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Despite the remarkable clinical responses achieved with immune checkpoint blockade therapy, the response rate is relatively low and only a subset of patients can benefit from the treatment. Aberrant RNA accumulation can mediate IFN signaling and stimulate an immune response, suggesting that targeting RNA decay machinery might sensitize tumor cells to immunotherapy. With this in mind, we identified an RNA exoribonuclease, XRN1, as a potential therapeutic target to suppress RNA decay and stimulate antitumor immunity. Silencing of XRN1 suppressed tumor growth in syngeneic immunocompetent mice and potentiated immunotherapy efficacy, while silencing of XRN1 alone did not affect tumor growth in immunodeficient mice. Mechanistically, XRN1 depletion activated IFN signaling and the viral defense pathway; both pathways play determinant roles in regulating immune evasion. Aberrant RNA-sensing signaling proteins (RIG-I/MAVS) mediated the expression of IFN genes, as depletion of each of them blunted the elevation of antiviral/IFN signaling in XRN1-silenced cells. Analysis of pan-cancer CRISPR-screening data indicated that IFN signaling triggered by XRN1 silencing is a common phenomenon, suggesting that the effect of XRN1 silencing may be extended to multiple types of cancers. Overall, XRN1 depletion triggers aberrant RNA-mediated IFN signaling, highlighting the importance of the aberrant RNA-sensing pathway in regulating immune responses. These findings provide the molecular rationale for developing XRN1 inhibitors and exploring their potential clinical application in combination with cancer immunotherapy.Significance:Targeting XRN1 activates an intracellular innate immune response mediated by RNA-sensing signaling and potentiates cancer immunotherapy efficacy, suggesting inhibition of RNA decay machinery as a novel strategy for cancer treatment.

Background Standard platinum-based therapy for ovarian cancer is inefficient against ovarian clear cell carcinoma (OCCC). OCCC is a distinct subtype of epithelial ovarian cancer. OCCC constitutes 25% of ovarian cancers in East Asia (Japan, Korea, China, Singapore) and 6–10% in Europe and North America. The cancer is characterized by frequent inactivation of ARID1A and 10% of cases of endometriosis progression to OCCC. The aim of this study was to identify drugs that are either FDA-approved or in clinical trials for the treatment of OCCC. Results High throughput screening of 166 compounds that are either FDA-approved, in clinical trials or are in pre-clinical studies identified several cytotoxic compounds against OCCC. ARID1A knockdown cells were more sensitive to inhibitors of either mTOR (PP242), dual mTOR/PI3K (GDC0941), ATR (AZD6738) or MDM2 (RG7388) compared to control cells. Also, compounds targeting BH3 domain (AZD4320) and SRC (AZD0530) displayed preferential cytotoxicity against ARID1A mutant cell lines. In addition, WEE1 inhibitor (AZD1775) showed broad cytotoxicity toward OCCC cell lines, irrespective of ARID1A status. Conclusions In a selection of 166 compounds we showed that inhibitors of ATR and WEE1 were cytotoxic against a panel of OCCC cell lines. These two drugs are already in other clinical trials, making them ideal candidates for treatment of OCCC.

Background Nodal staging systems vary substantially across solid tumors, implying heterogeneity in the behavior of nodal variables in various contexts. We hypothesized, in contradiction to this, that metastatic lymph node (LN) number is a universal and dominant predictor of outcome across solid tumors. Methods We performed a retrospective cohort analysis of 1 304 498 patients in the National Cancer Database undergoing surgery between 2004 and 2015 across 16 solid cancer sites. Multivariable Cox regression analyses were constructed using restricted cubic splines to model the association between nodal number and mortality. Recursive partitioning analysis (RPA) was used to derive nodal classification systems for each solid cancer based on metastatic LN count. The reproducibility of these findings was assessed in 1 969 727 patients from the Surveillance, Epidemiology, and End Results registry. Two-sided tests were used for all statistical analyses. Results Consistently across disease sites, mortality risk increased continuously with increasing number of metastatic LNs (P Conclusions Quantitative metastatic LN burden is a fundamental driver of mortality across solid cancers and should serve as a foundation for pathologic nodal staging across solid tumors.

Tumor organoid modeling has been recognized as a state-of-the-art system for in vitro research on cancer biology and precision oncology. Organoid culture technologies offer distinctive advantages, including faithful maintenance of physiological and pathological characteristics of human disease, self-organization into three-dimensional multicellular structures, and preservation of genomic and epigenomic landscapes of the originating tumor. These features effectively position organoid modeling between traditional cell line cultures in two dimensions and in vivo animal models as a valid, versatile, and robust system for cancer research. Here, we review recent advances in genomic and epigenomic characterization of tumor organoids and the novel findings obtained, highlight significant progressions achieved in organoid modeling of gene–drug interactions and genotype–phenotype associations, and offer perspectives on future opportunities for organoid modeling in basic and clinical cancer research.

While numerous factors affect prognosis in papillary thyroid carcinoma (PTC), the comparative impact of histologic grade has not been well described. Moreover, indications for external beam radiation therapy (EBRT) remain imprecise. We evaluate clinicopathologic characteristics and outcomes for PTC stratified by grade. We profiled histologic grade for PTC (well differentiated, moderately differentiated, poorly differentiated) via hospital (National Cancer Database) and population-based (Surveillance, Epidemiology, and End Results) registries. Cox regression was used to adjust for clinicopathologic covariates. Statistical interactions between subtypes and the effect of EBRT on survival were assessed. Collectively, worsening clinicopathologic factors (age, tumor size, extrathyroidal extension, nodal spread, M1 disease) and outcomes (disease-free survival, overall survival) correlated with less differentiated state, across all histologic grades (p

Long noncoding RNAs (lncRNAs) have important regulatory roles in cancer biology. Although some lncRNAs have well‐characterized functions, the vast majority of this class of molecules remains functionally uncharacterized. To systematically pinpoint functional lncRNAs, a computational approach was proposed for identification of lncRNA‐mediated competing endogenous RNAs (ceRNAs) through combining global and local regulatory direction consistency of expression. Using esophageal squamous cell carcinoma (ESCC) as model, we further identified many known and novel functional lncRNAs acting as ceRNAs (ce‐lncRNAs). We found that most of them significantly regulated the expression of cancer‐related hallmark genes. These ce‐lncRNAs were significantly regulated by enhancers, especially super‐enhancers (SEs). Landscape analyses for lncRNAs further identified SE‐associated functional ce‐lncRNAs in ESCC, such as HOTAIR, XIST, SNHG5, and LINC00094. THZ1, a specific CDK7 inhibitor, can result in global transcriptional downregulation of SE‐associated ce‐lncRNAs. We further demonstrate that a SE‐associated ce‐lncRNA, LINC00094 can be activated by transcription factors TCF3 and KLF5 through binding to SE regions and promoted ESCC cancer cell growth. THZ1 downregulated expression of LINC00094 through inhibiting TCF3 and KLF5. Our data demonstrated the important roles of SE‐associated ce‐lncRNAs in ESCC oncogenesis and might serve as targets for ESCC diagnosis and therapy., We developed the GloceRNA method for the identification of functional ce‐lncRNAs based on merging global and local regulatory direction consistency of expression associated with ceRNAs. GloceRNA identified many known and novel functional ce‐lncRNAs, which regulated the expression of a large number of cancer hallmark genes. We further identified novel SE‐associated ce‐lncRNAs and demonstrated their important roles in ESCC.

ZFP36L1 is a tandem zinc-finger RNA-binding protein that recognizes conserved adenylate-uridylate–rich elements (ARE) located in 3′untranslated regions (UTR) to mediate mRNA decay. We hypothesized that ZFP36L1 is a negative regulator of a posttranscriptional hub involved in mRNA half-life regulation of cancer-related transcripts. Analysis of in silico data revealed that ZFP36L1 was significantly mutated, epigenetically silenced, and downregulated in a variety of cancers. Forced expression of ZFP36L1 in cancer cells markedly reduced cell proliferation in vitro and in vivo, whereas silencing of ZFP36L1 enhanced tumor cell growth. To identify direct downstream targets of ZFP36L1, systematic screening using RNA pull-down of wild-type and mutant ZFP36L1 as well as whole transcriptome sequencing of bladder cancer cells {plus minus} tet-on ZFP36L1 was performed. A network of 1,410 genes was identified as potential direct targets of ZFP36L1. These targets included a number of key oncogenic transcripts such as HIF1A, CCND1, and E2F1. ZFP36L1 specifically bound to the 3′UTRs of these targets for mRNA degradation, thus suppressing their expression. Dual luciferase reporter assays and RNA electrophoretic mobility shift assays showed that wild-type, but not zinc-finger mutant ZFP36L1, bound to HIF1A 3′UTR and mediated HIF1A mRNA degradation, leading to reduced expression of HIF1A and its downstream targets. Collectively, our findings reveal an indispensable role of ZFP36L1 as a posttranscriptional safeguard against aberrant hypoxic signaling and abnormal cell-cycle progression. Significance: RNA-binding protein ZFP36L1 functions as a tumor suppressor by regulating the mRNA stability of a number of mRNAs involved in hypoxia and cell-cycle signaling.

BackgroundChemotherapy has long been shown to confer a survival benefit in patients with metastatic esophageal cancer. However, not all patients with metastatic disease receive chemotherapy.AimTo evaluate a large cancer database of metastatic esophageal cancer cases to identify predictors of receipt to chemotherapy and survival.MethodsWe interrogated the National Cancer Database (NCDB) between 2004-2015 and included patients with M1 disease who had received or did not receive chemotherapy. A logistic regression model was used to examine the associations between chemotherapy and potential confounders and a Cox proportional hazards model was employed to examine the effect of chemotherapy on overall survival (OS). Propensity score analyses were further performed to balance measurable confounders between patients treated with and without chemotherapy.ResultsA total of 29182 patients met criteria for inclusion in this analysis, with 21911 (75%) receiving chemotherapy and 7271 (25%) not receiving chemotherapy. The median follow-up was 69.45 mo. The median OS for patients receiving chemotherapy was 9.53 mo (9.33-9.72) vs 2.43 mo (2.27-2.60) with no chemotherapy. Year of diagnosis 2010-2014 [odds ratio (OR): 1.29, 95% confidence interval (CI): 1.17-1.43, P value < 0.001], median income > $46000 (OR: 1.49, 95%CI: 1.27-1.75, P value < 0.001), and node-positivity (OR: 1.35, 95%CI: 1.20-1.52, P < 0.001) were independent predictors of receiving chemotherapy, while female gender (OR: 0.86, 95%CI: 0.76-0.98, P = 0.019), black race (OR: 0.76, 95%CI: 0.67-0.93, P = 0.005), uninsured status (OR: 0.41, 95%CI: 0.33-0.52, P < 0.001), and high Charlson Comorbidity Index (CCI) (OR for CCI ≥ 2: 0.61, 95%CI: 0.50-0.74, P < 0.001) predicted for lower odds of receiving chemotherapy. Modeling the effect of chemotherapy on OS using a time-dependent coefficient showed that chemotherapy was associated with improved OS up to 10 mo, after which there is no significant effect on OS. Moreover, uninsured status [hazard ratio (HR): 1.20, 95%CI: 1.09-1.31, P < 0.001], being from the geographic Midwest (HR: 1.07, 95%CI: 1.01-1.14, P = 0.032), high CCI (HR for CCI ≥ 2: 1.16, 95%CI: 1.07-1.26, P < 0.001), and higher tumor grade (HR for grade 3 vs grade 1: 1.28, 95%CI: 1.14-1.44, P < 0.001) and higher T stage (HR for T1 vs T4: 0.89, 95%CI: 0.84-0.95, P < 0.001) were independent predictors of worse OS on multivariable analyses.ConclusionIn this large, retrospective NCDB analysis, we identified several socioeconomic and clinicopathologic predictors for receiving chemotherapy and OS in patients with metastatic esophageal cancer. The benefit of chemotherapy on OS is time-dependent and favors early initiation. Focused outreach in lower income and underinsured patients is critical as receipt of chemotherapy is associated with improved OS.

The Decipher 22-gene genomic classifier (GC) may help in post-radical prostatectomy (RP) decision making given its superior prognostic performance over clinicopathologic variables alone. However, most studies evaluating the GC have had a modest representation of African-American men (AAM). We evaluated the GC within a large Veteran Affairs cohort and compared its performance to CAPRA-S for predicting outcomes in AAM and non-AAM after RP. GC scores were generated for 548 prostate cancer (PC) patients, who underwent RP at the Durham Veteran Affairs Medical Center between 1989 and 2016. This was a clinically high-risk cohort and was selected to have either pT3a, positive margins, seminal vesicle invasion, or received post-RP radiotherapy. Multivariable Cox models and survival C-indices were used to compare the performance of GC and CAPRA-S for predicting the risk of metastasis and PC-specific mortality (PCSM). Median follow-up was 9 years, during which 37 developed metastasis and 20 died from PC. Overall, 55% (n = 301) of patients were AAM. In multivariable analyses, GC (high vs. intermediate and intermediate vs. low) was a significant predictor of metastasis in all men (all p

The genetic events occurring in recurrent nasopharyngeal carcinoma (rNPC) are poorly understood. Here, we performed whole-genome and whole-exome sequencing in 55 patients with rNPC and 44 primarily diagnosed NPC (pNPC), with 7 patients having paired rNPC and pNPC samples. Previously published pNPC exome data were integrated for analysis. rNPC and pNPC tissues had similar mutational burdens, however, the number of clonal mutations was increased in rNPC samples. TP53 and three NF-κB pathway components (TRAF3, CYLD, and NFKBIA) were significantly mutated in both pNPC and rNPC. Notably, mutations in TRAF3, CYLD, and NFKBIA were all clonal in rNPC, however, 55.6% to 57.9% of them were clonal in pNPC. In general, the number of clonal mutations in NF-κB pathway–associated genes was significantly higher in rNPC than in pNPC. The NF-κB mutational clonality was selected and/or enriched during NPC recurrence. The amount of NF-κB translocated to the nucleus in samples with clonal NF-κB mutants was significantly higher than that in samples with subclonal NF-κB mutants. Moreover, the nuclear abundance of NF-κB protein was significantly greater in pNPC samples with locoregional relapse than in those without relapse. Furthermore, high nuclear NF-κB levels were an independent negative prognostic marker for locoregional relapse-free survival in pNPC. Finally, inhibition of NF-κB enhanced both radiosensitivity and chemosensitivity in vitro and in vivo. In conclusion, NF-κB pathway activation by clonal mutations plays an important role in promoting the recurrence of NPC. Moreover, nuclear accumulation of NF-κB is a prominent biomarker for predicting locoregional relapse-free survival. Significance: This study uncovers genetic events that promote the progression and recurrence of nasopharyngeal carcinoma and has potential prognostic and therapeutic implications. See related commentary by Sehgal and Barbie, p. 5915

Background: While numerous factors determine prognosis in papillary thyroid carcinoma (PTC), distant metastasis (M1) represents one of the most dire. Escalating nodal burden and aggressive histolog...

Immunotherapy has been established as a standard in select molecular subgroups of treatment-refractory advanced gastric cancer. However, its role in resectable gastric cancer where perioperative systemic therapy is the standard remains unclear. We present a case of a man who was diagnosed with resectable gastric cancer that was microsatellite stable but programmed death-ligand 1 (PD-L1) and Epstein-Barr Virus (EBV)-positive. Given extenuating circumstances of the SARS-CoV-2 pandemic, preferences to limit exposure to the healthcare setting, and the unique tumor molecular features, neoadjuvant pembrolizumab and capecitabine was pursued after multidisciplinary discussion. He was able to achieve a complete response to this neoadjuvant regimen with no further signs of radiographic or pathologic disease on follow-up. We highlight a dramatic response to this novel approach that represents among the first cases to support a potentially viable neoadjuvant chemoimmunotherapy strategy to resectable gastric cancer. In select patients, perioperative immunotherapy-based therapy may constitute a promising strategy in resectable gastric cancer and warrants further investigation.

Modern disease staging systems have restructured human papillomavirus (HPV)-negative (HPV-) and HPV-positive (HPV+) oropharyngeal carcinoma (OPC) into distinct pathologic nodal systems. Given that quantitative lymph node (LN) burden is the dominant prognostic factor in most head and neck cancers, we investigated whether HPV- and HPV+ OPC warrant divergent pathologic nodal classification.Multivariable Cox regression models of OPC surgical patients identified via U.S. cancer registry data were constructed to determine associations between survival and nodal characteristics. Nonlinear associations between metastatic LN number and survival were modeled with restricted cubic splines. Recursive partitioning analysis (RPA) was used to derive unbiased nodal schema.Mortality risk escalated continuously with each successive positive LN in both OPC subtypes, with analogous slope. Survival hazard increased by 18.5% (hazard ratio [HR], 1.19 [95% CI, 1.16-1.21]; P .001) and 19.1% (HR, 1.19 [95% CI, 1.17-1.21]; P .001), with each added positive LN for HPV- and HPV+ OPC, respectively, up to identical change points of 5 positive LNs. Extranodal extension (ENE) was an independent predictor of HPV- OPC (HR, 1.55 [95% CI, 1.20-1.99]; P .001) and HPV+ OPC (HR 1.73 [95% CI, 1.36-2.20]; P .001) mortality. In RPA for both diseases, metastatic LN was the principal nodal covariate driving survival, with ENE as a secondary determinant. Given the similarities across analyses, we propose a concise, unifying HPV-/HPV+ OPC pathologic nodal classification schema: N1, 1-5 LN+/ENE-; N2, 1-5 LN+/ENE+; N3,5 LN+.HPV- and HPV+ OPC exhibit parallel relationships between nodal characteristics and relative mortality. In both diseases, metastatic LN number represents the principal nodal covariate governing survival, with ENE being an influential secondary element. A consolidated OPC pathologic nodal staging system that is based on these covariates may best convey prognosis.The current nodal staging system for oropharyngeal carcinoma (OPC) has divided human papillomavirus (HPV)-negative (HPV-) and HPV-positive (HPV+) OPC into distinct systems that rely upon criteria that establish them as separate entities, a complexity that may undermine the core objective of staging schema to clearly communicate prognosis. Our large-scale analysis revealed that HPV- and HPV+ pathologic nodal staging systems in fact mirror each other. Multiple analyses produced conspicuously similar nodal staging systems, with metastatic lymph node number and extranodal extension delineating the highest risk groups that shape prognosis. We propose unifying HPV- and HPV+ nodal systems to best streamline prognostication and maximize staging accuracy.

Although obesity is one of the strongest risk factors for esophageal adenocarcinoma, the molecular mechanisms underlying this association remain unclear. We recently identified four esophageal adenocarcinoma–specific master regulator transcription factors (MRTF) ELF3, KLF5, GATA6, and EHF. In this study, gene-set enrichment analysis of both esophageal adenocarcinoma patient samples and cell line models unbiasedly underscores fatty acid synthesis as the central pathway downstream of three MRTFs (ELF3, KLF5, GATA6). Further characterizations unexpectedly identified a transcriptional feedback loop between MRTF and fatty acid synthesis, which mutually activated each other through the nuclear receptor, PPARG. MRTFs cooperatively promoted PPARG transcription by directly regulating its promoter and a distal esophageal adenocarcinoma–specific enhancer, leading to PPARG overexpression in esophageal adenocarcinoma. PPARG was also elevated in Barrett’s esophagus, a recognized precursor to esophageal adenocarcinoma, implying that PPARG might play a role in the intestinal metaplasia of esophageal squamous epithelium. Upregulation of PPARG increased de novo synthesis of fatty acids, phospholipids, and sphingolipids as revealed by mass spectrometry–based lipidomics. Moreover, ChIP-seq, 4C-seq, and a high-fat diet murine model together characterized a novel, noncanonical, and cancer-specific function of PPARG in esophageal adenocarcinoma. PPARG directly regulated the ELF3 super-enhancer, subsequently activating the transcription of other MRTFs through an interconnected regulatory circuitry. Together, elucidation of this novel transcriptional feedback loop of MRTF/PPARG/fatty acid synthesis advances our understanding of the mechanistic foundation for epigenomic dysregulation and metabolic alterations in esophageal adenocarcinoma. More importantly, this work identifies a potential avenue for prevention and early intervention of esophageal adenocarcinoma by blocking this feedback loop. Significance: These findings elucidate a transcriptional feedback loop linking epigenomic dysregulation and metabolic alterations in esophageal adenocarcinoma, indicating that blocking this feedback loop could be a potential therapeutic strategy in high-risk individuals.

Gastrointestinal adenocarcinomas (GIAC) of the tubular gastrointestinal (GI) tract including esophagus, stomach, colon, and rectum comprise most GI cancers and share a spectrum of genomic features. However, the unified epigenomic changes specific to GIAC are poorly characterized. Using 907 GIAC samples from The Cancer Genome Atlas, we applied mathematical algorithms to large-scale DNA methylome and transcriptome profiles to reconstruct transcription factor (TF) networks and identify a list of functionally hyperactive master regulator (MR) TF shared across different GIAC. The top candidate HNF4A exhibited prominent genomic and epigenomic activation in a GIAC-specific manner. A complex interplay between the HNF4A promoter and three distal enhancer elements was coordinated by GIAC-specific MRTF including ELF3, GATA4, GATA6, and KLF5. HNF4A also self-regulated its own promoter and enhancers. Functionally, HNF4A promoted cancer proliferation and survival by transcriptional activation of many downstream targets, including HNF1A and factors of interleukin signaling, in a lineage-specific manner. Overall, our study provides new insights into the GIAC-specific gene regulatory networks and identifies potential therapeutic strategies against these common cancers. Significance: These findings show that GIAC-specific master regulatory transcription factors control HNF4A via three distal enhancers to promote GIAC cell proliferation and survival.

Purpose: Agents extracted from natural sources with antitumor property have attracted considerable attention from researchers and clinicians because of their safety, efficacy, and immediate availability. Degalactotigonin (DGT), extracted from Solanum nigrum L., has anticancer properties without serious side effects. Here, we explored whether DGT can inhibit the growth and metastasis of osteosarcoma. Experimental Design: MTT, colony formation, and apoptosis assays were performed to analyze the effects of DGT on osteosarcoma cell viability in vitro. The migration and invasion abilities were measured using a Transwell assay. Animal models were used to assess the roles of DGT in both tumor growth and metastasis of osteosarcoma. Gli1 expression and function were measured in osteosarcoma cells and clinical samples. After DGT treatment, Gli1 activation and the phosphorylation status of multiple cellular kinases were measured with a luciferase reporter and phospho-kinase antibody array. Results: DGT inhibited proliferation, induced apoptosis, and suppressed migration and invasion in osteosarcoma cells. DGT, injected intraperitoneally after tumor inoculation, significantly decreased the volume of osteosarcoma xenografts and dramatically diminished the occurrence of osteosarcoma xenograft metastasis to the lungs. Mechanistically, DGT inhibited osteosarcoma growth and metastasis through repression of the Hedgehog/Gli1 pathway, which maintains malignant phenotypes and is involved in the prognosis of osteosarcoma patients. DGT decreased the activity of multiple intracellular kinases that affect the survival of osteosarcoma patients, including GSK3β. In addition, DGT represses the Hedgehog/Gli1 pathway mainly through GSK3β inactivation. Conclusions: Our studies provide evidence that DGT can suppress the growth and metastasis of human osteosarcoma through modulation of GSK3β inactivation–mediated repression of the Hedgehog/Gli1 pathway. Clin Cancer Res; 24(1); 130–44. ©2017 AACR.

PURPOSE/OBJECTIVE(S) Nodal staging systems vary substantially across solid tumors, which implies significant heterogeneity in the behavior of nodal variables across cancers. We hypothesized, in contradiction to this, that quantitative metastatic lymph node (LN) burden is a universal and dominant predictor of mortality across solid cancers, and therefore is capable of both simplifying and improving nodal staging for many cancers. MATERIALS/METHODS Retrospective cohort analysis of 1,304,498 patients in the National Cancer Database with solid cancers undergoing surgery between 2004-2015 across the 16 most common cancer sites in the United States (bladder, breast, cervix, colorectal, endometrium, esophagus, gastric, head and neck, hepatobiliary, lung, melanoma, ovary, pancreas, prostate, renal, thyroid). Restricted cubic spline modeling was used to assess the independent association of LN number and survival across a continuous nonlinear spectrum. Recursive partitioning analysis (RPA) was used to develop 16 nodal classification systems based on quantitative nodal burden. The reproducibility of these findings was assessed in 1,969,727 patients from SEER-18. RESULTS Consistently across all disease sites and without exception, mortality risk increased continuously and steeply with each +LN to a change point of ≤5 LN, and continued to increase more modestly without plateau beyond this (P < 0.001 for all hazard ratios [HRs] per LN of the initial and secondary spline segments). Disease sites with the highest HR per LN of the initial spline segment were melanoma (1.92/LN), bladder (1.66/LN) and renal (1.57/LN). Conversely, sites with the lowest HR per LN of the initial spline segment were thyroid (1.13/LN), ovary (1.19/LN) and endometrium (1.21/LN). RPA-derived nodal classification systems for all 16 disease sites universally produced multiple prognostic groups (between 3-8 depending on site) with non-overlapping survival curves that captured a wide spectrum of mortality risk (all P < 0.001). These RPA-derived systems demonstrated improved concordance with mortality compared to current AJCC nodal staging systems in disease sites without pathologic nodal classification based on number of positive LNs including bladder, cervix, endometrium, lung, ovary, prostate, and renal. The RPA-derived systems were also validated in an independent database, producing groups with distinct all-cause mortality and cause-specific mortality (all P < 0.001). Defining high nodal burden as the RPA-derived group with the highest number of +LNs, this factor conveyed markedly higher mortality (HR range: 1.8-4.6) than any other nodal covariate, including extranodal extension, size, and location (HR range: 0.9-1.5) in multivariable analysis across all organ sites. CONCLUSION Quantitative metastatic LN burden is a fundamental driver of mortality across solid cancers. Given its reproducibility and objectiveness, +LN number should serve as a foundation for pathologic nodal staging across all solid tumors.

Nasopharyngeal carcinoma (NPC) is an invasive cancer with particularly high incidence in Southeast Asia and Southern China. The pathogenic mechanisms of NPC, particularly those involving epigenetic dysregulation, remain largely elusive, hampering clinical management of this malignancy. To identify novel druggable targets, we carried out an unbiased high-throughput chemical screening and observed that NPC cells were highly sensitive to inhibitors of cyclin-dependent kinases (CDK), especially THZ1, a covalent inhibitor of CDK7. THZ1 demonstrated pronounced antineoplastic activities both in vitro and in vivo. An integrative analysis using both whole-transcriptome sequencing and chromatin immunoprecipitation sequencing pinpointed oncogenic transcriptional amplification mediated by super-enhancers (SE) as a key mechanism underlying the vulnerability of NPC cells to THZ1 treatment. Further characterization of SE-mediated networks identified many novel SE-associated oncogenic transcripts, such as BCAR1, F3, LDLR, TBC1D2, and the long noncoding RNA TP53TG1. These transcripts were highly and specifically expressed in NPC and functionally promoted NPC malignant phenotypes. Moreover, DNA-binding motif analysis within the SE segments suggest that several transcription factors (including ETS2, MAFK, and TEAD1) may help establish and maintain SE activity across the genome. Taken together, our data establish the landscape of SE-associated oncogenic transcriptional network in NPC, which can be exploited for the development of more effective therapeutic regimens for this disease. Cancer Res; 77(23); 6614–26. ©2017 AACR.

Osteosarcoma (OS) has a high degree of chromosomal instability and total copy number (CN) changes. We examined 58 human OS samples including 40 primary tumors, 11 explants, and 7 cell lines using single nucleotide polymorphism (SNP) arrays, and revealed that 70% of the samples had one or more recurrent CN-neutral loss of heterozygosity (CNN‑LOH) also known as uniparental disomy (UPD). Importantly, 17% of the samples showed prominent homozygous deletion of 3q13.31, suggesting its role in tumorigenesis. We identified and characterized two novel lncRNAs, LOC285194 and BC040587, within this genomic locus, strongly suggesting their tumor suppressor activity. Frequent deletions and UPD suggest that OS often has mutant or non-expressed tumor suppressor genes including two lncRNAs.

Partial tandem duplication of MLL (MLL-PTD) characterizes acute myeloid leukemia (AML) patients often with a poor prognosis. To understand the order of occurrence of MLL-PTD in relation to other major AML mutations and to identify novel mutations that may be present in this unique AML molecular subtype, exome and targeted sequencing was performed on 85 MLL-PTD AML samples using HiSeq-2000. Genes involved in the cohesin complex (STAG2), a splicing factor (U2AF1) and a poorly studied gene, MGA were recurrently mutated, whereas NPM1, one of the most frequently mutated AML gene, was not mutated in MLL-PTD patients. Interestingly, clonality analysis suggests that IDH2/1, DNMT3A, U2AF1 and TET2 mutations are clonal and occur early, and MLL-PTD likely arises after these initial mutations. Conversely, proliferative mutations (FLT3, RAS), typically appear later, are largely subclonal and tend to be unstable. This study provides important insights for understanding the relative importance of different mutations for defining a targeted therapeutic strategy for MLL-PTD AML patients.

Objective Triple negative breast cancer (TNBC) is a subtype with poor prognosis and high heterogeneity. The aim of this study was to screen single nucleotide polymorphisms (SNPs) associated with the prognosis of TNBC patients and to explore the potential mechanism. Methods We selected SNPs(MAF≥1%) located on the 3' untranslated region (3'UTR) of differentially expressed genes in breast cancer through databases (Nextbio,ensemble,NCBI,MirSNP). We investigated the possible associations between 111 SNPs and progression risk among 323 TNBC patients using two-step case-control study with discovery cohort (n=162) and validation cohort (n=161). The Benjamini Hochberg false discovery rate (FDR) method was used to assess the statistical significance after correction for multiple comparisons. Kaplan-Meier analysis was applied in order to assess the association between disease free survival (DFS) and positive SNPs, as well as Body Mass Index(BMI). We also investigated the expression of FABP4 in adipocytes adjacent to TNBC tissues (n=35) using immunohistochemistry. The integrated optical density (IOD) was measured as an approximation of FABP4 expression. Mann-Whitney U test was used in an attempt to assess the correlations among SNPs in FABP4, FABP4 expression and TNBC prognosis. Results SNPs in FABP4, KRAS and NTRK2 genes associated with the recurrence risk of TNBC throughout the discovery cohort and validation cohort, while the statistical significance was retained after multiple comparisons only for FABP4 rs1054135. The G allele of rs1057035 was associated with decreased risk of disease progression (aHR 0.14, 95%CI0.03-0.66), as well as an increased DFS (P Conclusions Our study found a lipid metabolism related gene and an important SNP in the 3'UTR of FABP4 associated with TNBC prognosis, which may aid the screening of high-risk patients with TNBC recurrence and the development of novel chemotherapeutic agents. Citation Format: Yuan P, Wang W, Wu C, Lin D, Xu B. A single-nucleotide polymorphism in the 3'-UTR region of the adipocyte fatty acid binding protein 4 gene is associated with prognosis of triple negative breast cancer. [abstract]. In: Proceedings of the Thirty-Eighth Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2015 Dec 8-12; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2016;76(4 Suppl):Abstract nr P5-08-19.

// Haibo Sun 1, 2, * , De-Chen Lin 1, * , Xiao Guo 1, * , Behzad Kharabi Masouleh 3 , Sigal Gery 1 , Qi Cao 1 , Serhan Alkan 1 , Takayuki Ikezoe 4 , Chie Akiba 1 , Ronald Paquette 5 , Wenwen Chien 6 , Carsten Muller-Tidow 7 , Yang Jing 4 , Konstantin Agelopoulos 8 , Markus Muschen 9 , H. Phillip Koeffler 1, 6 1 Department of Hematology and Oncology, Cedars-Sinai Medical Center, Los Angeles, CA, USA 2 Department of Surgery, University of California San Francisco, San Francisco, CA, USA 3 Department of Oncology, Hematology and Stem Cell Transplantation, RWTH Aachen University Medical School, Aachen, Germany 4 Department of Hematology and Respiratory Medicine, Kochi University, Nankoku, Kochi, Japan 5 Department of Medicine, University of California at Los Angeles, Los Angeles, CA, USA 6 Cancer Science Institute of Singapore, National University of Singapore, Singapore 7 Department of Hematology and Oncology, University Hospital Halle, Halle, Germany 8 Department of Medicine, Hematology and Oncology, University of Muenster, Muenster, Germany 9 Department of Laboratory Medicine, University of California San Francisco, San Francisco, CA, USA * These authors have contributed equally to this work Correspondence to: Haibo Sun, e-mail: Haibo.Sun@ucsf.edu De-chen Lin, e-mail: De-chen.Lin@cshs.org Keywords: IRE1, ER stress, XBP1, unfolded protein response, micro RNA Received: July 13, 2015 Accepted: January 29, 2016 Published: February 25, 2016 ABSTRACT Survival of cancer cells relies on the unfolded protein response (UPR) to resist stress triggered by the accumulation of misfolded proteins within the endoplasmic reticulum (ER). The IRE1α-XBP1 pathway, a key branch of the UPR, is activated in many cancers. Here, we show that the expression of both mature and spliced forms of XBP1 ( XBP1s ) is up-regulated in acute myeloid leukemia (AML) cell lines and AML patient samples. IRE1α RNase inhibitors [MKC-3946, 2-hydroxy-1-naphthaldehyde (HNA), STF-083010 and toyocamycin] blocked XBP1 mRNA splicing and exhibited cytotoxicity against AML cells. IRE1α inhibition induced caspase-dependent apoptosis and G1 cell cycle arrest at least partially by regulation of Bcl-2 family proteins, G1 phase controlling proteins (p21 cip1 , p27 kip1 and cyclin D1), as well as chaperone proteins. Xbp1 deleted murine bone marrow cells were resistant to growth inhibition by IRE1α inhibitors. Combination of HNA with either bortezomib or AS 2 O 3 was synergistic in AML cytotoxicity associated with induction of p-JNK and reduction of p-PI3K and p-MAPK. Inhibition of IRE1α RNase activity increased expression of many miRs in AML cells including miR-34a. Inhibition of miR-34a conferred cellular resistance to HNA. Our results strongly suggest that targeting IRE1α driven pro-survival pathways represent an exciting therapeutic approach for the treatment of AML.

Background: Super-enhancers (SEs) are large clusters of transcriptionally active enhancers. Functionally, they are capable of driving the expression of key genes that control cell identity, which are also particularly important for tumor biology. This study is aimed at probing the biological function and mechanisms of the hyper-activation of the SEs associated core transcriptional regulatory circuitry (CRC) constituted by FOXA1-MAX-SP1 in hepatocellular carcinoma. Results: ChIP-seq analysis of H3K27ac showed that the expression of three transcription factors FOXA1, MAX and SP1 is driven by super-enhancers in hepatocellular carcinoma cells. Notably, these three transcription factors also regulate large number of super-enhancers, whose characteristics fit the mathematical model of the core transcriptional regulatory circuitry (CRC). ChIP-seq analysis of FOXA1, MAX, SP1, H3K27ac, H3K4me1 and H3K4me3 confirmed the occupancy of FOXA1, MAX and SP1 at super-enhancers and promoters of some genes, including FOXA1, MAX and SP1. Also, FOXA1, MAX and SP1 have a positive correlation with each other in human hepatocellular carcinoma tissue. In addition, CCK8 assay showed that FOXA1, MAX and SP1 could promote cell growth of HCC cells. Relative open chromatin at FOXA1, MAX and SP1 genes mirrored their more active transcriptional state in HCC patients from TCGA ATAC-seq data. The mRNA expression of these three TFs were increased, which predict a worse prognosis of HCC patients. Conclusion: FOXA1-MAX-SP1 regulate super-enhancers and may form a core transcriptional regulatory circuitry, so as to promote malignancy of hepatocellular carcinoma. They are potential therapeutic targets and prognostic biomarkers for hepatocellular carcinoma. This work was supported by the National Natural Science Foundation of China (81802812 and 81972658), the Natural Science Foundation of Guangdong Province (2018A030313129 and 2019A1515012114). Citation Format: Li Peng, Xiaoqing Yuan, Zhen Chen, Chaoyang Zhang, Duanqing Tian, Dong Yin, De-Chen Lin. Core transcriptional regulatory circuitry FOXA1-MAX-SP1 promotes malignancy of hepatocellular carcinoma [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 4686.

Background Clonal VDJ rearrangement of B/T cell receptors (B/TCRs) occurring during B/T lymphocyte development has been used as a marker to track the clonality of B/T cell populations. Methods We systematically profiled the B/T cell receptor repertoire of 936 cancer cell lines across a variety of cancer types as well as 462 Epstein-Barr Virus (EBV) transformed normal B lymphocyte lines using RNA sequencing data. Results Rearranged B/TCRs were readily detected in cell lines derived from lymphocytes, and subclonality or potential biclonality were found in a number of blood cancer cell lines. Clonal BCR/TCR rearrangements were detected in several blast phase CML lines and unexpectedly, one gastric cancer cell line (KE-97), reflecting a lymphoid origin of these cells. Notably, clonality was highly prevalent in EBV transformed B lymphocytes, suggesting either transformation only occurred in a few B cells or those with a growth advantage dominated the transformed population through clonal evolution. Conclusions Our analysis reveals the complexity and heterogeneity of the BCR/TCR rearrangement repertoire and provides a unique insight into the clonality of lymphocyte derived cell lines. Electronic supplementary material The online version of this article (10.1186/s12885-018-4840-5) contains supplementary material, which is available to authorized users.

Primary organoid cultures generated from patient biopsies comprise a novel improved platform for disease modeling, being genetically stable and closely recapitulating in vivo scenarios. Barrett esophagus (BE) is the major risk factor for esophageal adenocarcinoma. There has been a dearth of long-term in vitro expansion models of BE neoplastic transformation. We generated a long-term virus-free organoid expansion model of BE neoplasia from patient biopsies. Both wild-type and paired APC-knockout (APC(KO)) BE organoids genome-edited by CRISPR-Cas9 showed characteristic goblet cell differentiation. Autonomous Wnt activation was confirmed in APC(KO) organoids by overexpression of Wnt target genes and nuclear-translocated β-catenin expression after withdrawal of Wnt-3A and R-spondin-1. Wnt-activated organoids demonstrated histologic atypia, higher proliferative and replicative activity, reduced apoptosis, and prolonged culturability. Wnt-activated organoids also showed sustained protrusive migration ability accompanied by disrupted basement membrane reorganization and integrity. This CRISPR-Cas9 editing human-derived organoid model recapitulates the critical role of aberrant Wnt/β-catenin signaling activation in BE neoplastic transformation. This system can be used to study other ‘driver’ pathway alterations in BE-associated neoplasia, avoiding signaling noise presented in immortalized or cancer-derived cell lines.

Hepatocellular carcinoma (HCC) is one of the most dominant causes of neoplasm-related deaths worldwide. In this study, we identify and characterize HCCL5, a novel cytoplasmic long noncoding RNA (lncRNA), as a crucial oncogene in HCC. HCCL5 promoted cell growth, G1–S transition, invasion, and metastasis while inhibiting apoptosis of HCC cells both in vitro and in vivo. Moreover, HCCL5 was upregulated in TGF-β1-induced classical epithelial-to-mesenchymal transition (EMT) models, and this lncRNA in turn accelerated the EMT phenotype by upregulating the expression of transcription factors Snail, Slug, ZEB1, and Twist1. HCCL5 was transcriptionally driven by ZEB1 via a super-enhancer and was significantly and frequently overexpressed in human HCC tissues, correlating with worse overall survival of patients with HCC. Together, this study characterizes HCCL5 as a super-enhancer–driven lncRNA promoting HCC cell viability, migration, and EMT. Our data also suggest that HCCL5 may serve as a novel prognostic biomarker and therapeutic target in HCC. Significance: These findings identify the lncRNA HCCL5 as a super-enhancer–driven oncogenic factor that promotes the malignancy of hepatocellular carcinoma.

PARK2 (PARKIN) is an E3 ubiquitin ligase whose dysfunction has been associated with the progression of Parkinsonism and human malignancies, and its role in cancer remains to be explored. In this study, we report that PARK2 is frequently deleted and underexpressed in human glioma, and low PARK2 expression is associated with poor survival. Restoration of PARK2 significantly inhibited glioma cell growth both in vitro and in vivo, whereas depletion of PARK2 promoted cell proliferation. PARK2 attenuated both Wnt- and EGF-stimulated pathways through downregulating the intracellular level of β-catenin and EGFR. Notably, PARK2 physically interacted with both β-catenin and EGFR. We further found that PARK2 promoted the ubiquitination of these two proteins in an E3 ligase activity-dependent manner. Finally, inspired by these newly identified tumor-suppressive functions of PARK2, we tested and proved that combination of small-molecule inhibitors targeting both Wnt–β-catenin and EGFR–AKT pathways synergistically impaired glioma cell viability. Together, our findings uncover novel cancer-associated functions of PARK2 and provide a potential therapeutic approach to treat glioma. Cancer Res; 75(9); 1815–27. ©2015 AACR.

Relapse acute lymphoblastic leukemia (ALL) is the leading cause of childhood cancer deaths. Although relapse usually occurs in the bone marrow (medullary), extramedullary relapse occasionally occurs. Currently, the clonal origin and evolution of extramedullary relapse remain elusive. We selected two pediatric B-ALL patients who experienced testicular ALL relapse and interrogated their leukemic cells (diagnosis, remission, bone marrow relapse and testicular relapse) with whole exome sequencing. Case D483 (5.6 years old at diagnosis of ALL) developed bone marrow and testicular relapse 5 years after diagnosis of B-ALL. At diagnosis he was treated as an intermediate risk with hyperdiploid-ALL with the absence of any well-known ALL fusion-oncogene. Mutations of KRAS (G12D) and CREBBP (S1436C) were found in the founding leukemic clone at diagnosis and persisted in the bone marrow and testis at relapse). Mutation of CREBBP has been frequently found in ALL (particularly in hyperdiploid subtype) and is correlated with increased incidence of relapsed ALL. A MEF2B mutation (R17Q) was found in the bone marrow and testicular relapse sample. Missense mutation of this gene is frequently found in diffuse large B cell lymphoma (DLBCL); this protein regulates the expression of the proto-oncogene BCL6 and contributes to malignant transformation. Second child, case D727 (1.3 years old at diagnosis) harbored a MLL-AF9 fusion and was assigned as a high risk-ALL at diagnosis. Two NT5C2 mutations occurred at relapse, being present at different VAF in bone marrow and testicle: missense mutation R367Q was present with a VAF of 33.5% in bone marrow and 4.5% in testicle; while D407V was present with a VAF of 6.5% in bone marrow and 35.5% in the testicular relapse. NT5C2 encodes a 5'-nucleotidase involved in purine metabolism. The missense mutations (R367Q and D407V) identified here, have been reported as recurrent mutational hotspots of NT5C2 in relapse ALL and have been functionally validated. These mutations increase the 5'-IMP nucleotidase activity of NT5C2 protein leading to resistance to 6-mercaptopurine, a drug that was a component of the treatment regime of this patient. To understand the evolutionary trajectories of these two ALL cases, we analyzed clonal evolution based on their sequencing data. In patient D483, the relapse leukemia was directly evolved from the diagnosis leukemia clone: all of the mutations at diagnosis were persisted at relapse, and four mutated genes (MEF2B, KCNG1, AIM1, OTUD5) were acquired at both bone marrow and testicular relapse with different variant allele frequency (VAF). In patient D727, however, a faction of mutations present at diagnosis were subsequently lost at relapse, suggesting that relapsed leukemia arose from an ancestral subclone that developed before the overt leukemia at diagnosis. The mutational pattern and VAF cluster analysis results suggest that relapse in the patients' testicle represents an independently subclones from the relapse in their bone marrows. Taken together, our sequencing results suggest that relapse of patient D483 was directly evolved from the diagnosis leukemic clone; while the relapse leukemia cells (both bone marrow and testicle) of patient D727 was likely derived from a common ancestral clone, and the testicular relapse arose independently from the bone marrow relapse leukemia. Disclosures Lill: Sanofi: Speakers Bureau; California Cord Blood Services: Consultancy; Kite: Research Funding.

Current standard of care for patients with pediatric acute lymphoblastic leukemia (ALL) is mainly effective, with high remission rates after treatment. However, the genetic perturbations that give rise to this disease remain largely undefined, limiting the ability to address resistant tumors or develop less toxic targeted therapies. Here, we report the use of next-generation sequencing to interrogate the genetic and pathogenic mechanisms of 240 pediatric ALL cases with their matched remission samples. Commonly mutated genes fell into several categories, including RAS/receptor tyrosine kinases, epigenetic regulators, transcription factors involved in lineage commitment, and the p53/cell-cycle pathway. Unique recurrent mutational hotspots were observed in epigenetic regulators CREBBP (R1446C/H), WHSC1 (E1099K), and the tyrosine kinase FLT3 (K663R, N676K). The mutant WHSC1 was established as a gain-of-function oncogene, while the epigenetic regulator ARID1A and transcription factor CTCF were functionally identified as potential tumor suppressors. Analysis of 28 diagnosis/relapse trio patients plus 10 relapse cases revealed four evolutionary paths and uncovered the ordering of acquisition of mutations in these patients. This study provides a detailed mutational portrait of pediatric ALL and gives insights into the molecular pathogenesis of this disease. Cancer Res; 77(2); 390–400. ©2016 AACR.

// Shaowen Jin 1,2 , Kaimei Wang 3 , Kang Xu 1 , Junyao Xu 1 , Jian Sun 1 , Zhonghua Chu 4 , Dechen Lin 5 , Phillip H. Koeffler 5 , Jie Wang 1 and Dong Yin 2 1 Department of Hepatobiliary Surgery, Sun Yat-Sen Memorial Hospital, Sun Yat-Sen University, Guangzhou, China 2 Guangdong Provincial Key Laboratory of Malignant Tumor Epigenetics and Gene Regulation, Medical Research Center, Sun Yat-Sen Memorial Hospital, Sun Yat-Sen University, Guangzhou, China 3 Department of Pediatrics, Sun Yat-Sen Memorial Hospital, Sun Yat-Sen University, Guangzhou, China 4 Department of Gastrointestinal Surgery, Sun Yat-Sen Memorial Hospital, Sun Yat-Sen University, Guangzhou, China 5 Division of Hematology/Oncology, Cedars-Sinai Medical Center, University of California-Los Angeles (UCLA) School of Medicine Correspondence: Dong Yin, email: // Jie Wang, email: // Keywords : Hepatocellular carcinoma, PRL-1, SNP-Chip, Tumor metastasis, Prognostic marker Received : March 28, 2014 Accepted : May 17, 2014 Published : May 19, 2014 Abstract Our SNP-Chip data demonstrated 7/60 (12%) hepatocellular carcinoma (HCC) patients had PRL-1 copy number amplification. However, its biological functions and signaling pathways in HCC are deficient. Here, we investigated its oncogenic function and prognostic significance in HCC. PRL-1 protein levels were examined in 167 HCC samples by immunohistochemisty (IHC). The relationship of PRL-1 expression and clinicopathological features was assessed by correlation, Kaplan-Meier and Cox regression analyses. The oncogenic function of PRL-1 in HCC cells and its underlying mechanism were investigated by ectopic overexpression and knockdown model. PRL-1 levels in primary HCC and metastatic intravascular cancer thrombus were also determined by IHC. PRL-1 levels were frequently elevated in HCC tissues (81%), and elevated expression of PRL-1 was significantly associated with more aggressive phenotype and poorer prognosis in HCC patients ( p

Esophageal squamous cell carcinoma (ESCC) is a common and aggressive malignancy, with hitherto dismal clinical outcome. Genomic analyses of patient samples reveal a complex heterogeneous landscape for ESCC, which presents in both intertumor and intratumor forms, manifests at both genomic and epigenomic levels, and contributes significantly to tumor evolution, drug resistance, and metastasis. Here, we review the important molecular characteristics underlying ESCC heterogeneity, with an emphasis on genomic aberrations and their functional contribution to cancer evolutionary trajectories. We further discuss how novel experimental tools, including single-cell sequencing and three-dimensional organoids, may advance our understanding of tumor heterogeneity. Lastly, we suggest that deciphering the mechanisms governing tumor heterogeneity holds the potential to developing precision therapeutics for ESCC patients.

Understanding the intratumoral heterogeneity of hepatocellular carcinoma is instructive for developing personalized therapy and identifying molecular biomarkers. Here we applied whole-exome sequencing to 69 samples from 11 patients to resolve the genetic architecture of subclonal diversification. Spatial genomic diversity was found in all 11 hepatocellular carcinoma cases, with 29% of driver mutations being heterogeneous, including TERT, ARID1A, NOTCH2, and STAG2. Similar with other cancer types, TP53 mutations were always shared between all tumor regions, that is, located on the “trunk” of the evolutionary tree. In addition, we found that variants within several drug targets such as KIT, SYK, and PIK3CA were mutated in a fully clonal manner, indicating their therapeutic potentials for hepatocellular carcinoma. Temporal dissection of mutational signatures suggested that mutagenic processes associated with exposure to aristolochic acid and aflatoxin might play a more important role in early, as opposed to late, stages of hepatocellular carcinoma development. Moreover, we observed extensive intratumoral epigenetic heterogeneity in hepatocellular carcinoma based on multiple independent analytical methods and showed that intratumoral methylation heterogeneity might play important roles in the biology of hepatocellular carcinoma cells. Our results also demonstrated prominent heterogeneity of intratumoral methylation even in a stable hepatocellular carcinoma genome. Together, these findings highlight widespread intratumoral heterogeneity at both the genomic and epigenomic levels in hepatocellular carcinoma and provide an important molecular foundation for better understanding the pathogenesis of this malignancy. Cancer Res; 77(9); 2255–65. ©2017 AACR.

Purpose: Ewing sarcoma (EWS) is a devastating soft tissue sarcoma affecting predominantly young individuals. Tyrosine kinases (TK) and associated pathways are continuously activated in many malignancies, including EWS; these enzymes provide candidate therapeutic targets. Experimental Design: Two high-throughput screens (a siRNA library and a small-molecule inhibitor library) were performed in EWS cells to establish candidate targets. Spleen tyrosine kinase (SYK) phosphorylation was assessed in EWS patients and cell lines. SYK was inhibited by a variety of genetic and pharmacological approaches, and SYK-regulated pathways were investigated by cDNA microarrays. The transcriptional regulation of MALAT1 was examined by ChIP-qPCR, luciferase reporter, and qRT-PCR assays. Results: SYK was identified as a candidate actionable target through both high-throughput screens. SYK was highly phosphorylated in the majority of EWS cells, and SYK inhibition by a variety of genetic and pharmacologic approaches markedly inhibited EWS cells both in vitro and in vivo. Ectopic expression of SYK rescued the cytotoxicity triggered by SYK-depletion associated with the reactivation of both AKT and c-MYC. A long noncoding RNA, MALAT1, was identified to be dependent on SYK-mediated signaling. Moreover, c-MYC, a SYK-promoted gene, bound to the promoter of MALAT1 and transcriptionally activated MALAT1, which further promoted the proliferation of EWS cells. Conclusions: This study identifies a novel signaling involving SYK/c-MYC/MALAT1 as a promising therapeutic target for the treatment of EWS. Clin Cancer Res; 23(15); 4376–87. ©2017 AACR.

Ewing sarcoma (EWS) is an aggressive bone malignancy that mainly affects children and young adults. The mechanisms by which EWS (EWSR1) fusion genes drive the disease are not fully understood. CRM1 (XPO1) traffics proteins from the nucleus, including tumor suppressors and growth factors, and is overexpressed in many cancers. A small-molecule inhibitor of CRM1, KPT-330, has shown therapeutic promise, but has yet to be investigated in the context of EWS. In this study, we demonstrate that CRM1 is also highly expressed in EWS. shRNA-mediated or pharmacologic inhibition of CRM1 in EWS cells dramatically decreased cell growth while inducing apoptosis, cell-cycle arrest, and protein expression alterations to several cancer-related factors. Interestingly, silencing of CRM1 markedly reduced EWS–FLI1 fusion protein expression at the posttranscriptional level and upregulated the expression of the well-established EWS-FLI1 target gene, insulin-like growth factor binding protein 3 (IGFBP3), which inhibits IGF-1. Accordingly, KPT-330 treatment attenuated IGF-1–induced activation of the IGF-1R/AKT pathway. Furthermore, knockdown of IGFBP3 increased cell growth and rescued the inhibitory effects on IGF-1 signaling triggered by CRM1 inhibition. Finally, treatment of EWS cells with a combination of KPT-330 and the IGF-1R inhibitor, linsitinib, synergistically decreased cell proliferation both in vitro and in vivo. Taken together, these findings provide a strong rationale for investigating the efficacy of combinatorial inhibition of CRM1 and IGF-1R for the treatment of EWS. Cancer Res; 76(9); 2687–97. ©2016 AACR.

BCOR is a component of a variant Polycomb group repressive complex 1 (PRC1). Recently, we and others reported recurrent somatic BCOR loss-of-function mutations in myelodysplastic syndrome and acute myelogenous leukaemia (AML). However, the role of BCOR in normal hematopoiesis is largely unknown. Here, we explored the function of BCOR in myeloid cells using myeloid murine models with Bcor conditional loss-of-function or overexpression alleles. Bcor mutant bone marrow cells showed significantly higher proliferation and differentiation rates with upregulated expression of Hox genes. Mutation of Bcor reduced protein levels of RING1B, an H2A ubiquitin ligase subunit of PRC1 family complexes and reduced H2AK119ub upstream of upregulated HoxA genes. Global RNA expression profiling in murine cells and AML patient samples with BCOR loss-of-function mutation suggested that loss of BCOR expression is associated with enhanced cell proliferation and myeloid differentiation. Our results strongly suggest that BCOR plays an indispensable role in hematopoiesis by inhibiting myeloid cell proliferation and differentiation and offer a mechanistic explanation for how BCOR regulates gene expression such as Hox genes.

Esophageal cancer ranks sixth in terms of cancer-associated mortality (1). The majority (over 80%) of esophageal cancers are squamous cell carcinomas (ESCC) (2), which is the leading cause of cancer-related death in its epidemic regions, such as northern China. Even with the advent of modern therapeutic approaches, the 5 years survival of ESCC (

Purpose: To investigate the molecular mechanisms through which polo-like kinase-1 (PLK1) takes part in anoikis resistance of esophageal squamous cell carcinoma (ESCC) cells. Experimental Design: The role of PLK1 in cell anoikis resistance was examined by ectopic gene expression and siRNA-mediated knockdown. Glutathione S-transferase pull-down and co-immunoprecipitation assays were utilized to investigate PLK1-interacting proteins. Electrophoretic mobility shift assay, chromatin immunoprecipitation, and reporter gene assays were carried out to identify the transcription factors responsible for PLK1 expression during anoikis resistance. Results: We found that detachment of ESCC cells triggers the upregulation of PLK1. Elevated PLK1 expression contributes to protection against anoikis in cancer cells through the regulation of β-catenin expression. Moreover, we showed that, through direct binding to the PLK1 promoter, the NF-κB subunit RelA transcriptionally activates PLK1, which inhibits the ubiquitination and degradation of β-catenin. Inhibition of the NF-κB pathway restores the sensitivity of cancer cells to anoikis by downregulating PLK1/β-catenin expression. In addition, RelA gene amplification and protein overexpression was significantly correlated with PLK1 expression in ESCC tissues. Conclusions: Our findings suggest that upregulation of PLK1 triggered by cell detachment is regulated by RelA at the transcriptional level. PLK1 protects esophageal carcinoma cells from anoikis through modulation of β-catenin protein levels by inhibiting their degradation. Taken together, this study reveals critical mechanisms involved in the role of RelA/PLK1/β-catenin in anoikis resistance of ESCC cells. Clin Cancer Res; 17(13); 4285–95. ©2011 AACR.

Risk assessment of esophageal squamous cell carcinoma (ESCC) is currently based on clinicopathological parameters. To identify genomic markers that can predict overall survival in ESCC, we performed array comparative genomic hybridization (array CGH) on a screening set of 35 tumor samples from ESCC patients. Prognosis association of the genes selected on the basis of the array CGH results was further validated by real-time PCR in two independent sample sets (n = 151 and 84). Genomic analysis revealed seven high-level amplifications and two homozygous deletions. Gain of 11q13.2 and loss of 7q34 and 18q21.1-q23 were associated with poor outcome. Gain of 11q13.2 was an independent prognostic factor and was selected for further validation. In both validation sets of samples, copy number increase of CPT1A in 11q13.2 was correlated with short overall survival (P = 0.015, n = 151 and P = 0.044, n = 84). Multivariate analysis confirmed that CPT1A gain provided prognostic information in ESCC (HR, 1.643; 95% CI: 1.076-2.509; P = 0.022; HR, 2.488; 95% CI: 1.235-5.013; P = 0.011). Immunohistochemistry showed significant correlation between strong expression of CPT1A protein and poor outcome of ESCC patients (P = 0.018, n = 73). Our data suggest that gain of CPT1A may be a candidate prognostic factor.

Esophageal squamous cell carcinoma (ESCC) is the predominant histological subtype of esophageal cancer in Asia, characterized by high incidence and mortality rate. Although significant progress has been made in surgery and adjuvant chemoradiotherapy, the prognosis of the patients with this cancer still remains poor. Investigation into protein alterations that occurred in tumors can provide clues to discover new biomarkers for improving diagnosis and guiding targeted therapy. Hundreds of papers have appeared over the past several decades concerning protein alterations in ESCC. This review summarizes all the dysregulated proteins investigated in the disease from 187 published papers and analyzes their contributions to tumor development and progression. We document protein alterations associated with tumor metastasis and the transition from normal esophageal epithelia to dysplasia in order to reveal the most useful markers for prediction of clinical outcome, early detection, and identification of high-risk patients for targeted therapies. In particular, we discuss the largest and most rigorous studies on prognostic implications of proteins in ESCC, in which cyclin D1, p53, E-cadherin and VEGF appeared to have the strongest evidence as independent predictors of patient outcome.

The gene of SKP2, located on chromosome 5p13, plays a critical role in cell cycle progression, especially at the G1-S transition, putatively through its control of several cell cycle regulator proteins including p27kip1, p21cip1, p57kip2, p130, cyclin E, and c-Myc. Previous studies in this laboratory revealed that gain of chromosome 5p was often seen in esophageal squamous cell carcinoma (ESCC). In the present study, we examined the amplification status and expression level of SKP2 in ESCC and investigated its clinicopathologic significance. Amplification and elevated expression of SKP2 correlated significantly with tumor stage and positive lymph node metastasis (P < 0.05). The SKP2 protein expression level as determined by immunohistochemical staining showed a significant inverse correlation with p27 protein. In vivo assay showed that inhibition of SKP2 expression also decreased tumor growth and lung metastasis of ESCC cells. At the molecular level, knockdown of SKP2 by RNA interference inhibited cell migration and invasion ability. Knockdown of SKP2 expression sensitized cancer cells to anoikis, and a wobble mutant of SKP2 that is resistant to SKP2 small interfering RNA can rescue this effect. Expression level of pAkt decreased after SKP2 knockdown. Treatment of cells with phosphoinositidyl 3-kinase inhibitor (LY294002) and constitutively activator (insulin-like growth factor I) had significant effects on the anoikis of SKP2 RNA interference cells. These results show for the first time that SKP2 is amplified and overexpressed in ESCC. Elevated expression of SKP2 protected cancer cells from anoikis, and this effect was mediated, at least in part, by the phosphoinositidyl 3-kinase-Akt pathway. (Mol Cancer Res 2009;7(1):12–22)

ZBTB transcription factors orchestrate gene transcription during tissue development. However, their roles in glioblastoma multiforme (GBM) remain unexplored. Here, through a functional screening of ZBTB genes, we identify that BCL6 is required for GBM cell proliferation and is overexpressed in GBM samples. Using a somatic transgenic mouse model, Bcl6 confers the proliferative and invasive stimuli to glioma cells in vivo. Moreover, we discover AXL as a novel downstream target of BCL6. Through AXL, BCL6 enhances both MEK-ERK and S6K-RPS6 axes. Pharmacological inhibition of BCL6 activity effectively blocks GBM growth and inhibits AXL expression. Together, these findings uncover a novel glioma-promoting role of BCL6, and provide the rationale of targeting BCL6 as a potential therapeutic approach. Citation Format: Liang Xu, Ye CHEN, Marina Dutra-Clarke, Anand Mayakonda, Masaharu Hazawa, Steve E. Savinoff, Ngan Doan, Jonathan W. Said, William H. Yong, Henry Yang, Ling-Wen Ding, Yan-Yi Jiang, Jeffrey W. Tyner, Jianhong Ching, Jean-Paul Kovalik, Markus Müschen, Joshua J. Breunig, De-Chen Lin, Phillip Koeffler. BCL6 promotes glioma and serves as a novel therapeutic target [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 2569. doi:10.1158/1538-7445.AM2017-2569

Glioblastoma multiforme (GBM) remains the most aggressive brain malignancy with little improvement in prognosis or therapy for decades. Recently, we identified BCL6, also known as ZBTB27, to be a novel oncogene in GBM. In this study, we performed IHC analysis of 153 primary human glioma specimens and 8 normal brain samples. BCL6 expression is robustly elevated in tumor samples and positively correlated with glioma pathological grade. High BCL6 expression strongly predicts a worse prognosis of GBM patients. Depletion of BCL6 in human GBM cells reduced the incorporation of BrdU, promoted the cellular senescence and inhibited the growth of human GBM cells in vivo. Next, genome-wide occupancy of BCL6 in GBM cells was characterized by ChIP-seq assay. Genomic regions centered on BCL6 peaks are co-enriched with RNA-Pol II and flanked with strong H3K27ac and H3K4me3 modifications. MYC and two long non-coding RNAs MALAT1 and NEAT1 were identified as novel BCL6 targets in GBM. Moreover, pathway enrichment analysis of BCL6 peak-associated genes reveals a significant enrichment of JAK-STAT, TP53, ERBB and MAPK pathways. We demostrated further that BCL6 represses the TP53 pathway and promotes the JAK-STAT pathway activation in GBM cells. Together, our findings uncover potential downstream targets and provide a better understanding of BCL6 function in GBM. Citation Format: Ye Chen, Liang Xu, Marina Dutra-Clarke, Anand Mayakonda, De-Chen Lin, Lynnette Koh, Yuk Kien Chong, Edwin Sandanaraj, Vikas Madan, Henry Yang, Ngan Doan, Jonathan W. Said, William H. Yong, Markus Müschen, Beng Ti Ang, Carol Tang, Joshua J. Breunig, Phillip Koeffler. BCL6 modulates the TP53 and STAT pathways in glioma [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 1524. doi:10.1158/1538-7445.AM2017-1524

MGA is an incompletely studied gene with a high mutation frequency in MLL-PTD AML (9%) and in core bind factor AML (8%). This gene encodes a MAX-interacting protein and is believed to act as a transcription factor that suppresses MYC binding to its target. By in silico analysis, we found that MGA is expressed in normal myeloid hematopoietic cells and AML, and the expression level is comparable with TET2 or DNMT3A. Further data mining of TCGA revealed a high frequency of inactivating mutations of the MGA gene in a variety of cancers such as various adenocarcinomas. To interrogate functionally its role in leukemogenesis, lentiviral constructs containing either shRNA or CRISPR-sgRNA targeted to different regions of the MGA gene were generated. MGA expressing AML cell line EOL-1 was silenced by shRNA or CRISPER system. Silencing was confirmed by western blot (shRNA) and Sanger Sequencing (sgRNA). An increase of methylcellulose colony number (~30%) was observed in MGA silenced cell lines. Control EOL-1 cells or EOL-1 cells silenced with MGA CRISPR sgRNAs were injected into both flanks of NSG mice, and tumor masses were harvested 21 days after injection. Silencing of MGA by CRISPR-sgRNA consistently enhanced in vivo xenograft cell growth. In addition, western blot analyses revealed silencing of MGA in EOL-1 cells increased protein levels of Cyclin E1 and phos-RB (S807 phosphorylation inhibits the ability of RB to target protein allowing cell cycle progression), indicative of a proliferative advantage conferred by the silencing of MGA. MGA may be a potential regulator of the MYC pathway. We, therefore, examined whether silencing of MGA alters MYC transcriptional activity. Luciferase reporter assay was carried out in 293FT cells stabilized with either scramble or shRNA- targeting MGA. Luciferase activities were measured 48 h after transfection of cells with MYC activity reporter pMyc4ElbLuc and normalized to the corresponding co-transfected Renilla luciferase activity. A fourfold increase in luciferase activity was observed in MGA silenced cells when compared with non- targeting shRNA controls. Furthermore, Kaplan–Meier survival analysis was performed in the TCGA-AML patients by comparison of cases with highest versus lowest expression of MGA. P-values were calculated by log-rank test. MGA expression data and patient survival data were retrieved from TCGA-AML patients RNA seq, or microarray (70 AML patients). The MGA expression ‘high’ and ‘low’ groups were defined by 15% higher than the median or 15% lower than the median, respectively. AML patients with lower levels of MGA in their leukemic samples had a worse outcome compared with those whose leukemic cells expressed higher levels of MGA. Collectively, our results suggest that MGA may function as a potential tumor-suppressor in AML. Citation Format: Qiaoyang Sun, Lingwen Ding, Kar-Tong Tan, Wenwen Chien, Xinyi Loh, Jinfen Xiao, Anand Mayakonda, Dechen Lin, Yanyi Jiang, Henry Yang, Sigal Gery, H. Phillip Koeffler. MGA is a potential tumor suppressor in acute myeloid leukemia [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 806. doi:10.1158/1538-7445.AM2017-806

Studies have reported that the CCN family of proteins plays an important role in stimulating tumorigenesis. However, the relationship between the CCN protein family members and the features of hepatocellular carcinoma (HCC) remains unclear. The objective of this study was to determine the relationship between the expression levels of CCN protein family members and the features of HCC. Expression levels of the CCN family of proteins in 80-paired primary HCC samples and 11 normal liver samples were determined by a quantitative real-time PCR assay. Enhanced expression of nephroblastoma overexpressed protein (NOV) and decreased expression of Wnt-induced secreted protein 1 (WISP1), cysteine-rich protein 61 (CYR61) and connective tissue growth factor (CTGF) were found in HCC samples when compared to levels in matched non-cancerous tissues. No significant difference in WISP2 was found between matched-pair samples; only a few samples showed WISP3 expression. Furthermore, the expression levels of NOV, WISP1 and CYR61 were closely correlated with certain clinical features, including venous invasion, cellular differentiation, pTNM stage, disease-free survival and overall survival. Our results suggest that HCC progression may be enhanced by NOV and suppressed by WISP1 and CYR61. Our statistical analysis suggests that these proteins may be valuable in determining the prognosis of this deadly disease and directs attention to modulating the levels of these proteins as a potential mode of therapy.

Protein kinase Cι (PKCι) is an atypical PKC isoform and participates in multiple aspects of the transformed phenotype in human cancer cells. We previously reported that frequent amplification and overexpression of PKCι were correlated with lymph node metastasis in primary esophageal squamous cell carcinomas (ESCC). In the present study, short interfering RNA–mediated silencing of PKCι revealed that this enzyme was required for cell migration, invasion, and resistance to anoikis. In vivo experiments showed that PKCι suppression decreased tumor growth in esophageal cancer xenografts and lung metastases in nude mice. At the molecular level, knockdown of PKCι in suspended ESCC cells caused a decrease in S-phase kinase-associated protein 2 (SKP2) that had been reported to promote resistance to anoikis via the PI3K/AKT pathway. AKT phosphorylation was abolished after PKCι suppression, but AKT activation could be refreshed by PKCι upregulation, suggesting that PKCι enhanced cell resistance to anoikis via the PKCι-SKP2-PI3K/AKT pathway. Addition of the proteasome inhibitor MG132 prevented the decrease of SKP2 in PKCι silenced cells, and polyubiquitin-SKP2 was elevated after PKCι depletion, showing that PKCι might regulate the expression of SKP2 through the ubiquitin-proteasome pathway in suspended cells. Furthermore, overexpression of SKP2 in PKCι-downregulated cells restored cell resistance to anoikis. Most importantly, PKCι expression significantly correlated with SKP2 in 133 ESCC tissues (P = 0.031). Taken together, our data show that PKCι promotes tumorigenicity and metastasis of human esophageal cancer and that SKP2 is a candidate downstream effector of PKCι signaling in ESCC. Mol Cancer Res; 9(4); 390–402. ©2011 AACR.

PLK1 is essential for the maintenance of genomic stability during mitosis. In our study, we found that overexpression of PLK1 was an independent prognostic factor (RR=4.253, p=0.020) and significantly correlated with survivin, an antiapoptotic protein, in esophageal squamous cell carcinoma (ESCC). Reverse transcription-polymerase chain reaction and fluorescence in situ hybridization (FISH) revealed upregulation of PLK1 mRNA and amplification of PLK1 gene, respectively. Depletion of PLK1 activated the intrinsic apoptotic pathway, which was substantiated by loss of mitochondrial membrane potential, reduction of Mcl-1 and Bcl-2 as well as activation of caspase-9. Coimmunoprecipitation and confocal microscopy displayed that PLK1 was associated with survivin and PLK1 depletion led to downregulation of survivin. Cotransfection of survivin constructs could partially reverse PLK1-depletion-induced apoptosis. These data suggest that PLK1 might be a useful prognostic marker and a potential therapeutic target for ESCC. Survivin is probably involved in antiapoptotic function of PLK1.

PARK2 is an E3 ubiquitin ligase whose dysfunction has been associated with the progression of autosomal recessive juvenile Parkinson's disease and human malignancies. However, its role in cancer remains to be explored. In this study, we report that PARK2 is frequently deleted and underexpressed in human glioma, and low PARK2 expression is associated with poor survival in cohorts of both low-grade glioma and glioblastoma multiforme (GBM). Functional studies revealed a tumor-suppressive role of PARK2 in GBM cells. Restoration of PARK2 significantly inhibited glioma cell growth both in vitro and in vivo, while depletion of endogenous PARK2 promoted cell proliferation. cDNA microarray analysis showed that PARK2 expression strongly altered the activity of both the Wnt and EGFR pathways. Immunoblot analysis confirmed that ectopic expression of PARK2 suppressed the intracellular levels of β-catenin, EGFR, as well as their down-stream targets including Cyclin D1, TCF4, c-Myc, p-AKT and p-S6K, etc. Notably, PARK2 physically interacted with both β-catenin and EGFR, and promoted their ubiquitination in an E3-ligase dependent manner. Similar to the ligase-dead PARK2 mutant (C431S), three PARK2 mutants harboring cancer-derived somatic mutations (T173A, T240M and P294S) showed decreased ability to ubiquitinate either β-catenin or EGFR proteins. We further found that PARK2 attenuated the cellular response to both Wnt3a and EGF stimulation, suggesting PARK2 is a negative regulator of both the Wnt and EGFR pathways. Lastly, inspired by these newly identified functions of PARK2, we tested and proved that the combination of small-molecule inhibitors targeting both Wnt-β-catenin and EGFR-AKT pathways synergistically impaired glioma cell viability. In aggregate, our findings uncover novel, cancer-associated functions of PARK2 and provide a potential therapeutic approach to treat glioma. Citation Format: Liang Xu, De-Chen Lin, Ye Chen, Haiyan Yan, Masaharu Hazawa, Ngan Doan, Jonathan W. Said, Ling-Wen Ding, Li-Zhen Liu, Henry Yang, Shi-zhu Yu, Michael Kahn, Dong Yin, Phillip Koeffler. PARK2 is a negative regulator of Wnt and EGFR pathways in glioma. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 122. doi:10.1158/1538-7445.AM2015-122

Background Nasopharyngeal carcinoma (NPC) arises from the epithelial tissue of the nasopharynx. This cancer has remarkable ethnic and geographic distributions, with a particularly high prevalence in Southern China and Southeast Asia. Genetic susceptibility and Epstein-Barr virus infections have been implicated in the pathogenesis of NPC. However, genomic abnormalities of this neoplasm remain largely obscure, and no effective targeted therapy has been established. Therefore, a strong need exists to characterize the genetic alterations of this type of tumour for guiding the development of more effective and innovative therapeutic regimens. Methods Whole exomes sequencing (WES) was performed on 56 NPC germline/tumor pairs and 5 NPC cell lines. Transcriptome sequencing (RNA-seq) was conducted on 4 tumors from this cohort. An additional 61 NPC germline/tumor pairs and 5 non-paired primary tumors were further subjected to targeted regional deep-sequencing (TS). The somatic copy number variations (SCNV) of 52 primary tumors were profiled by SNP-array hybridization (50 of them also had WES data). Finally, both in vitro and in vivo biological and biochemical experiments were performed to evaluate the newly-identified mutations in NPC cell lines. Results We identified a total of 1,577 non-silent somatic mutations affecting 1,413 genes from WES, revealing a relatively low mutational rate and wide mutational diversity. Integration of the results from WES, TS and RNA-seq revealed a distinct mutational signature and a number of significantly mutated genes (such as TP53, ARID1A, MLL2, BAP1, PIK3CA, etc.) in NPC. Pathway enrichment analysis of genetic lesions identified several important cellular processes and pathways in NPC including chromatin modification, ERBB-PI3K signaling and autophagy machinery. We further characterized the biological functions of both ARID1A and BAP1 proteins in NPC cell lines. Depletion of wild-type endogenous ARID1A expression with shRNAs resulted in increased anchorage-independent colony formation, cell migration and xenograft growth in vivo, whereas ectopically expressed ARID1A suppressed both cell proliferation and migration. Similarly, ectopic expression of wild-type BAP1 suppressed anchorage-independent colony formation of NPC cells. Conclusion We characterized the genomic landscape of 128 NPC cases, and identified a number of novel driver genes with statistical and biological evidence. Integrated analysis showed enrichment of genetic lesions affecting chromatin modification, ERBB-PI3K signaling and autophagy machinery, offering opportunities for developing novel treatments. These results in aggregate provide an important genetic foundation for further study of the molecular pathology and etiology of this fatal disease. Citation Format: DECHEN LIN, Xuan Meng, Masaharu Hazawa, Yasunobu Nagata, Ana Maria Varela, Liang Xu, Yusuke Sato, Li-Zhen Liu, Ling-Wen Ding, Arjun Sharma, Boon Cher Goh, Soo Chin Lee, Bengt Fredrik Petersson, Feng Gang Yu, Paul Macary, Min Zin Oo, Soh Ha Chan, Henry Yang, Seishi Ogawa, H. Phillip Koeffler. The genomic landscape of nasopharyngeal carcinoma. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 3939. doi:10.1158/1538-7445.AM2015-3939