Non-coding Mutations Research Articles

Abstract 3933: Comprehensive molecular characterization of mitochondrial mutational landscape across the evolution of lung adenocarcinoma

Abstract The human mitochondrial genome is prone to genetic alterations, which lead to somatic mutations, changes in mtDNA copy number and altered expression of respiratory chain subunits. This is in part due to the fact that mtDNA has a limited ability to repair itself when it is damaged. Although a buildup of somatic mutations in mtDNA has been associated with increased cancer risk, the role of mtDNA alterations in malignant transformation of lung adenocarcinoma has not been addressed. Therefore, a clear understanding of changes in mtDNA along the pathway of lung tumorigenesis is critical for identifying molecular biomarkers related to carcinogenesis and tumor progression. Like all solid tumors, lung adenocarcinoma is thought to be initiated and to progress through a series of genetic alterations, including changes in mtDNA. Lungs resected for primary adenocarcinomas often harbor minute discrete foci of cytologically atypical pneumocyte proliferations designated as atypical adenomatous hyperplasia (AAH). Evidence suggests that AAH represents an initial step in the progression to adenocarcinoma in situ (AIS), minimally invasive adenocarcinoma (MIA) and ultimately fully invasive adenocarcinoma (ADA). In this study we used a unique cohort of 52 patients with matched specimens (n=133) representing different steps of disease progression (AAH, AIS, and MIA) coupled with novel ultra-deep mitochondrial sequencing (mtDNA-Seq) method to assess the mtDNA mutational landscape throughout the continuum of lung adenocarcinoma development. Utilizing a custom bioinformatics workflow, somatic mutations were detected in a subset of the premalignant non-invasive lesions, with overall higher mutational load observed in MIA and ACA specimens. Additionally, large degree of inter-patient heterogeneity were observed for mtDNA copy numbers in conjunction with the clonal histological evolution of pulmonary neoplasia. Moreover, an appreciable number of coding and non-coding mutations from regulatory regions of the mtDNA were detectable in the precursor AAH lesions and the continuum of LUAD progression. Majority of shared mutations showed heteroplasmic transition with increase in fractional abundance in ADA, compared to the AHH lesions, thereby indicating spatial clonal expansion as they progressed histologically. Here we report the first comprehensive characterization of mitochondrial mutational landscape in dynamic evolution of lung adenocarcinoma, and provide insight into the heterogeneity of clonal events associated with the progression from noninvasive to malignant disease. Citation Format: Alka Singh, Santanu Dasgupta, Ashwin L. Koppayi, Lisa Rooper, Vasudha Mishra, David Sidransky, Nishant Agrawal, Karthik Suresh, Evgeny Izumchenko. Comprehensive molecular characterization of mitochondrial mutational landscape across the evolution of lung adenocarcinoma [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 3933.

Abstract 7358: Role of noncoding genome architecture in esophageal cancer

Abstract Esophageal Cancer is a deadly disease with a tendency to be diagnosed at the later stages. Considered a rare disease, accounting for 1.1% of all cancers, EC comprises 2.6% of all cancer deaths. The survival rate for all stages is 21.7%. However, the survival rate decreases to 5.6 % for advanced-stage cancers. EC is more common in men and associated with older age and heavy alcohol and tobacco use. In the last 25 years, the death rate of EC has been decreasing slightly from 4.3 to 3.7. The death rates for lung, prostate, and colon cancers have decreased by half in the same time frame. Therefore, novel therapies and biomarkers are urgently needed to accelerate survival and reduce the death rates by at least half for patients with EC.We previously completed exome sequencing in a small sample size and observed that the mutations in the noncoding regions of the EC genome are highly affected. In this study, we perform exome sequencing in 110 paired esophageal tumors and normal tissues using an Agilent SSELXT HS Exon V8+UTR library enrichment kit. Twelve paired EC and normal tissues are sequenced using the PacBio Hifi system.In WES, we detected almost half of the noncoding region mutations in the 3UTR and 2kb downstream regions of the genome, significantly affecting 51 oncogenic or tumor suppressor genes. In addition, the involved genes are over-represented in critical signaling pathways and transcriptional regulation of pluripotent stem cells. The number of changes in the 5UTR and 2kb upstream are similar to the number of variations in the intronic regions. Apoptosis, stress response, and DNA double-strand break repair pathways are affected by 5UTR mutations. Mutations in the intronic regions frequently occurred in pro-tumorigenic immune regulation genes, such as IL-33 and IL-6, and oncogene-induced senescence.The combined effect of these genetic and coding region mutations on oncogenes, significant signaling pathways, cancer cell stemness, DNA damage repair pathways, and immune system regulation may significantly impact poor prognosis in EC patients. Further studies will verify the effectiveness of targeting these genes for developing candidate therapeutics. Citation Format: Hayriye Verda Erkizan, Robert Wadleigh. Role of noncoding genome architecture in esophageal cancer [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 7358.

Abstract 3057: The TARZN complex binds to de novo enhancer mutations and promotes oncogenic expression in T-ALL

Abstract TAL1 is overexpressed in 40-60% of T-cell acute lymphoblastic leukaemia (T-ALL) cases and forms an oncogenic core regulatory circuit (CRC) with other transcription factors such as LMO1, LMO2 and GATA3. In 5% of T-ALL cases an insertion of a consensus GT dinucleotide is observed upstream of the TAL1 gene, termed the “Mutation of TAL1 enhancer” (MuTE), driving TAL1 overexpression. Using an in vitro reconstitution DNA pull-down assay combined with quantitative mass spectrometry, we identified nine candidate proteins that preferentially bound to the MuTE sequence compared to the WT sequence. Among the candidates, we demonstrate by reciprocal immunoprecipitation experiments coupled to quantitative mass spectrometry that the RNA methyltransferase TARBP1 and the zinc finger proteins ZBTB2, ZBTB25 and ZNF639 formed a complex that we term TARZN. Interestingly, the TARZN complex also bound to de novo super enhancer sites upstream of the LMO1 and LMO2 genes in T-ALL cells, indicating a putative common mechanism between these different non-coding driver mutations. Furthermore, knock-down of all TARZN members resulted in lower TAL1 protein expression in MuTE-positive but not in MuTE-negative T-ALL cells. Overall, these data suggest that the TARZN complex cooperatively promotes oncogenic expression in T-ALL. Citation Format: Nurkaiyisah Zaal Anuar, Chai Yeen Goh, Boon Haow Chua, Shi Hao Tan, Joana R. Costa, Marc R. Mansour, Takaomi Sanda, Dennis Kappei. The TARZN complex binds to de novo enhancer mutations and promotes oncogenic expression in T-ALL [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 3057.

Abstract 4887: Unveiling the functional impact of glioma non-coding germline variants using large language models

Abstract Efforts to identify the risk for developing gliomas through Genome-Wide Association Studies (GWAS) face relevant challenges, such as accurately pinpointing the genomic positions of germline variants, primarily found in non-coding regions, with unclear functional implications in glioma development. In this context, the GET method, a large language model designed to unveil regulatory grammars across 213 human cell types, emerges as a valuable tool for elucidating the mechanisms of non-coding mutations. Our aims include: I) Validate the efficacy of the GET method using a well-documented case and prioritize relevant variants for analysis; II) Refine the genomic positions of these variants and exploring their linkage disequilibrium (LD); III) Identify potential motifs and genes that might be influenced by these high-impact variants. After examining published GWAS, 39 non-coding SNPs in gliomas were found. Adding 316 variants in high LD (R2 > 0.8) expanded the dataset to 355 variants for analysis. Using the GET method, we calculated impact scores (ISs) for each variant, considering changes in motif binding score (ΔBM) and motif gradient (GM) across human astrocytes. We focused on rare variants (risk allele frequency <0.25) with the higher odds ratios (OR). The table presents GET results for a well-known case-control, showing its role in increasing glioma risk through OCT4 motif disruption and enhanced MYC expression, as previously was confirmed experimentally. Moreover, it details results concerning the 5 rare germline variants exhibiting the highest ISs, including precise genomic positions and the most affected motifs and genes that could potentially be impacted by each variant. Using a large language model, this study overcomes the challenge of mere variant identification, aiming to establish a framework for uncovering the underlying mechanism of risk variants. While further validation is essential, this is a promising approach to explore the impact of germline mutations. Variant ID Genomic Position Altered Motif Affected Gene Ref allele Alt allele Case-control rs55705857 Chr8:129633445 OCT4+SOX2:POU MYC A G H-I variant #1 rs78378222 Chr17:7668433 HD/18 PER1 T G H-I variant #2 rs518394 Chr9:22019673 KLF/SP/2 CDKN2A G C H-I variant #3 rs79495512 Chr15:76199995 KLF/SP/2 LINGO1 T C H-I variant #4 rs78185702 Chr15:76317893 SMAD LINGO1 C T H-I variant #5 rs2538067 Chr7:54907724 CTCF EGFR G A Citation Format: Maria del Mar Alvarez-Torres, Xi Fu, Romella K Sagatelian, Alejandro L Buendia, Raul Rabadan. Unveiling the functional impact of glioma non-coding germline variants using large language models [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 4887.

Notch signaling genes and CD8+ T-cell dynamics: Their contribution to immune-checkpoint inhibitor therapy in oral squamous cell carcinoma: A retrospective study.

Aberrant Notch signaling pathway has been related with the tumorigenesis in head and neck region, involving oral cavity. Here, we report the correlation between mutations in the Notch signaling pathway and CD8+ T-cell infiltration via PD-L1, which lead to enhanced antitumor immunity and may target for immune-checkpoint inhibitors (ICIs) therapy. This retrospective study analyzed the results of immunohistochemical staining for PD-L1 and CD8+ T-cell infiltration in 10 patients and whole-exome sequencing (WES) was conducted on five of these patients to identify frequently mutated genes. Four of 10 patients were positive for PD-L1 and CD8+ T. By analyzing WES in three of these four patients, we notably identified the mutations of NOTCH1, FBXW7, and noncoding RNA intronic mutation in NOTCH2NLR in two of these three patients. This study may enable better selection of ICI therapy with CD8+ T-cell infiltration via PD-L1 expression for oral squamous cell carcinoma patients with mutations in Notch signaling pathway.

Functional dissection of human cardiac enhancers and noncoding de novo variants in congenital heart disease.

Rare coding mutations cause ∼45% of congenital heart disease (CHD). Noncoding mutations that perturb cis-regulatory elements (CREs) likely contribute to the remaining cases, but their identification has been problematic. Using a lentiviral massively parallel reporter assay (lentiMPRA) in human induced pluripotent stem cell-derived cardiomyocytes (iPSC-CMs), we functionally evaluated 6,590 noncoding de novo variants (ncDNVs) prioritized from the whole-genome sequencing of 750 CHD trios. A total of 403 ncDNVs substantially affected cardiac CRE activity. A majority increased enhancer activity, often at regions with undetectable reference sequence activity. Of ten DNVs tested by introduction into their native genomic context, four altered the expression of neighboring genes and iPSC-CM transcriptional state. To prioritize future DNVs for functional testing, we used the MPRA data to develop a regression model, EpiCard. Analysis of an independent CHD cohort by EpiCard found enrichment of DNVs. Together, we developed a scalable system to measure the effect of ncDNVs on CRE activity and deployed it to systematically assess the contribution of ncDNVs to CHD.

Deep whole-genome analysis of 494 hepatocellular carcinomas.

Over half of hepatocellular carcinoma (HCC) cases diagnosed worldwide are in China1-3. However, whole-genome analysis of hepatitis B virus (HBV)-associated HCC in Chinese individuals is limited4-8, with current analyses of HCC mainly from non-HBV-enriched populations9,10. Here we initiated the Chinese Liver Cancer Atlas (CLCA) project and performed deep whole-genome sequencing (average depth, 120×) of 494 HCC tumours. We identified 6 coding and 28 non-coding previously undescribed driver candidates. Five previously undescribedmutational signatures were found, including aristolochic-acid-associated indel and doublet base signatures, and a single-base-substitution signature that we termed SBS_H8. Pentanucleotide context analysis and experimental validation confirmed that SBS_H8 was distinct to the aristolochic-acid-associated SBS22. Notably, HBV integrations could take the form of extrachromosomal circular DNA, resulting in elevated copy numbers and gene expression. Our high-depth data also enabled us to characterize subclonal clustered alterations, including chromothripsis, chromoplexy and kataegis, suggesting that these catastrophic events could also occur in late stages of hepatocarcinogenesis. Pathway analysis of all classes of alterations further linked non-coding mutations to dysregulation of liver metabolism. Finally, we performed in vitro and in vivo assays to show that fibrinogen alpha chain (FGA), determined as both a candidate coding and non-coding driver, regulates HCC progression and metastasis. Our CLCA study depicts a detailed genomic landscape and evolutionary history of HCC in Chinese individuals, providing important clinical implications.

Integrative analysis of noncoding mutations identifies the druggable genome in preterm birth.

Preterm birth affects ~10% of pregnancies in the US. Despite familial associations, identifying at-risk genetic loci has been challenging. We built deep learning and graphical models to score mutational effects at base resolution via integrating the pregnant myometrial epigenome and large-scale patient genomes with spontaneous preterm birth (sPTB) from European and African American cohorts. We uncovered previously unidentified sPTB genes that are involved in myometrial muscle relaxation and inflammatory responses and that are regulated by the progesterone receptor near labor onset. We studied genomic variants in these genes in our recruited pregnant women administered progestin prophylaxis. We observed that mutation burden in these genes was predictive of responses to progestin treatment for preterm birth. To advance therapeutic development, we screened ~4000 compounds, identified candidate molecules that affect our identified genes, and experimentally validated their therapeutic effects on regulating labor. Together, our integrative approach revealed the druggable genome in preterm birth and provided a generalizable framework for studying complex diseases.

High UV damage and low repair, but not cytosine deamination, stimulate mutation hotspots at ETS binding sites in melanoma

Noncoding mutation hotspots have been identified in melanoma and many of them occur at the binding sites of E26 transformation-specific (ETS) proteins; however, their formation mechanism and functional impacts are not fully understood. Here, we used UV (Ultraviolet) damage sequencing data and analyzed cyclobutane pyrimidine dimer (CPD) formation, DNA repair, and CPD deamination in human cells at single-nucleotide resolution. Our data show prominent CPD hotspots immediately after UV irradiation at ETS binding sites, particularly at sites with a conserved TTCCGG motif, which correlate with mutation hotspots identified in cutaneous melanoma. Additionally, CPDs are repaired slower at ETS binding sites than in flanking DNA. Cytosine deamination in CPDs to uracil is suggested as an important step for UV mutagenesis. However, we found that CPD deamination is significantly suppressed at ETS binding sites, particularly for the CPD hotspot on the 5' side of the ETS motif, arguing against a role for CPD deamination in promoting ETS-associated UV mutations. Finally, we analyzed a subset of frequently mutated promoters, including the ribosomal protein genes RPL13A and RPS20, and found that mutations in the ETS motif can significantly reduce the promoter activity. Thus, our data identify high UV damage and low repair, but not CPD deamination, as the main mechanism for ETS-associated mutations in melanoma and uncover important roles of often-overlooked mutation hotspots in perturbing gene transcription.

Enhancer mutations modulate the severity of chemotherapy-induced myelosuppression.

Non-small cell lung cancer is often diagnosed at advanced stages, and many patients are still treated with classical chemotherapy. The unselective nature of chemotherapy often results in severe myelosuppression. Previous studies showed that protein-coding mutations could not fully explain the predisposition to myelosuppression. Here, we investigate the possible role of enhancer mutations in myelosuppression susceptibility. We produced transcriptome and promoter-interaction maps (using HiCap) of three blood stem-like cell lines treated with carboplatin or gemcitabine. Taking advantage of publicly available enhancer datasets, we validated HiCap results in silico and in living cells using epigenetic CRISPR technology. We also developed a network approach for interactome analysis and detection of differentially interacting genes. Differential interaction analysis provided additional information on relevant genes and pathways for myelosuppression compared with differential gene expression analysis at the bulk level. Moreover, we showed that enhancers of differentially interacting genes are highly enriched for variants associated with differing levels of myelosuppression. Altogether, our work represents a prominent example of integrative transcriptome and gene regulatory datasets analysis for the functional annotation of noncoding mutations.

Noncoding mutations drive persistence of a founder preleukemic clone which initiates late relapse in T-ALL

AbstractT-ALL relapse usually occurs early but can occur much later, which has been suggested to represent a de novo leukemia. However, we conclusively demonstrate late relapse can evolve from a pre-leukemic subclone harbouring a non-coding mutation that evades initial chemotherapy.

High-risk and silent clonal hematopoietic genotypes in patients with nonhematologic cancer

AbstractClonal hematopoiesis (CH) identified by somatic gene variants with variant allele fraction (VAF) ≥ 2% is associated with an increased risk of hematologic malignancy. However, CH defined by a broader set of genotypes and lower VAFs is ubiquitous in older individuals. To improve our understanding of the relationship between CH genotype and risk of hematologic malignancy, we analyzed data from 42 714 patients who underwent blood sequencing as a normal comparator for nonhematologic tumor testing using a large cancer-related gene panel. We cataloged hematologic malignancies in this cohort using natural language processing and manual curation of medical records. We found that some CH genotypes including JAK2, RUNX1, and XPO1 variants were associated with high hematologic malignancy risk. Chronic disease was predicted better than acute disease suggesting the influence of length bias. To better understand the implications of hematopoietic clonality independent of mutational function, we evaluated a set of silent synonymous and noncoding mutations. We found that silent CH, particularly when multiple variants were present or VAF was high, was associated with increased risk of hematologic malignancy. We tracked expansion of CH mutations in 26 hematologic malignancies sequenced with the same platform. JAK2 and TP53 VAF consistently expanded at disease onset, whereas DNMT3A and silent CH VAFs mostly decreased. These data inform the clinical and biological interpretation of CH in the context of nonhematologic cancer.

Noncoding mutations cause super-enhancer retargeting resulting in protein synthesis dysregulation during B cell lymphoma progression

Whole-genome sequencing of longitudinal tumor pairs representing transformation of follicular lymphoma to high-grade B cell lymphoma with MYC and BCL2 rearrangements (double-hit lymphoma) identified coding and noncoding genomic alterations acquired during lymphoma progression. Many of these transformation-associated alterations recurrently and focally occur at topologically associating domain resident regulatory DNA elements, including H3K4me3 promoter marks located within H3K27ac super-enhancer clusters in B cell non-Hodgkin lymphoma. One region found to undergo recurrent alteration upon transformation overlaps a super-enhancer affecting the expression of the PAX5/ZCCHC7 gene pair. ZCCHC7 encodes a subunit of the Trf4/5-Air1/2-Mtr4 polyadenylation-like complex and demonstrated copy number gain, chromosomal translocation and enhancer retargeting-mediated transcriptional upregulation upon lymphoma transformation. Consequently, lymphoma cells demonstrate nucleolar dysregulation via altered noncoding 5.8S ribosomal RNA processing. We find that a noncoding mutation acquired during lymphoma progression affects noncoding rRNA processing, thereby rewiring protein synthesis leading to oncogenic changes in the lymphoma proteome.

BTG2 Super-Enhancer Mutations Disrupt TFAP4 Binding and Deregulate BTG2 Expression in Diffuse Large B-Cell Lymphoma

Diffuse Large B-cell Lymphoma (DLBCL), the most frequent lymphoid malignancy, remains incurable in ~40% of patients. Coding-genome sequencing efforts identified several genes/pathways altered in this disease, as well as genetic subgroups of prognostic and therapeutic significance. However, the large non-coding portion of the genome remained largely unexplored. We recently identified a pervasive hypermutation mechanism targeting active super-enhancers (SEs) in >90% of DLBCLs and leading to dysregulation of multiple genes, including well-known lymphoma oncogenes (Bal et al., Nature 2022). This pervasive phenomenon exhibits hallmarks of AID activity, and we provided evidence of its oncogenic relevance by demonstrating that mutational hotspots in the BCL6, BCL2 and CXCR4 SEs impair the binding of specific transcriptional repressors, preventing their negative regulation and creating oncogenic dependencies in DLBCL cells. Here we aimed to define the pathogenic role of non-coding mutations targeting the intragenic SE (iSE) of the BTG2 gene, the second most commonly mutated in DLBCL (39% of samples analyzed, including 36/93 primary cases and 7/29 cell lines). BTG2 encodes a member of the B-cell translocation gene (BTG)/ Transducer of ErbB2.1(TOB) family involved in transcriptional co-activation and modulation of mRNA abundance. BTG2 is also targeted by somatic missense mutations in 6-11% of DLBCL samples, suggesting a major role in the pathogenesis of this disease, which has not been explored. In order to identify functionally relevant BTG2 SE mutations, we first screened an extended panel of 286 DLBCL samples for the presence of mutational clusters targeting predicted transcription factor binding motifs. We identified a 20bp sequence stretch that was significantly hypermutated in these tumors, as compared to other lymphoma types or to non-lymphoid tumors (n=64/286, 22%; vs 0/17 Burkitt Lymphomas; 18/284 Follicular Lymphomas; 1/240 Chronic Lymphocytic Leukemias, and 0/1596 other tumors; p<0.0001, Fisher's exact test). In particular, mutations of the C at position +904 to G or T were exclusively detected in 11% DLBCL samples (32/286, p<0.0001, Fisher's exact test). To define the consequences of BTG2 C904 mutations, we then investigated the impact of reverting the mutation to the wild type nucleotide in 2 mutant DLBCL cell lines, using the CRISPR-Cas9 technology. Correction of the mutation led to a significant counter selection of the corrected clones (~85% reduction in a clone recovery assay, compared to control; p<0.001, Fisher's exact test) and to reduced BTG2 mRNA expression (p<0.01, one-way ANOVA), suggesting oncogenic addiction. In silico motif prediction and in vitro DNA-binding assays using mutant vs wild type oligonucleotides in two different DLBCL cell lines, followed by mass spectrometry, identified the Transcription Factor AP4 (TFAP4) as a protein specifically bound to the wild type, but not to the mutant sequences. TFAP4 is an important regulator of B-cell proliferation and cell fate decisions, which can function as a transcriptional activator or repressor in germinal center B-cells and acts downstream of/in parallel with c-MYC. We confirmed that the C904G and C904T SE mutation impaired TFAP4 binding in isogenic DLBCL cell lines by ChIP-qPCR and led to increased BTG2 expression (p<0.001, one-way ANOVA). Moreover, loss of TFAP4 in CRISPR-Cas9 edited DLBCL cells led to higher BTG2 expression levels, documenting TFAP4 as a direct negative regulator of BTG2 expression (p<0.001, one-way ANOVA). Consistently, single cell analysis of normal GC B cells revealed an inverse correlation between the expression pattern of TFAP4 and BTG2 in cells primed to undergo plasma cell differentiation vs cells committed to dark zone re-entry, suggesting that these two genes may function as a molecular switch in the cell fate decision of GC B cells. The programs modulated by BTG2 and TFAP4 in the GC, and disrupted in tumors carrying deregulated BTG2 alleles, will be presented, as defined by RNA-seq analysis of 3 isogenic GC-derived cell lines followed by interrogation of over 100 clinically annotated primary DLBCL biopsies. Together, these data identify TFAP4 as a novel regulator of BTG2 and reveal a previously unappreciated mechanism by which mutations in the BTG2 SE disrupt this circuit and contribute to lymphomagenesis through deregulating BTG2 expression.

CNSC-40. HARNESSING EVOLUTIONARY CONSTRAINT IDENTIFIES POTENTIAL NON-CODING DRIVERS IN BRAIN TUMORS

Abstract The somatic mutational landscape of brain tumors has primarily focused on coding mutations for driver gene discovery. Non-coding mutations can drive cancer by targeting regulatory elements and be valuable biomarkers and therapeutic targets. However, their identification and functional impact have been elusive. Here, we apply evolutionary constraint (phyloP scores, a constraint metric from sequence alignment of 241 mammals) to whole genome sequencing data from three different brain tumors; pilocytic astrocytoma (PA), medulloblastoma (MB), and glioblastoma (GBM) to identify non-coding constraint mutations (NCCMs) with regulatory potential. NCCM rates (phyloP ≥ 1.2) differ significantly between these cancers, with low rates of NCCMs in PA reflecting its benign feature and high rates in MB and GBM, in support of their aggressivity and heterogeneity. Many of the NCCMs affect genes that are developmentally regulated and expressed in the brain, hence they could be potential drivers. In PA, a high NCCM frequency only affects the BRAF locus, while in MB, over 500 genes have high levels of NCCMs. Intriguingly, many of these genes are associated with different age of onset, such as NUAK1 in adult patients and HOXB1 in young patients, pointing to different molecular pathways in patient groups. NCCMs in the HOXB locus altered the expression of multiple genes of the cluster in MB cells. In GBM NCCMs with regulatory potential cluster as hotspots in PTEN, LRFN5 and MIR99AHG. We observe an enrichment of NCCMs in the regulatory regions of transcription factors including FOXA1, HOXA9 and EBF1. We conclude that since evolutionary constraint is agnostic to cell type or disease stage, it is a powerful predictor of function. Understanding changes in the non-coding cancer genomes will lead to more robust patient stratification of future treatment.

EPCO-08. CONVERGENCE OF NON-CODING SOMATIC MUTATIONS ON CANCER DRIVER PLEXUSES IN PEDIATRIC HIGH-GRADE GLIOMA

Abstract Central nervous system tumors are the most common pediatric malignancies after leukemia, and the most common cause of cancer death among persons ages 0 – 14 [1,2]. Despite its low incidence rate, pediatric high-grade gliomas (pHGGs) account for over 40% of all childhood brain tumor deaths [3, 4]. As such, it remains the focus of many research teams across the globe. The discovery of recurrent mutations in genes encoding histone H3.3 [5] was a breakthrough in the genetic basis of pHGGs and highlighted the relevance of epigenetic reconfiguration for oncogene activation. However, much work is still needed to uncover driver alterations responsible for tumor initiation or progression. pHGGs have frequent epigenetic dysregulation [6,7] and abundant somatic mutations in the noncoding regions of the genome [8], but the functional relevance of the noncoding somatic mutations has not been evaluated systematically. Here, we assess correlations between noncoding somatic mutations in pediatric brain-specific cis-regulatory elements (CREs) and changes in gene expression. We demonstrate the sparsity of recurrent mutations in CREs. Then illustrate the power of graph database in consolidating multi-omics datasets for efficient queries. Our network approach for charting the genomic, epigenetic, and transcriptomic landscape of pHGG allow us to 1) identify clusters of CREs associated with dysregulated gene expression, 2) nominate transcription factors whose binding affinities are recurrently impacted by somatic mutations found in pHGGs, and 3) establish regulatory networks potentially guiding pHGG tumor growth and progression.

EPCO-51. FUNCTIONAL ANALYSIS OF REGULATORY MUTATIONS IN PEDIATRIC BRAIN TUMORS

Abstract Pediatric brain tumors are the primary cause of cancer related mortality in children and are characterized by low mutations burden. The known somatic drivers reside in the coding regions of the genome. The role of non-coding mutations in brain tumors is important yet underexplored. In this study we investigate the impact of regulatory region mutations in pediatric brain tumors. We studied 1.3 million open chromatin regions (OCRs) genome-wide from 9 studies including developing/adult brain, and cancer. Within these OCRs we identified recurring mutations in whole genome sequences from 1003 pediatric brain tumors from CBTN and PCAWG cohorts. A total of 567 OCRs with significantly recurring mutations were found, showing enrichment in brain-specific enhancers and in repressed polycomb regions in stem cells. The mutations in these OCRs resulted in motif disruption or creation of 217 transcription factor binding sites. Differential expression analysis of mutated vs nonmutated samples identified perturbations of gene or protein expression in 67 (RNA) and 16 (protein) OCR-gene pairs. Furthermore, 17 mutated OCRs were found to correlate with alterations in gene expression programs, indicating a potential regulatory role affecting multiple downstream genes. We further performed network analysis to explore clusters of OCR associated genes that share biological networks and pathways, and identified 24 clusters. Among these, OCR mutation-associated genes in 6 clusters showed a survival impact in patients. These 6 clusters mapped to pathways including mRNA decay and degradation, longevity regulating pathway, autophagy, p53 pathway, HIF2-alpha transcription factor network, pathways in cancer among others. In summary, we report functionally important regulatory elements in the genome that impact the biological processes in pediatric brain tumors.

Can Whole Genome and Whole Transcriptome Sequencing Replace Standard Procedures in CLL Diagnostics?

Background: The advantages of genome-wide sequencing approaches over conventional methods in CLL diagnostics are a matter of debate and exact guidelines for the application of next-generation sequencing in a diagnostic context are currently missing. Aim: Compare the accuracy of whole genome sequencing (WGS) and whole transcriptome sequencing (WTS) in determining the presence of point mutations, IGHV mutational status (IGHVms), and chromosomal aberrations to conventional procedures in a clinical setting. Patients and Methods: The cohort comprised 317 CLL patients. Diagnosis was established following WHO guidelines. WGS (100x, 2x151bp) and WTS (5x10 7 reads, 2x101bp) data were generated on NovaSeq instruments. For routine diagnostics, sequencing was carried out by Sanger or targeted panel sequencing (1500x, 2x101bp). Chromosome banding analysis (CBA) and FISH were available for 231 patients. WGS data were analyzed for chromosomal aberrations and copy number alterations using Manta and GATK. IGHVms was determined by Sanger sequencing and/or fragment analysis for 248 patients. Analysis of IGHVms was carried out using IgCaller (WGS) and MiXCR (WTS). Results: Comparing copy number variants (CNV) and balanced structural variants (SV) between CBA and WGS revealed a high concordance. In detail, 297 CNV were detected by both techniques while 32 and 52 were detected only by CBA (small subclones) or by WGS, respectively. 33 balanced SV were detected by both while 8 and 3 were detected only by CBA and WGS, respectively. A complex karyotype (≥ 3 abnormalities) was observed by both techniques in 55 cases, while 5 and 10 cases were categorized as complex by CBA only or WGS only, respectively. Survival analysis of cases with 3 to 5 chromosomal aberrations predicted by routine or WGS revealed similar hazard ratios that were positively correlated with the number of aberrations. For comparing SNV calls from routine and WGS, we investigated genes with validated mutations in at least ten patients in routine diagnostics. This criterion was met by ATM, MYD88, NOTCH1, SF3B1, and TP53. We observed high concordance between routine and WGS data ( ATM 99.4%, MYD88 100%, NOTCH1 97.8%, SF3B1 100%, TP53 99.1%). Five mutations were detected in routine only and three in WGS only (routine only/WGS only: ATM 1/0, MYD88 0/0, NOTCH1 2/2, SF3B1 0/0, TP53 2/1) and found to be caused by low variant allele frequency (5 miscalls, VAF < 5%) and/or filtering out of low quality variants (5 cases). IGHV rearrangements analyzed by Sanger (248 patients), WGS (242/248 patients), and WTS (236/248 patients) data were compared for matches in the rearranged IGHV. IGHVms calling employed 98% of sequence identity as cutoff. Calling the rearranged IGHV was concordant in 93%, 95%, and 92% when comparing Sanger/MiXCR, Sanger/IgCaller, and MiXCR/IgCaller calls, respectively. For IGHVms, the corresponding percentages of concordance were 91%, 86%, and 83%. Mismatches in calling the IGHVms were found in borderline cases. Detection of stereotyped IGHV was fully concordant. In addition to reliably replicating routine results, we find that WGS/WTS data can also outperform routine procedures. In our hands, the highest hazard ratio (4.94) in survival analysis distinguishing mutated CLL (mCLL) from unmutated CLL (uCLL) is observed using WTS for IGHVms determination and 97% sequence identity as cutoff (208 patients, up to 30 years follow up). Survival for borderline cases (97.00-97.99% sequence identity) was statistically indistinguishable from uCLL in routine diagnostics as well as WGS/WTS. Furthermore, using WGS, CN-LOH regions and deletions below the detection limit of CBA (10 MB) can be detected. We observed such regions in samples with mutated TP53 (5 CN-LOH), ATM (2 CN-LOH, 4 deletions < 10MB), and deletions of minimally overlapping region chr8:21,700,000-23,600,000, whose loss was associated with poor survival (3/29 deletions < 3.1 MB). Conclusions: WGS/WTS data reliably replicate results obtained by standard procedures in a diagnostic setting. Sequencing offers advantages such as a unified workflow, better sensitivity for small deletions, CN-LOH detection, genome-wide analyses of coding and non-coding mutations, among others. Challenges of short read based sequencing regard the detection limit for point mutations and specificity/sensitivity in detecting chromosomal aberrations.

E4F1 and ZNF148 are transcriptional activators of the -57A > C and wild-type TERT promoter.

Point mutations within the TERT promoter are the most recurrent somatic noncoding mutations identified across different cancer types, including glioblastoma, melanoma, hepatocellular carcinoma, and bladder cancer. They are most abundant at -146C > T and -124C > T, and rarer at -57A > C, with the latter originally described as a familial case, but subsequently shown also to occur somatically. All three mutations create de novo E26-specific (ETS) binding sites and result in activation of the TERT gene, allowing cancer cells to achieve replicative immortality. Here, we used a systematic proteomics screen to identify transcription factors preferentially binding to the -146C > T, -124C > T, and -57A > C mutations. Although we confirmed binding of multiple ETS factors to the mutant -146C > T and -124C > T sequences, we identified E4F1 as a -57A > C-specific binder and ZNF148 as a TERT wild-type (WT) promoter binder that showed reduced interaction with the -124C > T allele. Both proteins are activating transcription factors that bind specifically to the -57A > C and WT (at position 124) TERT promoter sequence in corresponding cell lines, and up-regulate TERT transcription and telomerase activity. Our work describes new regulators of TERT gene expression with possible roles in cancer.

OR35-03 Uncovering Causative Mutations in Polycystic Ovary Syndrome

Abstract Disclosure: J. Srivastava: None. I. Ovcharenko: None. PCOS is a complex disorder manifesting as reproductive and metabolic abnormalities in women. The undefined etiology stems from our incomplete understanding of genetic aberrations contributing to the diseased phenotype. This has resulted in speculations surrounding the cause-consequence relationship between phenotypic features of PCOS and limited our knowledge of comprehensive genetic signatures of PCOS. Pleiotropic effects are salient features of genetic regulation in mammalian genomes. The contribution of a set of regulatory elements (REs) to upregulation of a single gene, or the genetic control of multigene loci by a single RE, give rise to heterogenous phenotypes. Enhancers in particular dictate the spatio-temporal scale of gene expression by controlling transcription factor (TF) binding events. The complex traits of PCOS are suggestive of pleiotropy originating from genetic and epigenetic changes that arise from nucleotide variations in REs of the genome. Genome Wide Association Studies (GWAS) have identified ∼100 risk variants associated with PCOS and implicated the involvement of several genes such as FSHB/R, THADA, DENND1A, etc. However, mechanisms by which they contribute to pathophysiology are unknown. Additionally, twin studies have implicated the heritability of PCOS to be ∼80% and yet, the heritability from GWAS variants accounts for less than 10%. Experimental data may account for the ‘missing heritability problem’ specifically through rare variants that are missed in GWAS. To address this issue, we developed a Deep Learning model of gene regulation in PCOS identifying hundreds of active REs orchestrating the activity of PCOS genes. We also established a foundation for quantifying the impact of single-nucleotide noncoding mutations on TF binding and RE activity. By combining thousands of experimental assays associated with a large number of biological events such as tissue-specific epigenetic maps of the human genome, multi-tissue profiling of TF binding and gene expression using data from high throughput experiments we devised a strategy to accurately identify functionally map causative mutations in the GWAS loci of PCOS genes. MPRA experimental data validates a large fraction of our predicted causative variants thus suggesting that our studies have a potential of a leap forward accurate dissection of genomic and epigenetic mechanisms of PCOS. Consolidated results of this analysis and experimental validation of our predictions will lead to detailing the regulatory mechanisms of PCOS. Presentation Date: Sunday, June 18, 2023