Hepatitis B virus (HBV) remains a major human pathogen, with over 240 million individuals suffering from chronic HBV infections. These can persist for decades due to the lack of therapies that can effectively target the stable viral covalently closed circular (ccc) DNA molecules present in infected hepatocytes. Using lentiviral transduction of a bacterial Cas9 gene and single guide RNAs (sgRNAs) specific for HBV, we observed effective inhibition of HBV DNA production in in vitro models of both chronic and de novo HBV infection. Cas9/sgRNA combinations specific for HBV reduced total viral DNA levels by up to ~1000-fold and HBV cccDNA levels by up to ~10-fold and also mutationally inactivated the majority of the residual viral DNA. Together, these data provide proof of principle for the hypothesis that CRISPR/Cas systems have the potential to serve as effective tools for the depletion of the cccDNA pool in chronically HBV infected individuals.

Highlights d Prostate cancers (PCs) with TP53 and RB1 loss exhibit very poor clinical outcomes d PCs with TP53/RB1 loss exhibit stem cell-like features and loss of AR activity d Loss of TP53/RB1 does not uniformly promote neuroendocrine transdifferentiation d TP53/RB1-null PCs exhibit replication stress and respond to PARP and ATR inhibition

Exposure to genotoxic agents, such as irradiation produces DNA damage, the toxicity of which is augmented when the DNA repair is impaired. Poly (ADP-ribose) polymerase (PARP) inhibitors were found to be “synthetic lethal” in cells deficient in BRCA1 and BRCA2 that impair homologous recombination. However, since many tumors, including prostate cancer (PCa) rarely have on such mutations, there is considerable interest in finding alternative determinants of PARP inhibitor sensitivity. We evaluated the effectiveness of radiation in combination with the PARP inhibitor, rucaparib in PCa cells. The combination index for clonogenic survival following radiation and rucaparib treatments revealed synergistic interactions in a panel of PCa cell lines, being strongest for LNCaP and VCaP cells that express ETS gene fusion proteins. These findings correlated with synergistic interactions for senescence activation, as indicated by β--galactosidase staining. Absence of PTEN and presence of ETS gene fusion thus facilitated activation of senescence, which contributed to decreased clonogenic survival. Increased radiosensitivity in the presence of rucaparib was associated with persistent DNA breaks, as determined by χ-H2AX, p53BP1, and Rad51 foci. VCaP cells, which harbor the TMPRSS2-ERG gene fusion and PC3 cells that stably express a similar construct (fusion III) showed enhanced sensitivity towards rucaparib, which, in turn, increased the radiation response to a similar extent as the DNA-PKcs inhibitor NU7441. Rucaparib radiosensitized PCa cells, with a clear benefit of low dose-rate radiation (LDR) administered over a longer period of time that caused enhanced DNA damage. LDR mimicking brachytherapy, which is used successfully in the clinic, was most effective when combined with rucaparib by inducing persistent DNA damage and senescence, leading to decreased clonogenic survival. This combination was most effective in the presence of the TMPRSS2-ERG and in the absence of PTEN, indicating clinical potential for brachytherapy in patients with intermediate and high risk PCa.

Prostate cancer (PC) initially depends on androgen receptor (AR) signaling for survival and growth. Therapeutics designed to suppress AR activity serve as the primary intervention for advanced disease. However, supraphysiological androgen (SPA) concentrations can produce paradoxical responses leading to PC growth inhibition. We sought to discern the mechanisms by which SPA inhibits PC and to determine if molecular context associates with antitumor activity. SPA produced an ARmediated, dose-dependent induction of DNA double-strand breaks, G 0 /G 1 cell-cycle arrest, and cellular senescence. SPA repressed genes involved in DNA repair and delayed the restoration of damaged DNA, which was augmented by poly (ADPribose) polymerase 1 inhibition. SPA-induced double-strand breaks were accentuated in BRCA2-deficient patients with PC, and combining SPA with poly (ADP-ribose) polymerase or DNA-dependent protein kinase inhibition further repressed growth. Next-generation sequencing was performed on biospecimens from patients with PC receiving SPA as part of ongoing phase II clinical trials. Patients with mutations in genes mediating homology-directed DNA repair were more likely to exhibit clinical responses to SPA. These results provide a mechanistic rationale for directing SPA therapy to patients with PC who have AR amplification or DNA repair deficiency and for combining SPA therapy with poly (ADP-ribose) polymerase inhibition.

Gastrointestinal stromal tumors (GIST) are caused by activating mutations in the KIT or PDGFRA receptor tyrosine kinase genes. Although >85% of GIST patients treated with the small-molecule inhibitor imatinib mesylate (Gleevec) achieve disease stabilization, complete remissions are rare and a substantial proportion of patients develop resistance to imatinib over time. Upregulation of soluble, non-chromatin-bound histone H2AX has an important role in imatinib-induced apoptosis of GIST cells. Additionally, H2AX levels in untreated GIST are maintained at low levels by a pathway that involves KIT, phosphoinositide 3-kinase, and the ubiquitin-proteasome system. In this study, we asked whether bortezomib-mediated inhibition of the ubiquitinproteasome machinery could lead to upregulation of histone H2AX and GIST cell death. We show that bortezomib rapidly triggers apoptosis in GIST cells through a combination of mechanisms involving H2AX upregulation and loss of KIT protein expression. Downregulation of KIT transcription was an underlying mechanism for bortezomib-mediated inhibition of KIT expression. In contrast, the nuclear factor-κB signaling pathway did not seem to play a major role in bortezomib-induced GIST cell death. Significantly, we found that bortezomib would induce apoptosis in two imatinib-resistant GIST cell lines as well as a short-term culture established from a primary imatinib-resistant GIST. Collectively, our results provide a rationale to test the efficacy of bortezomib in GIST patients with imatinib-sensitive or -resistant tumors.

Centrosome aberrations are a frequent finding in human tumors. However, very little is known about the molecular mechanisms leading to disruption of centrosome duplication control and the functional consequences of aberrant centrosome numbers. The high-risk human papillomavirus Type 16 (HPV-16) E6 and E7 oncoproteins are overexpressed in HPV-associated malignancies of the anogenital tract and have been instrumental in delineating different pathways of centrosome amplification. Whereas the E6 oncoprotein was found to provoke centrosome accumulation, the HPV-16 E7 oncoprotein triggers a genuine disruption of the centrosome duplication cycle. Importantly, the E7 oncoprotein can rapidly cause centrosome overduplication through a pathway that involves the concurrent formation of multiple daughters at single maternal centrioles (centriole flowers). Several lines of evidence suggest that cyclin E/CDK2 complexes and Polo-like kinase 4 (PLK4) are crucial players in this process. These findings underscore that the HPV-16 E7 oncoprotein is a unique tool to dissect normal and abnormal centriole biogenesis and the underlying molecular circuitry.

Remdesivir is a broad-spectrum antiviral nucleotide prodrug that has been clinically evaluated in Ebola virus patients and recently received emergency use authorization (EUA) for treatment of COVID-19. With approvals from the Federal Select Agent Program and the Centers for Disease Control and Prevention’s Institutional Biosecurity Board, we characterized the resistance profile of remdesivir by serially passaging Ebola virus under remdesivir selection; we generated lineages with low-level reduced susceptibility to remdesivir after 35 passages. We found that a single amino acid substitution, F548S, in the Ebola virus polymerase conferred low-level reduced susceptibility to remdesivir. The F548 residue is highly conserved in filoviruses but should be subject to specific surveillance among novel filoviruses, in newly emerging variants in ongoing outbreaks, and also in Ebola virus patients undergoing remdesivir therapy. Homology modeling suggests that the Ebola virus polymerase F548 residue lies in the F-motif of the polymerase active site, a region that was previously identified as susceptible to resistance mutations in coronaviruses. Our data suggest that molecular surveillance of this region of the polymerase in remdesivir-treated COVID-19 patients is also warranted.

Ebola virus (EBOV) infection is a major public health concern due to high fatality rates and limited effective treatments. Statins, widely used cholesterol-lowering drugs, have pleiotropic mechanisms of action and were suggested as potential adjunct therapy for Ebola virus disease (EVD) during the 2013-2016 outbreak in West Africa. Here, we evaluated the antiviral effects of statin (lovastatin) on EBOV infection Statin treatment decreased infectious EBOV production in primary human monocyte-derived macrophages and in the hepatic cell line Huh7. Statin treatment did not interfere with viral entry, but the viral particles released from treated cells showed reduced infectivity due to inhibition of viral glycoprotein processing, as evidenced by decreased ratios of the mature glycoprotein form to precursor form. Statin-induced inhibition of infectious virus production and glycoprotein processing was reversed by exogenous mevalonate, the rate-limiting product of the cholesterol biosynthesis pathway, but not by low-density lipoprotein. Finally, statin-treated cells produced EBOV particles devoid of the surface glycoproteins required for virus infectivity. Our findings demonstrate that statin treatment inhibits EBOV infection and suggest that the efficacy of statin treatment should be evaluated in appropriate animal models of EVD. Treatments targeting Ebola virus disease (EVD) are experimental, expensive, and scarce. Statins are inexpensive generic drugs that have been used for many years for the treatment of hypercholesterolemia and have a favorable safety profile. Here, we show the antiviral effects of statins on infectious Ebola virus (EBOV) production. Our study reveals a novel molecular mechanism in which statin regulates EBOV particle infectivity by preventing glycoprotein processing and incorporation into virus particles. Additionally, statins have anti-inflammatory and immunomodulatory effects. Since inflammation and dysregulation of the immune system are characteristic features of EVD, statins could be explored as part of EVD therapeutics.