Functional characterization of genetic alterations in cancer

The comprehensive identification of genetic alterations is critical to understanding the pathophysiology of cancer. Recent advances in sequencing technology have enabled the detailed description of cancer genomes. However, to translate these findings into a deeper understanding of cancer biology, analyzing the functional impact of cancer-associated genetic aberration is essential. Here I investigate how to accelerate the functional characterization of two classes of genetic alterations, point mutations and amplifications. The wide spectrum of point mutations that arise in cancer makes them challenging to study comprehensively. I have developed a scalable systematic method to experimentally infer the functional impact of cancer-associated gene variants. I performed pooled in vivo tumor formation assays and gene expression profiling using 474 mutant alleles curated from 5,338 human tumors. I identified 12 transforming alleles including two in genes (PIK3CB, POT1) that have not been previously shown to be tumorigenic. One rare KRAS allele, D33E, displayed tumorigenicity and constitutive activation of RAS effector pathways. By correlating gene expression changes induced upon expression of wild type and mutant alleles, I could infer the activity of specific alleles. These approaches enable the interrogation of cancer-associated alleles at scale and demonstrate that rare alleles may be functionally important. Frequently amplified regions in cancer often harbor oncogenic drivers. However, identifying the driver gene among many other amplified genes is challenging. In high-grade serous ovarian cancer (HGSOC), 1,825 genes are amplified across 63 amplicons. We employed systematic loss-of-function RNAi data to identify amplified genes that were essential in the ovarian lineage. We identified 50 amplified and essential genes and validated FRS2, an adaptor protein in FGFR pathway. FRS2-amplified cancer cell lines were dependent on FRS2 expression and FRS2 overexpression in immortalized cell lines was sufficient to promote anchorage independent growth and tumorigenesis in nude mice. This approach demonstrates that intersecting structural genomics with functional genomics can facilitate the discovery of driver genes in recurrently amplified regions. Collectively, the methods I present here provide a framework to study point mutations and amplifications to accelerate the interpretation of the cancer genome.Medical Science

High Throughput Interrogation of Somatic Mutations in High Grade Serous Cancer of the Ovary

BACKGROUND:Epithelial ovarian cancer is the most lethal of all gynecologic malignancies, and high grade serous ovarian cancer (HGSC) is the most common subtype of ovarian cancer. The objective of this study was to determine the frequency and types of point somatic mutations in HGSC using a mutation detection protocol called OncoMap that employs mass spectrometric-based genotyping technology. METHODOLOGY/PRINCIPAL FINDINGS:The Center for Cancer Genome Discovery (CCGD) Program at the Dana-Farber Cancer Institute (DFCI) has adapted a high-throughput genotyping platform to determine the mutation status of a large panel of known cancer genes. The mutation detection protocol, termed OncoMap has been expanded to detect more than 1000 mutations in 112 oncogenes in formalin-fixed paraffin-embedded (FFPE) tissue samples. We performed OncoMap on a set of 203 FFPE advanced staged HGSC specimens. We isolated genomic DNA from these samples, and after a battery of quality assurance tests, ran each of these samples on the OncoMap v3 platform. 56% (113/203) tumor samples harbored candidate mutations. Sixty-five samples had single mutations (32%) while the remaining samples had ≥ 2 mutations (24%). 196 candidate mutation calls were made in 50 genes. The most common somatic oncogene mutations were found in EGFR, KRAS, PDGRFα, KIT, and PIK3CA. Other mutations found in additional genes were found at lower frequencies (<3%). CONCLUSIONS/SIGNIFICANCE:Sequenom analysis using OncoMap on DNA extracted from FFPE ovarian cancer samples is feasible and leads to the detection of potentially druggable mutations. Screening HGSC for somatic mutations in oncogenes may lead to additional therapies for this patient population

TRAF2 is an NF-κB activating oncogene in epithelial cancers

Aberrant NF-κB activation is frequently observed in human cancers. Genome characterization efforts have identified genetic alterations in multiple components of the NF-κB pathway, some of which have been shown to be essential for cancer initiation and tumor maintenance. Here using patient tumors and cancer cell lines, we identify the NF-κB regulator, TRAF2 as an oncogene that is recurrently amplified and rearranged in 15% of human epithelial cancers. Suppression of TRAF2 in cancer cells harboring TRAF2 copy number gain inhibits proliferation, NF-κB activation, anchorage-independent growth and tumorigenesis. Cancer cells that are dependent on TRAF2 also require NF-κB for survival. The phosphorylation of TRAF2 at serine 11 is essential for the survival of cancer cells harboring TRAF2 amplification. Together these observations identify TRAF2 as a frequently amplified oncogene

IKK∊-Mediated Tumorigenesis Requires K63-Linked Polyubiquitination by a cIAP1/cIAP2/TRAF2 E3 Ubiquitin Ligase Complex

IκB kinase ∊ (IKK∊, IKBKE) is a key regulator of innate immunity and a breast cancer oncogene, amplified in ∼30% of breast cancers, that promotes malignant transformation through NF-κB activation. Here, we show that IKK∊ is modified and regulated by K63-linked polyubiquitination at lysine 30 and lysine 401. Tumor necrosis factor alpha and interleukin-1β stimulation induces IKK∊ K63-linked polyubiquitination over baseline levels in both macrophages and breast cancer cell lines, and this modification is essential for IKK∊ kinase activity, IKK∊-mediated NF-κB activation, and IKK∊-induced malignant transformation. Disruption of K63-linked ubiquitination of IKK∊ does not affect its overall structure but impairs the recruitment of canonical NF-κB proteins. A cIAP1/cIAP2/TRAF2 E3 ligase complex binds to and ubiquitinates IKK∊. Altogether, these observations demonstrate that K63-linked polyubiquitination regulates IKK∊ activity in both inflammatory and oncogenic contexts and suggests an alternative approach to targeting this breast cancer oncogene

Mutant KRAS regulates transposable element RNA and innate immunity via KRAB zinc-finger genes.

RAS genes are the most frequently mutated oncogenes in cancer, yet the effects of oncogenic RAS signaling on the noncoding transcriptome remain unclear. We analyzed the transcriptomes of human airway and bronchial epithelial cells transformed with mutant KRAS to define the landscape of KRAS-regulated noncoding RNAs. We find that oncogenic KRAS signaling upregulates noncoding transcripts throughout the genome, many of which arise from transposable elements (TEs). These TE RNAs exhibit differential expression, are preferentially released in extracellular vesicles, and are regulated by KRAB zinc-finger (KZNF) genes, which are broadly downregulated in mutant KRAS cells and lung adenocarcinomas in&nbsp;vivo. Moreover, mutant KRAS induces an intrinsic IFN-stimulated gene (ISG) signature that is often seen across many different cancers. Our results indicate that mutant KRAS remodels the repetitive noncoding transcriptome, demonstrating the broad scope of intracellular and extracellular RNAs regulated by this oncogenic signaling pathway

Genomic Profiling of Prostate Cancers from Men with African and European Ancestry

PurposeAfrican American (AFR) men have the highest mortality rate from prostate cancer (PCa) compared with men of other racial/ancestral groups. Differences in the spectrum of somatic genome alterations in tumors between AFR men and other populations have not been well-characterized due to a lack of inclusion of significant numbers in genomic studies.Experimental designTo identify genomic alterations associated with race, we compared the frequencies of somatic alterations in PCa obtained from four publicly available datasets comprising 250 AFR and 611 European American (EUR) men and a targeted sequencing dataset from a commercial platform of 436 AFR and 3018 EUR men.ResultsMutations in ZFHX3 as well as focal deletions in ETV3 were more frequent in tumors from AFR men. TP53 mutations were associated with increasing Gleason score. MYC amplifications were more frequent in tumors from AFR men with metastatic PCa, whereas deletions in PTEN and rearrangements in TMPRSS2-ERG were less frequent in tumors from AFR men. KMT2D truncations and CCND1 amplifications were more frequent in primary PCa from AFR men. Genomic features that could impact clinical decision making were not significantly different between the two groups including tumor mutation burden, MSI status, and genomic alterations in select DNA repair genes, CDK12, and in AR.ConclusionsAlthough we identified some novel differences in AFR men compared with other populations, the frequencies of genomic alterations in current therapeutic targets for PCa were similar between AFR and EUR men, suggesting that existing precision medicine approaches could be equally beneficial if applied equitably

NetSig: network-based discovery from cancer genomes

Approaches that integrate molecular network information and tumor genome data could complement gene-based statistical tests to identify likely new cancer genes, but are challenging to validate at scale and their predictive value remains unclear. We developed a robust statistic (NetSig) that integrates protein interaction networks and data from 4,742 tumor exomes and used it to accurately classify known driver genes in 60% of tested tumor types and to predict 62 new candidates. We designed a quantitative experimental framework to compare the in vivo tumorigenic potential of NetSig candidates, known oncogenes and random genes in mice showing that NetSig candidates induce tumors at rates comparable to known oncogenes and 10-fold higher than random genes. By reanalyzing nine tumor-inducing NetSig candidates in 242 patients with oncogene-negative lung adenocarcinomas, we find that two (AKT2 and TFDP2) are significantly amplified. Overall, we illustrate a scalable integrated computational and experimental workflow to expand discovery from cancer genomes