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CRISPR-based cancer dependency maps are accelerating advances in cancer precision medicine, but adequate functional maps are limited to the most common oncogenes. To identify opportunities for therapeutic intervention in other rarer subsets of cancer, we investigate the oncogene-specific dependencies conferred by the lung cancer oncogene, RIT1. Here, genome-wide CRISPR screening in KRAS, EGFR, and RIT1-mutant isogenic lung cancer cells identifies shared and unique vulnerabilities of each oncogene. Combining this genetic data with small-molecule sensitivity profiling, we identify a unique vulnerability of RIT1-mutant cells to loss of spindle assembly checkpoint regulators. Oncogenic RIT1M90I weakens the spindle assembly checkpoint and perturbs mitotic timing, resulting in sensitivity to Aurora A inhibition. In addition, we observe synergy between mutant RIT1 and activation of YAP1 in multiple models and frequent nuclear overexpression of YAP1 in human primary RIT1-mutant lung tumors. These results provide a genome-wide atlas of oncogenic RIT1 functional interactions and identify components of the RAS pathway, spindle assembly checkpoint, and Hippo/YAP1 network as candidate therapeutic targets in RIT1-mutant lung cancer., RIT1 mutations are mutually exclusive with other lung cancer drivers and lack targeted therapies. Here the authors examine genetic dependencies of mutant RIT1 with genome-wide CRISPR screens, revealing synergy between RIT1 and YAP1, and increased sensitivity to Aurora kinase inhibitors.

In recent work, we performed CRISPR/Cas9 screening in RIT1 (Ras-like in all tissues)-mutant cancer cells. We found that RIT1-mutant cells are vulnerable to loss of mitotic regulators, and mutant RIT1 synergizes with YAP1 (yes-associated protein 1) in oncogenesis. These findings can be leveraged to identify targeted therapies for RIT1-mutant cancer.

Most variants in most genes across most organisms have an unknown impact on the function of the corresponding gene. This gap in knowledge is especially acute in cancer, where clinical sequencing of tumors now routinely reveals patient-specific variants whose functional impact on the corresponding gene is unknown, impeding clinical utility. Transcriptional profiling was able to systematically distinguish these variants of unknown significance (VUS) as impactful vs. neutral in an approach called expression-based variant-impact phenotyping (eVIP). We profiled a set of lung adenocarcinoma-associated somatic variants using Cell Painting, a morphological profiling assay that captures features of cells based on microscopy using six stains of cell and organelle components. Using deep-learning-extracted features from each cell’s image, we found that cell morphological profiling (cmVIP) can predict variants’ functional impact and, particularly at the single-cell level, reveals biological insights into variants which can be explored in our public online portal. Given its low cost, convenient implementation, and single-cell resolution, cmVIP profiling therefore seems promising as an avenue for using non-gene-specific assays to systematically assess the impact of variants, including disease-associated alleles, on gene function.

Aberrant activation of the RAS family of guanosine triphosphatases (GTPases) is prevalent in lung adenocarcinoma, with somatic mutation of KRAS occurring in ~30% of tumors. We previously identified somatic mutations and amplifications of the gene encoding RAS family GTPase RIT1 in lung adenocarcinomas. To explore the biological pathways regulated by RIT1 and how they relate to the oncogenic KRAS network, we performed quantitative proteomic, phosphoproteomic, and transcriptomic profiling of isogenic lung epithelial cells in which we ectopically expressed wild-type or cancer-associated variants of RIT1 and KRAS. We found that both mutant KRAS and mutant RIT1 promoted canonical RAS signaling, and that overexpression of wild-type RIT1 partially phenocopied oncogenic RIT1 and KRAS, including induction of epithelial-to-mesenchymal transition. Our findings suggest that RIT1 protein abundance is a factor in its pathogenic function. Therefore, chromosomal amplification of wild-type RIT1 in lung and other cancers may be tumorigenic.

MGA, a transcription factor and member of the MYC network, is mutated or deleted in a broad spectrum of malignancies. As a critical test of a tumor suppressive role, we inactivated Mga in two mouse models of non-small cell lung cancer using a CRISPR-based approach. MGA loss significantly accelerated tumor growth in both models and led to de-repression of non-canonical Polycomb ncPRC1.6 targets, including genes involved in metastasis and meiosis. Moreover, MGA deletion in human lung adenocarcinoma lines augmented invasive capabilities. We further show that MGA-MAX, E2F6, and L3MBTL2 co-occupy thousands of promoters and that MGA stabilizes these ncPRC1.6 subunits. Lastly, we report that MGA loss also induces a pro-growth effect in human colon organoids. Our studies establish MGA as a bona fide tumor suppressor in vivo and suggest a tumor suppressive mechanism in adenocarcinomas resulting from widespread transcriptional attenuation of MYC and E2F target genes mediated by MGA-MAX associated with a non-canonical Polycomb complex.

SUMMARY RTK/RAS/RAF pathway alterations (RPAs) are a hallmark of lung adenocarcinoma (LUAD). In this study, we use whole-genome sequencing (WGS) of 85 cases found to be RPA(–) by previous studies from The Cancer Genome Atlas (TCGA) to characterize the minority of LUADs lacking apparent alterations in this pathway. We show that WGS analysis uncovers RPA(+) in 28 (33%) of the 85 samples. Among the remaining 57 cases, we observe focal deletions targeting the promoter or transcription start site of STK11 (n = 7) or KEAP1 (n = 3), and promoter mutations associated with the increased expression of ILF2 (n = 6). We also identify complex structural variations associated with high-level copy number amplifications. Moreover, an enrichment of focal deletions is found in TP53 mutant cases. Our results indicate that RPA(–) cases demonstrate tumor suppressor deletions and genome instability, but lack unique or recurrent genetic lesions compensating for the lack of RPAs. Larger WGS studies of RPA(–) cases are required to understand this important LUAD subset., In Brief Carrot-Zhang et al. perform whole-genome characterization of lung adenocarcinomas (LUADs) lacking RTK/RAS/RAF pathway alterations (RPAs) and identify mutations or structural variants in both coding and non-coding spaces that define a unique entity of RPA(–) LUADs and potentially explain the underlying biology of this disease., Graphical Abstract

Genome-scale CRISPR-Cas9 screens have enabled the identification of essential genes that can serve as cancer drug targets. However, single-gene knockout screens frequently underestimate the essentiality of paralogs, ancestrally duplicated genes that often functionally compensate for one another's loss; we have observed this directly in single-gene CRISPR screen data from a lung cancer cell line (P < 1E-16). Since the human genome is highly duplicated, our ability to identify novel drug targets is greatly limited. To identify paralog pairs essential for cancer cell survival, we developed a multiplexed CRISPR approach that uses paired guide RNAs to knock out human paralogs individually and in pairs. Our library includes 2,060 paralogs (1,030 pairs); the largest human paralog CRISPR library to date. We screened lung adenocarcinoma (PC9) and cervical carcinoma (HeLa) cell lines to identify synthetic lethal paralogs which have minimal growth effects when targeted individually but whose simultaneous loss leads to severely decreased growth. We found that 128 (16%) of the paralog pairs in our study were synthetic lethal and essential in at least one cell line. Gene set enrichment analysis revealed that our top paralogs are overrepresented in pathways related to cell cycle regulation, protein secretion, DNA repair, and PI3K-AKT signaling (FDR q-value < 2.52E-4). Importantly, 15 (18%) synthetic lethal paralog pairs have at least one member that is currently druggable. We validated our screen results using in vitro competition assays and DepMap CRISPR data analysis. For known synthetic lethal paralogs MAGOH and MAGOHB, competition assay data confirmed significantly reduced growth in the dual gene knockout condition versus single-gene knockouts (P < 0.05) and analysis of DepMap CRISPR score correlation with paralog expression showed that MAGOHB is more essential in cell lines with low MAGOH expression (P < 4.6E-41). For novel synthetic lethal paralogs CCNL1/CCNL2, PSMB5/PSMB8, and OXSR1/STK39, DepMap CRISPR score vs. expression correlation analysis again confirmed our findings as cell lines with low expression of paralog 1 showed lower CRISPR scores when paralog 2 was knocked out (P < 1.2E-8 for each pair), suggesting that our novel synthetic lethal paralogs are essential across a range of cancer cell lines. These interactions are also being validated via competition assay, and CRISPR knockout efficiency will be confirmed via Western blot and genomic DNA sequencing. Our studies point to a number of novel synthetic lethal paralogs that could serve as lung cancer drug targets. These paralogs could be targeted alone or in combination with existing therapies to suppress lung cancer growth and prevent acquired drug resistance. Paralog synthetic lethal therapies could make a major impact on clinical care by improving patient outcomes. Citation Format: Phoebe C. Parrish, James D. Thomas, Shriya Kamlapurkar, Robert K. Bradley, Alice H. Berger. Enabling cancer drug target discovery through genome-scale identification of synthetic lethal paralog pairs [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 2139.

Standard care of lung cancer is moving away from chemotherapy in favor of personalized approaches based on specific mutations in each tumor. Genome-sequencing studies have identified somatic mutations in the small GTPase RIT1 (Ras-like in all tissues) in lung adenocarcinoma patients. Of the identified mutations, M90I is the most recurrent. Thousands of patients per year are diagnosed with RIT1-driven cancer, but treatment options are limited. A targeted therapy for RIT1-driven disease could greatly improve patient outcomes. Little is known about how RIT1 drives cellular transformation. To genetically dissect signaling pathways downstream of RIT1, we performed a genome-wide CRISPR/Cas9 screen in isogenic PC9 lung adenocarcinoma cells in which cell survival is dependent on expression of RIT1M90I. We found that RIT1-mutant cells were highly dependent on components of the Spindle Assembly Checkpoint (SAC), including the Aurora kinases (A and B). The SAC is a surveillance mechanism that ensures proper chromosome segregation during mitosis. We hypothesized that RIT1M90I weakened the SAC and rendered cells vulnerable to loss of mitotic regulators. To explore this, we performed time-lapse imaging in HeLa H2B-GFP cells stably expressing RIT1M90I. In parental cells, the median duration of mitosis was 70.5 min (95% CI = 63 – 82 min), while in RIT1M90I-mutant cells, this was reduced to 48 min (95% CI = 45 – 51 min). Mitotic index was unaffected, suggesting that RIT1M90I does not regulate mitotic entry. This difference in mitotic timing was eliminated by treatment with reversine, an inhibitor that abolishes the SAC, demonstrating that RIT1M90I perturbs mitotic timing at the level of the SAC. If RIT1M90I weakens the SAC, we would expect higher prevalence of chromosomal abnormalities (such as chromosome bridges) in RIT1M90I-mutant cells. Indeed, analysis of fixed-cell populations indicated that RIT1M90I-mutant cells showed higher prevalence of mitotic abnormalities (79% of RIT1M90I-mutant cells compared to 46% of parental cells, 95% CI= 15 – 53). These data support the hypothesis that RIT1M90I weakens the SAC and imply that further SAC perturbation could be lethal. To explore this, we performed a small molecule screen of 160 clinically-relevant inhibitors in PC9-RIT1M90I and PC9-KRASG12V cells. Intriguingly, RIT1M90I-mutant cells were more sensitive than RAS-mutant cells to alisertib and barasertib, inhibitors of Aurora kinase A and B, respectively. Furthermore, alisertib or barasertib treatment abrogated soft agar colony formation in RIT1M90I-mutant cells but not in RAS-mutant cells. Together with our functional genomic analysis, we propose a model whereby expression of RIT1M90I weakens the SAC, thereby accelerating mitosis, increasing the abundance of mitotic abnormalities, and rendering cells vulnerable to genetic knockdown of SAC genes or small molecule inhibition of Aurora kinases A/B. Citation Format: Amanda Riley, Athea Vichas, Naomi T. Nkinsi, Phoebe C. Parrish, Shriya Kamlapurkar, Alice H. Berger. The spindle assembly checkpoint as a therapeutic vulnerability in RIT1-mutant lung adenocarcinoma [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 2269.

To compare lung adenocarcinoma (ADC) and lung squamous cell carcinoma (SqCC) and to identify new drivers of lung carcinogenesis, we examined the exome sequences and copy number profiles of 660 lung ADC and 484 lung SqCC tumor-normal pairs. Recurrent alterations in lung SqCCs were more similar to those of other squamous carcinomas than to alterations in lung ADCs. New significantly mutated genes included PPP3CA, DOT1L, and FTSJD1 in lung ADC, RASA1 in lung SqCC, and KLF5, EP300, and CREBBP in both tumor types. New amplification peaks encompassed MIR21 in lung ADC, MIR205 in lung SqCC, and MAPK1 in both. Lung ADCs lacking receptor tyrosine kinase-Ras-Raf pathway alterations had mutations in SOS1, VAV1, RASA1, and ARHGAP35. Regarding neoantigens, 47% of the lung ADC and 53% of the lung SqCC tumors had at least five predicted neoepitopes. Although targeted therapies for lung ADC and SqCC are largely distinct, immunotherapies may aid in treatment for both subtypes.

Recurrent broad-scale heterozygous deletions are frequently observed in human cancer. Here we tested the hypothesis that compound haploinsufficiency of neighboring genes at chromosome 8p promotes tumorigenesis. By targeting the mouse orthologs of human DOK2 and DUSP4 genes, which were co-deleted in approximately half of human lung adenocarcinomas, we found that compound-heterozygous deletion of Dok2 and Dusp4 in mice resulted in lung tumorigenesis with short latency and high incidence, and that their co-deletion synergistically activated MAPK signaling and promoted cell proliferation. Conversely, restoration of DOK2 and DUSP4 in lung cancer cells suppressed MAPK activation and cell proliferation. Importantly, in contrast to downregulation of DOK2 or DUSP4 alone, concomitant downregulation of DOK2 and DUSP4 was associated with poor survival in human lung adenocarcinoma. Therefore, our findings lend in vivo experimental support to the notion that compound haploinsufficiency, due to broad-scale chromosome deletions, constitutes a driving force in tumorigenesis.

Targeting somatically activated oncogenes has revolutionized the treatment of non–small cell lung cancer (NSCLC). Mutations in the gene mesenchymal–epithelial transition (MET) near the exon 14 splice sites are recurrent in lung adenocarcinoma and cause exon skipping (METΔ14). Here, we analyzed 4,422 samples from 12 different malignancies to estimate the rate of said exon skipping. METΔ14 mutation and transcript were most common in lung adenocarcinoma. Endogenously expressed levels of METΔ14 transformed human epithelial lung cells in a hepatocyte growth factor–dependent manner. In addition, overexpression of the orthologous mouse allele induced lung adenocarcinoma in a novel, immunocompetent mouse model. Met inhibition showed clinical benefit in this model. In addition, we observed a clinical response to crizotinib in a patient with METΔ14-driven NSCLC, only to observe new missense mutations in the MET activation loop, critical for binding to crizotinib, upon clinical progression. These findings support genomically selected clinical trials directed toward METΔ14 in a fraction of NSCLC patients, confirm second-site mutations for further therapeutic targeting prior to and beyond acquired resistance, and provide an in vivo system for the study of METΔ14 in an immunocompetent host. Cancer Res; 77(16); 4498–505. ©2017 AACR.

Lung adenocarcinoma is comprised of distinct mutational subtypes characterized by mutually exclusive oncogenic mutations in RTK/RAS pathway members KRAS, EGFR, BRAF and ERBB2, and translocations involving ALK, RET and ROS1. Identification of these oncogenic events has transformed the treatment of lung adenocarcinoma via application of therapies targeted toward specific genetic lesions in stratified patient populations. However, such mutations have been reported in only ∼55% of lung adenocarcinoma cases in the United States, suggesting other mechanisms of malignancy are involved in the remaining cases. Here we report somatic mutations in the small GTPase gene RIT1 in ∼2% of lung adenocarcinoma cases that cluster in a hotspot near the switch II domain of the protein. RIT1 switch II domain mutations are mutually exclusive with all other known lung adenocarcinoma driver mutations. Ectopic expression of mutated RIT1 induces cellular transformation in vitro and in vivo, which can be reversed by combined PI3K and MEK inhibition. These data identify RIT1 as a driver oncogene in a specific subset of lung adenocarcinomas and suggest PI3K and MEK inhibition as a potential therapeutic strategy in RIT1-mutated tumors.

Background: Discordance in hormone receptor (HR) status between primary (p) tumors and metastatic (m) recurrences has been widely described. Loss of estrogen and progesterone receptor expression occurs in ˜12% of asynchronous recurrences, leading to triple-negative (TN) status in the metastasis. Genomic mechanisms driving HR loss and its prognostic and therapeutic implications have not been fully elucidated. Methods: Targeted NGS (Oncopanel, OP) at Dana-Farber Cancer Institute using multiplexed copy number variation and mutation (mut) detection across the full coding regions of 300 genes and selected intronic regions of 35 genes was prospectively performed on either archival primary or metastatic samples collected in patients (pts) with metastatic breast cancer (MBC). Receptor status at initial diagnosis and recurrence were reviewed using a 1% cutoff to define HR-positivity and excluding HER2+ cases. Fisher´s exact test was used to compare frequency of alterations. Tumor mut burden (TMB) was computed normalizing the sum of reported exon mut in each pt by the exonic-bait-set size of the panel. Results: Between 8/2013-9/2016, 929 pts with MBC underwent OP testing. Of 517 pts diagnosed with primary HR+/HER2- breast cancer, at time of recurrence 388 remained HR+/HER2- (pHR+/mHR+), 39 switched to HR-/HER2- (pHR+/mTN, of which 23 (59%) had initial HR expression >10%), 10 switched to HER2+ and 80 had unknown metastatic receptor status. Comparison between primary samples in pHR+/mHR+ (n=245) and pHR+/mTN (n=24) showed that pHR+/mTN was significantly more likely to harbor mut in TP53, STK11 and MSH6, amplifications (amp) in CCNE1 and FGFR2, and less likely to have PIK3CA mut or CCND1 amp. Median TMB in primary pHR+/mHR+ was 6.05 mut/Mb (0-37.5) and 5.68 mut/Mb (1.2-10.9) in pHR+/mTN (p=0.45). Metastatic samples in pHR+/mTN (n=15) were enriched in ARID1A, CRTC2 and CDH1 mut compared to metastases (n=40) in pts who remained TN (pTN/mTN). Deletions in CDKN2A/2B and RB1, and mut in TP53, NOTCH2 and ERCC2 were more prevalent in recurrent tumors of pHR+/mTN than pHR+/mHR+. In metastases, TMB was higher in pHR+/mTN than pTN/mTN or pHR+/mHR+ (10.9 vs. 7.0 vs. 7.3 mut/Mb, respectively; p=0.002). Median OS from initial diagnosis was 9.4 yrs in pHR+/mTN, less than pHR+/mHR+ (15.9 yrs; p=0.009) and greater than pTN/mTN (4.3 yrs; p=0.008). Median OS from MBC diagnosis was 1.8 yrs in pHR+/mTN, less than pHR+/mHR+ (6.4 yrs; p=0.001) but not significantly different than pTN/mTN (1.5 yrs, p=0.3). pHR+/mHR+ (n=245)pHR+/mTN (n=24)p value NFreq (%)NFreq (%) MutTP536325.72083.3 Conclusion: Targeted NGS shows that alterations in DNA damage and cell-cycle regulation pathways in primary HR+ tumors are associated with HR loss in the metastatic setting. Primary tumors that lose HR appear more similar to basal-like than luminal tumors, despite >10% baseline HR expression in most pts, and once metastatic, survival is comparable to pTN/mTN. Metastases with HR loss have higher TMB than those that remain HR+ or TN throughout the course of the disease. These findings, if confirmed, may influence treatment and pt selection for clinical trials. Citation Format: Garrido-Castro AC, Hughes ME, Cherniack A, Barroso-Sousa R, Bychkovsky BL, Di Lascio S, Berger A, Mittendorf EA, Files JL, Guo H, Kumari P, Cerami E, Krop IE, Wagle N, Lindeman NI, MacConaill LE, Dillon DA, Winer EP, Lin NU. Genomic alterations associated with loss of HR expression in metastatic breast cancer [abstract]. In: Proceedings of the 2018 San Antonio Breast Cancer Symposium; 2018 Dec 4-8; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2019;79(4 Suppl):Abstract nr PD9-01.

Over the past decade, The Cancer Genome Atlas (TCGA) has profiled more than 11,000 tumors spanning 33 distinct cancer types. The TCGA PanCanAtlas is a collaborative project by the TCGA Research Network that aims to address relevant overarching questions in oncology based on a cross-cancer analysis of the full, uniformly reprocessed TCGA data set. Here, we present results from our analysis of genetic alterations in mitogenic signaling pathways across cancer. Genetic alterations in signaling pathways that control cell cycle progression, apoptosis, and cell growth are common hallmarks of cancer, but the extent, mechanisms, and co-occurrence of alterations in these pathways differ between individual tumors and tumor types. Using mutations and copy-number changes in 9,125 tumor samples profiled by TCGA, we analyzed the mechanisms and patterns of alterations in 10 canonical pathways: cell cycle, Hippo, Myc, Notch, beta-catenin / WNT, PI-3-Kinase / Akt, receptor-tyrosine kinase / RAS / MAP-kinase signaling, TP53, and TGF-beta signaling, as well as oxidative stress response. For each of these pathways, we propose an expert-curated description (or “template”) that includes the relevant (altered) genes and the connections between them, as well as a detailed catalogue of the driver mutations and copy number changes with known oncogenic relevance. We provide a high-level map of pathway alteration frequencies across tissues and relevant cancer subtypes as well as detailed frequencies of alteration at the gene level for each individual pathway. We also investigate relationships of co-occurrence and mutual exclusivity across pathways and evaluate therapeutic implications, including drug combinations. Forty-nine percent of tumors had at least one potentially targetable alteration in the evaluated pathways, and 31% of tumors had multiple targetable alterations, making them candidates for combination therapy. Our work delineates the full landscape of oncogenic alterations in mitogenic signaling pathways across cancer, and the pathway templates as well as the richly annotated data set that we provide will constitute an invaluable public resource for future use by the cancer genomics and precision oncology communities. Citation Format: Francisco Sanchez-Vega, Marco Mina, Joshua Armenia, Walid K. Chatila, Augustin Luna, Konnor La, Sofia Dimitriadoy, David L. Liu, Havish S. Kantheti, Zachary Heins, Angelica Ochoa, Benjamin Gross, Jianjiong Gao, Hongxin Zhang, Ritika Kundra, Cyriac Kandoth, Istemi Bahceci, Leonard Dervishi, Ugur Dogrusoz, Wanding Zhou, Hui Shen, Peter W. Laird, Alice H. Berger, Trever G. Bivona, Alexander J. Lazar, Gary Hammer, Thomas Giordano, Lawrence Kwong, Grant McArthur, Chenfei Huang, Mitchell J. Frederick, Frank McCormick, Matthew Meyerson, The Cancer Genome Atlas Research Network, Eliezer Van Allen, Andrew D. Cherniack, Giovanni Ciriello, Chris Sander, Nikolaus Schultz. The molecular landscape of oncogenic signaling pathways in The Cancer Genome Atlas [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 3302.

Genome-wide analyses of human lung adenocarcinoma have identified regions of consistent copy-number gain or loss, but in many cases the oncogenes and tumor suppressors presumed to reside in these loci remain to be determined. Here we identify the downstream of tyrosine kinase (Dok) family members Dok1, Dok2 and Dok3 as lung tumor suppressors. Single, double or triple compound loss of these genes in mice results in lung cancer, with penetrance and latency dependent on the number of lost Dok alleles. Cancer development is preceded by an aberrant expansion and signaling profile of alveolar type II cells and bronchioalveolar stem cells. In human lung adenocarcinoma, we identify DOK2 as a target of copy-number loss and mRNA downregulation and find that DOK2 suppresses lung cancer cell proliferation in vitro and in vivo. Given the genomic localization of DOK2, we propose it as an 8p21.3 haploinsufficient human lung tumor suppressor.

The ETS family of transcription factors has been repeatedly implicated in tumorigenesis. In prostate cancer, ETS family members, such as ERG, ETV1, ETV4, and ETV5, are frequently overexpressed due to chromosomal translocations, but the molecular mechanisms by which they promote prostate tumorigenesis remain largely undefined. Here, we show that ETS family members, such as ERG and ETV1, directly repress the expression of the checkpoint kinase 1 (CHK1), a key DNA damage response cell-cycle regulator essential for the maintenance of genome integrity. Critically, we find that ERG expression correlates with CHK1 downregulation in human patients and demonstrate that Chk1 heterozygosity promotes the progression of high-grade prostatic intraepithelial neoplasia into prostatic invasive carcinoma in Pten+/− mice. Importantly, CHK1 downregulation sensitizes prostate tumor cells to etoposide but not to docetaxel treatment. Thus, we identify CHK1 as a key functional target of the ETS proto-oncogenic family with important therapeutic implications. Significance: Genetic translocation and aberrant expression of ETS family members is a common event in different types of human tumors. Here, we show that through the transcriptional repression of CHK1, ETS factors may favor DNA damage accumulation and consequent genetic instability in proliferating cells. Importantly, our findings provide a rationale for testing DNA replication inhibitor agents in ETS-positive TP53-proficient tumors. Cancer Discov; 5(5); 550–63. ©2015 AACR. This article is highlighted in the In This Issue feature, p. 453

Recent genome sequencing efforts have identified millions of somatic mutations in cancer. However, the functional impact of most variants is poorly understood. Here we characterize 194 somatic mutations identified in primary lung adenocarcinomas. We present an expression-based variant-impact phenotyping (eVIP) method that uses gene expression changes to distinguish impactful from neutral somatic mutations. eVIP identified 69% of mutations analyzed as impactful and 31% as functionally neutral. A subset of the impactful mutations induces xenograft tumor formation in mice and/or confers resistance to cellular EGFR inhibition. Among these impactful variants are rare somatic, clinically actionable variants including EGFR S645C, ARAF S214C and S214F, ERBB2 S418T, and multiple BRAF variants, demonstrating that rare mutations can be functionally important in cancer.

Adenocarcinoma of the lung is the leading cause of cancer death worldwide. Here we report molecular profiling of 230 resected lung adenocarcinomas using messenger RNA, microRNA and DNA sequencing integrated with copy number, methylation and proteomic analyses. High rates of somatic mutation were seen (mean 8.9 mutations per megabase). Eighteen genes were statistically significantly mutated, including RIT1 activating mutations and newly described loss-of-function MGA mutations which are mutually exclusive with focal MYC amplification. EGFR mutations were more frequent in female patients, whereas mutations in RBM10 were more common in males. Aberrations in NF1, MET, ERBB2 and RIT1 occurred in 13% of cases and were enriched in samples otherwise lacking an activated oncogene, suggesting a driver role for these events in certain tumours. DNA and mRNA sequence from the same tumour highlighted splicing alterations driven by somatic genomic changes, including exon 14 skipping in MET mRNA in 4% of cases. MAPK and PI(3)K pathway activity, when measured at the protein level, was explained by known mutations in only a fraction of cases, suggesting additional, unexplained mechanisms of pathway activation. These data establish a foundation for classification and further investigations of lung adenocarcinoma molecular pathogenesis.

Recent cancer genome sequencing and analysis has identified millions of somatic mutations in cancer. However, the functional impact of most variants is poorly understood, limiting the use of this genetic knowledge for clinical decision-making. Here we describe a new high-throughput approach, expression-based variant impact phenotyping (eVIP), which uses gene expression changes to infer somatic mutation impact. We generated a lentiviral expression library representing 53 genes and 194 somatic mutations identified in primary lung adenocarcinomas. Next, we introduced this library into A549 lung adenocarcinoma cells and 96 hours later performed gene expression profiling using Luminex-based L1000 profiling. We built a computational pipeline, eVIP, to compare mutant and wild-type expression signatures to infer whether variants were gain-of-function, change-of-function, loss-of-function, or neutral. Overall, eVIP identified 69% of mutations as impactful whereas 31% appeared functionally neutral. A very high rate, 92%, of missense mutations in the KEAP1 and STK11 tumor suppressor genes were found to inactivate or diminish protein function. As a complementary approach, we assessed which mutations are epistatic to EGFR or capable of initiating xenograft tumor formation in vivo. A subset of the impactful mutations identified by eVIP could induce xenograft tumor formation in mice and/or confer resistance to cellular EGFR inhibition. Among these mutations were 20 rare or non-canonical somatic variants in clinically-actionable or -relevant oncogenes including EGFR S645C, ARAF S214C and S214F, ERBB2 S418T, and PIK3CA E600K. eVIP can, in principle, characterize any genetic variant, independent of prior knowledge of gene function. Further application of eVIP should significantly advance the pace of functional characterization of mutations identified from genome sequencing. Citation Format: Alice H. Berger, Angela N. Brooks, Xiaoyun Wu, Yashaswi Shrestha, Candace Chouinard, Federica Piccioni, Mukta Bagul, Atanas Kamburov, Marcin Imielinski, Larson Hogstrom, Cong Zhu, Xiaoping Yang, Sasha Pantel, Ryo Sakai, Nathan Kaplan, David Root, Rajiv Narayan, Ted Natoli, David Lahr, Itay Tirosh, Pablo Tamayo, Gad Getz, Bang Wong, John Doench, Aravind Subramanian, Todd R. Golub, Matthew Meyerson, Jesse S. Boehm. High-throughput phenotyping of lung cancer somatic mutations. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 4368.

SummaryLung adenocarcinoma, the most common subtype of non-small cell lung cancer, is responsible for more than 500,000 deaths per year worldwide. Here, we report exome and genome sequences of 183 lung adenocarcinoma tumor/normal DNA pairs. These analyses revealed a mean exonic somatic mutation rate of 12.0 events/megabase and identified the majority of genes previously reported as significantly mutated in lung adenocarcinoma. In addition, we identified statistically recurrent somatic mutations in the splicing factor gene U2AF1 and truncating mutations affecting RBM10 and ARID1A. Analysis of nucleotide context-specific mutation signatures grouped the sample set into distinct clusters that correlated with smoking history and alterations of reported lung adenocarcinoma genes. Whole-genome sequence analysis revealed frequent structural rearrangements, including in-frame exonic alterations within EGFR and SIK2 kinases. The candidate genes identified in this study are attractive targets for biological characterization and therapeutic targeting of lung adenocarcinoma.

It was originally proposed that tumour suppressor genes (TSGs) act in a recessive manner. Instead, numerous TSGs, including p53 and PTEN, exhibit haplo-insufficiency as a consequence of the dose-dependency of TSG function. Due to the challenges of identifying haplo-insufficient TSGs by human genetics analysis alone, mouse models play a pivotal role in firmly establishing the haplo-insufficiency of a gene, as in the recent identification of DOK2 as a haplo-insufficient lung TSG. In many cases, TSGs exhibit conditional or compound haplo-insufficiency, in which loss of one TSG allele is functionally important only in certain settings or after compound loss of other genes. The 5q deletion syndrome (5q(-) ) is a paradigm of compound haplo-insufficiency and demonstrates the importance of combinatorial interactions to elicit specific phenotypes. These concepts must be integrated into basic science studies to avoid delay in the identification of important TSGs. In the clinical realm, the challenges for molecular pathologists are the development of quantitative measures that can accurately and systematically ascertain the status of haplo-insufficient genes in tumour biopsies, and the use of this information to accurately predict prognosis and response to therapy.

This year, 2011, marks the forty-year anniversary of the statistical analysis of retinoblastoma that provided the first evidence that tumorigenesis can be initiated by as few as two mutations. This work provided the foundation for the two-hit hypothesis that explained the role of recessive tumour suppressor genes (TSGs) in dominantly inherited cancer susceptibility syndromes. However, four decades later, it is now known that even partial inactivation of tumour suppressors can critically contribute to tumorigenesis. Here we analyse this evidence and propose a continuum model of TSG function to explain the full range of TSG mutations found in cancer.

With the comprehensive analysis of cancer genomes approaching completion, the research community stands poised to rapidly advance genome-guided therapeutic hypotheses into clinical settings. However, for the vast majority of cancer patients, existing knowledge of the function(s) of the newly discovered mutant genes harbored by their tumor is incomplete or non-existent since most cancer mutations are exceedingly rare. As a result, we now have long lists of candidate alleles but a paucity of targets whose biology is sufficiently well understood to guide therapeutics. Here we present an interim progress report on a pilot effort aiming to create a generalizable framework to systematically map the molecular consequences of cancer variants at scale (Target Accelerator). First, we created an efficient pipeline to generate cancer variants and generated an initial library of 1300 mutant cDNA clones corresponding to variants in lung cancer and diffuse large B-cell lymphoma as well as those nominated by “pan-cancer” computational analyses. Second, we established an industry-scale, next-generation pipeline to generate new cancer models (Cell Line Factory) directly from patient samples. We have leveraged this pipeline to process over 330 samples from 208 patients across 16 cancer types, with over 60% growing through at least 5 population doublings. We show that tumor genomics can be retained in such patient-derived models and that drug testing to discover clinically validated dependencies within 3 months is feasible. In addition, we use combinatorial molecular barcoding to rapidly generate a panel of pathway-primed human tumorigenesis models that are suitable for massively parallel multiplexed tumorigenesis assays in vivo (TumorPlex). We hypothesized that this integrated framework could be utilized to generate meaningful functional hypotheses from cancer variants of unknown significance in a high-throughput manner. To test this hypothesis, we introduced over 1000 cancer mutations into cell models and created gene expression signatures together with phenotypic data. In lung cancer, we show that the mutational impact of mutant alleles with known and unknown functions can be rapidly assessed by comparing signatures of wild-type and mutant alleles. We show that this generalizable approach, which does not require prior knowledge, can place variants of unknown significance into dominant gain-of-function and loss-of-function categories. As a complementary approach, we have used TumorPlex assays to test the tumorigenic potential of 550 mutant alleles nominated by Pan-Cancer computational analyses and discovered unexpected variants in the KRAS, AKT1, MAP2K1, ERBB2, PIK3CB, NFE2L2, FAM200A and POT1 genes as being potently tumorigenic. These proof-of-concept studies demonstrate initial feasibility of mapping cancer variant function at scale. Importantly, they demarcate a path by which mapping variant function and predicting vulnerabilities might soon be possible on a patient-by-patient basis, achieving the promise of precision functional genomics. Citation Format: Alice H. Berger, Eejung Kim, Angela Brooks, Nina Ilic, Yashaswi Shrestha, Yuen-Yi Tseng, Xiaoyun Wu, Lihua Zou, Atanas Kamburov, Xiaoping Yang, Cong Zhu, Paula Keskula, Sara Seepo, Andrew Hong, Philip Kantoff, Keith L. Ligon, Levi A. Garraway, John G. Doench, David E. Root, Matthew Meyerson, William C. Hahn, Gad Getz, Todd R. Golub, Jesse S. Boehm. Towards precision functional genomics via next-generation functional mapping of cancer variants. [abstract]. In: Proceedings of the AACR Special Conference on Translation of the Cancer Genome; Feb 7-9, 2015; San Francisco, CA. Philadelphia (PA): AACR; Cancer Res 2015;75(22 Suppl 1):Abstract nr PR07.

Lung adenocarcinoma is comprised of distinct mutational subtypes characterized by mutually exclusive oncogenic alterations such as EGFR and KRAS mutations. Identification of these oncogenic events has transformed treatment strategies and drug development for lung adenocarcinoma. However, such mutations have been identified in only 50-60% of lung adenocarcinoma cases in the United States. We analyzed SNP array, exome, and RNA sequencing data from 230 primary lung adenocarcinomas to identify known and novel mutually exclusive driver events. MET exon 14 skipping was present in 10/230 tumors (4.3%) and only RNA sequencing data allowed robust detection of this event. To identify novel driver events, we performed a focused statistical analysis of the 87/230 tumors (38%) that lacked a mutation in MET or other known oncogenes. Copy number analysis revealed rare, focal amplifications of MET and ERBB2 that were enriched in this subset. Exome analysis revealed significant enrichment of TP53, KEAP1, NF1, and RIT1 mutations in the previously oncogene-negative subset. Of these mutated genes, only NF1 and RIT1 displayed mutual exclusivity with the other known and candidate driver events. Whereas TP53, KEAP1, and NF1 are well-documented cancer genes, little is known about the role of RIT1 in human cancer. RIT1 is a RAS-subfamily small GTPase with significant homology to HRAS, NRAS, and KRAS. Mutations in RIT1 were present in ∼2% of cases and clustered in the switch II domain. Ectopic expression of mutated RIT1 in murine fibroblasts and human immortalized lung epithelial cells conferred anchorage-independent growth and tumor forming capability. cDNA sequencing identified a p.M90I missense mutation in the NCI-H2110 cell line. Knockdown of RIT1 in NCI-H2110 cells revealed the role of RIT1 in regulation of PI3K and MEK signaling. Consistently, combination PI3K/mTOR/MEK inhibition could inhibit cellular transformation induced by ectopic expression of mutated RIT1, and PI3K/mTOR inhibition suppressed in vivo tumor growth of NCI-H2110 cells. Recently, RIT1 mutations were described in myeloid malignancies and Noonan syndrome. Our data, together with these reported observations, indicate that RIT1 is a RAS-pathway oncogene in lung adenocarcinoma. In addition, PI3K/mTOR inhibition with or without MEK inhibition should be explored as a therapeutic strategy for RIT1-mutated lung or other RIT1-mutated cancers. This work expands the number of U.S. lung adenocarcinoma tumors with identified driver events from 62% to 75% and nominates these additional alterations as candidate therapeutic targets. This abstract is also presented as Poster B26. Citation Format: Alice H. Berger, Angela N. Brooks, Marcin Imielinski, Andrew Cherniack, Fujiko Duke, Nathan Kaplan, Jeremiah Wala, Matthew Meyerson. NF1, MET, and RIT1 mutations are RAS-pathway driver events in lung adenocarcinoma. [abstract]. In: Proceedings of the AACR Special Conference on RAS Oncogenes: From Biology to Therapy; Feb 24-27, 2014; Lake Buena Vista, FL. Philadelphia (PA): AACR; Mol Cancer Res 2014;12(12 Suppl):Abstract nr PR08. doi: 10.1158/1557-3125.RASONC14-PR08

Recently, we identified somatic mutations in the RAS-family small GTPase gene RIT1 in 2.4% (10/413) of lung adenocarcinoma tumors. Mutation of RIT1 is mutually exclusive with other oncogenic mutations in the receptor tyrosine kinase/RAS pathway, suggesting that RIT1 mutations are rare driver mutations in lung adenocarcinoma. Oncogenic RIT1 mutations cluster in the switch II domain of the protein and are observed not only in lung adenocarcinoma but also in myeloid malignancies, breast carcinoma, cervical carcinoma, and in the germline of Noonan syndrome patients. Here we functionally characterize 26 RIT1 point mutations and in-frame insertions/deletions in murine fibroblasts and human immortalized lung epithelial cells and find that the majority of somatic RIT1 mutations are able to transform cells. Furthermore, we identified a human lung cancer cell line, NCI-H2110, with an endogenous RIT1 p.M90I mutation. Knockdown of RIT1 in NCI-H2110 cells revealed the role of RIT1 in regulation of PI3K and MEK signaling. Consistently, combination PI3K/MEK inhibition could inhibit cellular transformation induced by ectopic expression of mutated RIT1. Furthermore, PI3K inhibition suppressed in vivo growth of NCI-H2110 cells. These data identify RIT1 as an oncogene and nominate combined PI3K and MEK inhibition as a therapeutic strategy in RIT1-mutated cancers. This abstract is also presented as Poster A04. Citation Format: Alice H. Berger, Fujiko Duke, Marcin Imielinski, Nathan Kaplan, Jeremiah Wala, Geng-Xian Shi, Douglas A. Andres, Matthew Meyerson. Recurrent oncogenic mutations in the small GTPase RIT1 activate PI3K and MEK. [abstract]. In: Proceedings of the AACR-IASLC Joint Conference on Molecular Origins of Lung Cancer; 2014 Jan 6-9; San Diego, CA. Philadelphia (PA): AACR; Clin Cancer Res 2014;20(2Suppl):Abstract nr PR02.

Lung adenocarcinoma is comprised of distinct mutational subtypes characterized by mutually exclusive oncogenic mutations in RTK/RAS pathway members KRAS, EGFR, BRAF, and ERBB2, and translocations involving ALK, RET, and ROS1. Identification of these oncogenic events has transformed therapy of lung adenocarcinoma via application of therapies targeted towards specific genetic lesions in stratified patient populations. However, such mutations have been identified in only ∼55% of lung adenocarcinoma cases in the United States, suggesting other mechanisms of malignancy may be involved in the remaining cases. We analyzed exome sequencing data from 413 lung adenocarcinomas and identified somatic mutations in the small GTPase gene RIT1 in ∼2% of samples. Mutations in RIT1 cluster in a hotspot near the switch II domain of the protein that is homologous to the Q61 region in RAS genes. RIT1 switch II domain mutations are mutually exclusive with all other known lung adenocarcinoma driver mutations. Ectopic expression of mutated RIT1 induces activation of AKT and ERK signaling pathways and transforms NIH3T3 cells in vitro and in vivo. These data identify RIT1 as a driver oncogene in a specific subset of lung adenocarcinomas. Citation Information: Mol Cancer Ther 2013;12(11 Suppl):C140. Citation Format: Alice Berger, Marcin Imielinski, Fujiko Duke, Jeremiah Wala, Nathan Kaplan, Geng-Xian Shi, Douglas Andres, Matthew Meyerson. RIT1 mutations define a new genetic subset of lung adenocarcinoma. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2013 Oct 19-23; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2013;12(11 Suppl):Abstract nr C140.

An estimated 15-20% of all human malignancies is caused by infectious agents, such as human papillomavirus (HPV), Epstein-Barr virus (EBV) or Helicobacter pylori (H. pylori). The recent development of massively parallel next generation sequencing technology led to the discovery of Merkel cell polyomavirus, and more recently a significant enrichment of Fusobacterium nucleatum (F. nucleatum) and related Fusobacterium species in colorectal carcinomas compared to non-neoplastic colon tissue from the same patients. However, these studies involved relatively small number of samples and there are likely many additional cancer-associated microbes that are yet to be discovered. TCGA sequencing data from over 20 human cancer types provide an unprecedented opportunity for cancer-associated pathogen discovery. Using PathSeq, software for pathogen discovery by computational subtraction, we have begun analyzing TCGA sequencing data for the presence of viral, fungal or bacterial sequences. We will present our preliminary data from the analysis of mRNA-seq data including evidence for (1) a greater role of viral integration into tumor suppressor gene loci in HPV-driven tumors and (2) complex interplay between viruses, bacteria and host cancer cells in various cancer types. We will also discuss our discovery efforts for previously unannotated pathogens in the TCGA dataset. Citation Format: Joonil Jung, Chandra Sekhar Pedamallu, Aleksander D. Kostic, Samuel Freeman, Akinyemi I. Ojesina, Alice Berger, Ami Bhatt, Fujiko Duke, Gad Getz, Matthew Meyerson. Pan-cancer PathSeq analysis on TCGA data for pathogen discovery. [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 1501. doi:10.1158/1538-7445.AM2013-1501