Eejung Kim

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.

IκB Kinase ε Phosphorylates TRAF2 To Promote Mammary Epithelial Cell Transformation

ABSTRACT NF-κB transcription factors are central regulators of inflammation and when dysregulated contribute to malignant transformation. IκB kinase ε (IKKε; IKKi, encoded by IKBKE) is a breast oncogene that is amplified in 30% of breast cancers and drives transformation in an NF-κB-dependent manner. Here we demonstrate that IKKε interacts with and phosphorylates tumor necrosis factor receptor-associated factor 2 (TRAF2) at Ser11 in vitro and in vivo. This activity promotes Lys63-linked TRAF2 ubiquitination and NF-κB activation and is essential for IKKε transformation. Breast cancer cells that depend on IKKε expression for survival are also dependent on TRAF2. This work defines TRAF2 phosphorylation to be one key effector of IKKε-induced mammary epithelial cell transformation.

Systematic functional interrogation of rare cancer variants identifies oncogenic alleles

UNLABELLED Cancer genome characterization efforts now provide an initial view of the somatic alterations in primary tumors. However, most point mutations occur at low frequency, and the function of these alleles remains undefined. We have developed a scalable systematic approach to interrogate the function of cancer-associated gene variants. We subjected 474 mutant alleles curated from 5,338 tumors to pooled in vivo tumor formation assays and gene expression profiling. We identified 12 transforming alleles, including two in genes (PIK3CB, POT1) that have not been shown to be tumorigenic. One rare KRAS allele, D33E, displayed tumorigenicity and constitutive activation of known RAS effector pathways. By comparing gene expression changes induced upon expression of wild-type and mutant alleles, we inferred the activity of specific alleles. Because alleles found to be mutated only once in 5,338 tumors rendered cells tumorigenic, these observations underscore the value of integrating genomic information with functional studies. SIGNIFICANCE Experimentally inferring the functional status of cancer-associated mutations facilitates the interpretation of genomic information in cancer. Pooled in vivo screen and gene expression profiling identified functional variants and demonstrated that expression of rare variants induced tumorigenesis. Variant phenotyping through functional studies will facilitate defining key somatic events in cancer. Cancer Discov; 6(7); 714-26. ©2016 AACR.See related commentary by Cho and Collisson, p. 694This article is highlighted in the In This Issue feature, p. 681.

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

IκB kinase ε (IKKε, 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 IKKε is modified and regulated by K63-linked polyubiquitination at lysine 30 and lysine 401. Tumor necrosis factor alpha and interleukin-1β stimulation induces IKKε K63-linked polyubiquitination over baseline levels in both macrophages and breast cancer cell lines, and this modification is essential for IKKε kinase activity, IKKε-mediated NF-κB activation, and IKKε-induced malignant transformation. Disruption of K63-linked ubiquitination of IKKε 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 IKKε. Altogether, these observations demonstrate that K63-linked polyubiquitination regulates IKKε activity in both inflammatory and oncogenic contexts and suggests an alternative approach to targeting this breast cancer oncogene.

The Tyrosine Kinase Adaptor Protein FRS2 Is Oncogenic and Amplified in High-Grade Serous Ovarian Cancer

High-grade serous ovarian cancers (HGSOC) are characterized by widespread recurrent regions of copy-number gain and loss. Here, we interrogated 50 genes that are recurrently amplified in HGSOC and essential for cancer proliferation and survival in ovarian cancer cell lines. FRS2 is one of the 50 genes located on chromosomal region 12q15 that is focally amplified in 12.5% of HGSOC. We found that FRS2-amplified cancer cell lines are dependent on FRS2 expression, and that FRS2 overexpression in immortalized human cell lines conferred the ability to grow in an anchorage-independent manner and as tumors in immunodeficient mice. FRS2, an adaptor protein in the FGFR pathway, induces downstream activation of the Ras–MAPK pathway. These observations identify FRS2 as an oncogene in a subset of HGSOC that harbor FRS2 amplifications. Implications: These studies identify FRS2 as an amplified oncogene in a subset of HGSOC. FRS2 expression is essential to ovarian cancer cells that harbor 12q15 amplification. Mol Cancer Res; 13(3); 502–9. ©2014 AACR.

Exome Sequencing of African-American Prostate Cancer Reveals Loss-of-Function ERF Mutations

African-American men have the highest incidence of and mortality from prostate cancer. Whether a biological basis exists for this disparity remains unclear. Exome sequencing (n = 102) and targeted validation (n = 90) of localized primary hormone-naïve prostate cancer in African-American men identified several gene mutations not previously observed in this context, including recurrent loss-of-function mutations in ERF, an ETS transcriptional repressor, in 5% of cases. Analysis of existing prostate cancer cohorts revealed ERF deletions in 3% of primary prostate cancers and mutations or deletions in ERF in 3% to 5% of lethal castration-resistant prostate cancers. Knockdown of ERF confers increased anchorage-independent growth and generates a gene expression signature associated with oncogenic ETS activation and androgen signaling. Together, these results suggest that ERF is a prostate cancer tumor-suppressor gene. More generally, our findings support the application of systematic cancer genomic characterization in settings of broader ancestral diversity to enhance discovery and, eventually, therapeutic applications.Significance: Systematic genomic sequencing of prostate cancer in African-American men revealed new insights into prostate cancer, including the identification of ERF as a prostate cancer gene; somatic copy-number alteration differences; and uncommon PIK3CA and PTEN alterations. This study highlights the importance of inclusion of underrepresented minorities in cancer sequencing studies. Cancer Discov; 7(9); 973-83. ©2017 AACR.This article is highlighted in the In This Issue feature, p. 920.

NetSig: network-based discovery from cancer genomes

Methods that integrate molecular network information and tumor genome data could complement gene-based statistical tests to identify likely new cancer genes; but such approaches are challenging to validate at scale, and their predictive value remains unclear. We developed a robust statistic (NetSig) that integrates protein interaction networks with data from 4,742 tumor exomes. NetSig can accurately classify known driver genes in 60% of tested tumor types and predicts 62 new driver candidates. Using a quantitative experimental framework to determine in vivo tumorigenic potential in mice, we found that NetSig candidates induce tumors at rates that are comparable to those of known oncogenes and are ten-fold higher than those of 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. Our study presents a scalable integrated computational and experimental workflow to expand discovery from cancer genomes.

Expanding discovery from cancer genomes by integrating protein network analyses with in vivo tumorigenesis assays

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.

Abstract 5567: A scalable and integrated computational and experimental workflow to identify new driver genes in cancer genome data

High throughput sequencing has revolutionized the study of the cancer genome, enabling numerous discoveries in basic and clinical research. However, considerable sample sizes are required to find cancer driver genes with intermediate and low mutation frequencies, and for a large proportion of patients the molecular cause (e.g. driver gene(s)) of disease is unknown. Here, we describe an integrated computational and experimental workflow that combines cancer genome data, molecular network information, multiplexed in vivo tumorigenesis assays, and reanalysis of driver-gene-negative cancer patients to predict and validate new driver genes. We develop a statistic, network mutation burden, that combines molecular network information with data from 4,742 cancer genomes to accurately classify known driver genes across 21 tumor types and predict 62 driver gene candidates.Of these, 35 gene candidates were tested in multiplexed in vivo tumorigenesis cell assays using sensitized immortalized human embryonic kidney (HA1E-M) and immortalized human lung epithelial (SALE-Y) cell lines.Tumor formation in vivo was observed for 11 genes (2 in HA1E-M, 3 in SALE-Y, 6 in both). By reanalyzing 242 lung adenocarcinoma patients with an unknown molecular cause of disease we show that two of these candidates, TFDP2 and AKT2, are significantly amplified in multiple samples.Overall, we describe a scalable combined computational and experimental framework to predict and validate driver genes across many tumor types. Our proof-of-concept approach should become increasingly useful as the number of cancer genomes continues to grow. Citation Format: Heiko Horn, Michael S. Lawrence, Candace R. Chouinard, Yashaswi Shrestha, Jessica Xin Hu, Elizabeth Worstell, Emily Shea, Nina Ilic, Eejung Kim, Atanas Kamburov, Alireza Kashani, William C. Hahn, Joshua D. Campbell, Jesse S. Boehm, Gad Getz, Kasper Lage. A scalable and integrated computational and experimental workflow to identify new driver genes in cancer genome data [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 5567. doi:10.1158/1538-7445.AM2017-5567

Abstract 4863: Ubiquitination of the breast cancer oncogene IKKα is essential for transformation

Proceedings: AACR 103rd Annual Meeting 2012‐‐ Mar 31‐Apr 4, 2012; Chicago, IL IKBKE (IKKα) is a breast cancer oncogene that promotes human mammary cell transformation through the activation of the NF-κB pathway. Here we show that IKKα is modified by ubiquitination in the context of cell transformation. By mass spectrometry, we identified three lysine residues of IKKα that are ubiquitinated - Lysine30, Lysine401, and Lysine416. Mutation of all three lysine residues together eliminates IKKα ubiquitination. Single mutations of the three lysines revealed that K30 and K401 are essential for IKKα-induced malignant transformation. Although wildtype IKKα drives transformation in immortalized human cells, the IKKα K30R and IKKα K401R mutants fail to transform immortalized human cells. In addition, the IKKα K30R and IKKα K401R mutants fail to activate the NF-κB pathway and are unable to phosphorylate CYLD, a downstream target of IKKα. The ubiquitination of IKKα does not affect the protein stability of IKKα protein. Using an inducible ubiquitin-knockdown U2OS cell system, we show that IKKα specifically undergoes K63-linked polyubiquitination, a proteasome- and degradation-independent form of ubiquitination. Taken together, these data provide evidence that IKKα is subject to direct regulation by K63-linked polyubiquitination and that this ubiquitination is essential for IKKα-mediated oncogenic transformation. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 4863. doi:1538-7445.AM2012-4863