High Risk α-HPV E6 Impairs Translesion Synthesis by Blocking POLη Induction

High risk genus α human papillomaviruses (α-HPVs) express two versatile oncogenes (α-HPV E6 and E7) that cause cervical cancer (CaCx) by degrading tumor suppressor proteins (p53 and RB). α-HPV E7 also promotes replication stress and alters DNA damage responses (DDR). The translesion synthesis pathway (TLS) mitigates DNA damage by preventing replication stress from causing replication fork collapse. Computational analysis of gene expression in CaCx transcriptomic datasets identified a frequent increased expression of TLS genes. However, the essential TLS polymerases did not follow this pattern. These data were confirmed with in vitro and ex vivo systems. Further interrogation of TLS, using POLη as a representative TLS polymerase, demonstrated that α-HPV16 E6 blocks TLS polymerase induction by degrading p53. This doomed the pathway, leading to increased replication fork collapse and sensitivity to treatments that cause replication stress (e.g., UV and Cisplatin). This sensitivity could be overcome by the addition of exogenous POLη.

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The E6 Oncoproteins of High-Risk Papillomaviruses Bind to a Novel Putative GAP Protein, E6TP1, and Target It for Degradation

Molecular and Cellular Biology ◽

10.1128/mcb.19.1.733 ◽

1999 ◽

Vol 19 (1) ◽

pp. 733-744 ◽

Cited By ~ 138

Author(s):

Qingshen Gao ◽

Seetha Srinivasan ◽

Sarah N. Boyer ◽

David E. Wazer ◽

Vimla Band

Keyword(s):

High Risk ◽

P53 Protein ◽

Single Gene ◽

Human Papillomaviruses ◽

Dominant Negative ◽

Hpv16 E6 ◽

Hpv E6 ◽

E6 Oncoprotein ◽

E6 Protein

ABSTRACT The high-risk human papillomaviruses (HPVs) are associated with carcinomas of the cervix and other genital tumors. Previous studies have identified two viral oncoproteins, E6 and E7, which are expressed in the majority of HPV-associated carcinomas. The ability of high-risk HPV E6 protein to immortalize human mammary epithelial cells (MECs) has provided a single-gene model to study the mechanisms of E6-induced oncogenic transformation. In this system, the E6 protein targets the p53 tumor suppressor protein for degradation, and mutational analyses have shown that E6-induced degradation of p53 protein is required for MEC immortalization. However, the inability of most dominant-negative p53 mutants to induce efficient immortalization of MECs suggests the existence of additional targets of the HPV E6 oncoprotein. Using the yeast two-hybrid system, we have isolated a novel E6-binding protein. This polypeptide, designated E6TP1 (E6-targeted protein 1), exhibits high homology to GTPase-activating proteins for Rap, including SPA-1, tuberin, and Rap1GAP. The mRNA for E6TP1 is widely expressed in tissues and in vitro-cultured cell lines. The gene for E6TP1 localizes to chromosome 14q23.2-14q24.3 within a locus that has been shown to undergo loss of heterozygosity in malignant meningiomas. Importantly, E6TP1 is targeted for degradation by the high-risk but not the low-risk HPV E6 proteins both in vitro and in vivo. Furthermore, the immortalization-competent but not the immortalization-incompetent HPV16 E6 mutants target the E6TP1 protein for degradation. Our results identify a novel target for the E6 oncoprotein and provide a potential link between HPV E6 oncogenesis and alteration of a small G protein signaling pathway.

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R-Loops-Induced ATR Signaling As a Potential Therapeutic Target in Myelodysplastic Syndrome

Blood ◽

10.1182/blood-2019-126431 ◽

2019 ◽

Vol 134 (Supplement_1) ◽

pp. 4218-4218

Author(s):

Johann-Christoph Jann ◽

Johanna Flach ◽

Antje Knaflic ◽

Eva Altrock ◽

Nanni Schmitt ◽

...

Keyword(s):

Dna Damage ◽

Combination Therapy ◽

Research Funding ◽

Replication Fork ◽

Replication Stress ◽

Splicing Factor ◽

Cd34 Cells ◽

Pladienolide B ◽

Novel Therapeutic

Introduction: Somatic mutations in genes coding for splicing factors (e.g. SF3B1, U2AF1 and SRSF2) are found in about 50% of patients with Myelodysplastic Syndrome (MDS). These mutations have been shown to frequently occur early in the mutational hierarchy of the disease making them particularly attractive therapeutic targets. Recent research has revealed an association of splicing factor mutations (sfm) with elevated levels of DNA:RNA intermediates (R-loops), which induce replication stress and downstream activation of the ataxia telangiectasia and Rad3-related protein (ATR) pathway. The aim of this work was to investigate R-loops-associated ATR signaling as a novel therapeutic concept in primary CD34+ MDS patient cells carrying sfm, and to identify possible novel options for combination therapy. Methods: Using quantitative immunofluorescence microscopy we assessed levels of R-loops in primary CD34+ bone marrow cells isolated from MDS patients (n=23) with and without sfm. In addition, we evaluated the direct association of R-loops with induction of replication stress and activation of associated signaling by analyzing replication fork progression rates and phosphorylation of ATR target proteins. Furthermore, we determined the in vitro sensitivity of mutant (n=12) and non-mutant CD34+ (n=10) cells of MDS patients towards ATR inhibitors (VE-821 and AZD6738) alone and in combination with splicing modulator Pladienolide B and investigated the impact on DNA damage accumulation and apoptosis. We also performed these experiments in cord blood derived CD34+ cells overexpressing SRSF2P95H without other MDS-associated cellular alterations. Results: We found significantly elevated levels of R-loops in CD34+ cells from MDS patients carrying sfm compared to non-splicing factor mutated (non-sfm) MDS cells and healthy controls (mean MFI= 177 (sfm) vs. 78 (non-sfm) vs. 91 (healthy), p<0.0001). These were primarily found in the stem and progenitor as well as myeloid cell compartments, while lymphoid cells showed normal levels. Induced R-loops caused delayed replication fork dynamics as determined by fiber assay (mean fork speed= 0.282 kb/min (sfm) vs. 0.401 kb/min (non-sfm), p ≤0.0001), which in association with elevated levels of single-strand DNA marker replication protein A (RPA) strongly suggests the presence of replication stress in splicing factor mutated CD34+ cells. When exposed to ATR inhibitors in vitro, splicing factor mutant cells showed a significantly elevated sensitivity towards these drugs (normalized mean IC50= 0.89 µM (sfm) vs. 2.84 µM (non-sfm), p ≤0.001) associated with heightened levels of DNA damage and apoptosis, both of which were further increased by the addition of splicing modulator Pladienolide B. We also confirmed a direct correlation of R-loops associated ATR signaling with the presence of mutant SRSF2P95H by lentiviral overexpression in cord blood CD34+ cells. Conclusion: Overall, our results identify ATR as a promising novel therapeutic target in MDS with splicing factor mutations and provide a preclinical rationale for combination therapy with splicing modulator drugs. Disclosures Nolte: Novartis: Honoraria, Research Funding; Celgene: Honoraria, Research Funding.

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Inhibition of the translesion synthesis polymerase REV1 exploits replication gaps as a cancer vulnerability

Science Advances ◽

10.1126/sciadv.aaz7808 ◽

2020 ◽

Vol 6 (24) ◽

pp. eaaz7808 ◽

Cited By ~ 10

Author(s):

Sumeet Nayak ◽

Jennifer A. Calvo ◽

Ke Cong ◽

Min Peng ◽

Emily Berthiaume ◽

...

Keyword(s):

Electron Microscopy ◽

Dna Damage ◽

Stress Response ◽

Cancer Cell ◽

Small Molecule ◽

Replication Fork ◽

Translesion Synthesis ◽

Replication Stress ◽

Fiber Analysis ◽

Molecule Inhibitor

The replication stress response, which serves as an anticancer barrier, is activated not only by DNA damage and replication obstacles but also oncogenes, thus obscuring how cancer evolves. Here, we identify that oncogene expression, similar to other replication stress–inducing agents, induces single-stranded DNA (ssDNA) gaps that reduce cell fitness. DNA fiber analysis and electron microscopy reveal that activation of translesion synthesis (TLS) polymerases restricts replication fork slowing, reversal, and fork degradation without inducing replication gaps despite the continuation of replication during stress. Consistent with gap suppression (GS) being fundamental to cancer, we demonstrate that a small-molecule inhibitor targeting the TLS factor REV1 not only disrupts DNA replication and cancer cell fitness but also synergizes with gap-inducing therapies such as inhibitors of ATR or Wee1. Our work illuminates that GS during replication is critical for cancer cell fitness and therefore a targetable vulnerability.

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Transforming Properties of Beta-3 Human Papillomavirus E6 and E7 Proteins

mSphere ◽

10.1128/msphere.00398-20 ◽

2020 ◽

Vol 5 (4) ◽

Cited By ~ 1

Author(s):

Lucia Minoni ◽

Maria Carmen Romero-Medina ◽

Assunta Venuti ◽

Cécilia Sirand ◽

Alexis Robitaille ◽

...

Keyword(s):

High Risk ◽

Expression Profiles ◽

Expression Patterns ◽

Biological Properties ◽

Human Papillomaviruses ◽

Hpv E6 ◽

Hpv Types ◽

Beta 2 ◽

E6 And E7

ABSTRACT The beta human papillomaviruses (HPVs) are subdivided into 5 species (beta-1 to beta-5), and they were first identified in the skin. However, the beta-3 species appears to be more highly represented in the mucosal epithelia than in the skin. Functional studies have also highlighted that beta-3 HPV49 shares some functional similarities with mucosal high-risk (HR) HPV16. Here, we describe the characterization of the in vitro transforming properties of the entire beta-3 species, which includes three additional HPV types: HPV75, HPV76, and HPV115. HPV49, HPV75, and HPV76 E6 and E7 (E6/E7), but not HPV115 E6 and E7, efficiently inactivate the p53 and pRb pathways and immortalize or extend the life span of human foreskin keratinocytes (HFKs). As observed for HR HPV16, cell cycle deregulation mediated by beta-3 HPV E6/E7 expression leads to p16INK4a accumulation, whereas no p16INK4a was detected in beta-2 HPV38 E6/E7 HFKs. As shown for HPV49 E6, HPV75 and HPV76 E6s degrade p53 by an E6AP/proteasome-mediated mechanism. Comparative analysis of cellular gene expression patterns of HFKs containing E6 and E7 from HR HPV16, beta-3 HPV types, and beta-2 HPV38 further highlights the functional similarities of HR HPV16 and beta-3 HPV49, HPV75, and HPV76. The expression profiles of these four HPV HFKs show some similarities and diverge substantially from those of beta-3 HPV115 E6/E7 and beta-2 HPV38 E6/E7 HFKs. In summary, our data show that beta-3 HPV types share some mechanisms with HR HPV types and pave the way for additional studies aiming to evaluate their potential role in human pathologies. IMPORTANCE Human papillomaviruses are currently classified in different genera. Mucosal HPVs belonging to the alpha genus have been clearly associated with carcinogenesis of the mucosal epithelium at different sites. Beta HPV types have been classified as cutaneous. Although findings indicate that some beta HPVs from species 1 and 2 play a role, together with UV irradiation, in skin cancer, very little is known about the transforming properties of most of the beta HPVs. This report shows the transforming activity of E6 and E7 from beta-3 HPV types. Moreover, it highlights that beta-3 HPVs share some biological properties more extensively with mucosal high-risk HPV16 than with beta-2 HPV38. This report provides new paradigms for a better understanding of the biology of the different HPV types and their possible association with lesions at mucosal and/or cutaneous epithelia.

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Cloning and expression of the cDNA for E6-AP, a protein that mediates the interaction of the human papillomavirus E6 oncoprotein with p53.

Molecular and Cellular Biology ◽

10.1128/mcb.13.2.775 ◽

1993 ◽

Vol 13 (2) ◽

pp. 775-784 ◽

Cited By ~ 340

Author(s):

J M Huibregtse ◽

M Scheffner ◽

P M Howley

Keyword(s):

High Risk ◽

P53 Protein ◽

Growth Control ◽

Human Papillomaviruses ◽

Cloning And Expression ◽

Hpv16 E6 ◽

Reading Frame ◽

E6 Oncoprotein ◽

Protein Complex Formation

The E6 oncoproteins of the cancer-associated or high-risk human papillomaviruses (HPVs) target the cellular p53 protein. The association of E6 with p53 leads to the specific ubiquitination and degradation of p53 in vitro, suggesting a model by which E6 deregulates cell growth control by the elimination of the p53 tumor suppressor protein. Complex formation between E6 and p53 requires an additional cellular factor, designated E6-AP (E6-associated protein), which has a native and subunit molecular mass of approximately 100 kDa. Here we report the purification of E6-AP and the cloning of its corresponding cDNA, which contains a novel open reading frame encoding 865 amino acids. E6-AP, translated in vitro, has the following properties: (i) it associates with wild-type p53 in the presence of the HPV16 E6 protein and simultaneously stimulates the association of E6 with p53, (ii) it associates with the high-risk HPV16 and HPV18 E6 proteins in the absence of p53, and (iii) it induces the E6- and ubiquitin-dependent degradation of p53 in vitro.

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Modulation of microRNA-mRNA Target Pairs by Human Papillomavirus 16 Oncoproteins

mBio ◽

10.1128/mbio.02170-16 ◽

2017 ◽

Vol 8 (1) ◽

Cited By ~ 30

Author(s):

Mallory E. Harden ◽

Nripesh Prasad ◽

Anthony Griffiths ◽

Karl Munger

Keyword(s):

Gene Expression ◽

High Risk ◽

Human Papillomaviruses ◽

Cancer Development ◽

Cellular Gene ◽

Hpv16 E6 ◽

Hpv E6 ◽

E6 And E7 ◽

E7 Proteins ◽

Almost All

ABSTRACT The E6 and E7 proteins are the major oncogenic drivers encoded by high-risk human papillomaviruses (HPVs). While many aspects of the transforming activities of these proteins have been extensively studied, there are fewer studies that have investigated how HPV E6/E7 expression affects the expression of cellular noncoding RNAs. The goal of our study was to investigate HPV16 E6/E7 modulation of cellular microRNA (miR) levels and to determine the potential consequences for cellular gene expression. We performed deep sequencing of small and large cellular RNAs in primary undifferentiated cultures of human foreskin keratinocytes (HFKs) with stable expression of HPV16 E6/E7 or a control vector. After integration of the two data sets, we identified 51 differentially expressed cellular miRs associated with the modulation of 1,456 potential target mRNAs in HPV16 E6/E7-expressing HFKs. We discovered that the degree of differential miR expression in HFKs expressing HPV16 E6/E7 was not necessarily predictive of the number of corresponding mRNA targets or the potential impact on gene expression. Additional analyses of the identified miR-mRNA pairs suggest modulation of specific biological activities and biochemical pathways. Overall, our study supports the model that perturbation of cellular miR expression by HPV16 E6/E7 importantly contributes to the rewiring of cellular regulatory circuits by the high-risk HPV E6 and E7 proteins that contribute to oncogenic transformation. IMPORTANCE High-risk human papillomaviruses (HPVs) are the causative agents of almost all cervical cancers and many other cancers, including anal, vaginal, vulvar, penile, and oropharyngeal cancers. Despite the availability of efficacious HPV vaccines, it is critical to determine how HPVs cause cancer, as many people remain unvaccinated and the vaccine does not prevent cancer development in individuals who are already infected. Two HPV proteins, E6 and E7, are the major drivers of cancer development, and much remains to be learned about how the expression of these viral proteins reprograms infected cells, ultimately resulting in cancer development. Small, noncoding human RNAs, termed microRNAs (miRs), regulate gene expression and have been implicated in almost all human cancers, including HPV-associated cancers. Our study provides a comprehensive analysis of how E6 and E7 alter the expression of human miRs and how this potentially impacts cellular gene expression, which may contribute to cancer development.

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Cloning and expression of the cDNA for E6-AP, a protein that mediates the interaction of the human papillomavirus E6 oncoprotein with p53

Molecular and Cellular Biology ◽

10.1128/mcb.13.2.775-784.1993 ◽

1993 ◽

Vol 13 (2) ◽

pp. 775-784 ◽

Cited By ~ 1

Author(s):

J M Huibregtse ◽

M Scheffner ◽

P M Howley

Keyword(s):

High Risk ◽

P53 Protein ◽

Growth Control ◽

Human Papillomaviruses ◽

Cloning And Expression ◽

Hpv16 E6 ◽

Reading Frame ◽

E6 Oncoprotein ◽

Protein Complex Formation

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CX-5461 Enhances the Efficacy of APR-246 via Induction of DNA Damage and Replication Stress in Triple-Negative Breast Cancer

International Journal of Molecular Sciences ◽

10.3390/ijms22115782 ◽

2021 ◽

Vol 22 (11) ◽

pp. 5782

Author(s):

Ashwini Makhale ◽

Devathri Nanayakkara ◽

Prahlad Raninga ◽

Kum Kum Khanna ◽

Murugan Kalimutho

Keyword(s):

Breast Cancer ◽

Dna Damage ◽

Triple Negative Breast Cancer ◽

Combination Treatment ◽

Triple Negative ◽

Annexin V ◽

Replication Stress ◽

Breast Cancer Cell Lines ◽

Combination Strategy

Triple-negative breast cancer (TNBC) is an aggressive subtype of breast cancer lacking targeted therapy. Here, we evaluated the anti-cancer activity of APR-246, a P53 activator, and CX-5461, a RNA polymerase I inhibitor, in the treatment of TNBC cells. We tested the efficacy of individual and combination therapy of CX-5461 and APR-246 in vitro, using a panel of breast cancer cell lines. Using publicly available breast cancer datasets, we found that components of RNA Pol I are predominately upregulated in basal-like breast cancer, compared to other subtypes, and this upregulation is associated with poor overall and relapse-free survival. Notably, we found that the treatment of breast cancer cells lines with CX-5461 significantly hampered cell proliferation and synergistically enhanced the efficacy of APR-246. The combination treatment significantly induced apoptosis that is associated with cleaved PARP and Caspase 3 along with Annexin V positivity. Likewise, we also found that combination treatment significantly induced DNA damage and replication stress in these cells. Our data provide a novel combination strategy by utilizing APR-246 in combination CX-5461 in killing TNBC cells that can be further developed into more effective therapy in TNBC therapeutic armamentarium.

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