Kazimierz O. Wrzeszczynski

Identification of Tumor Suppressors and Oncogenes from Genomic and Epigenetic Features in Ovarian Cancer

The identification of genetic and epigenetic alterations from primary tumor cells has become a common method to identify genes critical to the development and progression of cancer. We seek to identify those genetic and epigenetic aberrations that have the most impact on gene function within the tumor. First, we perform a bioinformatic analysis of copy number variation (CNV) and DNA methylation covering the genetic landscape of ovarian cancer tumor cells. We separately examined CNV and DNA methylation for 42 primary serous ovarian cancer samples using MOMA-ROMA assays and 379 tumor samples analyzed by The Cancer Genome Atlas. We have identified 346 genes with significant deletions or amplifications among the tumor samples. Utilizing associated gene expression data we predict 156 genes with altered copy number and correlated changes in expression. Among these genes CCNE1, POP4, UQCRB, PHF20L1 and C19orf2 were identified within both data sets. We were specifically interested in copy number variation as our base genomic property in the prediction of tumor suppressors and oncogenes in the altered ovarian tumor. We therefore identify changes in DNA methylation and expression for all amplified and deleted genes. We statistically define tumor suppressor and oncogenic features for these modalities and perform a correlation analysis with expression. We predicted 611 potential oncogenes and tumor suppressors candidates by integrating these data types. Genes with a strong correlation for methylation dependent expression changes exhibited at varying copy number aberrations include CDCA8, ATAD2, CDKN2A, RAB25, AURKA, BOP1 and EIF2C3. We provide copy number variation and DNA methylation analysis for over 11,500 individual genes covering the genetic landscape of ovarian cancer tumors. We show the extent of genomic and epigenetic alterations for known tumor suppressors and oncogenes and also use these defined features to identify potential ovarian cancer gene candidates.

Functional Mutation of SMAC/DIABLO, Encoding a Mitochondrial Proapoptotic Protein, Causes Human Progressive Hearing Loss DFNA64

SMAC/DIABLO is a mitochondrial proapoptotic protein that is released from mitochondria during apoptosis and counters the inhibitory activities of inhibitor of apoptosis proteins, IAPs. By linkage analysis and candidate screening, we identified a heterozygous SMAC/DIABLO mutation, c.377C>T (p.Ser126Leu, refers to p.Ser71Leu in the mature protein) in a six-generation Chinese kindred characterized by dominant progressive nonsyndromic hearing loss, designated as DFNA64. SMAC/DIABLO is highly expressed in human embryonic ears and is enriched in the developing mouse inner-ear hair cells, suggesting it has a role in the development and homeostasis of hair cells. We used a functional study to demonstrate that the SMAC/DIABLO(S71L) mutant, while retaining the proapoptotic function, triggers significant degradation of both wild-type and mutant SMAC/DIABLO and renders host mitochondria susceptible to calcium-induced loss of the membrane potential. Our work identifies DFNA64 as the human genetic disorder associated with SMAC/DIABLO malfunction and suggests that mutant SMAC/DIABLO(S71L) might cause mitochondrial dysfunction.

Loss of DOK2 induces carboplatin resistance in ovarian cancer via suppression of apoptosis

OBJECTIVE Ovarian cancers are highly heterogeneous and while chemotherapy is the preferred treatment many patients are intrinsically resistant or quickly develop resistance. Furthermore, all tumors that recur ultimately become resistant. Recent evidence suggests that epigenetic deregulation may be a key factor in the onset and maintenance of chemoresistance. We set out to identify epigenetically silenced genes that affect chemoresistance. METHODS The epigenomes of a total of 45 ovarian samples were analyzed to identify epigenetically altered genes that segregate with platinum response, and further filtered with expression data to identify genes that were suppressed. A tissue culture carboplatin resistance screen was utilized to functionally validate this set of candidate platinum resistance genes. RESULTS Our screen correctly identified 19 genes that when suppressed altered the chemoresistance of the cells in culture. Of the genes identified in the screen we further characterized one gene, docking protein 2 (DOK2), an adapter protein downstream of tyrosine kinase, to determine if we could elucidate the mechanism by which it increased resistance. The loss of DOK2 decreased the level of apoptosis in response to carboplatin. Furthermore, in cells with reduced DOK2, the level of anoikis was decreased. CONCLUSIONS We have developed a screening methodology that analyzes the epigenome and informatically identifies candidate genes followed by in vitro culture screening of the candidate genes. To validate our screening methodology we further characterized one candidate gene, DOK2, and showed that loss of DOK2 induces chemotherapy resistance by decreasing the level of apoptosis in response to treatment.

The oncocytic subtype is genetically distinct from other pancreatic intraductal papillary mucinous neoplasm subtypes

In 2010, the World Health Organization reclassified the entity originally described as intraductal oncocytic papillary neoplasm as the ‘oncocytic subtype’ of intraductal papillary mucinous neoplasm. Although several key molecular alterations of other intraductal papillary mucinous neoplasm subtypes have been discovered, including common mutations in KRAS, GNAS, and RNF3, those of oncocytic subtype have not been well characterized. We analyzed 11 pancreatic ‘oncocytic subtype’ of intraductal papillary mucinous neoplasms. Nine pancreatic ‘oncocytic subtype’ of intraductal papillary mucinous neoplasms uniformly exhibited typical entity-defining morphology of arborizing papillae lined by layers of cells with oncocytic cytoplasm, prominent, nucleoli, and intraepithelial lumina. The remaining two were atypical. One lacked the arborizing papilla and had flat oncocytic epithelium only; the other one had focal oncocytic epithelium in a background of predominantly intestinal subtype intraductal papillary mucinous neoplasm. Different components of this case were analyzed separately. Formalin-fixed, paraffin-embedded specimens of all cases were microdissected and subjected to high-depth-targeted next-generation sequencing for a panel of 300 key cancer-associated genes in a platform that enabled the identification of sequence mutations, copy number alterations, and select structural rearrangements involving all targeted genes. Fresh frozen specimens of two cases were also subjected to whole-genome sequencing. For the nine typical pancreatic ‘oncocytic subtype’ of intraductal papillary mucinous neoplasms, the number of mutations per case, identified by next-generation sequencing, ranged from 1 to 10 (median=4). None of these cases had KRAS or GNAS mutations and only one had both RNF43 and PIK3R1 mutations. ARHGAP26, ASXL1, EPHA8, and ERBB4 genes were somatically altered in more than one of these typical ‘oncocytic subtype’ of intraductal papillary mucinous neoplasms but not in the other two atypical ones. In the neoplasm with flat oncocytic epithelium, the only mutated gene was KRAS. All components of the intestinal subtype intraductal papillary mucinous neoplasms with focal oncocytic epithelium manifested TP53, GNAS, and RNF43 mutations. In conclusion, this study elucidates that ‘oncocytic subtype’ of intraductal papillary mucinous neoplasm is not only morphologically distinct but also genetically distinct from other intraductal papillary mucinous neoplasm subtypes. Considering that now its biologic behavior is also being found to be different than other intraductal papillary mucinous neoplasm subtypes, ‘oncocytic subtype’ of intraductal papillary mucinous neoplasm warrants being recognized separately.

A quantitative proteomics-based signature of platinum sensitivity in ovarian cancer cell lines

Although DNA encodes the molecular instructions that underlie the control of cell function, it is the proteins that are primarily responsible for implementing those instructions. Therefore quantitative analyses of the proteome would be expected to yield insights into important candidates for the detection and treatment of disease. We present an iTRAQ (isobaric tag for relative and absolute quantification)-based proteomic analysis of ten ovarian cancer cell lines and two normal ovarian surface epithelial cell lines. We profiled the abundance of 2659 cellular proteins of which 1273 were common to all 12 cell lines. Of the 1273, 75 proteins exhibited elevated expression and 164 proteins had diminished expression in the cancerous cells compared with the normal cell lines. The iTRAQ expression profiles allowed us to segregate cell lines based upon sensitivity and resistance to carboplatin. Importantly, we observed no substantial correlation between protein abundance and RNA expression or epigenetic DNA methylation data. Furthermore, we could not discriminate between sensitivity and resistance to carboplatin on the basis of RNA expression and DNA methylation data alone. The present study illustrates the importance of proteomics-based discovery for defining the basis for the carboplatin response in ovarian cancer and highlights candidate proteins, particularly involved in cellular redox regulation, homologous recombination and DNA damage repair, which otherwise could not have been predicted from whole genome and expression data sources alone.

Integrative prediction of gene function and platinum-free survival from genomic and epigenetic features in ovarian cancer.

The identification of genetic and epigenetic alterations from primary tumor cells has become a common method to discover genes critical to the development, progression, and therapeutic resistance of cancer. We seek to identify those genetic and epigenetic aberrations that have the most impact on gene function within the tumor. First, we perform a bioinformatics analysis of copy number variation (CNV) and DNA methylation covering the genetic landscape of ovarian cancer tumor cells. We were specifically interested in copy number variation as our base genomic property in the prediction of tumor suppressors and oncogenes in the altered ovarian tumor. We identify changes in DNA methylation and expression specifically for all amplified and deleted genes. We statistically define tumor suppressor and oncogenic gene function from integrative analysis of three modalities: copy number variation, DNA methylation, and gene expression. Our method (1) calculates the extent of genomic and epigenetic alterations of defined tumor suppressor and oncogenic features for the functional prediction of significant ovarian cancer gene candidates and (2) identifies the functional activity or inactivity of known tumor suppressors and oncogenes in ovarian cancer. We applied our protocol on 42 primary serous ovarian cancer samples using MOMA-ROMA representational array assays. Additionally, we provide the basis for incorporating epigenetic profiles of ovarian tumors for the purposes of platinum-free survival prediction in the context of TCGA data.

Abstract 4361: Target and drug discovery for recalcitrant, rare and neglected cancers.

Efforts are underway to improve the treatment of recalcitrant, rare and neglected cancers through the discovery of potential therapeutic targets, the identification of possible therapeutic combinations, and the identification of genomic vulnerabilities, using state-of-the-art drug discovery, molecular characterization, and mechanism-of-action techniques. Our current focus is on sarcoma and small cell lung cancer. Sarcoma comprises approximately 1% of cancers inclusive of 50 subtypes and occurs in patients of all ages with a frequency spread evenly over the human age range. Small cell lung cancer (SCLC) constitutes approximately 15% of lung cancers, is extremely aggressive, has a high mortality rate and frequently recurs after conventional cytotoxic chemotherapy. We have established comprehensive panels of human sarcoma and small cell lung cancer cell lines (approximately 80 per tumor type). Cell lines are being screened for response to all of the FDA approved anticancer agents and to a library of investigational agents with a goal of identifying existing agents that may be suitable for novel clinical trials, plus identifying potential points of vulnerability for drug discovery. Analysis of selected somatic mutations showed that ATM is frequently mutated in both soft tissue and bone sarcoma. In addition, gene expression profiles were measured in sarcoma cell lines using Affymetrix Exon 1.0 ST arrays in an effort to facilitate identification of splice variants and fusion genes. The gene-level data from the Exon ST1 array compares well with published U133plus 2 expression profiles. Principal components analysis of genes in 48 sarcoma lines indicates that expression varies primarily by disease subtype. For instance, Ewing9s Sarcoma overexpressed genes including PRKCB, NPY5R and NPY1R, ITM2A plus adrenergic receptors B1, B3 and A1D, while ACTC1, IGF2 and CHRNA1 were dysregulated in rhabdomyosarcoma. Predicted associations between gene and miRNA expression included CSF1 with miR-128, thioredoxin reductase with miR-324-5p, MDM4 with miR-152, and PODXL with miR-199a-5p, suggesting potential regulatory relationships that might be exploited as cancer targets. In these lines, exome expression was found to be altered more than 10% in 232 genes, including known splice variant genes such as KLK11, UBE2C and sarcoma related fusion genes EWSR1 and ETV. Calculated fusion scores based on EWSR1-FLI1 expression profiles successfully segregated Ewing9s sarcoma from all other sarcoma cell lines. Association analysis between gene and microRNA expression and sensitivity to specific anticancer agents is ongoing. Our mission is to improve the treatment of these neglected cancers by providing a comprehensive public database of molecular signatures and sensitivities that can be leveraged by the scientific community at large. Funded by NCI Contract no. HHSN261200800001E. Citation Format: Anne Monks, Annamaria Rapisarda, Kazimierz O. Wrzeszczynski, E. Michael August, Eric C. Polley, Sudhir B. Kondapaka, Gurmeet Kaur, Dianne Newton, Beverley A. Teicher. Target and drug discovery for recalcitrant, rare and neglected cancers. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 4361. doi:10.1158/1538-7445.AM2013-4361

Analytical Validation of Clinical Whole-Genome and Transcriptome Sequencing of Patient-Derived Tumors: Clinical Application of Whole-Genome Sequencing for Reporting Targetable Variants in Cancer

We developed and validated a clinical whole-genome and transcriptome sequencing (WGTS) assay that provides a comprehensive genomic profile of a patients tumor. The ability to fully capture the mappable genome with sufficient sequencing coverage to precisely call DNA somatic single nucleotide variants, insertions/deletions, copy number variants, structural variants, and RNA gene fusions was analyzed. New York States Department of Health next-generation DNA sequencing guidelines were expanded for establishing performance validation applicable to whole-genome and transcriptome sequencing. Whole-genome sequencing laboratory protocols were validated for the Illumina HiSeq X Ten platform and RNA sequencing for Illumina HiSeq2500 platform for fresh or frozen and formalin-fixed, paraffin-embedded tumor samples. Various bioinformatics tools were also tested, and CIs for sensitivity and specificity thresholds in calling clinically significant somatic aberrations were determined. The validation was performed on a set of 125 tumor normal pairs. RNA sequencing was performed to call fusions and to confirm the DNA variants or exonic alterations. Here, we present our results and WGTS standards for variant allele frequency, reproducibility, analytical sensitivity, and present limit of detection analysis for single nucleotide variant calling, copy number identification, and structural variants. We show that The New York Genome Center WGTS clinical assay can provide a comprehensive patient variant discovery approach suitable for directed oncologic therapeutic applications.

Abstract 2714: Analytical validation of clinical whole genome and transcriptome sequencing of patient derived tumors: clinical application of whole genome sequencing for reporting targetable variants in cancer

Next Generation DNA Sequencing (NGS) technologies are currently being applied in the clinical setting for the treatment of disease. The goal is to use high-throughput sequencing to identify specific variants within each tumor and recommend personalized treatment approaches or clinical trials tailored to the individual’s disease and genomic profile. These assays are comprised of either predefined sequencing panels, where a handpicked set of clinically significant genes are examined within each patient, or are cancer type specific targeted sequencing protocols or whole exome platforms covering only the coding region of the patient’s genome. Whole genome sequencing allows hypothesis-free interrogation of both coding and non-coding regions of the genome revealing more potential therapeutic options than examining a small set of genes or genomic loci. The protocol eliminates sequence capture related bias observed in whole exome or panel sequencing. The New York Genome Center therefore has performed analytical validation of whole genome and transcriptome sequencing (WGTS) of patient derived tumors and matched normals for the purposes of clinical testing and have devised a clinical reporting strategy of significant driver and therapeutic associated mutations. Many clinical NGS guidelines are directed toward targeted panel or exome sequencing validation. Here, we expanded on New York State’s Department of Health NGS guidelines developing them into novel standards applicable to WGTS for the purposes of clinical test validation. We first sequenced a virtual tumor at very high coverage (300x) and downsampled to determine the optimum depth of sequencing necessary for high confidence somatic variant calling across the entire genome. We then validated whole genome sequencing laboratory protocols for DNA and RNA sequencing on a total of 50 specimens derived from fresh frozen (FF) and formalin-fixed paraffin-embedded (FFPE) tumor samples. We performed a series of experiments to assess the accuracy and reliability of the results based on our laboratory and bioinformatics protocols. We performed our validation on the 50 tumor normal pairs, a subset of which had known genomic profiles. Comparisons were also made for variant calling concordance and reproducibility between matched FF and FFPE tumors. Here, we present our validation results and clinical WGTS standards for depth of sequencing, reproducibility, sensitivity, and present limit of detection analysis for SNV calling, copy number identification and structural variants. RNA sequencing is performed to call fusion or exon skipping events and to confirm the DNA variants. The New York Genome Center WGTS clinical assay is intended to provide a more comprehensive patient variant discovery approach suitable for directed oncological therapeutic applications. Citation Format: Kazimierz O. Wrzeszczynski, Avinash Abhyankar, Vanessa Felice, Esra Dikoglu, Lukasz Kozon, Nicolas Robine, Anne-Katrin Emde, Olca Basturk, Umesh K. Bhanot, Alex Kentsis, Mahesh Mansukhani, Govind Bhagat, Vaidehi Jobanputra. Analytical validation of clinical whole genome and transcriptome sequencing of patient derived tumors: clinical application of whole genome sequencing for reporting targetable variants in 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 2714. doi:10.1158/1538-7445.AM2017-2714

Abstract A24: Hypoxia signaling pathway plays a role in ovarian cancer chemoresistance.

Epithelial ovarian cancer (EOC) is the leading cause of gynecologic cancer mortality worldwide. These cancers are highly heterogeneous and while chemotherapy is the preferred treatment many patients are intrinsically resistant or quickly develop resistance. Furthermore, all tumors that recur ultimately become resistant. Understanding the molecular alterations of primary tumor cells has become a common method to identify genes critical to the development of a drug-resistance phenotype and progression of cancer and could lead to the development of novel therapeutic strategies for successful treatment of patients. DNA copy number alterations are a common occurrence in all cancers. Specific chromosomal regions and focal points favor either gains or losses in DNA and are important in the regulation of various oncogenes and tumor suppressors. Contributions from The Cancer Genome Atlas as well as recent bioinformatic results have identified key players involved in ovarian cancer deregulation. Specifically, we examined the datasets to identify copy number alterations in genes that segregate with platinum response, and further filtered with expression data to identify genes that were amplified. Our analysis revealed correlations for expression and CNV for several genes. Of the genes identified in the screen we further characterized one gene involved in hypoxia, HIF1α. Hypoxia is a microenvironmental factor which plays a critical role in the development and progression of cancer, chemoresistance, and poor survival. Epithelial-to-mesenchymal transition (EMT) is the process by which adherent epithelial cells convert to motile mesenchymal cells and is now known to also occur in a variety of diseases including the progression of cancer. EMT may reflect an adaptation of cancer cells to survive cytotoxic drug activity, and may thus be responsible for chemosensitivity. Our results have indicated that the gain of HIF1α decreased the level of apoptosis in response to carboplatin. Furthermore, ovarian cell lines undergoing hypoxia are more invasive, have migratory ability, and display a transformed EMT phenotype. Taken together, we provide the evidence of a role for HIF1α as an important regulator of chemoresistance in ovarian cancer and hypothesize EMT as the underlying mechanisms. These results suggest that the inactivation of HIF-1α signaling by novel strategies may be a potential targeted therapeutic approach for overcoming EMT and chemoresistance induced by hypoxia. Note: This abstract was not presented at the conference. Citation Format: Noelle L. Cutter, Tyler Walther, Liam Gallagher, Robert Lucito, Kazimierz Wrzeszczynski. Hypoxia signaling pathway plays a role in ovarian cancer chemoresistance. [abstract]. In: Proceedings of the AACR Special Conference on Advances in Ovarian Cancer Research: Exploiting Vulnerabilities; Oct 17-20, 2015; Orlando, FL. Philadelphia (PA): AACR; Clin Cancer Res 2016;22(2 Suppl):Abstract nr A24.