CDK-independent role of D-type cyclins in regulating DNA mismatch repair.

Although mismatch repair (MMR) is essential for correcting DNA replication errors, it can also recognize other lesions, such as oxidized bases. In G0 and G1, MMR is kept in check through unknown mechanisms as it is error-prone during these cell cycle phases. We show that in mammalian cells, D-type cyclins are recruited to sites of oxidative DNA damage in a PCNA- and p21-dependent manner. D-type cyclins inhibit the proteasomal degradation of p21, which competes with MMR proteins for binding to PCNA, thereby inhibiting MMR. The ability of D-type cyclins to limit MMR is CDK4- and CDK6-independent and is conserved in G0 and G1. At the G1/S transition, the timely, cullin-RING ubiquitin ligase (CRL)-dependent degradation of D-type cyclins and p21 enables MMR activity to efficiently repair DNA replication errors. Persistent expression of D-type cyclins during S-phase inhibits the binding of MMR proteins to PCNA, increases the mutational burden, and promotes microsatellite instability.

D-type cyclins regulate DNA mismatch repair in the G1 and S phases of the cell cycle, maintaining genome stability.

The large majority of oxidative DNA lesions occurring in the G1 phase of the cell cycle are repaired by base excision repair (BER) rather than mismatch repair (MMR) to avoid long resections that can lead to genomic instability and cell death. However, the molecular mechanisms dictating pathway choice between MMR and BER have remained unknown. Here, we show that, during G1, D-type cyclins are recruited to sites of oxidative DNA damage in a PCNA- and p21-dependent manner. D-type cyclins shield p21 from its two ubiquitin ligases CRL1SKP2 and CRL4CDT2 in a CDK4/6-independent manner. In turn, p21 competes through its PCNA-interacting protein degron with MMR components for their binding to PCNA. This inhibits MMR while not affecting BER. At the G1/S transition, the CRL4AMBRA1-dependent degradation of D-type cyclins renders p21 susceptible to proteolysis. These timely degradation events allow the proper binding of MMR proteins to PCNA, enabling the repair of DNA replication errors. Persistent expression of cyclin D1 during S-phase increases the mutational burden and promotes microsatellite instability. Thus, the expression of D-type cyclins inhibits MMR in G1, whereas their degradation is necessary for proper MMR function in S.

REV7 Monomer Is Unable to Participate in Double Strand Break Repair and Translesion Synthesis but Suppresses Mitotic Errors.

Rev7 is a regulatory protein with roles in translesion synthesis (TLS), double strand break (DSB) repair, replication fork protection, and cell cycle regulation. Rev7 forms a homodimer in vitro using its HORMA (Hop, Rev7, Mad2) domain; however, the functional importance of Rev7 dimerization has been incompletely understood. We analyzed the functional properties of cells expressing either wild-type mouse Rev7 or Rev7K44A/R124A/A135D, a mutant that cannot dimerize. The expression of wild-type Rev7, but not the mutant, rescued the sensitivity of Rev7-/- cells to X-rays and several alkylating agents and reversed the olaparib resistance phenotype of Rev7-/- cells. Using a novel fluorescent host-cell reactivation assay, we found that Rev7K44A/R124A/A135D is unable to promote gap-filling TLS opposite an abasic site analog. The Rev7 dimerization interface is also required for shieldin function, as both Rev7-/- cells and Rev7-/- cells expressing Rev7K44A/R124A/A135D exhibit decreased proficiency in rejoining some types of double strand breaks, as well as increased homologous recombination. Interestingly, Rev7K44A/R124A/A135D retains some function in cell cycle regulation, as it maintains an interaction with Ras-related nuclear protein (Ran) and partially rescues the formation of micronuclei. The mutant Rev7 also rescues the G2/M accumulation observed in Rev7-/- cells but does not affect progression through mitosis following nocodazole release. We conclude that while Rev7 dimerization is required for its roles in TLS, DSB repair, and regulation of the anaphase promoting complex, dimerization is at least partially dispensable for promoting mitotic spindle assembly through its interaction with Ran.

Exploiting DNA repair defects in triple negative breast cancer to improve cell killing.

BACKGROUND: The lack of molecular targets for triple negative breast cancer (TNBC) has limited treatment options and reduced survivorship. Identifying new molecular targets may help improve patient survival and decrease recurrence and metastasis. As DNA repair defects are prevalent in breast cancer, we evaluated the expression and repair capacities of DNA repair proteins in preclinical models. METHODS: DNA repair capacity was analyzed in four TNBC cell lines, MDA-MB-157 (MDA-157), MDA-MB-231 (MDA-231), MDA-MB-468 (MDA-468), and HCC1806, using fluorescence multiplex host cell reactivation (FM-HCR) assays. Expression of DNA repair genes was analyzed with RNA-seq, and protein expression was evaluated with immunoblot. Responses to the combination of DNA damage response inhibitors and primary chemotherapy drugs doxorubicin or carboplatin were evaluated in the cell lines. RESULTS: Defects in base excision and nucleotide excision repair were observed in preclinical TNBC models. Gene expression analysis showed a limited correlation between these defects. Loss in protein expression was a better indicator of these DNA repair defects. Over-expression of PARP1, XRCC1, RPA, DDB1, and ERCC1 was observed in TNBC preclinical models, and likely contributed to altered sensitivity to chemotherapy and DNA damage response (DDR) inhibitors. Improved cell killing was achieved when primary therapy was combined with DDR inhibitors for ATM, ATR, or CHK1. CONCLUSION: Base excision and nucleotide excision repair pathways may offer new molecular targets for TNBC. The functional status of DNA repair pathways should be considered when evaluating new therapies and may improve the targeting for primary and combination therapies with DDR inhibitors.

Paradoxical Role of AT-rich Interactive Domain 1A in Restraining Pancreatic Carcinogenesis.

Background & Aims: ARID1A is postulated to be a tumor suppressor gene owing to loss-of-function mutations in human pancreatic ductal adenocarcinomas (PDAC). However, its role in pancreatic pathogenesis is not clear despite recent studies using genetically engineered mouse (GEM) models. We aimed at further understanding of its direct functional role in PDAC, using a combination of GEM model and PDAC cell lines. Methods: Pancreas-specific mutant Arid1a-driven GEM model (Ptf1a-Cre; KrasG12D; Arid1af/f or "KAC") was generated by crossing Ptf1a-Cre; KrasG12D ("KC") mice with Arid1af/f mice and characterized histologically with timed necropsies. Arid1a was also deleted using CRISPR-Cas9 system in established human and murine PDAC cell lines to study the immediate effects of Arid1a loss in isogenic models. Cell lines with or without Arid1a expression were developed from respective autochthonous PDAC GEM models, compared functionally using various culture assays, and subjected to RNA-sequencing for comparative gene expression analysis. DNA damage repair was analyzed in cultured cells using immunofluorescence and COMET assay. Results: Retention of Arid1a is critical for early progression of mutant Kras-driven pre-malignant lesions into PDAC, as evident by lower Ki-67 and higher apoptosis staining in "KAC" as compared to "KC" mice. Enforced deletion of Arid1a in established PDAC cell lines caused suppression of cellular growth and migration, accompanied by compromised DNA damage repair. Despite early development of relatively indolent cystic precursor lesions called intraductal papillary mucinous neoplasms (IPMNs), a subset of "KAC" mice developed aggressive PDAC in later ages. PDAC cells obtained from older autochthonous "KAC" mice revealed various compensatory ("escaper") mechanisms to overcome the growth suppressive effects of Arid1a loss. Conclusions: Arid1a is an essential survival gene whose loss impairs cellular growth, and thus, its expression is critical during early stages of pancreatic tumorigenesis in mouse models. In tumors that arise in the setting of ARID1A loss, a multitude of "escaper" mechanisms drive progression.

Adaptive mutability of colorectal cancers in response to targeted therapies.

The emergence of drug resistance limits the efficacy of targeted therapies in human tumors. The prevalent view is that resistance is a fait accompli: when treatment is initiated, cancers already contain drug-resistant mutant cells. Bacteria exposed to antibiotics transiently increase their mutation rates (adaptive mutability), thus improving the likelihood of survival. We investigated whether human colorectal cancer (CRC) cells likewise exploit adaptive mutability to evade therapeutic pressure. We found that epidermal growth factor receptor (EGFR)/BRAF inhibition down-regulates mismatch repair (MMR) and homologous recombination DNA-repair genes and concomitantly up-regulates error-prone polymerases in drug-tolerant (persister) cells. MMR proteins were also down-regulated in patient-derived xenografts and tumor specimens during therapy. EGFR/BRAF inhibition induced DNA damage, increased mutability, and triggered microsatellite instability. Thus, like unicellular organisms, tumor cells evade therapeutic pressures by enhancing mutability.

Defective base excision repair in the response to DNA damaging agents in triple negative breast cancer.

DNA repair defects have been increasingly focused on as therapeutic targets. In hormone-positive breast cancer, XRCC1-deficient tumors have been identified and proposed as targets for combination therapies that damage DNA and inhibit DNA repair pathways. XRCC1 is a scaffold protein that functions in base excision repair (BER) by mediating essential interactions between DNA glycosylases, AP endonuclease, poly(ADP-ribose) polymerase 1, DNA polymerase ß (POL ß), and DNA ligases. Loss of XRCC1 confers BER defects and hypersensitivity to DNA damaging agents. BER defects have not been evaluated in triple negative breast cancers (TNBC), for which new therapeutic targets and therapies are needed. To evaluate the potential of XRCC1 as an indicator of BER defects in TNBC, we examined XRCC1 expression in the TCGA database and its expression and localization in TNBC cell lines. The TCGA database revealed high XRCC1 expression in TNBC tumors and TNBC cell lines show variable, but mostly high expression of XRCC1. XRCC1 localized outside of the nucleus in some TNBC cell lines, altering their ability to repair base lesions and single-strand breaks. Subcellular localization of POL ß also varied and did not correlate with XRCC1 localization. Basal levels of DNA damage correlated with observed changes in XRCC1 expression, localization, and measure repair capacity. The results confirmed that XRCC1 expression changes indicate DNA repair capacity changes but emphasize that basal DNA damage levels along with protein localization are better indicators of DNA repair defects. Given the observed over-expression of XRCC1 in TNBC preclinical models and tumors, XRCC1 expression levels should be assessed when evaluating treatment responses of TNBC preclinical model cells.

Sodium sulfide selectively induces oxidative stress, DNA damage, and mitochondrial dysfunction and radiosensitizes glioblastoma (GBM) cells.

Glioblastoma (GBM) has a poor prognosis despite intensive treatment with surgery and chemoradiotherapy. Previous studies using dose-escalated radiotherapy have demonstrated improved survival; however, increased rates of radionecrosis have limited its use. Development of radiosensitizers could improve patient outcome. In the present study, we report the use of sodium sulfide (Na2S), a hydrogen sulfide (H2S) donor, to selectively kill GBM cells (T98G and U87) while sparing normal human cerebral microvascular endothelial cells (hCMEC/D3). Na2S also decreased mitochondrial respiration, increased oxidative stress and induced γH2AX foci and oxidative base damage in GBM cells. Since Na2S did not significantly alter T98G capacity to perform non-homologous end-joining or base excision repair, it is possible that GBM cell killing could be attributed to increased damage induction due to enhanced reactive oxygen species production. Interestingly, Na2S enhanced mitochondrial respiration, produced a more reducing environment and did not induce high levels of DNA damage in hCMEC/D3. Taken together, this data suggests involvement of mitochondrial respiration in Na2S toxicity in GBM cells. The fact that survival of LN-18 GBM cells lacking mitochondrial DNA (ρ0) was not altered by Na2S whereas the survival of LN-18 ρ+ cells was compromised supports this conclusion. When cells were treated with Na2S and photon or proton radiation, GBM cell killing was enhanced, which opens the possibility of H2S being a radiosensitizer. Therefore, this study provides the first evidence that H2S donors could be used in GBM therapy to potentiate radiation-induced killing.

WRN helicase is a synthetic lethal target in microsatellite unstable cancers.

Synthetic lethality-an interaction between two genetic events through which the co-occurrence of these two genetic events leads to cell death, but each event alone does not-can be exploited for cancer therapeutics1. DNA repair processes represent attractive synthetic lethal targets, because many cancers exhibit an impairment of a DNA repair pathway, which can lead to dependence on specific repair proteins2. The success of poly(ADP-ribose) polymerase 1 (PARP-1) inhibitors in cancers with deficiencies in homologous recombination highlights the potential of this approach3. Hypothesizing that other DNA repair defects would give rise to synthetic lethal relationships, we queried dependencies in cancers with microsatellite instability (MSI), which results from deficient DNA mismatch repair. Here we analysed data from large-scale silencing screens using CRISPR-Cas9-mediated knockout and RNA interference, and found that the RecQ DNA helicase WRN was selectively essential in MSI models in vitro and in vivo, yet dispensable in models of cancers that are microsatellite stable. Depletion of WRN induced double-stranded DNA breaks and promoted apoptosis and cell cycle arrest selectively in MSI models. MSI cancer models required the helicase activity of WRN, but not its exonuclease activity. These findings show that WRN is a synthetic lethal vulnerability and promising drug target for MSI cancers.

The CHD6 chromatin remodeler is an oxidative DNA damage response factor.

Cell survival after oxidative DNA damage requires signaling, repair and transcriptional events often enabled by nucleosome displacement, exchange or removal by chromatin remodeling enzymes. Here, we show that Chromodomain Helicase DNA-binding protein 6 (CHD6), distinct to other CHD enzymes, is stabilized during oxidative stress via reduced degradation. CHD6 relocates rapidly to DNA damage in a manner dependent upon oxidative lesions and a conserved N-terminal poly(ADP-ribose)-dependent recruitment motif, with later retention requiring the double chromodomain and central core. CHD6 ablation increases reactive oxygen species persistence and impairs anti-oxidant transcriptional responses, leading to elevated DNA breakage and poly(ADP-ribose) induction that cannot be rescued by catalytic or double chromodomain mutants. Despite no overt epigenetic or DNA repair abnormalities, CHD6 loss leads to impaired cell survival after chronic oxidative stress, abnormal chromatin relaxation, amplified DNA damage signaling and checkpoint hypersensitivity. We suggest that CHD6 is a key regulator of the oxidative DNA damage response.

Structural and functional characterization of the PNKP-XRCC4-LigIV DNA repair complex.

Non-homologous end joining (NHEJ) repairs DNA double strand breaks in non-cycling eukaryotic cells. NHEJ relies on polynucleotide kinase/phosphatase (PNKP), which generates 5΄-phosphate/3΄-hydroxyl DNA termini that are critical for ligation by the NHEJ DNA ligase, LigIV. PNKP and LigIV require the NHEJ scaffolding protein, XRCC4. The PNKP FHA domain binds to the CK2-phosphorylated XRCC4 C-terminal tail, while LigIV uses its tandem BRCT repeats to bind the XRCC4 coiled-coil. Yet, the assembled PNKP-XRCC4-LigIV complex remains uncharacterized. Here, we report purification and characterization of a recombinant PNKP-XRCC4-LigIV complex. We show that the stable binding of PNKP in this complex requires XRCC4 phosphorylation and that only one PNKP protomer binds per XRCC4 dimer. Small angle X-ray scattering (SAXS) reveals a flexible multi-state complex that suggests that both the PNKP FHA and catalytic domains contact the XRCC4 coiled-coil and LigIV BRCT repeats. Hydrogen-deuterium exchange indicates protection of a surface on the PNKP phosphatase domain that may contact XRCC4-LigIV. A mutation on this surface (E326K) causes the hereditary neuro-developmental disorder, MCSZ. This mutation impairs PNKP recruitment to damaged DNA in human cells and provides a possible disease mechanism. Together, this work unveils multipoint contacts between PNKP and XRCC4-LigIV that regulate PNKP recruitment and activity within NHEJ.