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Supplementary Data from Entinostat Decreases Immune Suppression to Promote Antitumor Responses in a HER2+ Breast Tumor Microenvironment

Supplementary Figure from Identifying Phased Mutations and Complex Rearrangements in Human Prostate Cancer Cell Lines through Linked-Read Whole-Genome Sequencing

Summary of sequence analysis of adenoid cystic carcinoma.

Supplementary Data from Identifying Phased Mutations and Complex Rearrangements in Human Prostate Cancer Cell Lines through Linked-Read Whole-Genome Sequencing

A limited number of cell lines have fueled the majority of preclinical prostate cancer research, but their genomes remain incompletely characterized. Here, we utilized whole-genome linked-read sequencing for comprehensive characterization of phased mutations and rearrangements in the most commonly used cell lines in prostate cancer research including PC3, LNCaP, DU145, CWR22Rv1, VCaP, LAPC4, MDA-PCa-2b, RWPE-1, and four derivative castrate-resistant (CR) cell lines LNCaP_Abl, LNCaP_C42b, VCaP-CR, and LAPC4-CR. Phasing of mutations allowed determination of “gene-level haplotype” to assess whether genes harbored heterozygous mutations in one or both alleles. Phased structural variant analysis allowed identification of complex rearrangement chains consistent with chromothripsis and chromoplexy. In addition, comparison of parental and derivative CR lines revealed previously known and novel genomic alterations associated with the CR phenotype.Implications:This study therefore comprehensively characterized phased genomic alterations in the commonly used prostate cancer cell lines, providing a useful resource for future prostate cancer research.

Axonal guidance signaling pathway alterations in adenoid cystic carcinoma.

Supplemental figure S4

The script for ChIP-Seq and RNA-Seq data integration

Figure S1. Experimental Figure Scheme. Figure S2. ChIP-Seq quality assessment for different samples and histone marks. Figure S3. Comparable ChIP-Seq and ChIP-qRT-PCR detection of histone enrichment for different histone marks near three reference genes. Figure S4. Comparable expression of reference genes. Figure S5. H3K9ac histone mark enrichment distribution near transcription start sites. Figure S6. H3K27ac histone mark enrichment distribution near transcription start sites. Figure S7. H3K9me3 histone mark enrichment distribution near transcription start sites. Figure S8. Correlation between tissue specific H3K9ac histone peak enrichment and expression of nearby genes. Figure S9. Correlation between tissue specific H3K9me3 histone peak enrichment and expression of nearby genes. Figure S10. Correlation between tissue specific H3K4me3 histone peak enrichment and expression of nearby genes for six study samples and two PDX-parental tissues. Figure S11. Correlation between tissue specific H3K27ac histone peak enrichment and expression of nearby genes for six study samples and two PDX-parental tissues. Figure S12. Correlation between tissue specific H3K9ac histone peak enrichment and expression of nearby genes for six study samples and two PDX-parental tissues. Figure S13. Correlation between tissue specific H3K9me3 histone peak enrichment and expression of nearby genes for six study samples and two PDX-parental tissues. Figure S14. Scheme for integration of ChIP-Seq Specific Histone Peaks with Expression Variation Analysis (EVA). Figure S15. Correlation of H3K27ac-enriched with genes differentially regulated in mesenchymal and classical HPV+ HNSCC subtypes.

Description of Supplemental Materials and Methods

List of 109 validated junctions associated with splice variants with location, gene symbol and category.

Purpose: Although most human cancers display a single histology, there are unusual cases where two or more distinct tissue types present within a primary tumor. One such example is metaplastic breast carcinoma, a rare but aggressive cancer with a heterogeneous histology, including squamous, chondroid, and spindle cells. Metaplastic carcinomas often contain an admixed conventional ductal invasive or in situ mammary carcinoma component, and are typically triple-negative for estrogen receptor, progesterone receptor, and HER-2 amplification/overexpression. An unanswered question is the origin of metaplastic breast cancers. While they may arise independently from their ductal components, their close juxtaposition favors a model that postulates a shared origin, either as two derivatives from the same primary cancer or one histology as an outgrowth of the other. Understanding the mechanism of development of these tumors may inform clinical decisions.Experimental Design: We performed exome sequencing for paired metaplastic and adjacent conventional invasive ductal carcinomas in 8 patients and created a pipeline to identify somatic variants and predict their functional impact, without having normal tissue. We then determined the genetic relationships between the histologically distinct compartments.Results: In each case, the tumor components have nearly identical landscapes of somatic mutation, implying that the differing histologies do not derive from genetic clonal divergence.Conclusions: A shared origin for tumors with differing histologies suggests that epigenetic or noncoding changes may mediate the metaplastic phenotype and that alternative therapeutic approaches, including epigenetic therapies, may be required for metaplastic breast cancers. Clin Cancer Res; 23(16); 4875–84. ©2017 AACR.

Chromatin alterations mediate mutations and gene expression changes in cancer. Chromatin immunoprecipitation followed by sequencing (ChIP-Seq) has been utilized to study genome-wide chromatin structure in human cancer cell lines, yet numerous technical challenges limit comparable analyses in primary tumors. Here we have developed a new whole-genome analytic pipeline to optimize ChIP-Seq protocols on patient-derived xenografts from human papillomavirus–related (HPV+) head and neck squamous cell carcinoma (HNSCC) samples. We further associated chromatin aberrations with gene expression changes from a larger cohort of the tumor and normal samples with RNA-Seq data. We detect differential histone enrichment associated with tumor-specific gene expression variation, sites of HPV integration in the human genome, and HPV-associated histone enrichment sites upstream of cancer driver genes, which play central roles in cancer-associated pathways. These comprehensive analyses enable unprecedented characterization of the complex network of molecular changes resulting from chromatin alterations that drive HPV-related tumorigenesis. Cancer Res; 77(23); 6538–50. ©2017 AACR.

Cell line authentication for SKN3

The incidence of HPV-related oropharyngeal squamous cell carcinoma (OPSCC) has increased more than 200% in the past 20 years. Recent genetic sequencing efforts have elucidated relevant genes in head and neck cancer, but HPV-related tumors have consistently shown few DNA mutations. In this study, we sought to analyze alternative splicing events (ASE) that could alter gene function independent of mutations. To identify ASE unique to HPV-related tumors, RNA sequencing was performed on 46 HPV-positive OPSCC and 25 normal tissue samples. A novel algorithm using outlier statistics on RNA-sequencing junction expression identified 109 splicing events, which were confirmed in a validation set from The Cancer Genome Atlas. Because the most common type of splicing event identified was an alternative start site (39%), MBD-seq genome-wide CpG methylation data were analyzed for methylation alterations at promoter regions. ASE in six genes showed significant negative correlation between promoter methylation and expression of an alternative transcriptional start site, including AKT3. The novel AKT3 transcriptional variant and methylation changes were confirmed using qRT-PCR and qMSP methods. In vitro silencing of the novel AKT3 variant resulted in significant growth inhibition of multiple head and neck cell lines, an effect not observed with wild-type AKT3 knockdown. Analysis of ASE in HPV-related OPSCC identified multiple alterations likely involved in carcinogenesis, including a novel, functionally active transcriptional variant of AKT3. Our data indicate that ASEs represent a significant mechanism of oncogenesis with untapped potential for understanding complex genetic changes that result in the development of cancer. Cancer Res; 77(19); 5248–58. ©2017 AACR.

R code for splice variant analysis and outlier statistics for identification of significant splice variants

Summary of sample cohort clinical data in tumors and normal samples including age, gender, race, smoking history, TNM stage, recurrence, follow up time, survival and HPV status

Supplemental table S1

SFigure 1. Distribution of ASEs present within each tumor. SFigure2. AKT3 splice variant expression by qRT-PCR in a panel of 20 cell lines. SFigure 3. Western blot analysis of canonical AKT1 pathway after transient knockdown of AKT3 splice variant. SFigure 4. Correlation of AKT3 splice variant with other PI3K/AKT pathway mutations in TCGA.

Cell line authentication for UM-SCC-047

Legends for supplemental figures S1-S15 and tables S1-S11

Table S1. Clinical data for patient-derived PDX1, PDX2, UPPP1, and UPPP2 samples used for ChIP-based analysis. Table S2. ChIP-DNA qRT-PCR primers-probe sequence Table S3. Sample-specific enrichment of H3K4me3 histone mark at 5''UTR of individual genes. Table S4. Sample-specific enrichment of H3K27ac histone mark at 5''UTR of individual genes. Table S5. Sample-specific enrichment of H3K9ac histone mark at 5''UTR of individual genes. Table S6. Sample-specific enrichment of H3K9me3 histone mark at 5''UTR of individual genes. Table S7. Gene set enrichment analysis of genes linked to H3K27ac-enrichment specific for tumor samples. Table S8. Gene set enrichment analysis of genes linked to H3K27ac-enrichment specific for normal samples. Table S9. Gene set enrichment analysis of genes linked to H3K4me3-enrichment specific for tumor samples. Table S10. Gene set enrichment analysis of genes linked to H3K4me3-enrichment specific for normal samples. Table S11. Correlation of H3K27ac-enriched with genes differentially regulated in HPV-KRT and HPV-IMU.

vcf files for 16 WES samples analyzed in this study

DiffBind script

Dysfunctional immune activation accumulates during chronic viral infection and contributes to disease pathogenesis. In HIV-1, immune activation is exacerbated by concurrent infection with hepatitis C virus (HCV), accelerating depletion of CD4+ T cells. HIV-1 suppression with antiretroviral therapy (ART) generally reconstitutes CD4+ T cell counts, while also reducing the proportion that is activated. Whether this immune reconstitution also reduces the complexity of the CD4+ T cell population is unknown. We sought to characterize the relationship between activated CD4+ T cell repertoire diversity and immune reconstitution following ART in HIV-1/HCV coinfection. We extracted T cell receptor (TCR) sequences from RNA sequencing data obtained from activated CD4+ T cells of HIV-1/HCV coinfected individuals before and after treatment with ART (clinical trial NCT01285050). There was notable heterogeneity in both the extent of CD4+ T cell reconstitution and in the change in activated CD4+ TCR repertoire diversity following ART. Decreases in activated CD4+ TCR repertoire diversity following ART were predictive of the degree of CD4+ T cell reconstitution. The association of decreased activated CD4+ TCR repertoire diversity and improved CD4+ T cell reconstitution may represent loss of nonspecifically activated TCR clonotypes, and possibly selective expansion of specifically activated CD4+ clones. These results provide insight into the dynamic relationship between activated CD4+ TCR diversity and CD4+ T cell recovery of HIV-1/HCV coinfected individuals after suppression of HIV-1 viremia.

Therapeutic combinations to alter immunosuppressive, solid tumor microenvironments (TME), such as in breast cancer, are essential to improve responses to immune checkpoint inhibitors (ICI). Entinostat, an oral histone deacetylase inhibitor, has been shown to improve responses to ICIs in various tumor models with immunosuppressive TMEs. The precise and comprehensive alterations to the TME induced by entinostat remain unknown. Here, we employed single-cell RNA sequencing on HER2-overexpressing breast tumors from mice treated with entinostat and ICIs to fully characterize changes across multiple cell types within the TME. This analysis demonstrates that treatment with entinostat induced a shift from a protumor to an antitumor TME signature, characterized predominantly by changes in myeloid cells. We confirmed myeloid-derived suppressor cells (MDSC) within entinostat-treated tumors associated with a less suppressive granulocytic (G)-MDSC phenotype and exhibited altered suppressive signaling that involved the NFκB and STAT3 pathways. In addition to MDSCs, tumor-associated macrophages were epigenetically reprogrammed from a protumor M2-like phenotype toward an antitumor M1-like phenotype, which may be contributing to a more sensitized TME. Overall, our in-depth analysis suggests that entinostat-induced changes on multiple myeloid cell types reduce immunosuppression and increase antitumor responses, which, in turn, improve sensitivity to ICIs. Sensitization of the TME by entinostat could ultimately broaden the population of patients with breast cancer who could benefit from ICIs.

Clearance of acute infection with hepatitis C virus (HCV) is associated with the chr19q13.13 region containing the rs368234815 (TT/ΔG) polymorphism. We fine-mapped this region to detect possible causal variants that may contribute to HCV clearance. First, we performed sequencing of IFNL1-IFNL4 region in 64 individuals sampled according to rs368234815 genotype: TT/clearance (N = 16) and ΔG/persistent (N = 15) (genotype-outcome concordant) or TT/persistent (N = 19) and ΔG/clearance (N = 14) (discordant). 25 SNPs had a difference in counts of alternative allele >5 between clearance and persistence individuals. Then, we evaluated those markers in an association analysis of HCV clearance conditioning on rs368234815 in two groups of European (692 clearance/1 025 persistence) and African ancestry (320 clearance/1 515 persistence) individuals. 10/25 variants were associated (P

Despite advancements in targeting the immune checkpoints program cell death protein 1 (PD-1), programmed death ligand 1 (PD-L1), and cytotoxic T lymphocyte–associated protein 4 (CTLA-4) for cancer immunotherapy, a large number of patients and cancer types remain unresponsive. Current immunotherapies focus on modulating an antitumor immune response by directly or indirectly expanding antitumor CD8 T cells. A complementary strategy might involve inhibition of Tregs that otherwise suppress antitumor immune responses. Here, we sought to identify functional immune molecules preferentially expressed on tumor-infiltrating Tregs. Using genome-wide RNA-Seq analysis of purified Tregs sorted from multiple human cancer types, we identified a conserved Treg immune checkpoint signature. Using immunocompetent murine tumor models, we found that antibody-mediated depletion of 4-1BB–expressing cells (4-1BB is also known as TNFRSF9 or CD137) decreased tumor growth without negatively affecting CD8 T cell function. Furthermore, we found that the immune checkpoint 4-1BB had a high selectivity for human tumor Tregs and was associated with worse survival outcomes in patients with multiple tumor types. Thus, antibody-mediated depletion of 4-1BB–expressing Tregs represents a strategy with potential activity across cancer types.

Mutations in mitochondrial DNA (mtDNA) have been linked to risk, progression, and treatment response of head and neck squamous cell carcinoma (HNSCC). Due to their clonal nature and high copy number, mitochondrial mutations could serve as powerful molecular markers for detection of cancer cells in bodily fluids, surgical margins, biopsies and lymph node (LN) metastasis, especially at sites where tumor involvement is not histologically apparent. Despite a pressing need for high-throughput, cost-effective mtDNA mutation profiling system, current methods for library preparation are still imperfect for detection of low prevalence heteroplasmic mutations. To this end, we have designed an ultra-deep amplicon-based sequencing library preparation approach that covers the entire mitochondrial genome. We sequenced mtDNA in 28 HNSCCs, matched LNs, surgical margins and bodily fluids, and applied multiregional sequencing approach on 14 primary tumors. Our results demonstrate that this quick, sensitive and cost-efficient method allows obtaining a snapshot on the mitochondrial heterogeneity, and can be used for detection of low frequency tumor-associated mtDNA mutations in LNs, sputum and serum specimens. These findings provide the foundation for using mitochondrial sequencing for risk assessment, early detection, and tumor surveillance.

A Molecular Features Set (MFS), is a result of vast diversity of bioinformatics pipelines. In case when MFS is used for further analysis to distinguish between phenotypes, it is often referred to as a signature. Lack of the “gold standard” for most experimental data modalities makes it hard to provide valid estimation for a particular MFS’s quality. Yet, this goal can partially be achieved by analyzing inner-sample Distance Matrix (DM) and their power to distinguish between phenotypes.The quality of a DM can be assessed by summarizing its power to quantify the differences of inner-phenotype and outer-phenotype distances. This estimation of the DM quality can be construed as a measure of the MFS’s quality.Here we propose Hobotnica, an approach to estimate MFS’s quality by their ability to stratify data, and assign them significance scores, that allows for collating various signatures and comparing their quality for contrasting groups.

Dysfunctional immune activation accumulates during chronic viral infection and contributes to disease pathogenesis. In HIV-1, immune activation is exacerbated by concurrent infection with hepatitis C virus (HCV), accelerating depletion of CD4(+) T cells. HIV-1 suppression with antiretroviral therapy (ART) generally reconstitutes CD4(+) T cell counts, while also reducing the proportion that is activated. Whether this immune reconstitution also reduces the complexity of the CD4(+) T cell population is unknown. We sought to characterize the relationship between activated CD4(+) T cell repertoire diversity and immune reconstitution following ART in HIV-1/HCV coinfection. We extracted T cell receptor (TCR) sequences from RNA sequencing data obtained from activated CD4(+) T cells of HIV-1/HCV coinfected individuals before and after treatment with ART (clinical trial NCT01285050). There was notable heterogeneity in both the extent of CD4(+) T cell reconstitution and in the change in activated CD4(+) TCR repertoire diversity following ART. Decreases in activated CD4(+) TCR repertoire diversity following ART were predictive of the degree of CD4(+) T cell reconstitution. The association of decreased activated CD4(+) TCR repertoire diversity and improved CD4(+) T cell reconstitution may represent loss of nonspecifically activated TCR clonotypes, and possibly selective expansion of specifically activated CD4(+) clones. These results provide insight into the dynamic relationship between activated CD4(+) TCR diversity and CD4(+) T cell recovery of HIV-1/HCV coinfected individuals after suppression of HIV-1 viremia.

A limited number of cell lines have fueled the majority of preclinical prostate cancer research, but their genomes remain incompletely characterized. Here, we utilized whole-genome linked-read sequencing for comprehensive characterization of phased mutations and rearrangements in the most commonly used cell lines in prostate cancer research including PC3, LNCaP, DU145, CWR22Rv1, VCaP, LAPC4, MDA-PCa-2b, RWPE-1, and four derivative castrate-resistant (CR) cell lines LNCaP_Abl, LNCaP_C42b, VCaP-CR, and LAPC4-CR. Phasing of mutations allowed determination of “gene-level haplotype” to assess whether genes harbored heterozygous mutations in one or both alleles. Phased structural variant analysis allowed identification of complex rearrangement chains consistent with chromothripsis and chromoplexy. In addition, comparison of parental and derivative CR lines revealed previously known and novel genomic alterations associated with the CR phenotype. Implications: This study therefore comprehensively characterized phased genomic alterations in the commonly used prostate cancer cell lines, providing a useful resource for future prostate cancer research.

A limited number of cell lines have fueled the majority of preclinical Prostate cancer (PCa) research. Despite tremendous effort in characterizing their molecular profiles, comprehensive whole genome sequencing with allelic phasing of somatic genome alterations has not been undertaken to date. Here, we utilized whole genome Linked-read sequencing to obtain haplotype information from the seven most commonly used PCa cell lines (PC3, LNCaP, DU145, CWR22Rv1, VCaP, LAPC4, MDA-PCa-2b), four castrate resistant (CR) subclones (LNCaP_Abl, LNCaP_C42b, VCaP-CR, LAPC4-CR), and an immortalized prostate epithelial line RWPE-1. Phasing of mutations allowed derivation of “Gene-level Haplotype” to assess whether a gene harbored heterozygous mutations in one or both alleles, providing a comprehensive catalogue of mono or bi-allelically inactivated genes. Phased structural variant analysis allowed identification of complex rearrangement chains consistent with chromothripsis and chromoplexy, with breakpoints occurred across a single allele, providing further evidence that complex SVs occurred in a concerted event, rather than through accumulation of multiple independent rearrangements. Additionally, comparison of parental and CR subclones revealed previously known and novel genomic alterations associated with the CR clones. This study therefore comprehensively characterized phased genomic alterations in the commonly used PCa cell lines and provided a useful resource for future cancer research.

We have recently performed the characterization of alternative splicing events (ASEs) in head and neck squamous cell carcinoma, which allows dysregulation of protein expression common for cancer cells. Such analysis demonstrated a high ASE prevalence among tumor samples, including tumor-specific alternative splicing in the GSN gene.In vitro studies confirmed that overall expression of either ASE-GSN or wild-type GSN (WT-GSN) isoform inversely correlated with cell proliferation, whereas the high ratio of ASE-GSN to WT-GSN correlated with increased cellular invasion. Additionally, a change in expression of either isoform caused compensatory changes in expression of the other isoform. Our results suggest that the overall expression and the balance between GSN isoforms are mediating factors in proliferation, while increased overall expression of ASE-GSN is specific to cancer tissues. As a result, we propose ASE-GSN can serve not only as a biomarker of disease and disease progression, but also as a neoantigen for head and neck squamous cell carcinoma treatment, for which only a limited number of disease-specific targeted therapies currently exist.

Candida glabrata is an opportunistic pathogen of humans, responsible for up to 30% of disseminated candidiasis. Adherence of C. glabrata to host cells is mediated by adhesin-like proteins (ALPs), about half of which are encoded in the subtelomeres. We performed a de novo assembly of two C. glabrata strains, BG2 and BG3993, using long single-molecule real-time (SMRT) reads, and constructed high-quality telomere-to-telomere assemblies of all 13 chromosomes to assess differences between C. glabrata strains. We documented variation between strains, and in agreement with earlier studies, found high (~0.5%-1%) frequencies of SNVs across the genome, including within subtelomeric regions. We documented changes in ALP gene structure and complement: there are large length differences in ALP genes in different strains, resulting from copy number variation in tandem repeats. We compared strains to characterize chromosome rearrangement events including within the poorly characterized subtelomeric regions. We show that rearrangements within the subtelomere regions all affect ALP-encoding genes, and 14/16 involve just the most terminal ALP gene. We present evidence that these rearrangements are mediated by break-induced replication. This study highlights the constrained nature of subtelomeric changes impacting ALP gene complement and subtelomere structure.

PD-1 blockade unleashes CD8 T cells1, including those specific for mutation-associated neoantigens (MANA), but factors in the tumour microenvironment can inhibit these T cell responses. Single-cell transcriptomics have revealed global T cell dysfunction programs in tumour-infiltrating lymphocytes (TIL). However, the majority of TIL do not recognize tumour antigens2, and little is known about transcriptional programs of MANA-specific TIL. Here, we identify MANA-specific T cell clones using the MANA functional expansion of specific T cells assay3 in neoadjuvant anti-PD-1-treated non-small cell lung cancers (NSCLC). We use their T cell receptors as a ‘barcode’ to track and analyse their transcriptional programs in the tumour microenvironment using coupled single-cell RNA sequencing and T cell receptor sequencing. We find both MANA- and virus-specific clones in TIL, regardless of response, and MANA-, influenza- and Epstein–Barr virus-specific TIL each have unique transcriptional programs. Despite exposure to cognate antigen, MANA-specific TIL express an incompletely activated cytolytic program. MANA-specific CD8 T cells have hallmark transcriptional programs of tissue-resident memory (TRM) cells, but low levels of interleukin-7 receptor (IL-7R) and are functionally less responsive to interleukin-7 (IL-7) compared with influenza-specific TRM cells. Compared with those from responding tumours, MANA-specific clones from non-responding tumours express T cell receptors with markedly lower ligand-dependent signalling, are largely confined to HOBIThigh TRM subsets, and coordinately upregulate checkpoints, killer inhibitory receptors and inhibitors of T cell activation. These findings provide important insights for overcoming resistance to PD-1 blockade., Single-cell RNA sequencing and T cell receptor sequencing are combined to identify transcriptional programs specific to mutation-associated neoantigen-specific T cells in non-small cell lung cancers treated with anti-PD-1, providing insights into resistance to PD-1 blockade.

Background In cancers, maintenance of telomeres often occurs through activation of the catalytic subunit of telomerase, encoded by TERT. Yet, most cancers show only modest levels of TERT gene expression, even in the context of activating hotspot promoter mutations (C228T and C250T). The role of epigenetic mechanisms, including DNA methylation, in regulating TERT gene expression in cancer cells is as yet not fully understood. Methods Here, we have carried out the most comprehensive characterization to date of TERT promoter methylation using ultra-deep bisulfite sequencing spanning the CpG island surrounding the core TERT promoter in 96 different human cell lines, including primary, immortalized and cancer cell types, as well as in control and reference samples. Results In general, we observed that immortalized and cancer cell lines were hypermethylated in a region upstream of the recurrent C228T and C250T TERT promoter mutations, while non-malignant primary cells were comparatively hypomethylated in this region. However, at the allele-level, we generally found that hypermethylation of promoter sequences in cancer cells is associated with repressed expression, and the remaining unmethylated alleles marked with open chromatin are largely responsible for the observed TERT expression in cancer cells. Conclusions Our findings suggest that hypermethylation of the TERT promoter alleles signals transcriptional repression of those alleles, leading to attenuation of TERT activation in cancer cells. This type of fine tuning of TERT expression may account for the modest activation of TERT expression in most cancers.

In cancers, maintenance of telomeres often occurs through activation of the catalytic subunit of telomerase, encoded byTERT. Yet, most cancers show only modest levels of telomerase gene expression, even in the context of activating hotspot promoter mutations (C228T and C250T). The role of epigenetic mechanisms, including DNA methylation, in regulating telomerase gene expression in cancer cells is not fully understood. Here, we have carried out the most comprehensive characterization to date ofTERTpromoter methylation using ultra-deep bisulfite sequencing spanning the CpG island surrounding the coreTERTpromoter in 96 different human cell lines. In general, we observed that immortalized and cancer cell lines were hypermethylated in a region upstream of the recurrent C228T and C250TTERTpromoter mutations, while non-malignant primary cells were comparatively hypomethylated in this region. However, at the allele-level, we generally observe hypermethylation of promoter sequences in cancer cells is associated with repressed expression, and the remaining unmethylated alleles marked with open chromatin are largely responsible for the observedTERTexpression in cancer cells. Our findings suggest that hypermethylation of theTERTpromoter alleles signals transcriptional repression of those alleles, leading to the attenuation ofTERTactivation in cancer cells.SIGNIFICANCEHypermethylation of theTERTpromoter alleles to attenuateTERTactivation in cancer cells may account for the modest activation ofTERTexpression in most cancers.

Background The Scholarly Concentrations program was established at Johns Hopkins University School of Medicine in 2009 with the aim of instilling passion for scholarship. Objective Our study aimed to determine whether the Scholarly Concentrations program achieves positive changes in medical student self-efficacy in conducting research and, if so, whether this results in future career aspirations toward scholarship. Design We used the Clinical Research Appraisal Inventory-Short Form (CRAI-SF) to assess changes in self-efficacy among students completing the Scholarly Concentrations program between 2014 and 2017. We calculated composite mean scores of six domains. We included outcomes on whether students published a manuscript, overall program perceptions, and likelihood of future research careers. We analyzed relationships between CRAI-SF scores and outcomes using paired t-tests and multivariable-adjusted logistic regression. Results A total of 419 students completed the Scholarly Concentrations program. All 6 CRAI domain scores showed significant improvements in self-efficacy between the pre-Scholarly Concentrations and post-Scholarly Concentrations ratings (range of changes 0.76–1.39, p

Motivation DNA sequencing archives have grown to enormous scales in recent years, and thousands of human genomes have already been sequenced. The size of these data sets has made searching the raw read data infeasible without high-performance data-query technology. Additionally, it is challenging to search a repository of short-read data using relational logic and to apply that logic across samples from multiple whole-genome sequencing samples. Results We have built a compact, efficiently-indexed database that contains the raw read data for over 250 human genomes, encompassing trillions of bases of DNA, and that allows users to search these data in real-time. The Terabase Search Engine enables retrieval from this database of all the reads for any genomic location in a matter of seconds. Users can search using a range of positions or a specific sequence that is aligned to the genome on the fly. Availability and implementation Public access to the Terabase Search Engine database is available at http://tse.idies.jhu.edu. Supplementary information Supplementary data are available at Bioinformatics online.

Accurately measuring mitochondrial DNA copy number (mtDNAcn) is important for improving our understanding of cancer biology. Prior studies have revealed differences in mtDNAcn between matched bulk tumor-normal pairs from a range of different organs. However, there is little consistency across tumor types; compared to their normal tissue counterparts, some cancers appear to have increased mtDNAcn, others appear to have decreased mtDNAcn, and in others, such as prostate cancer, studies have shown contradictory results. The lack of consistency of mtDNAcn changes in tumor-normal pairs may reflect a failure to address the marked heterogeneity in mtDNAcn across different cell types within normal tissues. In addition, tumor cell heterogeneity in mtDNAcn has also not been taken into account in most prior studies, which can further confound tumor-normal comparisons. Finally, little is known about mtDNAcn alterations in cancer precursor lesions. To address these technical limitations, we combined a recently introduced quantitative in situ hybridization method with immunohistochemistry in a multiplex assay. This enabled us to measure mtDNAcn using digital image analysis in specific cell populations of interest on formalin fixed paraffin embedded (FFPE) and frozen tissue samples. We determined that normal prostate basal cells have approximately 3-fold higher mtDNA levels than normal prostate luminal cells. Compared to the adjacent normal prostate glands, we found markedly increased levels of mtDNA in high-grade prostatic intraepithelial neoplasia (HGPIN), the presumptive precursor lesions to most prostate cancers. We also found increased levels as well as heterogeneity of mtDNAcn in invasive prostate cancer lesions, and more consistently elevated levels in castration-resistant metastatic prostate cancer (CRPC). Increased mtDNA copy number was also identified by whole genome sequencing and quantitative PCR from laser capture micro-dissected human prostate tumor-normal pairs. We also observed higher mtDNAcn in two other precancer lesion types, pancreatic intraepithelial neoplasia and colorectal adenomas. Our approach represents a technological advance as it facilitates mtDNAcn measurements in a manner that preserves morphology, allowing for the evaluation of specific cell populations of interest. These findings raise the hypothesis that increased mitochondrial mass and function may drive the development of precancerous lesions in the prostate, pancreas and colon, as well as invasive prostate cancer development and progression. Citation Format: Jiayu Chen, Qizhi Zheng, Jessica L. Hicks, Levent Trabzonlu, Emmanuel S. Antonarakis, Mark C. Markowski, Samuel R. Denmeade, Busra Ozbek, Anuj Gupta, Tatianna C. Larman, Ralph H. Hruban, Sarah Wheelan, Srinivasan Yegnasubramanian, Angelo M. De Marzo. Increased mitochondrial DNA copy number occurs during prostate cancer progression and in cancer precursor lesions across multiple organs [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 2404.