Recurrent Noncoding Mutations Research Articles

Abstract 1472: APOBEC-induced non-coding hotspot mutations near LEPROTL1 contribute to the tumorigenicity of bladder cancer via deregulation of LEPROTL1, DCTN6 and SARAF

Abstract In bladder cancer, APOBEC3 deaminases are major drivers of genomic mutagenesis, with most of these mutations occurring in synonymous or non-coding regions of the genome. The impact of these mutations on tumorigenesis has not been explored. This study aimed to investigate two non-coding hotspot mutations, likely induced by APOBEC3A near LEPROTL1—chr8: 29,952,919 G>A and chr8: 29,952,921 C>T—in bladder cancer. Using dual-luciferase reporter assays, we found that these mutations enhance both promoter and enhancer activities. Employing multi-omics approaches, including RNA-seq, ChiP-seq, and Hi-C, identified LEPROTL1, DCTN6, and SARAF as target genes co-regulated by a common enhancer where these hotspot non-coding mutations manifest. Isogenic cell lines, generated through CRISPR-mediated base editing to introduce these mutations, exhibited upregulation of target genes, and displayed oncogenic characteristics, such as increased cell growth, proliferation, invasiveness, and resistance to cisplatin. Moreover, these mutations exert their effect on cell proliferation by promoting cell cycle progression. These results suggest that recurrent non-coding somatic mutations upstream of LEPROTL1 disrupt the coregulation pattern of the three genes, thereby promoting bladder tumorigenesis. Citation Format: Bilal Lone, Kelly Butler, Alexandra Dobbins, Dhanusha Yesudhas, Arup Chakraborty, Evan Hickman, Salah Boudjadi, Andrea Apolo, Rouf Banday. APOBEC-induced non-coding hotspot mutations near LEPROTL1 contribute to the tumorigenicity of bladder cancer via deregulation of LEPROTL1, DCTN6 and SARAF [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 1472.

Identification of Recurrent Non-Coding Driver Mutations in Non-Hodgkin Lymphomas through Integrative Genomic Analysis of 777 Patients

IntroductionDiffuse large B-cell lymphoma (DLBCL) represents the most frequent NHL and comprises two molecular subgroups, namely germinal center B-cell-like (GCB) and activated B-cell-like (ABC) subgroups, each having distinct gene expression and genetic features. Follicular lymphoma (FL) is an indolent cancer that shares genetic features with DLBCL, particularly the GCB subgroup. Despite a growing knowledge about molecular features of these cancers. There remains a limited understanding of the recurrence of noncoding mutations that may impact gene function or expression in DLBCL, owing, in part, to their genetic complexity and a paucity of whole genome sequence (WGS) data. We sought to uncover novel mutation patterns through global meta-analysis of a large cohort of DLBCLs comprising 353 discovery cases, an independent set of 300 validation cases and comparison to genomes from 124 FLs.MethodsDLBCL exomes with matched constitutional DNA (n=200) from multiple studies and genomes from prior publications (40), ICGC (41) and 72 additional patients (n=153) were analyzed using a unified somatic single nucleotide variant (SNV) pipeline. Significantly mutated genes (SMGs) were identified using OncodriveFML. Recurrent regions affected by noncoding mutations (“peaks”) were determined using a novel approach - an extension of the rainfall plot computation followed by wavelet analysis. Structural variants (SV) were detected using Manta and copy number variations (CNVs) were determined using read-depth strategies. A separate set of 124 FL genomes was analyzed similarly. RNA-seq data were available for 143 of the DLBCL genomes and were processed using Salmon and analyzed for differential expression using DeSeq2. Using a custom capture panel, 82 genes were sequenced in some of the discovery cases and an independent DLBCL cohort comprising 300 cases. This allowed us to confirm and determine the recurrence of mutations and SVs affecting coding exons and UTRs. RNA-seq and SNP6.0 arrays were also applied to all validation cases to confirm patterns of coordinated expression.ResultsWe detected 52 SMGs (Fig. 1A) and 4382 mutation peaks among the DLBCL meta-cohort. Up to 24 of these have not previously been ascribed to DLBCL (Fig. 1A, purple). Most mutation peaks proximal to genes affected the first intron or promoter, consistent with AID activity. Many of the affected genes are known targets of aberrant somatic hypermutation though some novel targets of this process were identified. Non-genic peaks tended to affect putative distal enhancers of BCL6, IRF4 and PAX5. A subset of the noncoding regions and novel SMGs exhibited specificity for mutation within either ABC or GCB DLBCLs, including some genes involved in NF-kB signalling. Less than 10% of peaks were mutated among the FLs and these were largely those with a higher mutation burden in GCB DLBCL. We identified numerous genes recurrently affected by SVs, SNVs and CNVs and determined the subset of those with expression altered in the presence of such candidate regulatory mutations. For example, MEF2B and TOX both showed patterns indicative of tumour suppressor function, including a large number of focal deletions (Fig. 1C,D). In contrast, we observed patterns consistent with a role of mutations promoting the expression of each of TCF4, ΙΚΒΚΕ, NFKBIZ (two regulators of NF-κB) and the Fc γ receptor locus (Fig. 1B). TCF4 harbored mainly SV breakpoints that may disrupt some of the many annotated isoforms and leave others intact whereas NFKBIZ was predominantly affected by mutations in the 3'UTR. The FC γ locus contained numerous examples of focal gains and rearrangements involving this region and these were also detected in some of the FLs.ConclusionThis integrative genomic analysis offers numerous insights into the biology of DLBCL and features that distinguish it from indolent cancers arising in the germinal center. The novel coding and noncoding mutations found herein and those enriched in either DLBCL subgroup will aid to extend our understanding of the molecular underpinnings of these and their relationship to FL. Additional genes involved in NF-κB have been found to be deregulated through unique genetic mechanisms that have evaded detection though exome-based analyses. Further, the expression and sequence of Fc γ receptors is thought to impact the efficacy of anti-CD20 therapies, though the prevalence of somatic alterations has not been thoroughly explored. [Display omitted] DisclosuresSehn:Celgene: Consultancy, Honoraria; Amgen: Consultancy, Honoraria; Janssen: Consultancy, Honoraria; Abbvie: Consultancy, Honoraria; Roche/Genentech: Consultancy, Honoraria; Seattle Genetics: Consultancy, Honoraria. Steidl:Affimed Therapeutics: Consultancy; Juno Therapeutics: Consultancy. Connors:F Hoffmann-La Roche: Research Funding; NanoString Technologies, Amgen, Bayer, BMS, Cephalon, Roche, Genentech, Janssen, Lilly, Merck, Seattle Genetics, Takeda,: Research Funding; NanoString Technologies: Research Funding; Lilly: Research Funding; Janssen: Research Funding; Genentech: Research Funding; Cephalon: Research Funding; Bristol-Myers Squibb: Research Funding; Bayer Healthcare: Research Funding; Seattle Genetics: Research Funding; Merck: Research Funding; Amgen: Research Funding; Takeda: Research Funding. Scott:Janssen: Consultancy, Honoraria; Celgene: Consultancy, Honoraria; BCCA: Patents & Royalties: Patent describing molecular subtyping of DLBCL licensed to NanoString Technologies. Patent describing measurement of the proliferation signature in MCL.. Morin:Epizyme, Inc: Consultancy.

Disruption of the m-SWI/SNF Complex Mediated By Recurrent Non-Coding Mutations in BCL7A Induces Tumor Cell Proliferation in Multiple Myeloma

Multiple myeloma (MM) is a biologically heterogeneous plasma cell malignancy, however, the mechanisms underlying this complexity are incompletely understood. By deep (70x) whole-genome sequencing (WGS) of 312 primary MM patients, we observed mutations in about 62% of patients in the 5' untranslated region and intron 1 of the 12q24.3 region which cytogenetically marks the BCL7A gene. Integration of WGS with RNA-seq data suggested a widespread loss of BCL7A expression in MM cells as compared to normal plasma cells (PC). To understand the effect of noncoding mutations in BCL7A promoter we performed in-vitro luciferase reporter assay in MM cell lines (H929, MM1S and KK1) devoid of BCL7A mutation in 5'UTR and found that the normalized luciferase reporter activity indicated that the mutation in the promoter significantly reduced BCL7A promoter activity. Next, we recapitulated the loss of BCL7A observed in MM patients, in a panel of MM cell lines using shRNA based genetic interference and observed the appearance of a more proliferative phenotype. The effects of BCL7A loss in MM cells were further confirmed using CRISPR-Cas9 system. BCL7A-KO (knock-out) cells had higher proliferative rate compared to wild type (WT) cells and lentiviral add back of BCL7A plasmid reversed this effect. This phenomenon was validated in a in vivo model, where we found that mice bearing BCL7A KO MM cells developed tumors faster than mice injected with control or add-back cells, suggesting that BCL7A loss increases tumorigenesis in vivo. On the other hand, ectopic expression of BCL7A significantly reduced cell viability and colony formation over time, inducing apoptotic cell death and impacting genes involved in chromatin and chromosome organization, DNA repair and cell cycle. Interestingly, BCL7A is an important member of the m-SWI/SNF chromatin remodeling complex. Using comparative mass spectrometry analysis, we observed that BCL7A expression is essential for the formation of a functional canonical m-SWI/SNF complex in MM cells, controlling the incorporation of active sub-units into the complex. These data suggest that loss-of-function mutations in the BCL7A region may disrupt the formation of SWI/SNF complex activating a transcriptional program that leads to MM cell growth and viability. We therefore evaluated the transcriptomic changes in MM cells upon BCL7A perturbation in a panel of MM cell lines by whole genome RNA-seq. Integrated analysis of modulated genes identified a set of 23 genes significantly upregulated in the presence of BCL7A depletion and downregulated in presence of BCL7A ectopic expression. To investigate whether these genes are involved in the phenotypic and functional effects observed in MM after BCL7A depletion, we performed LOF (loss-of-function) studies (si-RNA screen) using these genes in scrambled and BCL7A KD (knock-down) MM cells. Among others, we observed that MM cells are highly sensitive to the inhibition of RPS3A (V-Fos transformation effector protein) only in the context of BCL7A loss. Our RNA-seq data revealed that RPS3A is highly expressed in primary MM cells, and its expression significantly correlates with low expression of BCL7A in MM patients. Importantly, we observed that RPS3A binds to the core m-SWI/SNF complex proteins; SMARCC2 and SMARCB1 in the absence of BCL7A. RPS3A gene encodes a ribosomal protein that is a component of the 40S subunit and its overexpression transforms NIH3T3 mouse fibroblasts and induces tumor formation in nude mice and also used as an expression marker in squamous cell carcinoma. In conclusion, we here report biological consequences of a frequent non-coding mutation of BCL7A in MM and loss-of-function mutations in the BCL7A region may disrupt the formation of SWI/SNF complex activating a transcriptional process that leads to MM cell growth and viability. Altogether this study shed lights on a new pathogenic mechanism that may drive MM growth with potential translational implication, that may be applicable to other hematological diseases. Disclosures Anderson: Oncopep and C4 Therapeutics.: Other: Scientific Founder of Oncopep and C4 Therapeutics.; Bristol Myers Squibb: Membership on an entity's Board of Directors or advisory committees; Sanofi-Aventis: Membership on an entity's Board of Directors or advisory committees; Janssen: Membership on an entity's Board of Directors or advisory committees; Gilead: Membership on an entity's Board of Directors or advisory committees; Millenium-Takeda: Membership on an entity's Board of Directors or advisory committees; Celgene: Membership on an entity's Board of Directors or advisory committees. Fulciniti:NIH: Research Funding. Munshi:BMS: Consultancy; OncoPep: Consultancy, Current equity holder in private company, Membership on an entity's Board of Directors or advisory committees, Patents & Royalties; C4: Current equity holder in private company; Janssen: Consultancy; Adaptive: Consultancy; Legend: Consultancy; Amgen: Consultancy; AbbVie: Consultancy; Karyopharm: Consultancy; Takeda: Consultancy.

Abstract PO-04: Noncoding mutations in mantle cell lymphoma disrupt regulation of HNRNPH1 by alternative splicing

Abstract Non-Hodgkin lymphomas (NHL) are a collection of cancers with each malignancy having distinct clinical management and prognosis. Mantle cell lymphoma (MCL) is particularly genetically heterogeneous and is considered incurable. Through a combination of exome, genome, and targeted sequencing of MCL tumors, we identified recurrent mutations in HNRNPH1 (heterogeneous nuclear ribonucleoprotein H1). These mutations are largely intronic or silent and are associated with a putative cis regulatory region involving a single exon. In RNA-seq data from matched cases, we identified variable representation of two distinct HNRNPH1 isoforms. Based on the reading frame of the affected exons, canonical splicing is predicted to produce a functional protein, while alternative splicing introduces a premature termination codon leading to nonsense-mediated decay. We observed a significantly higher proportion of canonical transcripts in MCL tumors bearing HNRNPH1 mutations, leading us to conclude that mutations in HNRNPH1 significantly alter its splicing. Furthermore, increased canonical splicing results in higher HNRNPH1 protein abundance as determined by immunohistochemical analysis of an MCL tissue microarray. HNRNPH1 mutation status and splicing ratio are associated with shorter survival of MCL patients. We developed an in vitro reporter minigene and introduced three specific mutations corresponding to the patient-identified mutations in HNRNPH1. These mutations lead to an increase in canonical splicing and translation of the HNRNPH1 minigene-derived peptide. Additionally, when HNRNPH1 is overexpressed, we observe a decrease of this peptide, implicating HNRNPH1 in the regulation of its own splicing. This is supported by preliminary data indicating that HNRNPH1 binds its own RNA. Beyond its own splicing, HNRNPH1 is likely also involved in the splicing of additional splicing factors. Overexpression of HNRNPH1 affects the splicing of SRSF3 and HNRNPDL, and many other targets are likely to be identified. This work elucidates a functional role for recurrent noncoding HNRNPH1 mutations and implicates HNRNPH1 expression and splicing of its downstream targets in lymphomagenesis. We continue to explore trans regulatory targets of HNRNPH1 using in vitro and cell-based models. While splicing is a growing field of interest in lymphoma biology, the unique pattern and consequences of these largely silent mutations specifically implicate alternative splicing as an oncogenic mechanism in MCL. Citation Format: Krysta M. Coyle, Quratulain Qureshi, Prasath Pararajalingam, Nicole Thomas, Timothy E. Audas, Ryan D. Morin. Noncoding mutations in mantle cell lymphoma disrupt regulation of HNRNPH1 by alternative splicing [abstract]. In: Proceedings of the AACR Virtual Meeting: Advances in Malignant Lymphoma; 2020 Aug 17-19. Philadelphia (PA): AACR; Blood Cancer Discov 2020;1(3_Suppl):Abstract nr PO-04.

Genomic Instability Signature of Palindromic Non-Coding Somatic Mutations in Bladder Cancer.

Simple SummaryBladder cancer is the tenth most common cancer worldwide, and its incidence has increased markedly in recent decades. However, current prognostic factors are insufficient to predict outcome at the individual level whereas non-coding somatic alterations remain weakly explored. The goal of this study was to identify clinical biomarkers in non-coding regions for bladder cancer patients. We identified a new type of frequent non-coding somatic genomic instability, specific to bladder tumors. This mutational signature is a promising candidate clinical biomarker for the early detection of relapse and a major low-cost alternative to the TMB to monitor the response to immunotherapy for bladder cancer patients.Numerous pan-genomic studies identified alterations in protein-coding genes and signaling pathways involved in bladder carcinogenesis, while non-coding somatic alterations remain weakly explored. The goal of this study was to identify clinical biomarkers in non-coding regions for bladder cancer patients. We have previously identified in bladder tumors two non-coding mutational hotspots occurring at high frequencies (≥30%). These mutations are located close to the GPR126 and PLEKHS1 genes, at the guanine or the cytosine of a TGAACA core motif flanked, on both sides, by a stretch of palindromic sequences. Here, we hypothesize that such a pattern of recurrent non-coding mutations could be a signature of somatic genomic instability specifically involved in bladder cancer. We analyzed 26 additional mutable non-coding sites with the same core motif in a cohort of 103 bladder cancers composed of 44 NMIBC cases and 59 MIBC cases using high-resolution melting (HRM) and Sanger sequencing. Five bladder cancers were additionally analyzed for protein-coding gene mutations using a targeted NGS panel composed of 571 genes. Expression levels of three members of the APOBEC3 family genes were assessed using real-time quantitative RT-PCR. Non-coding somatic mutations were observed for at least one TGAACA core motif locus in 62.1% (64/103) of bladder tumor samples. These non-coding mutations co-occurred in the bladder tumors but were absent in prostate tumor, HPV-positive Head and Neck Squamous Cell Carcinoma, and high microsatellite instability (MSI-H) colorectal tumor series. This signature of palindromic non-coding somatic mutations, specific to bladder tumors, was not associated with patients’ outcome and was more frequent in females. Interestingly, this signature was associated with high tumor mutational burden (TMB) and high expression levels of APOBEC3B and interferon inducible genes. We identified a new type of somatic genomic instability targeting the TGAACA core motif loci flanked by palindromic sequences in bladder cancer. This mutational signature is a promising candidate clinical biomarker for the early detection of relapse and a major low-cost alternative to the TMB to monitor the response to immunotherapy for bladder cancer patients.

Coding and noncoding drivers of mantle cell lymphoma identified through exome and genome sequencing

AbstractMantle cell lymphoma (MCL) is an uncommon B-cell non-Hodgkin lymphoma (NHL) that is incurable with standard therapies. The genetic drivers of this cancer have not been firmly established, and the features that contribute to differences in clinical course remain limited. To extend our understanding of the biological pathways involved in this malignancy, we performed a large-scale genomic analysis of MCL using data from 51 exomes and 34 genomes alongside previously published exome cohorts. To confirm our findings, we resequenced the genes identified in the exome cohort in 191 MCL tumors, each having clinical follow-up data. We confirmed the prognostic association of TP53 and NOTCH1 mutations. Our sequencing revealed novel recurrent noncoding mutations surrounding a single exon of the HNRNPH1gene. In RNA-seq data from 103 of these cases, MCL tumors with these mutations had a distinct imbalance of HNRNPH1 isoforms. This altered splicing of HNRNPH1 was associated with inferior outcomes in MCL and showed a significant increase in protein expression by immunohistochemistry. We describe a functional role for these recurrent noncoding mutations in disrupting an autoregulatory feedback mechanism, thereby deregulating HNRNPH1 protein expression. Taken together, these data strongly imply a role for aberrant regulation of messenger RNA processing in MCL pathobiology.

Novel Mutation Hotspots within Non-Coding Regulatory Regions of the Chronic Lymphocytic Leukemia Genome

Mutations in non-coding DNA regions are increasingly recognized as cancer drivers. These mutations can modify gene expression in cis or by inducing high-order chormatin structure modifications with long-range effects. Previous analysis reported the detection of recurrent and functional non-coding DNA mutations in the chronic lymphocytic leukemia (CLL) genome, such as those in the 3′ untranslated region of NOTCH1 and in the PAX5 super-enhancer. In this report, we used whole genome sequencing data produced by the International Cancer Genome Consortium in order to analyze regions with previously reported regulatory activity. This approach enabled the identification of numerous recurrently mutated regions that were frequently positioned in the proximity of genes involved in immune and oncogenic pathways. By correlating these mutations with expression of their nearest genes, we detected significant transcriptional changes in genes such as PHF2 and S1PR2. More research is needed to clarify the function of these mutations in CLL, particularly those found in intergenic regions.

Pathway and network analysis of more than 2500 whole cancer genomes

The catalog of cancer driver mutations in protein-coding genes has greatly expanded in the past decade. However, non-coding cancer driver mutations are less well-characterized and only a handful of recurrent non-coding mutations, most notably TERT promoter mutations, have been reported. Here, as part of the ICGC/TCGA Pan-Cancer Analysis of Whole Genomes (PCAWG) Consortium, which aggregated whole genome sequencing data from 2658 cancer across 38 tumor types, we perform multi-faceted pathway and network analyses of non-coding mutations across 2583 whole cancer genomes from 27 tumor types compiled by the ICGC/TCGA PCAWG project that was motivated by the success of pathway and network analyses in prioritizing rare mutations in protein-coding genes. While few non-coding genomic elements are recurrently mutated in this cohort, we identify 93 genes harboring non-coding mutations that cluster into several modules of interacting proteins. Among these are promoter mutations associated with reduced mRNA expression in TP53, TLE4, and TCF4. We find that biological processes had variable proportions of coding and non-coding mutations, with chromatin remodeling and proliferation pathways altered primarily by coding mutations, while developmental pathways, including Wnt and Notch, altered by both coding and non-coding mutations. RNA splicing is primarily altered by non-coding mutations in this cohort, and samples containing non-coding mutations in well-known RNA splicing factors exhibit similar gene expression signatures as samples with coding mutations in these genes. These analyses contribute a new repertoire of possible cancer genes and mechanisms that are altered by non-coding mutations and offer insights into additional cancer vulnerabilities that can be investigated for potential therapeutic treatments.

Non-Coding Mutations in Urothelial Bladder Cancer: Biological and Clinical Relevance and Potential Utility as Biomarkers

BACKGROUND & OBJECTIVE: Whole genome sequencing has identified recurrent non-coding mutations that may be important in carcinogenesis. We investigate the frequency of 5 such non-coding mutation hotspots in urothelial bladder cancers (UBCs) and as

Recurrent Non-Coding Mutations in BCL7A May Have Significant Functional Consequence in Multiple Myeloma

Multiple myeloma (MM) is a complex hematological malignancy characterized by gene pathway deregulations. Initial sequencing approaches have failed to identify any single frequent (>25%) mutation in the coding genome. We, therefore performed a deep (average coverage > 80X) whole genome sequencing (WGS) on 260 MM samples (208 newly diagnosed and 52 first relapse after uniform treatment) to comprehensively identify recurrent somatic alterations in non-coding regions. We have identified the most frequently involved genes affected by perturbation in neighboring non-coding region and integrate their expression using our matching deep RNA-seq data from the same patients. One of the most prominent examples is mutations in the 5' untranslated region and intron 1 of the BCL7A gene in 76% of myeloma patients. Integration of WGS with RNA-seq data confirmed significant downregulation of its expression (p values < 1e-5) in the MM cells as compared to normal plasma cells (PC). This led us to investigate the consequences of BCL-7A loss in MM. To evaluate the role of BCL7A in MM, using gain of- (GOF) and loss-of-function (LOF) approaches, we have utilized a large panel of MM cell lines with differential expression of BCL7A at the RNA and protein levels. Ectopic expression of BCL7A in a panel of 3 MM cell lines with low basal levels of BCL7a significantly reduced cell viability and colony formation over time. Inhibition of cell viability was associated with induction of apoptotic cell death in the BCL7A overexpressing cells compared to control cells. LOF studies in 3 MM cell lines with relatively higher expression of BCL7a using 3 BCL7A-specific shRNA constructs showed a more proliferative phenotype, with increased growth and viability and enhanced colony formation. The effects of BCL7A loss in MM cells were further confirmed using CRISPR-Cas9 system. BCL7a-KO cells had higher proliferative rate compared to WT cells and add back of lentiviral BCL7a plasmid reversed this effect. BCL7A is part of the SWI/SNF chromatin remodeling complex. Mutations in the genes encoding m-SWI/SNF subunits are found in more than 20% of human cancers, with subunit- and complex-specific functions. We confirmed that when expressed, BCL7A interacts with BCL11A into the SWI/SNF complex in MM cells. Comparative, mass spectrometry analysis in fact revealed SMARCC2 (BAF170), an integral subunit of SWI/SNF complex, to bind with BCL7A-BCL11A complex. However, BCL7A loss causes decreased SMARCC2 incorporation into SWI/SNF, thus suggesting that presence of BCL7A is crucial in the formation of SWI/SNF complex in MM cells and might play an important role in chromatin remodeling. Interestingly, oncogenes DEK (DNA binding oncogene) and TPD52 (tumor protein D52) involved in cancer cell proliferation and chromatin remodeling formed complex with BCL11A in BCL7A KO MM cells. Additionally, several anti-apoptotic proteins such as ANXA-1 and BCL2 are in complex with BCL11A when BCL7A is lost, suggesting the formation of an anti-apoptotic complex with consequences on MM cell survival. Currently ongoing studies are investigating the molecular mechanism of non-coding mutations impacting BCL7A expression and pathways affected by its downregulation with impact on MM cell growth and survival. In conclusion, we report biological consequences of a frequent (>75% patients) non-coding mutation in MM with cellular and molecular effects of BCL7A loss in which implicates a functional role of the m-SWI/SNF complex in driving a MM cell proliferative phenotype. Disclosures Anderson: Gilead Sciences: Other: Advisory Board; Janssen: Other: Advisory Board; Sanofi-Aventis: Other: Advisory Board; C4 Therapeutics: Other: Scientific founder ; OncoPep: Other: Scientific founder . Munshi:Abbvie: Consultancy; Abbvie: Consultancy; Amgen: Consultancy; Amgen: Consultancy; Adaptive: Consultancy; Adaptive: Consultancy; Celgene: Consultancy; Janssen: Consultancy; Janssen: Consultancy; Takeda: Consultancy; Takeda: Consultancy; Oncopep: Consultancy; Oncopep: Consultancy; Celgene: Consultancy.

Mutations Affecting RNA Binding Proteins Are a Novel Feature of Mantle Cell Lymphoma

Objectives Mantle cell lymphoma (MCL) is an uncommon B-cell non-Hodgkin lymphoma that is incurable with standard therapies. The genetic drivers of this cancer have not been firmly established and the features known to contribute to differences in clinical course remain limited. We sought to extend our understanding of the molecular etiology of this malignancy using an integrative genomic analysis of diagnostic biopsies. Methods We performed exome sequencing on 51 frozen MCL tumors and analyzed these alongside previously published exome cohorts. We sequenced tumour genomes and matched constitutional DNA from 34 frozen MCLs, along with matched constitutional DNA, to more broadly identify the pattern of non-coding mutations. Based on mutations identified in this discovery cohort, we re-sequenced 18 recurrently-mutated genes in 212 archival MCLs, each having clinical follow-up data. We also performed RNA-seq on 110 of these cases and analyzed these data for alternative splicing and differential expression, including the differential splicing of HNRNPH1 in the context of recurrent intronic mutations. We investigated the functional and phenotypic effect of mutations and deregulated HNRNPH1 protein through ectopic expression of full-length HNRNPH1 and a mini-gene containing the exons and introns affected by mutations. Using custom droplet digital PCR (ddPCR) assays, we validated alternative splicing patterns in HNRNPH1 itself and other targets identified through re-analysis of available CLIP-seq data. Results In addition to confirming the prognostic association of TP53 and NOTCH1 mutations in MCL, we identified two additional genes associated with outcome: EWSR1 with poor outcome (HR = 5.6) and MEF2B with good outcome (HR = 0.2). By comparing mutation patterns to diffuse large B-cell lymphoma (DLBCL), we identified an MCL-specific missense hot spot in MEF2B, non-specific truncating mutations in EWSR1, and truncating mutations affecting the DAZAP1 C-terminus in both MCL and DLBCL. The DAZAP1 mutations are predicted to alter protein sub-cellular localization and disrupt protein-protein interactions. We also identified the focal recurrence of non-coding mutations surrounding a single exon of the HNRNPH1 gene that were largely restricted to MCL. These mutations affected a region bound by HNRNPH1 protein and disrupted the preferred binding motif of this protein. Intronic mutations were significantly associated with alternative splicing of the HNRNPH1 mRNA and appear to disrupt a negative regulatory loop that normally limits the level of HNRNPH1. Using cell-based assays, we have evaluated the role of HNRNPH1 in cell survival and proliferation. Our interrogation of alternative splicing events in downstream targets implicate HNRNPH1 as a master splicing regulator which may broadly perturb the transcriptome and proteome to favor lymphomagenesis in MCL. Conclusions We discovered three novel MCL-related genes with roles in RNA trafficking or splicing, namely EWSR1, DAZAP1, and HNRNPH1. Mutations in these RNA-binding proteins were identified in 49 of 291 (17%) samples analyzed. Our results improve the current understanding of the MCL mutational landscape, highlight the similarities and differences between MCL and DLBCL, and strongly implicate a role for aberrant regulation of RNA metabolism in MCL pathobiology. We elucidated a functional role for recurrent non-coding HNRNPH1 mutations specific to MCL and identified multiple downstream targets. We continue to explore putative trans targets of HNRNPH1, a novel oncoprotein in MCL. Disclosures Steidl: Seattle Genetics: Consultancy; Roche: Consultancy; Bristol-Myers Squibb: Research Funding; Bayer: Consultancy; Nanostring: Patents & Royalties: Filed patent on behalf of BC Cancer; Juno Therapeutics: Consultancy; Tioma: Research Funding. Connors:Bristol-Myers Squibb: Consultancy; Seattle Genetics: Honoraria, Research Funding; Takeda Pharmaceuticals: Honoraria. Villa:Roche, Abbvie, Celgene, Seattle Genetics, Lundbeck, AstraZeneca, Nanostring, Janssen, Gilead: Consultancy, Honoraria. Johnson:Roche: Consultancy, Employment, Honoraria, Membership on an entity's Board of Directors or advisory committees, Other: Travel fees, gifts, and others, Research Funding; Abbvie: Consultancy, Employment, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Merck: Consultancy, Honoraria; BMS: Consultancy, Honoraria; BD Biosciences: Other: Provided a significant proportion of the antibodies used in this project free of cost.; Seattle Genetics: Honoraria; Lundbeck: Employment, Honoraria, Membership on an entity's Board of Directors or advisory committees, Other: Travel fees, gifts, and others, Research Funding. Scott:Janssen: Consultancy, Research Funding; NanoString: Patents & Royalties: Named inventor on a patent licensed to NanoSting [Institution], Research Funding; Celgene: Consultancy; Roche/Genentech: Research Funding.

Recurrent noncoding U1snRNA mutations drive cryptic splicing in SHH medulloblastoma.

Summary ParagraphRecurrent somatic single nucleotide variants (SNVs) in cancer are largely confined to protein coding genes, and are rare in most pediatric cancers1–3. We report highly recurrent hotspot mutations of U1 spliceosomal small nuclear RNAs (snRNAs) in ~50% of Sonic Hedgehog medulloblastomas (Shh-MB), which were not present across other medulloblastoma subgroups. This U1-snRNA hotspot mutation (r.3a>g), was identified in <0.1% of 2,442 cancers across 36 other tumor types. Largely absent from infant Shh-MB, the mutation occurs in 97% of adults (Shhδ), and 25% of adolescents (Shhα). The U1-snRNA mutation occurs in the 5′ splice site binding region, and snRNA mutant tumors have significantly disrupted RNA splicing with an excess of 5′ cryptic splicing events. Mutant U1-snRNA mediated alternative splicing inactivates tumor suppressor genes (PTCH1), and activates oncogenes (GLI2, CCND2), represents a novel target for therapy, and constitutes a highly recurrent and tissue-specific mutation of a non-protein coding gene in cancer.

Recurrent Noncoding Mutations in Skin Cancers: UV Damage Susceptibility or Repair Inhibition as Primary Driver?

Somatic mutations arising in human skin cancers are heterogeneously distributed across the genome, meaning that certain genomic regions (e.g., heterochromatin or transcription factor binding sites) have much higher mutation densities than others. Regional variations in mutation rates are typically not a consequence of selection, as the vast majority of somatic mutations in skin cancers are passenger mutations that do not promote cell growth or transformation. Instead, variations in DNA repair activity, due to chromatin organization and transcription factor binding, have been proposed to be a primary driver of mutational heterogeneity in melanoma. However, as discussed in this review here, recent studies indicate that chromatin organization and transcription factor binding also significantly modulate the rate at which UV lesions form in DNA. The authors propose that local variations in lesion susceptibility may be an important driver of mutational hotspots in melanoma and other skin cancers, particularly at binding sites for ETS transcription factors.

Identification of recurrent noncoding mutations in B-cell lymphoma using capture Hi-C

AbstractThe search for driver mutations is critical to the understanding of many malignancies and has yielded important targets for therapeutic intervention. To date, most of these studies have focused on coding regions of the genome. Cornish and colleagues explore the potential role of noncoding regions of the genome in a large cohort of lymphoma patients.

Promoter capture Hi-C-based identification of recurrent noncoding mutations in colorectal cancer

Efforts are being directed to systematically analyze the non-coding regions of the genome for cancer-driving mutations1-6. cis-regulatory elements (CREs) represent a highly enriched subset of the non-coding regions of the genome in which to search for such mutations. Here we use high-throughput chromosome conformation capture techniques (Hi-C) for 19,023 promoter fragments to catalog the regulatory landscape of colorectal cancer in cell lines, mapping CREs and integrating these with whole-genome sequence and expression data from The Cancer Genome Atlas7,8. We identify a recurrently mutated CRE interacting with the ETV1 promoter affecting gene expression. ETV1 expression influences cell viability and is associated with patient survival. We further refine our understanding of the regulatory effects of copy-number variations, showing that RASL11A is targeted by a previously identified enhancer amplification1. This study reveals new insights into the complex genetic alterations driving tumor development, providing a paradigm for employing chromosome conformation capture to decipher non-coding CREs relevant to cancer biology.

Abstract 1418: Identification of recurrent regulatory mutations in breast cancer

Abstract Since the identification of recurrent TERT promoter mutations in melanoma resulting in increased TERT expression, there has been increased interest in identifying recurrent regulatory non-coding mutations (Horn et al. 2013, Huang et al. 2013). Several studies have attempted pan-cancer analyses in order to identify these types of mutations, but often the results suffer from low coverage of regulatory regions or do not extend to breast cancer. While some breast cancer specific studies have identified some significantly mutated promoters and lncRNAs, they have often failed to incorporate transcriptome data to assess the impact and relevance of mutations on the expression of genes within tumors (Nik-Zainal et al. 2016). In order to address this, we assembled and generated a data set consisting of 458 breast cancer cases with matched tumor/normal pairs. This cohort consists of 22.4% luminal A, 19% luminal B, 16.4% HER2-enriched, 21% basal-like, 0.8% normal-like, and 20.4% unknown with regards to molecular subtype. This is important due to different breast cancer subtypes having dissimilar phenotypes and varying rates of gene coding mutations. This data set has a mix of whole genome, exome, transcriptome, and custom capture sequencing. We designed a custom capture reagent that covers regions assembled from regulatory databases, 5' untranslated regions, 500 bases upstream and downstream of transcription start sites, and 50,000 bases upstream and downstream of 178 genes that have been implicated as being important in breast cancer (Lesurf et al. 2016). While this custom capture region is similar in size to an exome, it has advantages over whole genome and exome sequencing, particularly with respect to coverage in GC-rich promoter regions. With these data, we predict that we will be able to identify novel, regulatory coding and non-coding drivers of breast cancer that would not be discovered without integrated analysis of the DNA- and RNA-seq data for each tumor. Instrument data were processed using the McDonnell Genome Institute somatic variant calling pipeline that includes 5 SNV callers and 3 indel callers. We then used these steps to filter variants: min. 20x coverage in both the tumor and normal sample, min. 2.5% tumor variant allele frequency, min. 3 variant supporting reads in the tumor sample, max. 10% variant allele frequency in the normal sample. We also filtered against gnomAD and a panel of normals. Rheinbay et al. 2017 identified recurrently mutated promoter regions for nine genes: TBC1D12, ZNF143, ALDOA, NEAT1, RMRP, CITED2, FOXA1, CTNNB1, LEPROTL1. Our preliminary analysis has revealed that we also see mutations within these regions. We plan to present on the significance of mutations within these previously seen regions, based on recurrence and transcriptome changes, as well as novel recurrent regulatory regions that our analysis reveals, particularly with respect to molecular subtype. Citation Format: Kelsy C. Cotto, Arpad Danos, Robert Lesurf, Morag Park, Malachi Griffith, Obi L. Griffith. Identification of recurrent regulatory mutations in breast cancer [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 1418.

Abstract 2281: Flexible discovery of recurrent coding and non-coding mutations in tumor whole genomes

Abstract Analysis of somatic protein-coding mutations, particularly via tumor exome sequencing, has driven discovery of novel cancer genes in recent years. However, ~97% of the human genome is non-coding and not addressable by exome-targeting methods. Tumor whole genome sequencing, by contrast, allows for genome-wide discovery of somatic events and may identify novel non-coding driver mutations. In conjunction, flexible tools employing principled statistical methods are needed for discovery of recurrent somatic driver mutations from tumor whole genomes. Here, we developed a computational toolset that addresses these needs and describe its application to a novel cohort of lung adenocarcinoma tumors generated by the APOLLO (Applied Proteogenomics OrganizationaL Learning and Outcomes) consortium. Our toolset flexibly computes feature-wise and hotspot somatic mutation enrichment statistics in both protein-coding and non-coding (e.g. promoters, enhancers) genomic regions. Additionally, we incorporated an affinity propagation-based clustering procedure that groups test regions by user-specified genomic covariates that can influence local mutation properties. We applied our toolset to several TCGA datasets of tumor exome sequencing. We found strong agreement between our results and those of other tools: among the top 15 genes called at FDR &amp;lt; 0.01 by MutSigCV, we observed a median overlap of 75.7% with genes ranked in the top 15 by our methods. We next applied our toolset to tumor whole genome sequencing data from our APOLLO lung adenocarcinoma cohort. During analysis, we simultaneously controlled for several genomic covariates, including GC/CpG content, replication timing, and proximal mRNA expression. Gene-centric analysis identified several significantly mutated genes, including KRAS, STK11, and TP53 (FDR &amp;lt; 0.005). We examined matched mRNA-seq data from this cohort and found significant over-expression of KRAS mRNA in samples possessing a non-silent coding mutation in this gene (p &amp;lt; 0.02). We additionally assessed somatic mutation enrichment in promoters, 3' UTRs, non-coding genes, and lung-specific enhancers. From these results, we found significant enrichment of somatic mutations in the body of the long non-coding RNA NEAT1. Among the tumors with somatic NEAT1 mutations, a single sample was hypermutated at this locus and corresponded with the lowest observed RNA expression for this lncRNA in our cohort (TPM of 12.2 vs. overall mean TPM of 84.2). In conclusion, we developed a flexible toolset for interrogating both coding and non-coding landscapes from tumor whole genomes. We applied our methods to a novel cohort of lung adenocarcinoma tumors and identified recurrently mutated genes and non-coding regions, including the NEAT1 lncRNA. Citation Format: Anthony R. Soltis, Coralie Viollet, Harvey B. Pollard, Christopher A. Moskaluk, Robert F. Browning, Clifton L. Dalgard, Craig D. Shriver, Matthew D. Wilkerson. Flexible discovery of recurrent coding and non-coding mutations in tumor whole genomes [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 2281.

Abstract 2457: Investigating the functional significance of novel, recurrent noncoding mutations of TBC1D12 in bladder cancer

Abstract Somatic noncoding mutations remain widely unexplored despite examples of critical importance such as driver mutations in the promoter of the TERT gene. We recently identified novel somatic mutations in the regulatory Kozak sequence (just upstream of the translation start site) of TBC1D12 in ~15% of bladder cancer patients, making it one of the most frequently mutated noncoding regions in any cancer (Araya et. al 2016). TBC1D12 was not previously implicated in cancer, and little is currently known about its function. It is a member of the TBC1 superfamily, suggesting a role in intracellular trafficking as a RAB-specific GTPase-activating protein (GAP). To investigate the functional implications of TBC1D12 mutations in bladder cancer, we undertook a combined computational and experimental approach. We analyzed clinical correlates, identified downstream effector candidates, and assessed the impact of these mutations on gene expression. To determine the clinical relevance of the TBC1D12 mutation, we did correlation and survival analyses of clinical data of bladder cancer patients from TCGA. We found that TBC1D12 mutations confer slightly worse survival but were not linked to other clinical correlates. To identify potential downstream effectors, we used reverse phase protein array (RPPA) data from 127 patients to search for proteins that were differentially expressed or phosphorylated between patients with or without TBC1D12 mutations. We found differential expression of GSK3 and differential phosphorylation of p90RSK and c-Jun, suggesting three potential downstream effector candidates. Hierarchical clustering of RPPA data for these three proteins revealed 4 subgroups with distinct molecular phenotypes. Furthermore, most of the TBC1D12 mutants were clustered within one molecular subtype. To experimentally test these downstream effector candidates and identify new ones, we modulated the levels of TBC1D12 in bladder cancer cell line Ku-19-19 using overexpression plasmids or siRNAs. After validating that these transfections were effective in changing TBC1D12 expression, we performed RNA-Seq experiments. Results from this study are expected to be presented at the meeting. In summary, these studies suggest that the novel and recurrent noncoding mutations in TBC1D12 may affect patient survival and alter the expression or post-translational modification of downstream targets. Potential molecular mechanisms associated with the presence of TBC1D12 mutations will be discussed, and a series of follow-up studies are underway. These studies also demonstrate that applying both computational and experimental approaches can help develop and test evidence-based hypotheses, contributing to an understanding of the clinical and functional impact of TBC1D12. Citation Format: Angela S. Li, Jason A. Reuter, Can Cenik, Michael P. Synder. Investigating the functional significance of novel, recurrent noncoding mutations of TBC1D12 in bladder cancer [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 2457. doi:10.1158/1538-7445.AM2017-2457

Recurrent noncoding regulatory mutations in pancreatic ductal adenocarcinoma.

The contributions of coding mutations to tumorigenesis are relatively well known; however, little is known about somatic alterations in noncoding DNA. Here we describe GECCO (Genomic Enrichment Computational Clustering Operation) to analyze somatic noncoding alterations in 308 pancreatic ductal adenocarcinomas (PDAs) and identify commonly mutated regulatory regions. We find recurrent noncoding mutations are enriched in PDA pathways, including axon guidance and cell adhesion, and novel processes including transcription and homeobox genes. We identify mutations in protein binding sites correlating with differential expression of proximal genes and experimentally validate effects of mutations on expression. We developed an expression modulation score that quantifies the strength of gene regulation imposed by each class of regulatory elements, and find the strongest elements are most frequently mutated, suggesting a selective advantage. Our detailed single-cancer analysis of noncoding alterations identifies regulatory mutations as candidates for diagnostic and prognostic markers, and suggests novel mechanisms for tumor evolution.

Abstract A14: Convergence analysis of regulatory mutations into immuno-modulatory pathways across 14 tumor types

Abstract Evasion of immune system surveillance is a hallmark of cancer. Cancer cells may secrete cytokines like TGF-β which hampers cytotoxic T-cells and natural killer cells in addition to recruiting tumor-infiltrating regulatory T-cells endowed with immunosuppressive potential. Little is known about the genomic basis for immune evasion especially in the context of dysregulation and rewiring of the immune-related circuitry of tumor cells. A vast majority of mutations in cancer frequently occur in non-coding regions. The functional impact of these mutations in mediating interactions with the tumor microenvironment have largely been unexplored. Given that recent efforts have implicated non-coding elements in various disease association studies, it can be expected that a significant number of recurrent non-coding mutations in cancer have a regulatory effect. Pan-Cancer analysis has improved the discovery and analysis of these regulatory mutations while avoiding the type I and type II errors made in several tissue-specific cancer projects. By leveraging recent pan-cancer whole-genome sequencing efforts, we have been able to characterize the non-coding mutational profiles of 505 samples, spread across 14 tumor types. These methods amplify the power to detect heterogeneous signals of positive selection thereby enhancing our ability to distinguish ‘driver’ from ‘passenger’ alterations. This study aims at applying a pan-cancer genome-wide approach towards identifying regulatory mutations that potentially impact immune modulation and evasion. We first infer the genome-wide position- and sample-specific probabilities of mutation from somatic mutations calls. A multinomial logistic regression model describes the relationship between the mutation rate and a set of explanatory variables such as the sample ID, replication timing, the genomic context and the local mutation rate. The predicted site-specific probabilities, when overlaid with tissue-specific annotations from the Roadmap Epigenomics consortium and GENCODE, allow us to derive a test statistic for each cis non-coding region that yields information regarding the likelihood that the region of interest is a “driver region”. By utilizing published databases on enhancer-gene links, we extend this framework to comprehensively characterize distal enhancer regions mapped with their target genes. We then systematically identify key immunomodulatory networks enriched for non-coding mutations and evaluate these in a pan-cancer context. A number of immune-related genes are found to harbor an excess of non-coding mutations suggesting higher-order regulatory convergence. We assign a significance score to this convergence by factoring the proximity to the target gene as well as the genomic instability modeled through local copy number changes. Therefore, we integrate individual low-frequency alterations into high-frequency recurrent events across different tumor types. We believe that the proposed model presents an unbiased method towards characterizing the impact of regulatory mutations towards immunomodulation, immune suppression and evasion. We anticipate that this approach will serve as a template for future functional non-coding mutational dissections in tumor-related studies. Citation Format: Karthik Murugadoss, Malene Rasmussen, Alvin Shi, Manolis Kellis. Convergence analysis of regulatory mutations into immuno-modulatory pathways across 14 tumor types. [abstract]. In: Proceedings of the AACR Special Conference on Tumor Immunology and Immunotherapy; 2016 Oct 20-23; Boston, MA. Philadelphia (PA): AACR; Cancer Immunol Res 2017;5(3 Suppl):Abstract nr A14.