Author Manuscript Published OnlineFirst on May 11, 2016; DOI: 10.1158/1078-0432.CCR-15-1050 Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. Molecular Pathways: Targeting DNA Repair Pathway Defects Enriched in Metastasis Niall M. Corcoran, Michael J. Clarkson, Ryan Stuchbery and Christopher M. Hovens Department of Surgery, Division of Urology, Royal Melbourne Hospital and University of Melbourne, Parkville 3050, and The Epworth Prostate Centre, Epworth Hospital, Richmond 3121, Victoria, Australia. Corresponding Author: Christopher M. Hovens, 5th Floor Clinical Sciences Building, Royal Melbourne Hospital, University of Melbourne, Parkville, 3050, VIC Australia. Phone: +613 93427705; Fax: +613 93466488; E-mail: [email protected] Running Title: DNA Repair in Metastasis Disclosure of Potential Conflicts of Interest No potential conflicts of interest were disclosed. 1 Downloaded from clincancerres.aacrjournals.org on September 28, 2021. © 2016 American Association for Cancer Research. Author Manuscript Published OnlineFirst on May 11, 2016; DOI: 10.1158/1078-0432.CCR-15-1050 Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. Abstract The maintenance of a pristine genome, free from errors, is necessary to prevent cellular transformation and degeneration. When errors in DNA are detected, DNA damage response (DDR) genes and their regulators are activated to effect repair. When these DDR pathways are themselves mutated or aberrantly downregulated, cancer and neurodegenerative disorders can ensue. Multiple lines of evidence now indicate however that defects in key regulators of DNA repair pathways are highly enriched in human metastasis specimens and hence may be a key step in the acquisition of metastasis and the ability of localized disease to disseminate. Some of the key regulators of checkpoints in the DNA damage response are the TP53 protein and the PARP enzyme family and targeting of these pathways, especially through PARP inhibition, are now being exploited therapeutically to effect significant clinical responses in subsets of individuals particularly in ovarian and prostate cancer, including those with a marked metastatic burden. Targeting DNA repair deficient tumors with drugs that take advantage of the fundamental differences between normal repair proficient cells and repair deficient tumors offers new avenues for treating advanced disease in the future. 2 Downloaded from clincancerres.aacrjournals.org on September 28, 2021. © 2016 American Association for Cancer Research. Author Manuscript Published OnlineFirst on May 11, 2016; DOI: 10.1158/1078-0432.CCR-15-1050 Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. Background The DNA Damage Response Pathway One of the hallmarks of the human cancer genome is the prevalence of apparently random aberrations in the normal order of genetic information on the chromosomes. This genomic instability is directly linked to the acquired loss of function in any one of six DNA damage repair (DDR) pathways (Fig. 1), including those of mismatch repair (MMR) (1, 2), homologous recombination repair (HMR) (3, 4), non-homologous end joining (NHEJ) (5-7), translesion DNA synthesis (TLS)(8), base excision repair (BER) (9) or nucleotide excision repair (NER) pathways (10-12). The molecular machinery governing these processes is both detailed and complex and beyond the scope of this review. We refer the reader wishing a more thorough exposition of the biochemistry of DNA repair to a number of excellent recent reviews (13-16). Instead we will focus on those DNA repair mechanisms and key proteins that have been specifically linked to advanced disease and metastasis, such as DNA damage checkpoint control and TP53, as well as other repair proteins such as PARP that are exploitable therapeutically. We will then explore the potential for clinically targeting these pathways, as well as their effect on cellular function (in particular the generation of neo-antigens) to impede disease progression and improve patient survival. DNA Damage Checkpoints and TP53 Once DNA damage has occurred it is vital that the cell does not proceed through the S- phase of the cell cycle and permanently ‘fix’ damage in daughter cells. To prevent this, once DNA damage has been detected, cell cycle checkpoints are activated, orchestrated by two master regulators, ATM and ATR, kinases that phosphorylate key checkpoint effector proteins leading to cell cycle arrest. Loss of function or imbalance in either ATM or ATR as well as in some key mediators such as BRCA1, has been linked to aggressive and metastatic tumors in preclinical models of breast cancer and in clinical prostate cancer (17-19), underlying the importance of cell cycle checkpoint pathways in the spread of tumor cells. One important downstream target of ATM/ATR is TP53, the key effector of apoptosis and senescence following DNA damage. Loss of TP53 function renders cells unable to induce apoptosis or senescence programs in response to DNA damage and therefore primes these 3 Downloaded from clincancerres.aacrjournals.org on September 28, 2021. © 2016 American Association for Cancer Research. Author Manuscript Published OnlineFirst on May 11, 2016; DOI: 10.1158/1078-0432.CCR-15-1050 Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. cells for transformation. It is not surprising then that mutation in TP53 is the most commonly occurring mutation in human cancers with over 50% of all tumors having aberrations in the TP53 gene. However it has become increasingly apparent that distinct defects in TP53 produce differing cellular effects. For instance, the metastatic potential of tumors is associated with missense mutations in the DNA binding domain, which are predicted to result in ‘gain of function’, as opposed to the more frequently observed loss of function mutations that lead to a hypofunction or absence of TP53 in cells. For instance, whilst TP53 null mice do form tumors they rarely metastasize or display an invasive phenotype (20, 21). Conversely, mice expressing missense mutations in the ‘hotspot’ DNA binding domain of the TP53 protein, display a markedly higher incidence of metastatic carcinomas and osteosarcomas (22, 23). The presence of TP53 mutations confers a poor prognosis for breast cancer patients, a surrogate for metastasis formation (24). Recent work also suggests that the late acquisition of missense TP53 mutations in subclonal populations of tumor cells is also the driver of metastatic expansion in clinical prostate cancer (25). Regulating DNA Repair Pathways: The PARP Family The PARP family of post-translational modifying enzymes regulate protein function and co- factor binding by catalyzing the covalent attachment of one or more ADP-ribose units to client substrates (26). Three family members (PARP 1-3) play a critical role in DNA repair. Following single and double-strand breaks (DSBs), PARP proteins bind to damaged DNA and promote ADP-ribosylation of both themselves and a number of other chromatin proteins, thereby activating either base excision repair or homologous recombination repair (27). In addition, poly ADP-ribosylated PARP blocks access to free DNA ends by proteins involved in the error prone non-homologous end-joining repair of DSBs. The PARP proteins are also involved in the repair of single strand breaks; however if PARP function is impeded then persistent single strand breaks can lead to the formation of replication cycle-induced double-strand breaks (14). Such inhibition in the presence of a pre-exiting DNA repair defect can result in catastrophic genomic instability and cellular demise and is discussed further below. 4 Downloaded from clincancerres.aacrjournals.org on September 28, 2021. © 2016 American Association for Cancer Research. Author Manuscript Published OnlineFirst on May 11, 2016; DOI: 10.1158/1078-0432.CCR-15-1050 Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. DNA Repair Pathway Aberrations in Human Metastases Whilst a number of individual studies have reported the prevalence of DNA repair pathway aberrations in metastatic samples from individual datasets (25, 28-30), to date no systematic analysis across large combined metastatic datasets have been reported. To address this issue we screened DNA copy number and mutation data (31) across 6 different human metastasis datasets comprising in total 317 metastatic samples across 3 different tumor types, namely, melanoma(32), colorectal (33) and prostate cancer(25, 28-30). We used a comprehensive list of 180 DNA repair pathway genes across 18 different categories (34) which are either directly involved or act as modifiers of DNA repair protein function. In a very high proportion of the metastatic specimens, at least one alteration in a DNA repair gene was identified (Fig. 2). Across the tumor types, 70% of prostate cancer metastases exhibited either a deletion, amplification or point mutation in a DNA repair pathway gene, and this proportion was even higher for the melanoma (75%) and colorectal (82%) metastases (Fig. 2). There were direct parallels in the spectrum of DNA repair pathway genes undergoing point mutation in prostate cancer with those seen in the colorectal and melanoma datasets. Across the entire metastatic datasets, mutations in the TP53 gene dominated, with 65% of the metastases harbouring point defects