Beta human papillomavirus 8 E6 allows colocalization of non-homologous end joining and homologous recombination repair factors.

Beta human papillomavirus (ß-HPV) are hypothesized to make DNA damage more mutagenic and potentially more carcinogenic. Double strand breaks (DSBs) are the most deleterious DNA lesion. They are typically repaired by homologous recombination (HR) or non-homologous end joining (NHEJ). HR occurs after DNA replication while NHEJ can occur at any point in the cell cycle. HR and NHEJ are not thought to occur in the same cell at the same time. HR is restricted to cells in phases of the cell cycle where homologous templates are available, while NHEJ occurs primarily during G1. ß-HPV type 8 protein E6 (8E6) attenuates both repair pathways. We use a series of immunofluorescence microscopy and flow cytometry experiments to better define the impact of this attenuation. We found that 8E6 causes colocalization of HR factors (RPA70 and RAD51) with an NHEJ factor (activated DNA-PKcs or pDNA-PKcs) at persistent DSBs. 8E6 also causes RAD51 foci to form during G1. The initiation of NHEJ and HR at the same lesion could lead to antagonistic DNA end processing. Further, HR cannot be readily completed in an error-free manner during G1. Both aberrant repair events would cause deletions. To determine if these mutations were occurring, we used next generation sequencing of the 200kb surrounding a CAS9-induced DSB. 8E6 caused a 21-fold increase in deletions. Chemical and genetic inhibition of p300 as well as an 8E6 mutant that is incapable of destabilizing p300 demonstrates that 8E6 is acting via p300 destabilization. More specific chemical inhibitors of DNA repair provided mechanistic insight by mimicking 8E6-induced dysregulation of DNA repair in a virus-free system. Specifically, inhibition of NHEJ causes RAD51 foci to form in G1 and colocalization of RAD51 with pDNA-PKcs.

Beta Human Papillomavirus 8 E6 Induces Micronucleus Formation and Promotes Chromothripsis.

Cutaneous beta genus human papillomaviruses (ß-HPVs) are suspected to promote the development of nonmelanoma skin cancer (NMSC) by destabilizing the host genome. Multiple studies have established the genome destabilizing capacities of ß-HPV proteins E6 and E7 as a cofactor with UV. However, the E6 protein from ß-HPV8 (HPV8 E6) induces tumors in mice without UV exposure. Here, we examined a UV-independent mechanism of HPV8 E6-induced genome destabilization. We showed that HPV8 E6 reduced the abundance of anaphase bridge resolving helicase, Bloom syndrome protein (BLM). The diminished BLM was associated with increased segregation errors and micronuclei. These HPV8 E6-induced micronuclei had disordered micronuclear envelopes but retained replication and transcription competence. HPV8 E6 decreased antiproliferative responses to micronuclei and time-lapse imaging revealed HPV8 E6 promoted cells with micronuclei to complete mitosis. Finally, whole-genome sequencing revealed that HPV8 E6 induced chromothripsis in nine chromosomes. These data provide insight into mechanisms by which HPV8 E6 induces genome instability independent of UV exposure. IMPORTANCE Some beta genus human papillomaviruses (ß-HPVs) may promote skin carcinogenesis by inducing mutations in the host genome. Supporting this, the E6 protein from ß-HPV8 (8 E6) promotes skin cancer in mice with or without UV exposure. Many mechanisms by which 8 E6 increases mutations caused by UV have been elucidated, but less is known about how 8 E6 induces mutations without UV. We address that knowledge gap by showing that 8 E6 causes mutations stemming from mitotic errors. Specifically, 8 E6 reduces the abundance of BLM, a helicase that resolves and prevents anaphase bridges. This hinders anaphase bridge resolution and increases their frequency. 8 E6 makes the micronuclei that can result from anaphase bridges more common. These micronuclei often have disrupted envelopes yet retain localization of nuclear-trafficked proteins. 8 E6 promotes the growth of cells with micronuclei and causes chromothripsis, a mutagenic process where hundreds to thousands of mutations occur in a chromosome.

The potential of PCNA inhibition as a therapeutic strategy in cervical cancer.

Cervical cancers are the fourth most common and most deadly cancer in women worldwide. Despite being a tremendous public health burden, few novel approaches to improve care for these malignancies have been introduced. We discuss the potential for proliferating cell nuclear antigen (PCNA) inhibition to address this need as well as the advantages and disadvantages for compounds that can therapeutically inhibit PCNA with a specific focus on cervical cancer.

HPV 16 E7 alters translesion synthesis signaling.

A subset of human papillomaviruses (HPVs) are the cause of virtually every cervical cancer. These so-called "high-risk" HPVs encode two major oncogenes (HPV E6 and E7) that are necessary for transformation. Among "high-risk" HPVs, HPV16 causes most cervical cancers and is often used as a representative model for oncogenic HPVs. The HPV16 E7 oncogene facilitates the HPV16 lifecycle by binding and destabilizing RB, which ensures the virus has access to cellular replication machinery. RB destabilization increases E2F1-responsive gene expression and causes replication stress. While HPV16 E6 mitigates some of the deleterious effects associated with this replication stress by degrading p53, cells undergo separate adaptations to tolerate the stress. Here, we demonstrate that this includes the activation of the translesion synthesis (TLS) pathway, which prevents replication stress from causing replication fork collapse. We show that significantly elevated TLS gene expression is more common in cervical cancers than 15 out of the 16 the other cancer types that we analyzed. In addition to increased TLS protein abundance, HPV16 E7 expressing cells have a reduced ability to induct a critical TLS factor (POLη) in response to replication stress-inducing agents. Finally, we show that increased expression of at least one TLS gene is associated with improved survival for women with cervical cancer.

Predicting the Prognostic Value of POLI Expression in Different Cancers via a Machine Learning Approach.

Translesion synthesis (TLS) is a cell signaling pathway that facilitates the tolerance of replication stress. Increased TLS activity, the particularly elevated expression of TLS polymerases, has been linked to resistance to cancer chemotherapeutics and significantly altered patient outcomes. Building upon current knowledge, we found that the expression of one of these TLS polymerases (POLI) is associated with significant differences in cervical and pancreatic cancer survival. These data led us to hypothesize that POLI expression is associated with cancer survival more broadly. However, when cancers were grouped cancer type, POLI expression did not have a significant prognostic value. We presented a binary cancer random forest classifier using 396 genes that influence the prognostic characteristics of POLI in cervical and pancreatic cancer selected via graphical least absolute shrinkage and selection operator. The classifier was then used to cluster patients with bladder, breast, colorectal, head and neck, liver, lung, ovary, melanoma, stomach, and uterus cancer when high POLI expression was associated with worsened survival (Group I) or with improved survival (Group II). This approach allowed us to identify cancers where POLI expression is a significant prognostic factor for survival (p = 0.028 in Group I and p = 0.0059 in Group II). Multiple independent validation approaches, including the gene ontology enrichment analysis and visualization tool and network visualization support the classification scheme. The functions of the selected genes involving mitochondrial translational elongation, Wnt signaling pathway, and tumor necrosis factor-mediated signaling pathway support their association with TLS and replication stress. Our multidisciplinary approach provides a novel way of identifying tumors where increased TLS polymerase expression is associated with significant differences in cancer survival.

Beta Human Papillomavirus 8E6 Attenuates LATS Phosphorylation after Failed Cytokinesis.

Beta genus human papillomaviruses (ß-HPVs) cause cutaneous squamous cell carcinomas (cSCCs) in a subset of immunocompromised patients. However, ß-HPVs are not necessary for tumor maintenance in the general population. Instead, they may destabilize the genome in the early stages of cancer development. Supporting this idea, ß-HPV's 8E6 protein attenuates p53 accumulation after failed cytokinesis. This paper offers mechanistic insight into how ß-HPV E6 causes this change in cell signaling. An in silico screen and characterization of HCT 116 cells lacking p300 suggested that the histone acetyltransferase is a negative regulator of Hippo pathway (HP) gene expression. HP activation restricts growth in response to stimuli, including failed cytokinesis. Loss of p300 resulted in increased HP gene expression, including proproliferative genes associated with HP inactivation. ß-HPV 8E6 expression recapitulates some of these phenotypes. We used a chemical inhibitor of cytokinesis (dihydrocytochalasin B [H2CB]) to induce failed cytokinesis. This system allowed us to show that ß-HPV 8E6 reduced activation of large tumor suppressor kinase (LATS), an HP kinase. LATS is required for p53 accumulation following failed cytokinesis. These phenotypes were dependent on ß-HPV 8E6 destabilizing p300 and did not completely attenuate the HP. It did not alter H2CB-induced nuclear exclusion of the transcription factor YAP. ß-HPV 8E6 also did not decrease HP activation in cells grown to a high density. Although our group and others have previously described inhibition of DNA repair, to the best of our knowledge, this marks the first time that a ß-HPV E6 protein has been shown to hinder HP signaling.IMPORTANCE ß-HPVs contribute to cSCC development in immunocompromised populations. However, it is unclear if these common cutaneous viruses are tumorigenic in the general population. Thus, a more thorough investigation of ß-HPV biology is warranted. If ß-HPV infections do promote cSCCs, they are hypothesized to destabilize the cellular genome. In vitro data support this idea by demonstrating the ability of the ß-HPV E6 protein to disrupt DNA repair signaling events following UV exposure. We show that ß-HPV E6 more broadly impairs cellular signaling, indicating that the viral protein dysregulates the HP. The HP protects genome fidelity by regulating cell growth and apoptosis in response to a myriad of deleterious stimuli, including failed cytokinesis. After failed cytokinesis, ß-HPV 8E6 attenuates phosphorylation of the HP kinase (LATS). This decreases some, but not all, HP signaling events. Notably, ß-HPV 8E6 does not limit senescence associated with failed cytokinesis.

Humeral Head Ossification Growth Prediction: Can This Method Be Reproduced?

BACKGROUND: Radiographically assessing skeletal maturity is of paramount importance to guide appropriate management for patients with adolescent idiopathic scoliosis. The reported advantages of the Humeral Head Ossification System (HHOS) are an even distribution around peak growth age (PGA), the presence of an optimal view of the humeral head in most scoliosis radiographs, and an interobserver and intraobserver reliability of 0.95 and 0.96, respectively. The goal of this study was to determine whether similar reliability could be achieved at an institution other than the one at which the HHOS was originally described. METHODS: Six raters used the HHOS to stage the humeral head on 30 deidentified posteroanterior spine radiographs. This process was repeated 2 weeks later. All raters were provided with a document demonstrating the radiographic parameters of each stage, as well as annotations to aid with classification. Intraclass coefficients were calculated. A secondary analysis was then performed grouping stages 1-2 as "pre-PGA" and 3-5 as "post-PGA." RESULTS: Fair to moderate interobserver and intraobserver reliability was achieved when determining each individual HHOS stage. This improved to good to excellent when the humeral was classified as pre-PGA or post-PGA. Subanalysis did not show any correlation between intraobserver reliability and level of orthopaedic experience. CONCLUSIONS: We found fair to moderate interobserver and intraobserver reliability with the HHOS and this was independent of level of orthopaedic experience. This is much lower than reported by the authors who developed the system, and suggests that the HHOS might not be as easy to incorporate into a scoliosis management algorithm as previously noted. The improved reliability achieved when staging the humeral head as pre-PGA or post-PGA might be the true benefit of this system. LEVEL OF EVIDENCE: Level III-diagnostic.

Level of Experience Does Not Influence the Accuracy of Radiographic and Ultrasound Measurements of Magnetically Controlled Growing Rod Distractions.

BACKGROUND: Magnetically controlled growing rods (MCGR) have become a popular surgical option for the treatment of early-onset scoliosis. Both radiographs and ultrasound are currently used to measure the amount of length achieved when MCGRs are distracted. Previous studies have investigated the intraobserver and interobserver reliability of radiographic and ultrasound measurements of MCGR distraction. Some authors have reported that there is a "learning curve" in measuring MCGR lengthening with ultrasound, suggesting that new users require several months of experience before they can accurately perform the measurements. The goal of this study was to determine whether surgical experience of the rater is associated with the accuracy of radiographic and ultrasound measurements of MCGR distraction. METHODS: Six raters evaluated 29 deidentified radiographs and 30 ultrasound images from early-onset scoliosis patients with MCGR. Raters had varying levels of experience, ranging from a senior fellowship-trained pediatric orthopaedic surgeon to a junior orthopaedic surgery resident. Raters measured the amount of rod distraction in 2 sessions spaced 2 weeks apart. All raters were provided with a document demonstrating the radiographic and ultrasound measurement techniques before the first round of measurements. Intraclass correlation coefficients were calculated. RESULTS: Excellent intraobserver and interobserver agreement was achieved for both radiographic and ultrasound measurements of MCGR distraction. Subanalysis based on experience level showed that excellent intraobserver agreement was maintained with no evidence of decreased reliability in raters with less experience. CONCLUSIONS: Excellent intraobserver and interobserver agreement was obtained with radiographic and ultrasound measurements of MCGR distraction, regardless of the experience level of the rater. Posting a document with the radiographic and ultrasound measurement techniques in the orthopaedic surgery clinic, and perhaps also the radiology reading room may help avoid inaccurate measurements of distraction length secondary to a learning curve. LEVEL OF EVIDENCE: Level III-diagnostic.

High-Risk Alphapapillomavirus Oncogenes Impair the Homologous Recombination Pathway.

Persistent high-risk genus human Alphapapillomavirus (HPV) infections cause nearly every cervical carcinoma and a subset of tumors in the oropharyngeal tract. During the decades required for HPV-associated tumorigenesis, the cellular genome becomes significantly destabilized. Our analysis of cervical tumors from four separate data sets found a significant upregulation of the homologous-recombination (HR) pathway genes. The increased abundance of HR proteins can be replicated in primary cells by expression of the two HPV oncogenes (E6 and E7) required for HPV-associated transformation. HPV E6 and E7 also enhanced the ability of HR proteins to form repair foci, and yet both E6 and E7 reduce the ability of the HR pathway to complete double-strand break (DSB) repair by about 50%. The HPV oncogenes hinder HR by allowing the process to begin at points in the cell cycle when the lack of a sister chromatid to serve as a homologous template prevents completion of the repair. Further, HPV E6 attenuates repair by causing RAD51 to be mislocalized away from both transient and persistent DSBs, whereas HPV E7 is only capable of impairing RAD51 localization to transient lesions. Finally, we show that the inability to robustly repair DSBs causes some of these lesions to be more persistent, a phenotype that correlates with increased integration of episomal DNA. Together, these data support our hypothesis that HPV oncogenes contribute to the genomic instability observed in HPV-associated malignancies by attenuating the repair of damaged DNA.IMPORTANCE This study expands the understanding of HPV biology, establishing a direct role for both HPV E6 and E7 in the destabilization of the host genome by blocking the homologous repair of DSBs. To our knowledge, this is the first time that both viral oncogenes were shown to disrupt this DSB repair pathway. We show that HPV E6 and E7 allow HR to initiate at an inappropriate part of the cell cycle. The mislocalization of RAD51 away from DSBs in cells expressing HPV E6 and E7 hinders HR through a distinct mechanism. These observations have broad implications. The impairment of HR by HPV oncogenes may be targeted for treatment of HPV+ malignancies. Further, this attenuation of repair suggests HPV oncogenes may contribute to tumorigenesis by promoting the integration of the HPV genome, a common feature of HPV-transformed cells. Our data support this idea since HPV E6 stimulates the integration of episomes.

ß-HPV 5 and 8 E6 disrupt homology dependent double strand break repair by attenuating BRCA1 and BRCA2 expression and foci formation.

Recent work has explored a putative role for the E6 protein from some ß-human papillomavirus genus (ß-HPVs) in the development of non-melanoma skin cancers, specifically ß-HPV 5 and 8 E6. Because these viruses are not required for tumor maintenance, they are hypothesized to act as co-factors that enhance the mutagenic capacity of UV-exposure by disrupting the repair of the resulting DNA damage. Supporting this proposal, we have previously demonstrated that UV damage signaling is hindered by ß-HPV 5 and 8 E6 resulting in an increase in both thymine dimers and UV-induced double strand breaks (DSBs). Here we show that ß-HPV 5 and 8 E6 further disrupt the repair of these DSBs and provide a mechanism for this attenuation. By binding and destabilizing a histone acetyltransferase, p300, ß-HPV 5 and 8 E6 reduce the enrichment of the transcription factor at the promoter of two genes critical to the homology dependent repair of DSBs (BRCA1 and BRCA2). The resulting diminished BRCA1/2 transcription not only leads to lower protein levels but also curtails the ability of these proteins to form repair foci at DSBs. Using a GFP-based reporter, we confirm that this reduced foci formation leads to significantly diminished homology dependent repair of DSBs. By deleting the p300 binding domain of ß-HPV 8 E6, we demonstrate that the loss of robust repair is dependent on viral-mediated degradation of p300 and confirm this observation using a combination of p300 mutants that are ß-HPV 8 E6 destabilization resistant and p300 knock-out cells. In conclusion, this work establishes an expanded ability of ß-HPV 5 and 8 E6 to attenuate UV damage repair, thus adding further support to the hypothesis that ß-HPV infections play a role in skin cancer development by increasing the oncogenic potential of UV exposure.

Manipulation of cellular DNA damage repair machinery facilitates propagation of human papillomaviruses.

In general, the interplay among viruses and DNA damage repair (DDR) pathways can be divided based on whether the interaction promotes or inhibits the viral lifecycle. The propagation of human papillomaviruses is both promoted and inhibited by DDR proteins. As a result, HPV proteins both activate repair pathways, such as the ATM and ATR pathways, and inhibit other pathways, most notably the p53 signaling pathway. Indeed, the role of HPV proteins, with regard to the DDR pathways, can be divided into two broad categories. The first set of viral proteins, HPV E1 and E2 activate a DNA damage response and recruit repair proteins to viral replication centers, where these proteins are likely usurped to replicate the viral genome. Because the activation of the DDR response typically elicits a cell cycle arrest that would impeded the viral lifecycle, the second set of HPV proteins, HPV E6 and E7, prevents the DDR response from pausing cell cycle progression or inducing apoptosis. This review provides a detailed account of the interactions among HPV proteins and DDR proteins that facilitate HPV propagation.

Beta human papillomavirus E6 expression inhibits stabilization of p53 and increases tolerance of genomic instability.

UNLABELLED: Infections with the beta genus of human papillomaviruses (ß-HPVs) may contribute to the development of nonmelanoma skin cancers. However, ß-HPV genomes are found at too low a copy number in tumors for the virus to be necessary for tumor maintenance. Instead, they are hypothesized to destabilize the host genome by allowing the persistence of mutations that can drive tumorigenesis independently of the viral genome. Supporting this premise is our previous finding that the expression of some ß-HPV E6 proteins can attenuate p53 signaling in response to DNA damage. We show that ß-HPV E6 proteins can prevent the stabilization of p53 in response to two types of genome-destabilizing events, aberrant mitosis and dysregulated centrosome duplication. The inability to stabilize p53 in response to these stimuli allows cells expressing HPV5, HPV8, or HPV38 E6 to remain proliferatively active, leading to further genome deterioration in a proportion of these cells. These phenotypes are lost by the introduction of a mutation into the p300 binding domain of HPV8 E6 or by the transfection of mutated p300 that is resistant to the degradation mediated by HPV5 or HPV8 E6. These findings expand the understanding of the role played by p300 in promoting the faithful resolution of mitotic figures as well as proper centrosome duplication. Finally, we describe a phenomenon by which binucleated cells are resolved via cytokinesis into two cells, each with one nucleus. These data support the hypothesis that ß-HPV infections may promote tumorigenesis via genome destabilization. IMPORTANCE: The work described in this report provides support for the hypothesis that ß-HPV infections may contribute to nonmelanoma skin cancer by increasing the likelihood that tumorigenic mutations are introduced into the host cell's genome. We demonstrate that expression of the E6 proteins from some of these viruses increases the tolerance of two genome-destabilizing events, aberrant cell division and dysregulated centrosome duplication. Typically, these mutagenic occurrences elicit the stabilization of the tumor suppressor p53, which prevents further propagation of cells containing these errors. We show that the expression of ß-HPV E6 restricts this stabilization of p53, leading not only to continued cellular proliferation but also to further accumulation of similar mutagenic events. Finally, in addition to supporting a role for ß-HPV infections in certain skin cancers, we present studies with a mutated ß-HPV E6 protein suggesting that the histone acetyltransferase p300 plays a role in promoting genome stability during replication.

HPV 5 and 8 E6 abrogate ATR activity resulting in increased persistence of UVB induced DNA damage.

The role of the E6 oncoprotein from high-risk members of the α human papillomavirus genus in anogenital cancer has been well established. However, far less is known about the E6 protein from the ß human papillomavirus genus (ß-HPVs). Some ß-HPVs potentially play a role in non-melanoma skin cancer development, although they are not required for tumor maintenance. Instead, they may act as a co-factor that enhances the carcinogenic potential of UV damage. Indeed, the E6 protein from certain ß-HPVs (HPV 5 and 8) promotes the degradation of p300, a histone acetyl transferase involved in UV damage repair. Here, we show that the expression of HPV 5 and 8 E6 increases thymine dimer persistence as well as the likelihood of a UVB induced double strand break (DSB). Importantly, we provide a mechanism for the increased DNA damage by showing that both extended thymine dimer persistence as well as elevated DSB levels are dependent on the ability of HPV 8 E6 to promote p300 degradation. We further demonstrate that HPV 5 and 8 E6 expression reduces the mRNA and protein levels of ATR, a PI3 kinase family member that plays a key role in UV damage signaling, but that these levels remain unperturbed in cells expressing a mutated HPV 8 E6 incapable of promoting p300 degradation. We confirm that the degradation of p300 leads to a reduction in ATR protein levels, by showing that ATR levels rebound when a p300 mutant resistant to HPV 8 mediated degradation and HPV 8 E6 are co-transfected. Conversely, we show that ATR protein levels are reduced when p300 is targeted for degradation by siRNA. Moreover, we show the reduced ATR levels in HPV 5 and 8 E6 expressing cells results in delayed ATR activation and an attenuated ability of cells to phosphorylate, and as a result accumulate, p53 in response to UVB exposure, leading to significantly reduced cell cycle arrest. In conclusion, these data demonstrate that ß-HPV E6 expression can enhance the carcinogenic potential of UVB exposure by promoting p300 degradation, resulting in a reduction in ATR levels, which leads to increased thymine dimer persistence and increased UVB induced DSBs.

Blinatumomab consolidation post-autologous stem cell transplantation in patients with diffuse large B-cell lymphoma.

ABSTRACT: Outcomes in patients with relapsed diffuse large B-cell lymphoma (DLBCL) who undergo autologous stem cell transplant (auto-SCT) are poor. Blinatumomab is a CD3/CD19 bispecific T-cell engager that directs cytotoxic T cells to CD19+ cells. Here, we performed a pilot study of blinatumomab consolidation after auto-SCT for 14 patients with DLBCL or transformed follicular lymphoma. All patients underwent standard-of-care auto-SCT with carmustine, etoposide, cytarabine, and melphalan (BEAM) conditioning followed by 1 cycle (4 weeks continuous infusion) of blinatumomab consolidation starting at day 42 after auto-SCT. All 14 patients treated on study completed BEAM auto-SCT and 1 cycle of posttransplant blinatumomab. Five patients developed grade 1 cytokine release syndrome (CRS), with no grade 2 or higher CRS. Immune effector cell-associated neurotoxicity syndrome was not observed. Patients were followed up for 3 years after auto-SCT, with median follow-up of 37 (range, 12-65) months. One-hundred days after auto-SCT (1 month after blinatumomab consolidation), 12 patients (86%) had achieved complete remission. At 1 year after auto-SCT, 7 patients (50%) remained in CR, and 1 patient had died of progressive disease. Patients who relapsed had a lower CD8:CD4 T-cell ratio before starting blinatumomab than patients who remained in remission. This pilot study demonstrates blinatumomab consolidation after auto-SCT is safe and well tolerated. Strategies to increase the CD8:CD4 ratio and use additional cycles of consolidation in a larger randomized trial are needed to confirm the efficacy of consolidation with blinatumomab after auto-SCT. This trial was registered at www.clinicaltrials.gov as #NCT03072771.

Interactions among human papillomavirus proteins and host DNA repair factors differ during the viral life cycle and virus-induced tumorigenesis.

This review focuses on the impact of human papillomavirus (HPV) oncogenes on DNA repair pathways with a particular focus on how these relationships change as productive HPV infections transition to malignant lesions. We made specific efforts to incorporate advances in the understanding of HPV and DNA damage repair over the last 4 years. We apologize for any articles that we missed in compiling this report.

Inhibition of p300 increases cytotoxicity of cisplatin in pancreatic cancer cells.

Pancreatic cancer is a notoriously deadly disease with a five-year survival rate around 10 percent. Since early detection of these tumors is difficult, pancreatic cancers are often diagnosed at advanced stages. At this point, genotoxic chemotherapeutics can be used to manage tumor growth. However, side effects of these drugs are severe, limiting the amount of treatment that can be given and resulting in sub-optimal dosing. Thus, there is an urgent need to identify chemo-sensitizing agents that can lower the effective dose of genotoxic agents and as a result reduce the side effects. Here, we use transformed and non-transformed pancreatic cell lines to evaluate DNA repair inhibitors as chemo-sensitizing agents. We used a novel next generation sequencing approach to demonstrate that pancreatic cancer cells have a reduced ability to faithfully repair DNA damage. We then determine the extent that two DNA repair inhibitors (CCS1477, a small molecule inhibitor of p300, and ART558, a small molecule inhibitor of polymerase theta) can exploit this repair deficiency to make pancreatic cancer cells more sensitive to cisplatin, a commonly used genotoxic chemotherapeutic. Immunofluorescence microscopy and cell viability assays show that CCS1477 delayed repair and significantly sensitized pancreatic cancer cells to cisplatin. The increased toxicity was not seen in a non-transformed pancreatic cell line. We also found that while ART558 sensitizes pancreatic cancer cells to cisplatin, it also sensitized non-transformed pancreatic cancer cells.

Beta human papillomavirus 8E6 promotes alternative end joining.

Double strand breaks (DSBs) are one of the most lethal DNA lesions in cells. The E6 protein of beta-human papillomavirus (HPV8 E6) impairs two critical DSB repair pathways: homologous recombination (HR) and non-homologous end joining (NHEJ). However, HPV8 E6 only delays DSB repair. How DSBs are repaired in cells with HPV8 E6 remains to be studied. We hypothesize that HPV8 E6 promotes a less commonly used DSB repair pathway, alternative end joining (Alt-EJ). Using CAS9-based Alt-EJ reporters, we show that HPV8 E6 promotes Alt-EJ. Further, using small molecule inhibitors, CRISPR/CAS9 gene knockout, and HPV8 E6 mutant, we find that HPV8 E6 promotes Alt-EJ by binding p300, an acetyltransferase that facilitates DSB repair by HR and NHEJ. At least some of this repair occurs through a subset of Alt-EJ known as polymerase theta dependent end joining. Finally, whole genome sequencing analysis showed HPV8 E6 caused an increased frequency of deletions bearing the microhomology signatures of Alt-EJ. This study fills the knowledge gap of how DSB is repaired in cells with HPV8 E6 and the mutagenic consequences of HPV8 E6 mediated p300 destabilization. Broadly, this study supports the hypothesis that beta-HPV promotes cancer formation by increasing genomic instability.

Duvelisib for Critically Ill Patients With Coronavirus Disease 2019: An Investigator-Initiated, Randomized, Placebo-Controlled, Double-Blind Pilot Trial.

Background: Despite improvements in prevention and treatment, severe coronavirus disease 2019 (COVID-19) is associated with high mortality. Phosphoinositide 3-kinase (PI3K) pathways contribute to cytokine and cell-mediated lung inflammation. We conducted a randomized, placebo-controlled, double-blind pilot trial to determine the feasibility, safety, and preliminary activity of duvelisib, a PI3Kδγ inhibitor, for the treatment of COVID-19 critical illness. Methods: We enrolled adults aged ≥18 years with a primary diagnosis of COVID-19 with hypoxic respiratory failure, shock, and/or new cardiac disease, without improvement after at least 48 hours of corticosteroid. Participants received duvelisib (25 mg) or placebo for up to 10 days. Participants had daily semi-quantitative viral load measurements performed. Dose modifications were protocol driven due to adverse events (AEs) or logarithmic change in viral load. The primary endpoint was 28-day overall survival (OS). Secondary endpoints included hospital and intensive care unit length of stay, 60-day OS, and duration of critical care interventions. Safety endpoints included viral kinetics and AEs. Exploratory endpoints included serial cytokine measurements and cytometric analysis. Results: Fifteen patients were treated in the duvelisib cohort, and 13 in the placebo cohort. OS at 28 days was 67% (95% confidence interval [CI], 38%-88%) compared to 62% (95% CI, 32%-86%) for placebo (P = .544). Sixty-day OS was 60% versus 46%, respectively (hazard ratio, 0.66 [95% CI, .22-1.96]; P = .454). Other secondary outcomes were comparable. Duvelisib was associated with lower inflammatory cytokines. Conclusions: In this pilot study, duvelisib did not significantly improve 28-day OS compared to placebo for severe COVID-19. Duvelisib appeared safe in this critically ill population and was associated with reduction in cytokines implicated in COVID-19 and acute respiratory distress syndrome, supporting further investigation. Clinical Trials Registration: NCT04372602.

Beta HPV Deregulates Double-Strand Break Repair.

Beta human papillomavirus (beta HPV) infections are common in adults. Certain types of beta HPVs are associated with nonmelanoma skin cancer (NMSC) in immunocompromised individuals. However, whether beta HPV infections promote NMSC in the immunocompetent population is unclear. They have been hypothesized to increase genomic instability stemming from ultraviolet light exposure by disrupting DNA damage responses. Implicit in this hypothesis is that the virus encodes one or more proteins that impair DNA repair signaling. Fluorescence-based reporters, next-generation sequencing, and animal models have been used to test this primarily in cells expressing beta HPV E6/E7. Of the two, beta HPV E6 appears to have the greatest ability to increase UV mutagenesis, by attenuating two major double-strand break (DSB) repair pathways, homologous recombination, and non-homologous end-joining. Here, we review this dysregulation of DSB repair and emerging approaches that can be used to further these efforts.