Combining EHMT and PARP Inhibition: A Strategy to Diminish Therapy-Resistant Ovarian Cancer Tumor Growth while Stimulating Immune Activation.

Despite the success of Poly-ADP-ribose polymerase inhibitors (PARPi) in the clinic, high rates of resistance to PARPi presents a challenge in the treatment of ovarian cancer, thus it is imperative to find therapeutic strategies to combat PARPi resistance. Here, we demonstrate that inhibition of epigenetic modifiers Euchromatic histone lysine methyltransferases 1/2 (EHMT1/2) reduces the growth of multiple PARPi-resistant ovarian cancer cell lines and tumor growth in a PARPi-resistant mouse model of ovarian cancer. We found that combinatory EHMT and PARP inhibition increases immunostimulatory dsRNA formation and elicits several immune signaling pathways in vitro. Using epigenomic profiling and transcriptomics, we found that EHMT2 is bound to transposable elements, and that EHMT inhibition leads to genome-wide epigenetic and transcriptional derepression of transposable elements. We validated EHMT-mediated activation of immune signaling and upregulation of transposable element transcripts in patient-derived, therapy-naïve, primary ovarian tumors, suggesting potential efficacy in PARPi-sensitive disease as well. Importantly, using multispectral immunohistochemistry, we discovered that combinatory therapy increased CD8 T cell activity in the tumor microenvironment of the same patient-derived tissues. In a PARPi-resistant syngeneic murine model, EHMT and PARP inhibition combination inhibited tumor progression and increased Granzyme B+ cells in the tumor. Together, our results provide evidence that combinatory EHMT and PARP inhibition stimulates a cell autologous immune response in vitro, is an effective therapy to reduce PARPi resistant ovarian tumor growth in vivo, and promotes anti-tumor immunity activity in the tumor microenvironment of patient-derived ex vivo tissues of ovarian cancer.

CASC4/GOLM2 drives high grade serous carcinoma anoikis resistance through the recycling of EGFR.

Ovarian cancer is the deadliest gynecological malignancy, and accounts for over 150,000 deaths per year worldwide. The high grade serous ovarian carcinoma (HGSC) subtype accounts for almost 70% of ovarian cancers and is the deadliest. HGSC originates in the fimbria of the fallopian tube and disseminates through the peritoneal cavity. HGSC survival in peritoneal fluid requires cells to resist anoikis (anchorage-independent apoptosis). Most anoikis resistant mechanisms are dependent on microenvironment interactions with cell surface-associated proteins, such as integrins and receptor tyrosine kinases (RTKs). We previously identified the gene CASC4 as a driver of anoikis resistance. CASC4 is predicted to be a Golgi-associated protein that may regulate protein trafficking to the plasma membrane, but CASC4 is largely uncharacterized in literature; thus, we sought to determine how CASC4 confers anoikis resistance to HGSC cells. Mining of publicly available ovarian cancer datasets (TCGA) showed that CASC4 is associated with worse overall survival and increased resistance to platinum-based chemotherapies. For experiments, we cultured three human HGSC cell lines (PEO1, CaOV3, OVCAR3), and a murine HGSC cell line, (ID8) with shRNA-mediated CASC4 knockdowns (CASC4 KD) in suspension, to recapitulate the peritoneal fluid environment in vitro. CASC4 KD significantly inhibited cell proliferation and colony formation ability, and increased apoptosis. A Reverse Phase Protein Assay (RPPA) showed that CASC4 KD resulted in a broad re-programming of membrane-associated proteins. Specifically, CASC4 KD led to decreased protein levels of the RTK Epidermal Growth Factor Receptor (EGFR), an initiator of several oncogenic signaling pathways, leading us to hypothesize that CASC4 drives HGSC survival through mediating recycling and trafficking of EGFR. Indeed, loss of CASC4 led to a decrease in both EGFR membrane localization, reduced turnover of EGFR, and increased EGFR ubiquitination. Moreover, a syngeneic ID8 murine model of ovarian cancer showed that knocking down CASC4 leads to decreased tumor burden and dissemination.

Targeting BRPF3 moderately reverses olaparib resistance in high grade serous ovarian carcinoma.

PARP inhibitors (PARPi) kill cancer cells by stalling DNA replication and preventing DNA repair, resulting in a critical accumulation of DNA damage. Resistance to PARPi is a growing clinical problem in the treatment of high grade serous ovarian carcinoma (HGSOC). Acetylation of histone H3 lysine 14 (H3K14ac) and associated histone acetyltransferases (HATs) and epigenetic readers have known functions in DNA repair and replication. Our objectives are to examine their expression and activities in the context of PARPi-resistant HGSOC, and to determine if targeting H3K14ac or associated proteins has therapeutic potential. Using mass spectrometry profiling of histone modifications, we observed increased H3K14ac enrichment in PARPi-resistant HGSOC cells relative to isogenic PARPi-sensitive lines. By reverse-transcriptase quantitative PCR and RNA-seq, we also observed altered expression of numerous HATs in PARPi-resistant HGSOC cells and a PARPi-resistant PDX model. Knockdown of HATs only modestly altered PARPi response, although knockdown and inhibition of PCAF significantly increased resistance. Pharmacologic inhibition of HBO1 depleted H3K14ac but did not affect PARPi response. However, knockdown and inhibition of BRPF3, a bromodomain and PHD-finger containing protein that is known to interact in a complex with HBO1, did reduce PARPi resistance. This study demonstrates that depletion of H3K14ac does not affect PARPi response in HGSOC. Our data suggest that the bromodomain function of HAT proteins, such as PCAF, or accessory proteins, such as BRPF3, may play a more direct role compared to direct HATs function in PARPi response.

Novel chromobox 2 inhibitory peptide decreases tumor progression.

BACKGROUND: The Polycomb Repressor Complex 1 (PRC1) is an epigenetic regulator of differentiation and development, consisting of multiple subunits including RING1, BMI1, and Chromobox. The composition of PRC1 dictates its function and aberrant expression of specific subunits contributes to several diseases including cancer. Specifically, the reader protein Chromobox2 (CBX2) recognizes the repressive modifications including histone H3 lysine 27 tri-methylation (H3K27me3) and H3 lysine 9 dimethylation (H3K9me2). CBX2 is overexpressed in several cancers compared to the non-transformed cell counterparts, it promotes both cancer progression and chemotherapy resistance. Thus, inhibiting the reader function of CBX2 is an attractive and unique anti-cancer approach. RESEARCH DESIGN & METHODS: Compared with other CBX family members, CBX2 has a unique A/T-hook DNA binding domain that is juxtaposed to the chromodomain (CD). Using a computational approach, we constructed a homology model of CBX2 encompassing the CD and A/T hook domain. We used the model as a basis for peptide design and identified blocking peptides that are predicted to directly bind the CD and A/T-hook regions of CBX2. These peptides were tested in vitro and in vivo models. CONCLUSION: The CBX2 blocking peptide significantly inhibited both 2D and 3D growth of ovarian cancer cells, downregulated a CBX2 target gene, and blunted tumor growth in vivo.

Combination CDC-like kinase inhibition (CLK)/Dual-specificity tyrosine-regulated kinase (DYRK) and taxane therapy in CTNNB1-mutated endometrial cancer.

SM08502 (cirtuvivint) is a novel pan CDC-like kinase (CLK) and Dual specificity tyrosine kinase (DYRK) inhibitor that targets mRNA splicing and is optimized for Wnt pathway inhibition. Previous evaluation of single agent CLK/DYRK inhibition (SM04690) demonstrated inhibition of tumor progression and ß-catenin/TCF transcriptional activity in CTNNB1-mutant endometrial cancer (EC). In-vitro analysis of SM08502 similarly decreases Wnt transcriptional activity and cellular proliferation while increasing cellular apoptosis. SM08502 is an active single-agent therapy with IC50's in the nanomolar range for all EC cell lines evaluated. Combination of SM08502 with paclitaxel has synergistic effect in vitro, as demonstrated by Combination Index <1, and inhibits tumor progression in four endometrial cancer models (HEC265, Ishikawa, Ishikawa-S33Y, and SNGM). In our in vivo mouse models, Ishikawa demonstrated significantly lower tumor volumes of combination vs SM08502 alone (Repeated Measures one-way ANOVA, p = 0.04), but not vs paclitaxel alone. HEC265, SNGM, and Ishikawa-S33Y tumors all had significantly lower tumor volumes with combination SM08502 and paclitaxel compared to single-agent paclitaxel (Repeated Measures one-way ANOVA, p = 0.01, 0.004, and 0.0008, respectively) or single-agent SM08502 (Repeated Measures one-way ANOVA, p = 0.002, 0.005, and 0.01, respectively) alone. Mechanistically, treatment with SM08502 increases alternative splicing (AS) events compared to treatment with paclitaxel. AS regulation is an important post-transcriptional mechanism associated with the oncogenic process in many cancers, including EC. Results from these studies have led to a Phase I evaluation of this combination in recurrent EC.

Combinatory EHMT and PARP inhibition induces an interferon response and a CD8 T cell-dependent tumor regression in PARP inhibitor-resistant models.

Euchromatic histone lysine methyltransferases 1 and 2 (EHMT1/2), which catalyze demethylation of histone H3 lysine 9 (H3K9me2), contribute to tumorigenesis and therapy resistance through unknown mechanisms of action. In ovarian cancer, EHMT1/2 and H3K9me2 are directly linked to acquired resistance to poly-ADP-ribose polymerase (PARP) inhibitors and are correlated with poor clinical outcomes. Using a combination of experimental and bioinformatic analyses in several PARP inhibitor resistant ovarian cancer models, we demonstrate that combinatory inhibition of EHMT and PARP is effective in treating PARP inhibitor resistant ovarian cancers. Our in vitro studies show that combinatory therapy reactivates transposable elements, increases immunostimulatory dsRNA formation, and elicits several immune signaling pathways. Our in vivo studies show that both single inhibition of EHMT and combinatory inhibition of EHMT and PARP reduces tumor burden, and that this reduction is dependent on CD8 T cells. Together, our results uncover a direct mechanism by which EHMT inhibition helps to overcome PARP inhibitor resistance and shows how an epigenetic therapy can be used to enhance anti-tumor immunity and address therapy resistance.

The Capacity of the Ovarian Cancer Tumor Microenvironment to Integrate Inflammation Signaling Conveys a Shorter Disease-free Interval.

PURPOSE: Ovarian cancer has one of the highest deaths to incidence ratios across all cancers. Initial chemotherapy is effective, but most patients develop chemoresistant disease. Mechanisms driving clinical chemo-response or -resistance are not well-understood. However, achieving optimal surgical cytoreduction improves survival, and cytoreduction is improved by neoadjuvant chemotherapy (NACT). NACT offers a window to profile pre- versus post-NACT tumors, which we used to identify chemotherapy-induced changes to the tumor microenvironment. EXPERIMENTAL DESIGN: We obtained matched pre- and post-NACT archival tumor tissues from patients with high-grade serous ovarian cancer (patient, n = 6). We measured mRNA levels of 770 genes (756 genes/14 housekeeping genes, NanoString Technologies), and performed reverse phase protein array (RPPA) on a subset of matched tumors. We examined cytokine levels in pre-NACT ascites samples (n = 39) by ELISAs. A tissue microarray with 128 annotated ovarian tumors expanded the transcriptional, RPPA, and cytokine data by multispectral IHC. RESULTS: The most upregulated gene post-NACT was IL6 (16.79-fold). RPPA data were concordant with mRNA, consistent with elevated immune infiltration. Elevated IL6 in pre-NACT ascites specimens correlated with a shorter time to recurrence. Integrating NanoString (n = 12), RPPA (n = 4), and cytokine (n = 39) studies identified an activated inflammatory signaling network and induced IL6 and IER3 (immediate early response 3) post-NACT, associated with poor chemo-response and time to recurrence. CONCLUSIONS: Multiomics profiling of ovarian tumor samples pre- and post-NACT provides unique insight into chemo-induced changes to the tumor microenvironment. We identified a novel IL6/IER3 signaling axis that may drive chemoresistance and disease recurrence.

Targeting Fatty Acid Oxidation to Promote Anoikis and Inhibit Ovarian Cancer Progression.

Epithelial-derived high-grade serous ovarian cancer (HGSOC) is the deadliest gynecologic malignancy. Roughly 80% of patients are diagnosed with late-stage disease, which is defined by wide-spread cancer dissemination throughout the pelvic and peritoneal cavities. HGSOC dissemination is dependent on tumor cells acquiring the ability to resist anoikis (apoptosis triggered by cell detachment). Epithelial cell detachment from the underlying basement membrane or extracellular matrix leads to cellular stress, including nutrient deprivation. In this report, we examined the contribution of fatty acid oxidation (FAO) in supporting anoikis resistance. We examined expression Carnitine Palmitoyltransferase 1A (CPT1A) in a panel of HGSOC cell lines cultured in adherent and suspension conditions. With CPT1A knockdown cells, we evaluated anoikis by caspase 3/7 activity, cleaved caspase 3 immunofluorescence, flow cytometry, and colony formation. We assessed CPT1A-dependent mitochondrial activity and tested the effect of exogenous oleic acid on anoikis and mitochondrial activity. In a patient-derived xenograft model, we administered etomoxir, an FAO inhibitor, and/or platinum-based chemotherapy. CPT1A is overexpressed in HGSOC, correlates with poor overall survival, and is upregulated in HGSOC cells cultured in suspension. CPT1A knockdown promoted anoikis and reduced viability of cells cultured in suspension. HGSOC cells in suspension culture are dependent on CPT1A for mitochondrial activity. In a patient-derived xenograft model of HGSOC, etomoxir significantly inhibited tumor progression. IMPLICATIONS: Targeting FAO in HGSOC to promote anoikis and attenuate dissemination is a potential approach to promote a more durable antitumor response and improve patient outcomes.

Amplification of the Mutation-Carrying BRCA2 Allele Promotes RAD51 Loading and PARP Inhibitor Resistance in the Absence of Reversion Mutations.

Patients harboring germline breast cancer susceptibility genes 1 and 2 (BRCA1/2) mutations are predisposed to developing breast, pancreatic, and ovarian cancers. BRCA2 plays a critical role in homologous recombination (HR) DNA repair and deleterious mutations in BRCA2 confer sensitivity to PARP inhibition. Recently, the PARP inhibitors olaparib and rucaparib were FDA approved for the treatment of metastatic breast cancer and patients with recurrent ovarian cancer with mutations in BRCA1/2. Despite their initial antitumor activity, the development of resistance limits the clinical utility of PARP inhibitor therapy. Multiple resistance mechanisms have been described, including reversion mutations that restore the reading frame of the BRCA2 gene. In this study, we generated olaparib- and rucaparib-resistant BRCA2-mutant Capan1 cell lines. We did not detect secondary reversion mutations in the olaparib- or rucaparib-resistant clones. Several of the resistant clones had gene duplication and amplification of the mutant BRCA2 allele, with a corresponding increase in expression of a truncated BRCA2 protein. In addition, HR-mediated DNA repair was rescued, as evidenced by the restoration of RAD51 foci formation. Using mass spectrometry, we identified Disruptor Of Telomeric silencing 1-Like (DOT1L), as an interacting partner of truncated BRCA2. RNAi-mediated knockdown of BRCA2 or DOT1L was sufficient to resensitize cells to olaparib. The results demonstrate that independent of a BRCA2 reversion, mutation amplification of a mutant-carrying BRCA2 contributes to PARP inhibitor resistance.

Histone methyltransferases EHMT1 and EHMT2 (GLP/G9A) maintain PARP inhibitor resistance in high-grade serous ovarian carcinoma.

BACKGROUND: Euchromatic histone-lysine-N-methyltransferases 1 and 2 (EHMT1/2, aka GLP/G9A) catalyze dimethylation of histone H3 lysine 9 (H3K9me2) and have roles in epigenetic silencing of gene expression. EHMT1/2 also have direct roles in DNA repair and are implicated in chemoresistance in several cancers. Resistance to chemotherapy and PARP inhibitors (PARPi) is a major cause of mortality in high-grade serous ovarian carcinoma (HGSOC), but the contribution of the epigenetic landscape is unknown. RESULTS: To identify epigenetic mechanisms of PARPi resistance in HGSOC, we utilized unbiased exploratory techniques, including RNA-Seq and mass spectrometry profiling of histone modifications. Compared to sensitive cells, PARPi-resistant HGSOC cells display a global increase of H3K9me2 accompanied by overexpression of EHMT1/2. EHMT1/2 overexpression was also observed in a PARPi-resistant in vivo patient-derived xenograft (PDX) model. Genetic or pharmacologic disruption of EHMT1/2 sensitizes HGSOC cells to PARPi. Cell death assays demonstrate that EHMT1/2 disruption does not increase PARPi-induced apoptosis. Functional DNA repair assays show that disruption of EHMT1/2 ablates homologous recombination (HR) and non-homologous end joining (NHEJ), while immunofluorescent staining of phosphorylated histone H2AX shows large increases in DNA damage. Propidium iodide staining and flow cytometry analysis of cell cycle show that PARPi treatment increases the proportion of PARPi-resistant cells in S and G2 phases, while cells treated with an EHMT1/2 inhibitor remain in G1. Co-treatment with PARPi and EHMT1/2 inhibitor produces an intermediate phenotype. Immunoblot of cell cycle regulators shows that combined EHMT1/2 and PARP inhibition reduces expression of specific cyclins and phosphorylation of mitotic markers. These data suggest DNA damage and altered cell cycle regulation as mechanisms of sensitization. RNA-Seq of PARPi-resistant cells treated with EHMT1/2 inhibitor showed significant gene expression changes enriched in pro-survival pathways that remain unexplored in the context of PARPi resistance, including PI3K, AKT, and mTOR. CONCLUSIONS: This study demonstrates that disrupting EHMT1/2 sensitizes HGSOC cells to PARPi, and suggests a potential mechanism through DNA damage and cell cycle dysregulation. RNA-Seq identifies several unexplored pathways that may alter PARPi resistance. Further study of EHMT1/2 and regulated genes will facilitate development of novel therapeutic strategies to successfully treat HGSOC.

Multi-Omic Approaches Identify Metabolic and Autophagy Regulators Important in Ovarian Cancer Dissemination.

High-grade serous ovarian cancers (HGSOCs) arise from exfoliation of transformed cells from the fallopian tube, indicating that survival in suspension, and potentially escape from anoikis, is required for dissemination. We report here the results of a multi-omic study to identify drivers of anoikis escape, including transcriptomic analysis, global non-targeted metabolomics, and a genome-wide CRISPR/Cas9 knockout (GeCKO) screen of HGSOC cells cultured in adherent and suspension settings. Our combined approach identified known pathways, including NOTCH signaling, as well as novel regulators of anoikis escape. Newly identified genes include effectors of fatty acid metabolism, ACADVL and ECHDC2, and an autophagy regulator, ULK1. Knockdown of these genes significantly inhibited suspension growth of HGSOC cells, and the metabolic profile confirmed the role of fatty acid metabolism in survival in suspension. Integration of our datasets identified an anoikis-escape gene signature that predicts overall survival in many carcinomas.

Activation of Wnt signaling promotes olaparib resistant ovarian cancer.

Epithelial ovarian cancer (EOC) has one of the highest death to incidence ratios among all cancers. High grade serous ovarian carcinoma (HGSOC) is the most common and deadliest EOC histotype due to the lack of therapeutic options following debulking surgery and platinum/taxane-based chemotherapies. For recurrent chemosensitive HGSOC, poly(ADP)-ribose polymerase inhibitors (PARPi; olaparib, rucaparib, or niraparib) represent an emerging treatment strategy. While PARPi are most effective in homologous recombination DNA repair-deficient (HRD) HGSOCs, recent studies have observed a significant benefit in non-HRD HGSOCs. However, all HGSOC patients are likely to acquire resistance. Therefore, there is an urgent clinical need to understand PARPi resistance and to introduce novel combinatorial therapies to manage PARPi resistance and extend HGSOC disease-free intervals. In a panel of HGSOC cell lines, we established matched olaparib sensitive and resistant cells. Transcriptome analysis of the matched olaparib-sensitive vs -resistant cells revealed activation of the Wnt signaling pathway and consequently increased TCF transcriptional activity in PARPi-resistant cells. Forced activation of canonical Wnt signaling in several PARPi-sensitive cells via WNT3A reduced olaparib and rucaparib sensitivity. PARPi resistant cells were sensitive to inhibition of Wnt signaling using the FDA-approved compound, pyrvinium pamoate, which has been shown to promote downregulation of ß-catenin. In both an HGSOC cell line and a patient-derived xenograft model, we observed that combining pyrvinium pamoate with olaparib resulted in a significant decrease in tumor burden. This study demonstrates that Wnt signaling can mediate PARPi resistance in HGSOC and provides a clinical rationale for combining PARP and Wnt inhibitors.

Peripheral AAV Injection for Retrograde Transduction of Dorsal Root and Trigeminal Ganglia.

Adeno-associated Virus (AAV) vectors are useful vehicles for delivering transgenes to a number of different tissues and organs in vivo. To date, most of these applications deliver the vectors to their target by either infusion into the bloodstream or direct injection into the target tissue. Recently there has been progress in delivering AAV vectors to neurons of the peripheral nervous system (PNS) following application of vectors to the peripheral epithelium, such as the skin or eye. This delivery only requires treatment of the epithelium to access the underlying nerve termini, and following treatment the vectors are transported retrogradely to the cell bodies of these neurons in the ganglia, such as dorsal root ganglia (DRG) or trigeminal ganglia (TG). Here we describe the methodology for highly efficient transduction of mouse DRG and rabbit TG following application of AAV vectors to the foot, or to the cornea, respectively.

Type I Protein Arginine Methyltransferases Overexpression Promotes Transformation and Potentiates Her2/Neu-Driven Tumorigenesis.

Bao and colleagues demonstrate that type I protein arginine methyltransferases (PRMT) are directly involved in mammary gland transformation and tumor progression. Notably, several distinct phenotypes require further investigation such as PRMT1/CARM1-induced transformation, CARM1-mediated delay in tumorigenesis, and PRMTs potentiation of Her2-dependent tumors. The PRMT overexpression transgenic mouse models should encourage and facilitate further mechanistic interrogation and the development of PRMT-directed therapies.See related article by Bao et al., p. 21.

CBX2 identified as driver of anoikis escape and dissemination in high grade serous ovarian cancer.

High grade serous ovarian carcinoma (HGSOC) is often diagnosed at an advanced stage. Chromobox 2 (CBX2), a polycomb repressor complex subunit, plays an oncogenic role in other cancers, but little is known about its role in HGSOC. We hypothesize that CBX2 upregulation promotes HGSOC via induction of a stem-like transcriptional profile and inhibition of anoikis. Examination of Gene Expression Omnibus (GEO) datasets and The Cancer Genome Atlas (TCGA) established that increased CBX2 expression conveyed chemoresistance and worse disease-free and overall survival. In primary HGSOC tumors, we observed CBX2 expression was significantly elevated compared to benign counterparts. In HGSOC cell lines, forced suspension promoted CBX2 expression. Subsequently, CBX2 knockdown inhibited anchorage-independent proliferation and potentiated anoikis-dependent apoptosis. Furthermore, CBX2 knockdown re-sensitized cells to platinum-based chemotherapy. Forced suspension promoted increased ALDH activity and ALDH3A1 expression and CBX2 knockdown led to a decrease in both ALDH activity and ALDH3A1 expression. Investigation of CBX2 expression on a HGSOC tissue microarray revealed CBX2 expression was apparent in both primary and metastatic tissues. CBX2 is an important regulator of stem-ness, anoikis escape, HGSOC dissemination, and chemoresistance and potentially serves as a novel therapeutic target.

In Vivo Knockdown of the Herpes Simplex Virus 1 Latency-Associated Transcript Reduces Reactivation from Latency.

During herpes simplex virus (HSV) latency, most viral genes are silenced, with the exception of one region of the genome encoding the latency-associated transcript (LAT). This long noncoding RNA was originally described as having a role in enhancing HSV-1 reactivation. However, subsequent evidence showing that the LAT blocked apoptosis and promoted efficient establishment of latency suggested that its effects on reactivation were secondary to establishment. Here, we utilized an adeno-associated virus (AAV) vector to deliver a LAT-targeting hammerhead ribozyme to HSV-1-infected neurons of rabbits after the establishment of HSV-1 latency. The rabbits were then induced to reactivate latent HSV-1. Using this model, we show that decreasing LAT levels in neurons following the establishment of latency reduced the ability of the virus to reactivate. This demonstrates that the HSV-1 LAT RNA has a role in reactivation that is independent of its function in establishment of latency. In addition, these results suggest the potential of AAV vectors expressing LAT-targeting ribozymes as a potential therapy for recurrent HSV disease such as herpes stromal keratitis, a leading cause of infectious blindness.IMPORTANCE Herpes simplex virus (HSV) establishes a lifelong infection and remains dormant (latent) in our nerve cells. Occasionally HSV reactivates to cause disease, with HSV-1 typically causing cold sores whereas HSV-2 is the most common cause of genital herpes. The details of how HSV reactivates are largely unknown. Most of HSV's genes are silent during latency, with the exception of RNAs made from the latency-associated transcript (LAT) region. While viruses that make less LAT do not reactivate efficiently, these viruses also do not establish latency as efficiently. Here we deliver a ribozyme that can degrade the LAT to the nerve cells of latently infected rabbits using a gene therapy vector. We show that this treatment blocks reactivation in the majority of the rabbits. This work shows that the LAT RNA is important for reactivation and suggests the potential of this treatment as a therapy for treating HSV infections.

Depletion of the Insulator Protein CTCF Results in Herpes Simplex Virus 1 Reactivation In Vivo.

Herpes simplex virus 1 (HSV-1) establishes a lifelong latent infection in host peripheral neurons, including the neurons of the trigeminal ganglia (TG). HSV-1 can reactivate from neurons to cause recurrent infection. During latency, the insulator protein CTCF occupies DNA binding sites on the HSV-1 genome, and these sites have been previously characterized as functional enhancer-blocking insulators. Previously, CTCF was found to be dissociated from wild-type virus postreactivation but not in mutants that do not reactivate, indicating that CTCF eviction may also be an important component of reactivation. To further elucidate the role of CTCF in reactivation of HSV-1, we used recombinant adeno-associated virus (rAAV) vectors to deliver a small interfering RNA targeting CTCF to peripheral neurons latent with HSV-1 in rabbit TG. Our data show that CTCF depletion resulted in long-term and persistent shedding of infectious virus in the cornea and increased ICP0 expression in the ganglia, indicating that CTCF depletion facilitates HSV-1 reactivation.IMPORTANCE Increasing evidence has shown that the insulator protein CTCF regulates gene expression of DNA viruses, including the gammaherpesviruses. While CTCF occupation and insulator function control gene expression in DNA viruses, CTCF eviction has been correlated to increased lytic gene expression and the dissolution of transcriptional domains. Our previous data have shown that in the alphaherpesvirus HSV-1, CTCF was found to be dissociated from the HSV-1 genome postreactivation, further indicating a global role for CTCF eviction in the transition from latency to reactivation in HSV-1 genomes. Using an rAAV8, we targeted HSV-1-infected peripheral neurons for CTCF depletion to show that CTCF depletion precedes the shedding of infectious virus and increased lytic gene expression in vivo, providing the first evidence that CTCF depletion facilitates HSV-1 reactivation.

PARP inhibitors: Clinical utility and possibilities of overcoming resistance.

PARP inhibitors represent a major breakthrough in ovarian cancer care. Almost half of all ovarian cancers have deficiencies in the homologous recombination (HR) DNA repair pathway, namely BRCA1/2 mutations. Given the limited therapeutic options for recurrent ovarian cancer patients there has been a significant effort to develop novel therapies to exploit DNA repair deficiencies. In 2005 and 2006, inhibiting PARP enzymes was first observed to be highly effective against cancers with HR deficiencies. PARP inhibitors are being utilized in the clinic to manage recurrent ovarian cancers that display defects in the HR repair pathway. However, PARP inhibitors also show significant clinical benefit in patients without HR deficiencies. There are currently three FDA-approved PARP inhibitors for recurrent ovarian cancer and an additional two PARP inhibitors being evaluated in late stage clinical trials. Given the expanding clinical use of PARP inhibitors and the high likelihood of acquired resistance, there is a significant need for clinical strategies to manage PARP inhibitor resistant disease. This review will examine PARP inhibitors in the context of: indications and toxicities, novel biomarkers to predict response, targeted-therapy resistance, and potential approaches to manage resistant disease.

Adeno-associated Virus Vectors Efficiently Transduce Mouse and Rabbit Sensory Neurons Coinfected with Herpes Simplex Virus 1 following Peripheral Inoculation.

UNLABELLED: Following infection of epithelial tissues, herpes simplex virus 1 (HSV-1) virions travel via axonal transport to sensory ganglia and establish a lifelong latent infection within neurons. Recent studies have revealed that, following intraganglionic or intrathecal injection, recombinant adeno-associated virus (rAAV) vectors can also infect sensory neurons and are capable of stable, long-term transgene expression. We sought to determine if application of rAAV to peripheral nerve termini at the epithelial surface would allow rAAV to traffic to sensory ganglia in a manner similar to that seen with HSV. We hypothesized that footpad or ocular inoculation with rAAV8 would result in transduction of dorsal root ganglia (DRG) or trigeminal ganglia (TG), respectively. To test this, we inoculated the footpads of mice with various amounts of rAAV as well as rAAV capsid mutants. We demonstrated that this method of inoculation can achieve a transduction rate of >90% of the sensory neurons in the DRG that innervate the footpad. Similarly, we showed that corneal inoculation with rAAV vectors in the rabbit efficiently transduced >70% of the TG neurons in the optic tract. Finally, we demonstrated that coinfection of mouse footpads or rabbit eyes with rAAV vectors and HSV-1 resulted in colocalization in nearly all of the HSV-1-positive neurons. These results suggest that rAAV is a useful tool for the study of HSV-1 infection and may provide a means to deliver therapeutic cargos for the treatment of HSV infections or of dysfunctions of sensory ganglia. IMPORTANCE: Adeno-associated virus (AAV) has been shown to transduce dorsal root ganglion sensory neurons following direct intraganglionic sciatic nerve injection and intraperitoneal and intravenous injection as well as intrathecal injection. We sought to determine if rAAV vectors would be delivered to the same sensory neurons that herpes simplex virus (HSV-1) infects when applied peripherally at an epithelial surface that had been treated to expose the underlying sensory nerve termini. For this study, we chose two well-established HSV-1 infection models: mouse footpad infection and rabbit ocular infection. The results presented here provide the first description of AAV vectors transducing neurons following delivery at the skin/epithelium/eye. The ability of AAV to cotransduce HSV-1-infected neurons in both the mouse and the rabbit provides an opportunity to experimentally explore and disrupt host and viral proteins that are integral to the establishment of HSV-1 latency, to the maintenance of latency, and to reactivation from latency in vivo.

A limited innate immune response is induced by a replication-defective herpes simplex virus vector following delivery to the murine central nervous system.

Herpes simplex virus type 1 (HSV-1)-based vectors readily transduce neurons and have a large payload capacity, making them particularly amenable to gene therapy applications within the central nervous system (CNS). Because aspects of the host responses to HSV-1 vectors in the CNS are largely unknown, we compared the host response of a nonreplicating HSV-1 vector to that of a replication-competent HSV-1 virus using microarray analysis. In parallel, HSV-1 gene expression was tracked using HSV-specific oligonucleotide-based arrays in order to correlate viral gene expression with observed changes in host response. Microarray analysis was performed following stereotactic injection into the right hippocampal formation of mice with either a replication-competent HSV-1 or a nonreplicating recombinant of HSV-1, lacking the ICP4 gene (ICP4-). Genes that demonstrated a significant change (P < .001) in expression in response to the replicating HSV-1 outnumbered those that changed in response to mock or nonreplicating vector by approximately 3-fold. Pathway analysis revealed that both the replicating and nonreplicating vectors induced robust antigen presentation but only mild interferon, chemokine, and cytokine signaling responses. The ICP4- vector was restricted in several of the Toll-like receptor-signaling pathways, indicating reduced stimulation of the innate immune response. These array analyses suggest that although the nonreplicating vector induces detectable activation of immune response pathways, the number and magnitude of the induced response is dramatically restricted compared to the replicating vector, and with the exception of antigen presentation, host gene expression induced by the nonreplicating vector largely resembles mock infection.