential biology of anti-CTLA-4 and anti-PD-1 by integrating transcriptomic and clinical characterization from large cohorts of patients with metastatic melanoma treated with anti-CTLA-4, anti-PD-1, or combination blockade. Methods: We performed a standardized meta-analysis of all available cohorts of ICB-treated (anti-PD-1 alone, anti-CTLA-4 alone, and combination) melanoma patients with sequencing of pretreatment tumor and clinical annotations (n=572; 268 evaluated post quality-control filtering, cutaneous). Raw data in the form of FASTQs were obtained for all cohorts, and data were reprocessed using standardized pipelines. Immune infiltrate was characterized using single-sample gene set enrichment analysis (ssGSEA) scores from 18 immune cell type signatures, and GSEA was performed on differentially expressed genes between responders and non-responders for each treatment cohort accounting for differences in immune infiltrate. To dissect the cell types and drivers of nominated signatures, we analyzed scRNAseq from a separate cohort of pretreatment metastatic melanoma. Results: We found that immune infiltrate was strongly predictive of response to combination blockade (OR=6.16, p Conclusion: High rates of response to combination in patients with high levels of immune infiltrate suggests that immune high patients may differentially benefit from combination blockade. Identifying features of non-responding patients stratified by immune infiltrate can lead to new biological insights into ICB. Citation Format: Amy Y. Huang, Natalie Vokes, Cora Ricker, Tyler Aprati, Emily Robitschek, Jiekun Yang, Li-Lun Ho, Kyriakitsa Galani, Kelly P. Burke, Giuseppe Tarantino, Jiajia Chen, Arlene H. Sharpe, Eliezer M. Van Allen, Manolis Kellis, Genevieve M. Boland, David Liu. Multiomic meta-analysis of differential response to PD-1 and CTLA-4 blockade in metastatic melanoma [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 3271.

Pancreatic ductal adenocarcinoma (PDAC) remains one of the deadliest cancers. Uncovering mechanisms responsible for the heterogeneous clinical features of this disease is an essential step toward developing improved and more specific therapeutic approaches. Here, we sought to identify transcriptional regulators of aggressive PDAC growth through in vivo CRISPR screening of epigenetic and transcription factors in an orthotopic model. We identified the ISL LIM homeobox 2 (ISL2) gene as a tumor suppressor whose depletion enhances the proliferation of human PDAC cells in vitro and in vivo and cooperates with activated KRAS to initiate PDAC in a murine model. Conversely, the upregulation of ISL2 expression through CRISPR-mediated locus-specific epigenetic editing results in reduced cell proliferation. Importantly, ISL2 is epigenetically silenced through DNA methylation in ~60% of PDAC tumors, which correlates with poor patient outcome. Functional studies showed that ISL2 loss rewires metabolic gene expression, and consequently potentiates oxidative phosphorylation while reducing glycolysis. This metabolic shift creates selective vulnerability to small molecule inhibitors of mitochondrial respiration and fatty acid oxidation. Collectively, these findings reveal ISL2 as a novel tumor suppressor whose inactivation drives metabolic reprogramming in an aggressive PDAC subset and point to potential therapeutic vulnerabilities in these tumors.

Predicting the response and identifying additional targets that will improve the efficacy of chemotherapy is a major goal in cancer research. Through large-scale in vivo and in vitro CRISPR knockout screens in pancreatic ductal adenocarcinoma cells, we identified genes whose genetic deletion or pharmacologic inhibition synergistically increase the cytotoxicity of MEK signaling inhibitors. Furthermore, we show that CRISPR viability scores combined with basal gene expression levels could model global cellular responses to the drug treatment. We develop drug response evaluation by in vivo CRISPR screening (DREBIC) method and validated its efficacy using large-scale experimental data from independent experiments. Comparative analyses demonstrate that DREBIC predicts drug response in cancer cells from a wide range of tissues with high accuracy and identifies therapeutic vulnerabilities of cancer-causing mutations to MEK inhibitors in various cancer types., Predicting the response to chemotherapy is a major goal of cancer research. Here the authors use CRISPR knockout screens in pancreatic ductal adenocarcinoma cells to identify deletions synergistic with MEK inhibitors.

Chemoresistance is driven by unique regulatory networks in the genome that are distinct from those necessary for cancer development. Here, we investigate the contribution of enhancer elements to cisplatin resistance in ovarian cancers. Epigenome profiling of multiple cellular models of chemoresistance identified unique sets of distal enhancers, super-enhancers (SE), and their gene targets that coordinate and maintain the transcriptional program of the platinum-resistant state in ovarian cancer. Pharmacologic inhibition of distal enhancers through small-molecule epigenetic inhibitors suppressed the expression of their target genes and restored cisplatin sensitivity in vitro and in vivo. In addition to known drivers of chemoresistance, our findings identified SOX9 as a critical SE-regulated transcription factor that plays a critical role in acquiring and maintaining the chemoresistant state in ovarian cancer. The approach and findings presented here suggest that integrative analysis of epigenome and transcriptional programs could identify targetable key drivers of chemoresistance in cancers. Significance: Integrative genome-wide epigenomic and transcriptomic analyses of platinum-sensitive and -resistant ovarian lines identify key distal regulatory regions and associated master regulator transcription factors that can be targeted by small-molecule epigenetic inhibitors.

Whole-genome sequencing efforts of tumors and normal tissues have identified numerous genetic mutations, both somatic and germline, that do not overlap with coding genomic sequences. Attributing a functional role to these noncoding mutations and characterizing them using experimental methods has been more challenging compared with coding mutations. In this review, we provide a brief introduction to the world of noncoding mutations. We discuss recent progress in identifying noncoding mutations and the analytic and experimental approaches utilized to interpret their functional roles. We also highlight the potential mechanisms by which a noncoding mutation may exert its effect and discuss future challenges and opportunities.

Background The mutational processes underlying non-coding cancer mutations and their biological significance in tumor evolution are poorly understood. To get better insights into the biological mechanisms of mutational processes in breast cancer, we integrate whole-genome level somatic mutations from breast cancer patients with chromatin states and transcription factor binding events. Results We discover that a large fraction of non-coding somatic mutations in estrogen receptor (ER)-positive breast cancers are confined to ER binding sites. Notably, the highly mutated estrogen receptor binding sites are associated with more frequent chromatin loop contacts and the associated distal genes are expressed at higher level. To elucidate the functional significance of these non-coding mutations, we focus on two of the recurrently mutated estrogen receptor binding sites. Our bioinformatics and biochemical analysis suggest loss of DNA-protein interactions due to the recurrent mutations. Through CRISPR interference, we find that the recurrently mutated regulatory element at the LRRC3C-GSDMA locus impacts the expression of multiple distal genes. Using a CRISPR base editor, we show that the recurrent C→T conversion at the ZNF143 locus results in decreased TF binding, increased chromatin loop formation, and increased expression of multiple distal genes. This single point mutation mediates reduced response to estradiol-induced cell proliferation but increased resistance to tamoxifen-induced growth inhibition. Conclusions Our data suggest that ER binding is associated with localized accumulation of somatic mutations, some of which affect chromatin architecture, distal gene expression, and cellular phenotypes in ER-positive breast cancer. Electronic supplementary material The online version of this article (10.1186/s13059-018-1572-4) contains supplementary material, which is available to authorized users.