The human papillomavirus (HPV) E7 oncoprotein is a primary driver of HPV-mediated carcinogenesis. The E7 proteins from diverse HPV bind to the host cellular non-receptor protein tyrosine phosphatase type 14 (PTPN14) and direct it for degradation through the activity of the E7-associated host E3 ubiquitin ligase UBR4. Herein we show that a highly conserved arginine residue in the C-terminal domain of diverse HPV E7 mediates interaction with PTPN14. We found that disruption of PTPN14 binding through mutation of the C-terminal arginine did not impact the ability of several high-risk HPV E7 proteins to bind and degrade the retinoblastoma tumor suppressor or activate E2F target gene expression. HPVs infect human keratinocytes and we previously reported that both PTPN14 degradation by HPV16 E7 and PTPN14 CRISPR knockout repress keratinocyte differentiation-related genes. Now we have found that blocking PTPN14 binding through mutation of the conserved C-terminal arginine rendered both HPV16 and HPV18 E7 unable to repress differentiation-related gene expression. We then confirmed that the HPV18 E7 variant that could not bind PTPN14 was also impaired in repressing differentiation when expressed from the complete HPV18 genome. Finally, we found that the ability of HPV18 E7 to extend the lifespan of primary human keratinocytes required PTPN14 binding. CRISPR/Cas9 knockout of PTPN14 rescued keratinocyte lifespan extension in the presence of the PTPN14 binding-deficient HPV18 E7 variant. These results support the model that PTPN14 degradation by high-risk HPV E7 leads to repression of differentiation and contributes to its carcinogenic activity. IMPORTANCE The E7 oncoprotein is a primary driver of HPV-mediated carcinogenesis. HPV E7 binds the putative tumor suppressor PTPN14 and targets it for degradation using the ubiquitin ligase UBR4. PTPN14 binds to a C-terminal arginine highly conserved in diverse HPV E7. Our previous efforts to understand how PTPN14 degradation contributes to the carcinogenic activity of high-risk HPV E7 used variants of E7 unable to bind to UBR4. Now, by directly manipulating E7 binding to PTPN14 and using a PTPN14 knockout rescue experiment we clearly demonstrate that the degradation of PTPN14 is required for high-risk HPV18 E7 to extend keratinocyte lifespan. Our data show that PTPN14 binding by HPV16 E7 and HPV18 E7 represses keratinocyte differentiation. HPV-positive cancers are frequently poorly differentiated and the HPV life cycle depends upon keratinocyte differentiation. The finding that PTPN14 binding by HPV E7 impairs differentiation has significant implications for HPV-mediated carcinogenesis and the HPV life cycle.

Epstein-Barr virus (EBV) causes lymphomas and epithelial cell cancers. Though generally silent in B lymphocytes, this widely prevalent virus can cause endemic Burkitt lymphoma and post-transplant lymphoproliferative disorders/lymphomas in immunocompromised hosts. By learning how EBV breaches barriers to cell proliferation, we hope to undermine those strategies to treat EBV lymphomas and potentially other cancers. We had previously found that EBV, through activation of cellular STAT3 prevents phosphorylation of Chk1, and thereby, suppresses activation of the intra-S phase cell-cycle checkpoint, a potent barrier to oncogene-driven proliferation. This observation prompted us to examine the consequences on DNA repair since homologous recombination repair, the most error-free form, requires phosphoChk1. We now report that the defect in Chk1 phosphorylation also curtails RAD51 nucleation, and thereby, homologous recombination repair of DNA double strand breaks. The resulting reliance on error-prone microhomology-mediated end-joining (MMEJ) repair makes EBV-transformed cells susceptible to PARP inhibition and simultaneous accrual of genome-wide deletions and insertions resulting from synthesis-dependent MMEJ. Analysis of transcriptomic and drug susceptibility data from hundreds of cancer lines reveals a STAT3-dependent gene-set predictive of susceptibility of cancers to synthetic lethal PARP inhibition. These findings i) demonstrate how the tumor virus EBV reshapes cellular DNA

Objectives: Acute Respiratory Infection (ARI) is the most common cause of childhood morbidity and mortality in developing countries, including Haiti. Our objective was to detect pathogens found in children with ARI in rural Haiti to help develop evidence-based guidelines for treatment and prevention. Methods: Retrospective study of students with ARI at four schools in rural Haiti. Viral and/or bacterial pathogens were identified by qPCR in 177 nasal swabs collected from April 2013 through November 2015.Results: Most common viruses detected were Rhinovirus (36%), Influenza A (16%) and Adenovirus (7%), and bacteria were Streptococcus pneumoniae (58%) and Staphylococcus aureus (28%). Compared to older children, children aged 3-5 years had more Influenza A (28% vs. 9%, p = 0.002) and Adenovirus detected (14% vs. 3%, p = 0.01). Similarly, S. pneumoniae was greatest in children 3-5 years old (71% 3-5yrs; 58% 6-15 years; 25% 16-20 years; p = 0.008). Children 3-10 years old presented with fever more than children 11-20 years old (22% vs 7%; p = 0.02) and were more often diagnosed with pneumonia (28% vs 4%, p < 0.001). Conclusions: Younger children had increased fever, pneumonia, and detection of Influenza A and S. pneumoniae. These data support the need for influenza and pneumococcus vaccination in early childhood in Haiti.

EEPD1 (endonuclease/exonuclease/phosphatase family domain-containing 1) is an uncharacterized human protein that we found to be transcriptionally increased upon induction of DNA double strand breaks (DSBs). A549 cells with EEPD1 repressed by siRNA arrested at the G1/S and G2/M transitions and had markedly increased sensitivity to various DSB-inducing agents, such as camptothecin, VP-16, hydroxyurea (HU), and ionizing irradiation (IR). There are two major types of DSB repair, non-homologous end-joining (NHEJ) and homologous recombination (HR), which is essential for replication fork repair. Using the EJ5 NHEJ reporter system, EEPD1 repression resulted in a 2-fold increase in NHEJ repair. However, formation of 53BP1 foci was unaffected in these cells, indicating that the initial step towards NHEJ was intact. However, in the HT256 HR reporter system, EEPD1 repression produced a 5-fold decrease in HR repair, indicating that EEPD1 mediates HR and inhibits NHEJ, placing it at the decision point for DSB repair. Immunofluorescence assays of HR components were performed in the presence of HU. EEPD1 repression significantly decreased γ-H2Ax, RAD51, and RPA32 foci formation, so placing EEPD1 above these HR components. The initial activity that commits a DSB to HR repair and away from NHEJ is 5′ end resection. This generates the 3′ ss DNA that results in ATR/Chk1 activation as well as RPA32 and γ-H2Ax foci. To assess this, a ss BRDU end resection assay was done after IR and there was a 4-fold decrease in 5′ end resection when EEPD1 was repressed. Consistently, there was also a decrease in ATR and Chk1 phosphorylation. Co-immunoprecipitation studies found that EEPD1 interacts with Ku80 and NBS1. In vitro assays using purified recombinant EEPD1 protein found its 5′ endonuclease activity was distinct from both Exo1 and DNA2, the two major mediators of 5′ end resection. This activity was, however, repressed by the Ku complex, indicating antagonism between EEPD1 and Ku. Cells with decreased EEPD1 expression showed severe nuclear anomalies, such as micronuclei, nucleoplasmic bridges that may have been caused by unopposed NHEJ when the functional HR was not available. Metaphase analysis in these cells also showed a significant increase in chromosomal aberrations, especially after IR and HU. These data suggest a novel model of collapsed replication fork repair pathway choice, where the free DS end binds the MRN complex, and NBS1 recruits EEPD1, inhibiting NHEJ and promoting HR via initiation of 5′ end resection. This is supported by the finding that EEPD1 is required for RPA32/ATRIP activation of ATR/Chk1, and all subsequent steps in repairing collapsed replication forks. Without such repair, the collapsed forks are end-joined to create chromosome fusions, which generate the nucleoplasmic bridging during mitosis. Lung and endometrial cancers have significant rates of EEPD1 mutation, further indicating that EEPD1 might be a novel tumor suppressor. Citation Format: Yuehan Wu, Suk-Hee Lee, Elizabeth A. Williamson, Brian L. Reinert, Gayathri Srinivasan, Sudha Singh, Aruna-Shanker Jaiswal, Silvia Tornaletti, Alexis C. Brantly, Robert A. Hromas. The 5′ endonuclease EEPD1 maintains genomic stability by mediating DNA repair pathway choice. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 2427. doi:10.1158/1538-7445.AM2014-2427