Epstein–Barr-virus-associated Gastric Cancer (EBVaGC) comprises approximately 10% of global gastric cancers and is known to be the most hypermethylated of all tumor types. EBV infection has been shown to directly induce the hypermethylation of both the host and viral genome following initial infection of gastric epithelial cells. Many studies have been completed in an attempt to identify genes that frequently become hypermethylated and therefore significant pathways that become silenced to promote tumorigenesis. It is clear that EBV-induced hypermethylation silences key tumor suppressor genes, cell cycle genes and cellular differentiation factors to promote a highly proliferative and poorly differentiated cell population. EBV infection has been shown to induce methylation in additional malignancies including Nasopharyngeal Carcinoma and Burkitt’s Lymphoma though not to the same level as in EBVaGC. Lastly, some genes silenced in EBVaGC are common to other heavily methylated tumors such as colorectal and breast tumors; however, some genes are unique to EBVaGC and can provide insights into the major pathways involved in tumorigenesis.

Deciphering the molecular pathogenesis of virally induced cancers is challenging due, in part, to the heterogeneity of both viral gene expression and host gene expression. Epstein-Barr virus (EBV) is a ubiquitous herpesvirus prevalent in B-cell lymphomas of immune-suppressed individuals. EBV infection of primary human B cells leads to their immortalization into lymphoblastoid cell lines (LCLs), serving as a model of these lymphomas. In previous studies, reports from our laboratory have described a temporal model for immortalization with an initial phase characterized by expression of Epstein-Barr nuclear antigens (EBNAs), high levels of c-Myc activity, and hyperproliferation in the absence of the latent membrane proteins (LMPs), called latency IIb. This is followed by the long-term outgrowth of LCLs expressing the EBNAs along with the LMPs, particularly NFκB-activating LMP1, defining latency III. However, LCLs express a broad distribution of LMP1 such that a subset of these cells express LMP1 at levels similar to those seen in latency IIb, making it difficult to distinguish these two latency states. In this study, we performed mRNA sequencing (mRNA-Seq) on early EBV-infected latency IIb cells and latency III LCLs sorted by NFκB activity. We found that latency IIb transcriptomes clustered independently from latency III independently of NFκB. We identified and validated mRNAs defining these latency states. Indeed, we were able to distinguish latency IIb cells from LCLs expressing low levels of LMP1 using multiplex RNA-fluorescencein situhybridization (RNA-FISH) targeting EBVEBNA2orLMP1and humanCCR7orMGST1. This report defines latency IIb as a bona fide latency state independent from latency III and identifies biomarkers for understanding EBV-associated tumor heterogeneity.IMPORTANCEEBV is a ubiquitous pathogen, with >95% of adults harboring a life-long latent infection in memory B cells. In immunocompromised individuals, latent EBV infection can result in lymphoma. The established expression profile of these lymphomas is latency III, which includes expression of all latency genes. However, single-cell analysis of EBV latent gene expression in these lymphomas suggests heterogeneity where most cells express the transcription factor, EBNA2, and only a fraction of the cells express membrane protein LMP1. Our work describes an early phase after infection where the EBNAs are expressed without LMP1, called latency IIb. However, LMP1 levels within latency III vary widely, making these states hard to discriminate. This may have important implications for therapeutic responses. It is crucial to distinguish these states to understand the molecular pathogenesis of these lymphomas. Ultimately, better tools to understand the heterogeneity of these cancers will support more-efficacious therapies in the future.

PIK3CA is the second most mutated gene in cancer leading to aberrant PI3K/AKT/mTOR signaling and increased translation, proliferation, and survival. Some 4-25% of gastric cancers display activating PIK3CA mutations including 80% of EBV-associated GCs. Small molecules including pan-PI3K and dual PI3K/mTOR inhibitors have shown moderate success clinically, due to broad on-target/off-tissue effects. Thus, isoform specific and mutant selective inhibitors have been of significant interest. However, drug resistance is a problem and has affected success of new drugs. There has been a concerted effort to define mechanisms of resistance and identify potent combinations in many tumor types, though gastric cancer is comparatively understudied. In this study we identified modulators of the response to the PI3K-alpha-specific inhibitor, BYL719, in PIK3CA mutant GCs. We found that loss of NEDD9 or inhibition of BCL-XL conferred hyper-sensitivity to BYL719, through increased cell cycle arrest and cell death, respectively. Additionally, we discovered that loss of CBFB conferred resistance to BYL719. CBFB loss led to up-regulation of the protein kinase PIM1, which can phosphorylate and activate several overlapping downstream substrates as AKT thereby maintaining pathway activity in the presence of PI3K-alpha inhibition. The addition of a pan-PIM inhibitor re-sensitized resistant cells to BYL719. Our data provide clear mechanistic insights into PI3K-alpha inhibitor response in PIK3CA mutant gastric tumors and can inform future work as mutant selective inhibitors are in development for diverse tumor types. Implications: Loss of either NEDD9 or BCL-XL confers hyper-sensitivity to PI3K-alpha inhibition while loss of CBFB confers resistance through a CBFB/PIM1 signaling axis.

Epstein–Barr virus (EBV) was the first discovered human tumor virus and is the etiological agent of B-cell lymphomas and also epithelial cancers. Indeed, nearly 10% of gastric cancers worldwide are EBV-positive and display unique molecular, epigenetic and clinicopathological features. EBV-positive gastric cancers display the highest rate of host genome methylation of all tumor types studied and harbor recurrent mutations activating PI3Kα, silencing ARID1A and amplifying PD-L1. While EBV infection of B cells can be studied efficiently, de novo epithelial cell infection is much more difficult. We propose that new culture models including 3D-based gastric organoids and xenografts can bring new insight into EBV- induced gastric carcinogenesis and will lead to improved precision medicine-based therapies for patients with EBV-positive gastric cancer.

<p>S1. BYL719 and DMSO beta scores S2. Ribbon plots of non-gastric cancer cell lines included in screen. S3. Dasatinib renders AGS cells hyper-sensitive to BYL719 S4. Loss of BCL-XL induces cell death in BYL719 treated MKN1 S5. BCL-XL inhibitor A-1331852 is the only inhibitor capable of increasing BYL719-induced cell death and inhibiting cell growth. S6. CBFB binding co-factor RUNX1 is down-regulated at protein and mRNA level in BYL719 resistant clones. S7. Pan-PIM inhibition or PIM1 KO re-sensitizes CBFB KO and resistant clones to BYL719.</p>