Contribution of the DNA binding domain of p53 to regulation of its stability

Tumour suppressor p53 is frequently mutated in cancers. While wild type p53 is normally a rapidly degraded protein, mutant forms of p53 are stabilised and accumulate to high levels in tumour cells. Several studies have shown that mutant p53 acquires oncogenic properties and actively contributes to tumourigenesis. It is therefore important to understand how the stability of mutant p53 is regulated. This thesis shows that mutant and wild type p53 are ubiquitinated and degraded through overlapping but distinct pathways. While Mdm2 can drive the degradation of both mutant and wild type p53, this study suggests that the ability of Mdm2 to function as a ubiquitin ligase is less important in the degradation of mutant p53, which is heavily ubiquitinated in an Mdm2-independent manner. The contribution of Mdm2 to the degradation of mutant p53 may reflect an ability of Mdm2 to deliver the ubiquitinated mutant p53 to the proteasome. Ubiquitination does not efficiently target mutant p53 for the proteasomal degradation, however ubiquitinated p53 mutants localize to the cytoplasm. This thesis suggests the role for the chaperone-associated ubiquitin ligase CHIP in ubiquitination of mutant p53, although other unidentified ubiquitin ligases appear to contribute. Interaction of mutant p53 with its family member p73 decreases ubiquitination, suggesting p73 can play a role in regulation of stability of mutant p53

Parallel CRISPR-Cas9 screens clarify impacts of p53 on screen performance

CRISPR-Cas9 genome engineering has revolutionised high-throughput functional genomic screens. However, recent work has raised concerns regarding the performance of CRISPR-Cas9 screens using TP53 wild-type human cells due to a p53-mediated DNA damage response (DDR) limiting the efficiency of generating viable edited cells. To directly assess the impact of cellular p53 status on CRISPR-Cas9 screen performance, we carried out parallel CRISPR-Cas9 screens in wild-type and TP53 knockout human retinal pigment epithelial cells using a focused dual guide RNA library targeting 852 DDR-associated genes. Our work demonstrates that although functional p53 status negatively affects identification of significantly depleted genes, optimal screen design can nevertheless enable robust screen performance. Through analysis of our own and published screen data, we highlight key factors for successful screens in both wild-type and p53-deficient cells

Neddylation promotes ubiquitylation and release of Ku from DNA-damage sites.

The activities of many DNA-repair proteins are controlled through reversible covalent modification by ubiquitin and ubiquitin-like molecules. Nonhomologous end-joining (NHEJ) is the predominant DNA double-strand break (DSB) repair pathway in mammalian cells and is initiated by DSB ends being recognized by the Ku70/Ku80 (Ku) heterodimer. By using MLN4924, an anti-cancer drug in clinical trials that specifically inhibits conjugation of the ubiquitin-like protein, NEDD8, to target proteins, we demonstrate that NEDD8 accumulation at DNA-damage sites is a highly dynamic process. In addition, we show that depleting cells of the NEDD8 E2-conjugating enzyme, UBE2M, yields ionizing radiation hypersensitivity and reduced cell survival following NHEJ. Finally, we demonstrate that neddylation promotes Ku ubiquitylation after DNA damage and release of Ku and Ku-associated proteins from damage sites following repair. These studies provide insights into how the NHEJ core complex dissociates from repair sites and highlight its importance for cell survival following DSB induction.We thank Thimo Kurz (University of Dundee, UK) for providing MLN4924 and Kate Dry, Rimma Berlotserkovskaya (S.P.J.’s laboratory), and Eric Lightcap (Takeda Pharmaceuticals) for critical reading of the manuscript. We thank Sylvie Urbe and Michael Clague (University of Liverpool, UK) for providing the GFP-CSN5 plasmid, the Division of Signal Transduction Therapy (University of Dundee, UK) for providing UBE2M and UBE2F plasmids, Matthew Petroski (Sanford-Burnham Medical Research Institute, US) for providing FLAG-UBA3 wild-type (WT) and FLAG-UBA3-A171T constructs, and Nico Dantuma (Karolinska Institute, Sweden) and Changshun Shao (Rutgers University) for providing CUL4A and CUL4B plasmids, respectively. We also thank Nicola Lawrence, Alex Sossick, and Richard Butler (Gurdon Institute, Cambridge, UK) for help with microscopy, Volocity, and Fiji. Research in the S.P.J.’s laboratory is funded by Cancer Research UK programme grant C6/A11224, the European Research Council, and the European Community Seventh Framework Programme grant agreement no. HEALTH-F2-2010-259893 (DDResponse). Core funding is provided by CRUK (C6946/A14492) and the Wellcome Trust (WT092096). S.P.J. receives his salary from the University of Cambridge, UK, supplemented by CRUK. N.L. is funded by CRUK programme grant C6/A11224, J.S.B. is funded by a Wellcome Trust Clinical Fellowship (WT083416), and Y.G. and M.S.-C. are funded by European Research Council grant DDREAM. S.B. was funded by an EMBO long-term fellowship ALTF 93-2010, Cancer Research UK, and a post-doctoral grant from Ligue Nationale Contre le Cancer. P.B. is supported by the Emmy Noether Programme of the German Research Foundation (DFG, BE 5342/1-1).This is the final published version. It first appeared at http://www.sciencedirect.com/science/article/pii/S2211124715003496

Impact of DNA damage repair alterations on prostate cancer progression and metastasis

Prostate cancer is among the most common diseases worldwide. Despite recent progress with treatments, patients with advanced prostate cancer have poor outcomes and there is a high unmet need in this population. Understanding molecular determinants underlying prostate cancer and the aggressive phenotype of disease can help with design of better clinical trials and improve treatments for these patients. One of the pathways often altered in advanced prostate cancer is DNA damage response (DDR), including alterations in BRCA1/2 and other homologous recombination repair (HRR) genes. Alterations in the DDR pathway are particularly prevalent in metastatic prostate cancer. In this review, we summarise the prevalence of DDR alterations in primary and advanced prostate cancer and discuss the impact of alterations in the DDR pathway on aggressive disease phenotype, prognosis and the association of germline pathogenic1 alterations in DDR genes with risk of developing prostate cancer

Ubiquitination and Degradation of Mutant p53▿

While wild-type p53 is normally a rapidly degraded protein, mutant forms of p53 are stabilized and accumulate to high levels in tumor cells. In this study, we show that mutant and wild-type p53 proteins are ubiquitinated and degraded through overlapping but distinct pathways. While Mdm2 can drive the degradation of both mutant and wild-type p53, our data suggest that the ability of Mdm2 to function as a ubiquitin ligase is less important in the degradation of mutant p53, which is heavily ubiquitinated in an Mdm2-independent manner. Our initial attempts to identify ubiquitin ligases that are responsible for the ubiquitination of mutant p53 have suggested a role for the chaperone-associated ubiquitin ligase CHIP (C terminus of Hsc70-interacting protein), although other unidentified ubiquitin ligases also appear to contribute. The contribution of Mdm2 to the degradation of mutant p53 may reflect the ability of Mdm2 to deliver the ubiquitinated mutant p53 to the proteasome

Preventing and Overcoming Resistance to PARP Inhibitors: A Focus on the Clinical Landscape

Poly(ADP-ribose) polymerase (PARP) inhibitors (PARPi) are now a first-line maintenance treatment in ovarian cancer and have been approved in other cancer types, including breast, pancreatic and prostate. Despite their efficacy, and as is the case for other targeted therapies, resistance to PARPi has been reported clinically and is generating a growing patient population of unmet clinical need. Here, we discuss the mechanisms of resistance that have been described in pre-clinical models and focus on those that have been already identified in the clinic, highlighting the key challenges to fully characterise the clinical landscape of PARPi resistance and proposing ways of preventing and overcoming it

Parallel CRISPR-Cas9 screens clarify impacts of p53 on screen performance

CRISPR-Cas9 genome engineering has revolutionised high-throughput functional genomic screens. However, recent work has raised concerns regarding the performance of CRISPR-Cas9 screens using TP53 wild-type human cells due to a p53-mediated DNA damage response (DDR) limiting the efficiency of generating viable edited cells. To directly assess the impact of cellular p53 status on CRISPR-Cas9 screen performance, we carried out parallel CRISPR-Cas9 screens in wild-type and TP53 knockout human retinal pigment epithelial cells using a focused dual guide RNA library targeting 852 DDR-associated genes. Our work demonstrates that although functional p53 status negatively affects identification of significantly depleted genes, optimal screen design can nevertheless enable robust screen performance. Through analysis of our own and published screen data, we highlight key factors for successful screens in both wild-type and p53-deficient cells

Proteomic investigations reveal a role for RNA processing factor THRAP3 in the DNA damage response

The regulatory networks of the DNA damage response (DDR) encompass many proteins and posttranslational modifications. Here, we use mass spectrometry-based proteomics to analyze the systems-wide response to DNA damage by parallel quantification of the DDR-regulated phosphoproteome, acetylome and proteome. We show that phosphorylation-dependent signaling networks are regulated more strongly compared to acetylation. Among the phosphorylated proteins identified are many putative substrates of DNA-PK, ATM and ATR kinases, but a majority of phosphorylated proteins do not share the ATM/ATR/DNA-PK target consensus, suggesting an important role of downstream kinases in amplifying DDR signals. We show that the splicing-regulator phosphatase PPM1G is recruited to sites of DNA damage, while the splicing-associated protein THRAP3 is excluded from these regions. Moreover, THRAP3 depletion causes cellular hypersensitivity to DNA damaging agents, thus suggesting an important link between RNA metabolism and DNA repair. Our results broaden the knowledge of DNA damage signaling networks and identify novel components of the DDR