Repression of Transcription at DNA Breaks Requires Cohesin throughout Interphase and Prevents Genome Instability.

Cohesin subunits are frequently mutated in cancer, but how they function as tumor suppressors is unknown. Cohesin mediates sister chromatid cohesion, but this is not always perturbed in cancer cells. Here, we identify a previously unknown role for cohesin. We find that cohesin is required to repress transcription at DNA double-strand breaks (DSBs). Notably, cohesin represses transcription at DSBs throughout interphase, indicating that this is distinct from its known role in mediating DNA repair through sister chromatid cohesion. We identified a cancer-associated SA2 mutation that supports sister chromatid cohesion but is unable to repress transcription at DSBs. We further show that failure to repress transcription at DSBs leads to large-scale genome rearrangements. Cancer samples lacking SA2 display mutational patterns consistent with loss of this pathway. These findings uncover a new function for cohesin that provides insights into its frequent loss in cancer.

Epigenetic changes in histone acetylation underpin resistance to the topoisomerase I inhibitor irinotecan.

The topoisomerase I (TOP1) inhibitor irinotecan triggers cell death by trapping TOP1 on DNA, generating cytotoxic protein-linked DNA breaks (PDBs). Despite its wide application in a variety of solid tumors, the mechanisms of cancer cell resistance to irinotecan remains poorly understood. Here, we generated colorectal cancer (CRC) cell models for irinotecan resistance and report that resistance is neither due to downregulation of the main cellular target of irinotecan TOP1 nor upregulation of the key TOP1 PDB repair factor TDP1. Instead, the faster repair of PDBs underlies resistance, which is associated with perturbed histone H4K16 acetylation. Subsequent treatment of irinotecan-resistant, but not parental, CRC cells with histone deacetylase (HDAC) inhibitors can effectively overcome resistance. Immunohistochemical analyses of CRC tissues further corroborate the importance of histone H4K16 acetylation in CRC. Finally, the resistant clones exhibit cross-resistance with oxaliplatin but not with ionising radiation or 5-fluoruracil, suggesting that the latter two could be employed following loss of irinotecan response. These findings identify perturbed chromatin acetylation in irinotecan resistance and establish HDAC inhibitors as potential therapeutic means to overcome resistance.

Development of an oligonucleotide-based fluorescence assay for the identification of tyrosyl-DNA phosphodiesterase 1 (TDP1) inhibitors.

Topoisomerase 1 (TOP1) generates transient nicks in the DNA to relieve torsional stress encountered during the cellular processes of transcription, replication, and recombination. At the site of the nick there is a covalent linkage of TOP1 with DNA via a tyrosine residue. This reversible TOP1-cleavage complex intermediate can become trapped on DNA by TOP1 poisons such as camptothecin, or by collision with replication or transcription machinery, thereby causing protein-linked DNA single- or double-strand breaks and resulting in cell death. Tyrosyl-DNA phosphodiesterase 1 (TDP1) is a key enzyme involved in the repair of TOP1-associated DNA breaks via hydrolysis of 3'-phosphotyrosine bonds. Inhibition of TDP1 is therefore an attractive strategy for targeting cancer cells in conjunction with TOP1 poisons. Existing methods for monitoring the phosphodiesterase activity of TDP1 are generally gel based or of high cost. Here we report a novel, oligonucleotide-based fluorescence assay that is robust, sensitive, and suitable for high-throughput screening of both fragment and small compound libraries for the detection of TDP1 inhibitors. We further validated the assay using whole cell extracts, extending its potential application to determine of TDP1 activity in clinical samples from patients undergoing chemotherapy.

Ubiquitin ligase UBR3 regulates cellular levels of the essential DNA repair protein APE1 and is required for genome stability.

APE1 (Ref-1) is an essential human protein involved in DNA damage repair and regulation of transcription. Although the cellular functions and biochemical properties of APE1 are well characterized, the mechanism involved in regulation of the cellular levels of this important DNA repair/transcriptional regulation enzyme, remains poorly understood. Using an in vitro ubiquitylation assay, we have now purified the human E3 ubiquitin ligase UBR3 as a major activity that polyubiquitylates APE1 at multiple lysine residues clustered on the N-terminal tail. We further show that a knockout of the Ubr3 gene in mouse embryonic fibroblasts leads to an up-regulation of the cellular levels of APE1 protein and subsequent genomic instability. These data propose an important role for UBR3 in the control of the steady state levels of APE1 and consequently error free DNA repair.

PBRM1 Deficiency Confers Synthetic Lethality to DNA Repair Inhibitors in Cancer.

Inactivation of Polybromo 1 (PBRM1), a specific subunit of the PBAF chromatin remodeling complex, occurs frequently in cancer, including 40% of clear cell renal cell carcinomas (ccRCC). To identify novel therapeutic approaches to targeting PBRM1-defective cancers, we used a series of orthogonal functional genomic screens that identified PARP and ATR inhibitors as being synthetic lethal with PBRM1 deficiency. The PBRM1/PARP inhibitor synthetic lethality was recapitulated using several clinical PARP inhibitors in a series of in vitro model systems and in vivo in a xenograft model of ccRCC. In the absence of exogenous DNA damage, PBRM1-defective cells exhibited elevated levels of replication stress, micronuclei, and R-loops. PARP inhibitor exposure exacerbated these phenotypes. Quantitative mass spectrometry revealed that multiple R-loop processing factors were downregulated in PBRM1-defective tumor cells. Exogenous expression of the R-loop resolution enzyme RNase H1 reversed the sensitivity of PBRM1-deficient cells to PARP inhibitors, suggesting that excessive levels of R-loops could be a cause of this synthetic lethality. PARP and ATR inhibitors also induced cyclic GMP-AMP synthase/stimulator of interferon genes (cGAS/STING) innate immune signaling in PBRM1-defective tumor cells. Overall, these findings provide the preclinical basis for using PARP inhibitors in PBRM1-defective cancers. SIGNIFICANCE: This study demonstrates that PARP and ATR inhibitors are synthetic lethal with the loss of PBRM1, a PBAF-specific subunit, thus providing the rationale for assessing these inhibitors in patients with PBRM1-defective cancer. GRAPHICAL ABSTRACT: http://cancerres.aacrjournals.org/content/canres/81/11/2888/F1.large.jpg.

CHD7 and 53BP1 regulate distinct pathways for the re-ligation of DNA double-strand breaks.

Chromatin structure is dynamically reorganized at multiple levels in response to DNA double-strand breaks (DSBs). Yet, how the different steps of chromatin reorganization are coordinated in space and time to differentially regulate DNA repair pathways is insufficiently understood. Here, we identify the Chromodomain Helicase DNA Binding Protein 7 (CHD7), which is frequently mutated in CHARGE syndrome, as an integral component of the non-homologous end-joining (NHEJ) DSB repair pathway. Upon recruitment via PARP1-triggered chromatin remodeling, CHD7 stimulates further chromatin relaxation around DNA break sites and brings in HDAC1/2 for localized chromatin de-acetylation. This counteracts the CHD7-induced chromatin expansion, thereby ensuring temporally and spatially controlled 'chromatin breathing' upon DNA damage, which we demonstrate fosters efficient and accurate DSB repair by controlling Ku and LIG4/XRCC4 activities. Loss of CHD7-HDAC1/2-dependent cNHEJ reinforces 53BP1 assembly at the damaged chromatin and shifts DSB repair to mutagenic NHEJ, revealing a backup function of 53BP1 when cNHEJ fails.

The SWI/SNF chromatin remodelling complex: Its role in maintaining genome stability and preventing tumourigenesis.

Genes encoding subunits of the two SWI/SNF chromatin remodelling complexes (BAF and PBAF) are mutated in almost 20% of all human cancers. In addition to a role in regulating transcription, recent work from our laboratory and others identified roles for both complexes in DNA damage responses and the maintenance of sister chromatid cohesion, which may have profound impacts on genome stability and contribute to its role as a tumour suppressor. Here, we review some of the transcription-independent functions of the SWI/SNF chromatin remodelling complex and discuss these in light of their potential relevance to tumourigenesis.

Clinical and cellular roles for TDP1 and TOP1 in modulating colorectal cancer response to irinotecan.

Colorectal cancer is the third most common cancer in the world. Despite surgery, up to 50% of patients relapse with incurable disease. First-line chemotherapy uses the topoisomerase 1 (TOP1) poison irinotecan, which triggers cell death by trapping TOP1 on DNA. The removal of TOP1 peptide from TOP1-DNA breaks is conducted by tyrosyl-DNA phosphodiesterase 1 (TDP1). Despite putative roles for TDP1 and TOP1 in colorectal cancer, their role in cellular and clinical responses to TOP1-targeting therapies remains unclear. Here, we show varying expression levels of TOP1 and TDP1 polypeptides in multiple colorectal cancer cell lines and in clinical colorectal cancer samples. TDP1 overexpression or TOP1 depletion is protective. Conversely, TDP1 depletion increases DNA-strand breakage and hypersensitivity to irinotecan in a TOP1-dependent manner, presenting a potential therapeutic opportunity in colorectal cancer. TDP1 protein levels correlate well with mRNA and with TDP1 catalytic activity. However, no correlation is observed between inherent TDP1 or TOP1 levels alone and irinotecan sensitivity, pointing at their limited utility as predictive biomarkers in colorectal cancer. These findings establish TDP1 as a potential therapeutic target for the treatment of colorectal cancer and question the validity of TOP1 or TDP1 on their own as predictive biomarkers for irinotecan response.

TDP1/TOP1 Ratio as a Promising Indicator for the Response of Small Cell Lung Cancer to Topotecan.

BACKGROUND AND OBJECTIVE: Small cell lung cancer (SCLC) is one of the most challenging tumors to treat due to high proliferation rate, early metastatic dissemination and rapid development of chemotherapy resistance. The current treatment protocols involve the use of topoisomerase 1 (TOP1) poisons such as irinotecan and topotecan in combination with platinum-based compounds. TOP1 poisons kill cancer cells by trapping TOP1 on DNA, generating lethal DNA double-strand breaks. A potential mechanism employed by cancer cells to resist killing by TOP1 poisons is to overexpress enzymes involved in the repair of TOP1-DNA breaks. Tyrosyl DNA phosphodiesterase 1 (TDP1) is a key player in this process and despite its importance, no data is currently available to correlate TDP1 protein and mRNA levels with catalytic activity in SCLC. In addition, it is not known if TDP1 and TOP1 protein levels correlate with the cellular response of SCLC to TOP1 based therapies. METHODS AND RESULTS: We report a remarkable variation in TDP1 and TOP1 protein levels in a panel of SCLC cell lines. TDP1 protein level correlates well with TDP1 mRNA and TDP1 catalytic activity, as measured by two newly developed independent activity assays, suggesting the potential utility of immunohistochemistry in assessing TDP1 levels in SCLC tissues. We further demonstrate that whilst TDP1 protein level alone does not correlate with topotecan sensitivity, TDP1/TOP1 ratio correlates well with sensitivity in 8 out of 10 cell lines examined. CONCLUSION: This study provides the first cellular analyses of TDP1 and TOP1 in SCLC and suggests the potential utility of TDP1/TOP1 ratio to assess the response of SCLC to topotecan. The establishment and validation of an easy-to-use TDP1 enzymatic assay in cell extracts could be exploited as a diagnostic tool in the clinic. These findings may help in stratifying patients that are likely to benefit from TOP1 poisons and TDP1 inhibitors currently under development.