Oncogenic Kaposi’s sarcoma-associated herpesvirus (KSHV) employs a biphasic life cycle consisting of latency and lytic replication to achieve lifelong infection. Despite its essential role in KSHV persistence and tumorigenicity, much remains unknown about how KSHV lytic reactivation is regulated. Leveraging high-throughput transcriptomics, we identify microRNA-31-5p (miRNA-31-5p) as a key regulator of KSHV lytic reactivation, capable of modulating expression of KSHV lytic genes and progression through the lytic cascade. Mechanistically, miR-31-5p controls the KSHV lytic switch by regulating expression of the RNA-binding protein KHDRBS3. miR-31-5p-mediated KHDRBS3 repression results in decreased nascent transcription of crucial viral lytic genes including the main KSHV transcription factor RTA. We characterize KHDRBS3 as a major host factor that is critical for KSHV lytic replication and uncover its key role in KSHV lytic gene transcription. Our results highlight a pivotal role for the miR-31-5p/KHDRBS3 axis in modulating KSHV reactivation and provide insights into gene-expression regulation of lytic KSHV.

Oncogenic Kaposi's sarcoma-associated herpesvirus (KSHV), an etiological agent of Kaposi's sarcoma and primary effusion lymphoma, employs a biphasic life cycle consisting of latency and lytic replication to achieve lifelong infection. Despite its essential role in KSHV persistence and tumorigenicity, much remains unknown about how KSHV lytic reactivation is regulated. Leveraging high-throughput transcriptomics, we identify microRNA-31-5p (miR-31-5p) as a key regulator of KSHV lytic reactivation capable of restricting KSHV entry into the lytic replication cycle. Ectopic expression of miR-31-5p impairs KSHV lytic gene transcription and production of lytic viral proteins, culminating in dramatic reduction of infectious virion production during KSHV reactivation. miR-31-5p overexpression also markedly reduces the expression of critical viral early genes, including the master regulator of the latent-lytic switch, KSHV replication and transcription activator (RTA) protein. Through mechanistic studies, we demonstrate that miR-31-5p represses KSHV lytic reactivation by directly targeting the KH domain protein KHDRBS3, an RNA-binding protein known to regulate RNA processing including alternative splicing. Our study highlights KHDRBS3 as an essential proviral host factor that is key to the successful completion of KSHV lytic replication and suggests its novel function in viral lytic gene transcription during KSHV reactivation. Taken together, these findings reveal a previously unrecognized role for the miR-31-5p/KHDRBS3 axis in regulating the KSHV latency-lytic replication switch and provide insights into gene expression regulation of lytic KSHV, which may be leveraged for lytic cycle-targeted therapeutic strategies against KSHV-associated malignancies.

The landscape of non-coding mutations in cancer progression and immune evasion is largely unexplored. Here, we identify transcrptome-wide somatic and germline 3' untranslated region (3'-UTR) variants from 375 gastric cancer patients from The Cancer Genome Atlas. By performing gene expression quantitative trait loci (eQTL) and immune landscape QTL (ilQTL) analysis, we discover 3'-UTR variants with cis effects on expression and immune landscape phenotypes, such as immune cell infiltration and T cell receptor diversity. Using a massively parallel reporter assay, we distinguish between causal and correlative effects of 3'-UTR eQTLs in immune-related genes. Our approach identifies numerous 3'-UTR eQTLs and ilQTLs, providing a unique resource for the identification of immunotherapeutic targets and biomarkers. A prioritized ilQTL variant signature predicts response to immunotherapy better than standard-of-care PD-L1 expression in independent patient cohorts, showcasing the untapped potential of non-coding mutations in cancer.

Supplementary Figure from Transcriptome Profiling of ADAR1 Targets in Triple-Negative Breast Cancer Cells Reveals Mechanisms for Regulating Growth and Invasion

AbstractADARs catalyze adenosine-to-inosine (A-to-I) editing of double-stranded RNA and regulate global gene expression output through interactions with RNA and other proteins. ADARs play important roles in development and disease, and previous work has shown that ADAR1 is oncogenic in a growing list of cancer types. Here we show that ADAR1 is a critical gene for triple-negative breast cancer cells, as ADAR1 loss results in reduced growth (viability and cell cycle progression), invasion, and mammosphere formation. Whole transcriptome sequencing analyses demonstrate that ADAR1 regulates both coding and noncoding targets by altering gene expression level, A-to-I editing, and splicing. We determine that a recoding edit in filamin B (FLNB chr3:58156064) reduces the tumor suppressive activities of the protein to promote growth and invasion. We also show that several tumor suppressor miRNAs are upregulated upon ADAR1 loss and suppress cell-cycle progression and invasion. This work describes several novel mechanisms of ADAR1-mediated oncogenesis in triple-negative breast cancer, providing support to strategies targeting ADAR1 in this aggressive cancer type that has few treatment options.Implications:Targeting ADAR1 and thus downstream FLNB editing and miRNA regulation represents a possible novel therapeutic strategy in triple-negative breast cancer.

Background Adenosine deaminases acting on RNA (ADARs) modify many cellular RNAs by catalyzing the conversion of adenosine to inosine (A-to-I), and their deregulation is associated with several cancers. We recently showed that A-to-I editing is elevated in thyroid tumors and that ADAR1 is functionally important for thyroid cancer cell progression. The downstream effectors regulated or edited by ADAR1 and the significance of ADAR1 deregulation in thyroid cancer remain, however, poorly defined. Methods We performed whole transcriptome sequencing to determine the consequences of ADAR1 deregulation for global gene expression, RNA splicing and editing. The effects of gene silencing or RNA editing were investigated by analyzing cell viability, proliferation, invasion and subnuclear localization, and by protein and gene expression analysis. Results We report an oncogenic function for CDK13 in thyroid cancer and identify a new ADAR1-dependent RNA editing event that occurs in the coding region of its transcript. CDK13 was significantly over-edited (c.308A > G) in tumor samples and functional analysis revealed that this editing event promoted cancer cell hallmarks. Finally, we show that CDK13 editing increases the nucleolar abundance of the protein, and that this event might explain, at least partly, the global change in splicing produced by ADAR1 deregulation. Conclusions Overall, our data support A-to-I editing as an important pathway in cancer progression and highlight novel mechanisms that might be used therapeutically in thyroid and other cancers.

Cancer immunotherapies targeting the interaction between Programmed death 1 (PD-1) and Programmed death ligand 1 (PD-L1) have recently been approved for the treatment of multiple cancer types, including gastric cancer. However, not all patients respond to these therapies, while some eventually acquire resistance. A partial predictive biomarker for positive response to PD-1/PD-L1 therapy is PD-L1 expression, which has been shown to be under strict post-transcriptional control in cancer. By fractionating the PD-L1 3' untranslated region (3'UTR) into multiple overlapping fragments, we identified a small 100-nucleotide-long cis-acting region as being necessary and sufficient for post-transcriptional repression of PD-L1 expression in gastric cancer. In parallel, we performed a correlation analysis between PD-L1 expression and all host miRNAs in stomach cancer patient samples. A single miRNA, miR-105-5p, was predicted to bind to the identified cis-acting 3'UTR region and to negatively correlate with PD-L1 expression. Overexpression of miR-105-5p in gastric cancer cell lines resulted in decreased expression of PD-L1, both at the total protein and surface expression levels, and induced CD8+ T cell activation in co-culture assays. Finally, we show that expression of miR-105-5p in gastric cancer is partly controlled by DNA methylation of a cancer- and germline-specific promoter of its host gene, GABRA3. Dysregulation of miR-105-5p is observed in many cancer types and this study shows the importance of this miRNA in controlling the immunogenicity of cancer cells, thus highlighting it as a potential biomarker for PD-1/PD-L1 therapy and target for combinatorial immunotherapy.

Gastric cancer (GC) is the fifth most common cancer worldwide. In approximately 10% of GC cases, cancer cells show ubiquitous and monoclonal Epstein-Barr virus (EBV) infection. A significant feature of EBV-associated GC (EBVaGC) is high lymphocytic infiltration and high expression of immune checkpoint proteins, including programmed death-ligand 1 (PD-L1). This highlights EBVaGC as a strong candidate for immune checkpoint blockade therapy. Indeed, several recent studies have shown that EBV positivity in GC correlates with positive response to programmed cell death protein 1 (PD-1)/PD-L1 blockade therapy. Understanding the mechanisms that control PD-L1 expression in EBVaGC can indicate new predictive biomarkers for immunotherapy, as well as therapeutic targets for combination therapy. Various mechanisms have been implicated in PD-L1 expression regulation, including structural variations, post-transcriptional control, oncogenic activation of intrinsic signaling pathways, and increased sensitivity to extrinsic signals. This review provides the most recent updates on the multilayered control of PD-L1 expression in EBVaGC.