Human papillomaviruses (HPVs) are the etiological agents of cervical cancer and other human malignancies. HPVs are classified into high-and low-risk genotypes according to their association with cancer. Host cell transformation by high-risk HPVs relies in part on the ability of the viral E6 protein to induce the degradation of p53. We report the development of a cellular assay that accurately quantifies the p53 degradation activity of E6 in vivo, based on the fusion of p53 to Renilla luciferase (RLuc-p53). This assay was used to measure the p53 degradation activities of E6 proteins from 29 prevalent HPV types and variants of HPV type 16 (HPV16) and HPV33 by determining the amount of E6 expression vector required to reduce by half the levels of RLuc-p53 (50% effective concentration [EC 50 ]). These studies revealed an unexpected variability in the p53 degradation activities of different E6 proteins, even among active types whose EC 50 s span more than 2 log units. Differences in activity were greater between types than between variants and did not correlate with differences in the intracellular localization of E6, with most being predominantly nuclear. Protein and mRNA expression of the 29 E6 proteins was also examined. For 16 high-risk types, spliced transcripts that encode shorter E6*I proteins of variable sizes and abundances were detected. Mutation of the splice donor site in five different E6 proteins increased their p53 degradation activity, suggesting that mRNA splicing can limit the activity of some highrisk E6 types. The quantification of p53 degradation in vivo represents a novel tool to systematically compare the oncogenic potentials of E6 proteins from different HPV types and variants.

Purpose: KRAS mutation (MT) is a major oncogenic driver in PDAC. A small subset of PDACs harbor KRAS-wild-type (WT). We aim to characterize the molecular profiles of KRAS-WT PDAC to uncover new pathogenic drivers and offer targeted treatments. Experimental Design: Tumor tissue obtained from surgical or biopsy material was subjected to next-generation DNA/RNA sequencing, microsatellite-instability (MSI) and mismatch-repair (MMR) status determination. Results: Of the 2,483 patients (male 53.7%, median-age 66 years) studied, 266 tumors (10.7%) were KRAS-WT. The most frequently mutated gene in KRAS-WT-PDAC was TP53 (44.5%), followed by BRAF (13.0%) . Multiple mutations within the DNA-damage-repair (BRCA2, ATM, BAP1, RAD50, FANCE, PALB2), chromatin-remodeling (ARID1A, PBRM1, ARID2, KMT2D, KMT2C, SMARCA4, SETD2), and cell-cycle-control pathways (CDKN2A, CCND1, CCNE1) were detected frequently. There was no statistically-significant difference in PDL1-expression between KRAS-WT (15.8%) and MT (17%) tumors. However, KRAS-WT-PDAC were more likely to be MSI-high (4.7% vs 0.7%; p<0.05), TMB-high (4.5% vs 1%; p<0.05), and exhibit increased infiltration of CD8+ T-cells, NK-cells and myeloid dendritic cells. KRAS-WT-PDACs exhibited gene fusions of BRAF (6.6%), FGFR2 (5.2%), ALK (2.6%), RET (1.3%) and NRG1 (1.3%), as well as amplification of FGF3 (3%), ERBB2 (2.2%), FGFR3 (1.8%), NTRK (1.8%) and MET (1.3%). Real-world evidence reveals a survival advantage of KRAS-WT patients in overall cohorts as well as in patients treated with gemcitabine/nab-paclitaxel or 5FU/oxaliplatin. Conclusions: KRAS-WT PDAC represents 10.7% of PDAC and is enriched with targetable alterations, including immuno-oncologic markers. Identification of KRAS-WT patients in clinical practice may expand therapeutic options in a clinically meaningful manner.

Several proteins and RNAs expressed by mammalian viruses have been reported to interfere with RNA interference (RNAi) activity. We investigated the ability of the HIV-1-encoded RNA elements Trans-Activation Response (TAR) and RevResponse Element (RRE) to alter RNAi. MicroRNA let7-based assays showed that RRE is a potent suppressor of RNAi activity, while TAR displayed moderate RNAi suppression. We demonstrate that RRE binds to TAR-RNA Binding Protein (TRBP), an essential component of the RNA Induced Silencing Complex (RISC). The binding of TAR and RRE to TRBP displaces small interfering (si)RNAs from binding to TRBP. Several stem-deleted RRE mutants lost their ability to suppress RNAi activity, which correlated with a reduced ability to compete with siRNA-TRBP binding. A lentiviral vector expressing TAR and RRE restricted RNAi, but RNAi was restored when Rev or GagPol were coexpressed. Adenoviruses are restricted by RNAi and encode their own suppressors of RNAi, the Virus-Associated (VA) RNA elements. RRE enhanced the replication of wild-type and VA-deficient adenovirus. Our work describes RRE as a novel suppressor of RNAi that acts by competing with siRNAs rather than by disrupting the RISC. This function is masked in lentiviral vectors co-expressed with viral proteins and thus will not affect their use in gene therapy. The potent RNAi suppressive effects of RRE identified in this study could be used to enhance the expression of RNAi restricted viruses used in oncolysis such as adenoviruses.