Platinum-induced DNA Damage Research Articles

Predictive modeling of gene mutations for the survival outcomes of epithelial ovarian cancer patients.

Epithelial ovarian cancer (EOC) has a low overall survival rate, largely due to frequent recurrence and acquiring resistance to platinum-based chemotherapy. EOC with homologous recombination (HR) deficiency has increased sensitivity to platinum-based chemotherapy because platinum-induced DNA damage cannot be repaired. Mutations in genes involved in the HR pathway are thought to be strongly correlated with favorable response to treatment. Patients with these mutations have better prognosis and an improved survival rate. On the other hand, mutations in non-HR genes in EOC are associated with increased chemoresistance and poorer prognosis. For this reason, accurate predictions in response to treatment and overall survival remain challenging. Thus, analyses of 360 EOC cases on NCI's The Cancer Genome Atlas (TCGA) program were conducted to identify novel gene mutation signatures that were strongly correlated with overall survival. We found that a considerable portion of EOC cases exhibited multiple and overlapping mutations in a panel of 31 genes. Using logistical regression modeling on mutational profiles and patient survival data from TCGA, we determined whether specific sets of deleterious gene mutations in EOC patients had impacts on patient survival. Our results showed that six genes that were strongly correlated with an increased survival time are BRCA1, NBN, BRIP1, RAD50, PTEN, and PMS2. In addition, our analysis shows that six genes that were strongly correlated with a decreased survival time are FANCE, FOXM1, KRAS, FANCD2, TTN, and CSMD3. Furthermore, Kaplan-Meier survival analysis of 360 patients stratified by these positive and negative gene mutation signatures corroborated that our regression model outperformed the conventional HR genes-based classification and prediction of survival outcomes. Collectively, our findings suggest that EOC exhibits unique mutation signatures beyond HR gene mutations. Our approach can identify a novel panel of gene mutations that helps improve the prediction of treatment outcomes and overall survival for EOC patients.

Abstract A035: Genome-wide CRISPRi screening reveals determinants of platinum sensitivity and resistance in high-grade serous ovarian cancer

Abstract A035: Genome-wide CRISPRi screening reveals determinants of platinum sensitivity and resistance in high-grade serous ovarian cancer

Chloroxine overrides DNA damage tolerance to restore platinum sensitivity in high-grade serous ovarian cancer

High-grade serous cancer (HGSC) accounts for ~67% of all ovarian cancer deaths. Although initially sensitive to platinum chemotherapy, resistance is inevitable and there is an unmet clinical need for novel therapies that can circumvent this event. We performed a drug screen with 1177 FDA-approved drugs and identified the hydroxyquinoline drug, chloroxine. In extensive validation experiments, chloroxine restored sensitivity to both cisplatin and carboplatin, demonstrating broad synergy in our range of experimental models of platinum-resistant HGSC. Synergy was independent of chloroxine’s predicted ionophore activity and did not relate to platinum uptake as measured by atomic absorption spectroscopy. Further mechanistic investigation revealed that chloroxine overrides DNA damage tolerance in platinum-resistant HGSC. Co-treatment with carboplatin and chloroxine (but not either drug alone) caused an increase in γH2AX expression, followed by a reduction in platinum-induced RAD51 foci. Moreover, this unrepaired DNA damage was associated with p53 stabilisation, cell cycle re-entry and triggering of caspase 3/7-mediated cell death. Finally, in our platinum-resistant, intraperitoneal in vivo model, treatment with carboplatin alone resulted in a transient tumour response followed by tumour regrowth. In contrast, treatment with chloroxine and carboplatin combined, was able to maintain tumour volume at baseline for over 4 months. In conclusion, our novel results show that chloroxine facilitates platinum-induced DNA damage to restore platinum sensitivity in HGSC. Since chloroxine is already licensed, this exciting combination therapy could now be rapidly translated for patient benefit.

Clinicopathological and Functional Evaluation Reveal NBS1 as a Predictor of Platinum Resistance in Epithelial Ovarian Cancers.

Platinum resistance seriously impacts on the survival outcomes of patients with ovarian cancers. Platinum-induced DNA damage is processed through DNA repair. NBS1 is a key DNA repair protein. Here, we evaluated the role of NBS1 in ovarian cancers. NBS1 expression was investigated in clinical cohorts (protein level (n = 331) and at the transcriptomic level (n = 1259)). Pre-clinically, sub-cellular localization of NBS1 at baseline and following cisplatin therapy was tested in platinum resistant (A2780cis, PEO4) and sensitive (A2780, PEO1) ovarian cancer cells. NBS1 was depleted and cisplatin sensitivity was investigated in A2780cis and PEO4 cells. Nuclear NBS1 overexpression was associated with platinum resistance (p = 0.0001). In univariate and multivariate analysis, nuclear NBS1 overexpression was associated with progression free survival (PFS) (p-values = 0.003 and 0.017, respectively) and overall survival (OS) (p-values = 0.035 and 0.009, respectively). NBS1 mRNA overexpression was linked with poor PFS (p = 0.011). Pre-clinically, following cisplatin treatment, we observed nuclear localization of NBS1 in A2780cis and PEO4 compared to A2780 and PEO1 cells. NBS1 depletion increased cisplatin cytotoxicity, which was associated with accumulation of double strand breaks (DSBs), S-phase cell cycle arrest, and increased apoptosis. NBS1 is a predictor of platinum sensitivity and could aid stratification of ovarian cancer therapy.

Retraction Note: The melanocortin signaling cAMP axis accelerates repair and reduces mutagenesis of platinum-induced DNA damage

Editor's Note: this Article has been retracted; the Retraction Note is available at https://doi.org/10.1038/s41598-020-80467-y.

Genomic profiling of platinum-resistant ovarian cancer: The road into druggable targets.

Ovarian cancer is the most lethal gynecologic cancer. High-grade serous carcinoma (HGSC) is the most frequent histologic subtype and while it is a highly platinum-sensitive cancer at initial treatment, nearly 90 % of stage IIIC patients recur in 5 years and eventually become resistant to platinum treatment. Historically, the definition of platinum-resistant disease is based on the time interval between last platinum therapy and recurrence shorter than 6 months. Nowadays the use of sophisticated imaging techniques and serum markers to detect recurrence makes the accuracy of this clinical definition less clear and even more debatable as we begin to better understand the molecular landscape of HGSC and markers of platinum resistance and sensitivity. HGSC is characterized by a low frequency of recurrent mutations, great genomic instability with widespread copy number variations, universal TP53 mutations, and homologous recombination deficiency in more than 50 % of cases. Platinum agents form DNA adducts and intra- and inter-strand cross-links in the DNA. Most of DNA repair pathways are involved at some point in the repair of platinum induced DNA damaging, most notably homologous recombination, Fanconi Anemia, and nucleotide excision repair pathways. Mechanisms of platinum resistance are related mostly to the limitation of platinum-DNA adduct formation by changing cellular pharmacology, and to the prevention of cell death after DNA damage due to alterations in DNA repair pathways and cell cycle regulation. Understanding these mechanisms of sensitivity and resistance may help to define the utility of platinum re-challenge in each situation and guide new therapeutic opportunities. Moreover, the discovery of mechanisms of synthetic lethality related to alterations in DNA repair and cell cycle regulation pathways has opened up a new avenue for drug therapy in the last decade. In the present article, we review pathways involved in platinum-induced DNA damage repair and their relationship with genomic alterations present in HGSC. Moreover, we report new treatment strategies that are underway to target these alterations.

Single Nucleotide Polymorphisms in MiRNA Binding Sites of Nucleotide Excision Repair-Related Genes Predict Clinical Benefit of Oxaliplatin in FOLFOXIRI Plus Bevacizumab: Analysis of the TRIBE Trial.

Background: The nucleotide excision repair (NER) pathway participates in platinum-induced DNA damage repair. Single nucleotide polymorphisms (SNPs) in miRNA-binding sites in the NER genes RPA2 and GTF2H1 are associated with the risk of colorectal cancer (CRC). Here, we analyzed whether RPA2 and GTF2H1 SNPs predict the efficacy of oxaliplatin in metastatic CRC (mCRC) patients. Patients and methods: Genomic DNA was extracted from blood samples from 457 patients with mCRC enrolled in the TRIBE trial, which compared first-line FOLFOXIRI plus bevacizumab (BEV) (n = 230, discovery cohort) and first-line FOLFIRI plus BEV (n = 227, control cohort). SNPs were analyzed by PCR-based direct sequencing. Results: In the FOLFOXIRI + BEV-treated cohort expressing wild-type KRAS, progression-free survival (PFS) was shorter for the RPA2 rs7356 C/C variant subgroup than the any T allele subgroup in univariate analysis (9.1 versus 13.3 months respectively, hazard ratio (HR) 2.32, 95% confidence interval (CI): 1.07–5.03, p = 0.020) and this remained significant in multivariable analysis (HR 2.97, 95%CI: 1.27–6.94, p = 0.012). A similar trend was observed for overall survival. In contrast, patients expressing mutant RAS and RPA2 rs7356 C/C variant had longer PFS with FOLFOXIRI + BEV than with FOLFIRI + BEV (12.1 versus 7.6 months, HR 0.23, 95%CI: 0.09–0.62, p = 0.002) but no superiority of FOLFOXIRI + BEV was observed for the RAS mutant, RPA2 rs7356 any T variant subgroup (11.7 versus 9.6 months, HR 0.77, 95%CI: 0.56–1.07, p = 0.12) or the RAS wild-type, RPA2 rs7356 C/C variant subgroup. Conclusion: RPA2 SNPs may serve as predictive and prognostic markers of oxaliplatin responsiveness in a RAS status-dependent manner in mCRC patients receiving FOLFOXIRI + BEV.

Nanoparticle-Mediated Gene Silencing for Sensitization of Lung Cancer to Cisplatin Therapy.

Platinum-based chemotherapy remains a mainstay treatment for the management of advanced non-small cell lung cancer. A key cellular factor that contributes to sensitivity to platinums is the 5′-3′ structure-specific endonuclease excision repair cross-complementation group 1 (ERCC1)/ xeroderma pigmentosum group F (XPF). ERCC1/XPF is critical for the repair of platinum-induced DNA damage and has been the subject of intense research efforts to identify small molecule inhibitors of its nuclease activity for the purpose of enhancing patient response to platinum-based chemotherapy. As an alternative to small molecule inhibitors, small interfering RNA (siRNA) has often been described to be more efficient in interrupting protein–protein interactions. The goal of this study was therefore to determine whether biocompatible nanoparticles consisting of an amphiphilic triblock copolymer (polyethylenimine-polycaprolactone-polyethylene glycol (PEI-PCL-PEG)) and carrying siRNA targeted to ERCC1 and XPF made by microfluidic assembly are capable of efficient gene silencing and able to sensitize lung cancer cells to cisplatin. First, we show that our PEI-PCL-PEG micelleplexes carrying ERCC1 and XPF siRNA efficiently knocked down ERCC1/XPF protein expression to the same extent as the standard siRNA transfection reagent, Lipofectamine. Second, we show that our siRNA-carrying nanoparticles enhanced platinum sensitivity in a p53 wildtype model of non-small cell lung cancer in vitro. Our results suggest that nanoparticle-mediated targeting of ERCC1/XPF is feasible and could represent a novel therapeutic strategy for targeting ERCC1/XPF in vivo.

Abstract GMM-055: BMI1 AND RING1A ARE INVOLVED IN H2A UBIQUITINATION AT SITES OF PLATINUM-INDUCED DAMAGE

Abstract Ovarian cancer is the 5th leading cause of death among women with the five year survival rate for ovarian cancer patients being only 46%. The standard of care for treatment of ovarian cancer patients is debulking surgery followed by platinum-taxane based chemotherapy. However, the major obstacle in the treatment of these patients with platinum based agents is development of chemoresistance. The platinum agents, cisplatin and carboplatin damage DNA by forming adducts with adjacent guanines, a high density of which are found in the promoter CpG islands of genes. Aberrant promoter DNA hypermethylation of genes and their subsequent transcriptional repression has been associated with development of cisplatin resistance. However, the mechanism of initiation of this aberrant DNA methylation and subsequent transcriptional repression is not known. Our overall hypothesis is that platinum induced DNA damage or repair of the damage leads to recruitment of repressive proteins to sites of damage. These repressive proteins transiently repress transcription in vicinity of damage to promote repair. However, similar to our findings with enzyme induced double strand breaks, repressive proteins may be retained at some key loci causing persistent transcriptional repression and gene silencing contributing to the development of platinum resistance. Our preliminary data demonstrates that BMI1, a member of polycomb repressive complex 1, localizes to sites of damage after cisplatin treatment. We also observe mono-ubiquitination of H2AX after cisplatin treatment and we hypothesize that this ubiquitination is occurring on K119. Mono-ubiquitination of H2A/H2AX at K119 has been associated with transcriptional repression and gene silencing during development and differentiation and also during repair of ionizing radiation and enzyme induced double strand breaks. Our data suggests that knockdown of RING1A reduces the cisplatin induced H2AX ubiquitination. We are currently determining which repair pathways and/or proteins result in recruitment of BMI1, RING1A to sites of damage. Importantly, after acute cisplatin treatment we also observe a reduction in expression of candidate genes which are known to be methylated in cisplatin resistant cells. We plan to determine if RING1A mediated ubiquitination is responsible for reduction in expression of target genes after acute cisplatin treatment and their methylation in cisplatin resistant cells. Understanding the mechanism of platinum induced gene silencing will enable us to design therapies to avert the development of drug resistant ovarian cancers. Citation Format: Shruthi Sriramkumar, Heather M. O'Hagan. BMI1 AND RING1A ARE INVOLVED IN H2A UBIQUITINATION AT SITES OF PLATINUM-INDUCED DAMAGE [abstract]. In: Proceedings of the 12th Biennial Ovarian Cancer Research Symposium; Sep 13-15, 2018; Seattle, WA. Philadelphia (PA): AACR; Clin Cancer Res 2019;25(22 Suppl):Abstract nr GMM-055.

Abstract 2576: BMI1 and RING1A are involved in H2A ubiquitination at sites of platinum-induced damage

Abstract Ovarian cancer (OC) is a lethal gynecological malignancy with a 5-year survival rate of 46%. Surgical debulking followed by platinum and taxane based chemotherapy is the standard of care for OC patients. However, the major obstacle in the use of these agents for treatment of OC patients is development of chemoresistance. An epigenetic mechanism commonly associated with development of platinum resistance is promoter DNA methylation and associated gene silencing. Transcriptional silencing of tumor suppressor genes like MutL homolog 1 (MLH1) by this mechanism has been well established in development of platinum resistance. However, the mechanism of initiation of this aberrant promoter DNA methylation is not known. Platinum agents are DNA damaging agents which crosslink guanines and form intra-strand or inter-strand adducts. We hypothesize that platinum induced DNA damage or repair of the damage results in recruitment of proteins involved in transcriptional repression to sites of damage. Recruitment of repressive proteins results in transient repression of transcription in the vicinity of damage to promote repair. However, similar to our findings with enzyme induced double strand breaks, repair of damage can occasionally result in retention of repressive proteins at sites of damage causing persistent transcriptional repression and gene silencing. Such persistent repression of key loci potentially contributes to the development of platinum resistance. We demonstrate that treatment of platinum sensitive OC cells with the IC50 dose of cisplatin for 8 hours resulted in ubiquitination of H2A/H2AX by western blot analysis. We hypothesize that this ubiquitination occurs on K119 of H2A/H2AX. H2A/H2AX ubiquitination at K119 mediated by Polycomb repressive complex 1 (PRC1) has been associated with transcriptional repression and gene silencing during development and differentiation and also during DNA repair. We observe a reduction in platinum induced H2AX ubiquitination on knockdown of PRC1 complex member RING1A using western blot analysis. Using immunofluorescence, we observe that BMI1, another PRC1 complex member, localizes to sites of platinum induced DNA damage. Platinum induced lesions are repaired by different repair pathways including – Nucleotide excision repair pathway (NER), Fanconi Anemia repair pathway (FA) and Homologous recombination repair (HRR). We demonstrate that knockdown of proteins in global genome NER and HRR pathways results in a decrease in platinum induced ubiquitination. Through on-going studies we seek to further elucidate the mechanism of recruitment of BMI1, RING1A to sites of platinum induced DNA damage and their role in long-term gene silencing. Understanding this mechanism will enable us to design inhibitors to prevent the occurrence of platinum resistant tumors in OC patients. Citation Format: Shruthi Sriramkumar, Heather M. O'Hagan. BMI1 and RING1A are involved in H2A ubiquitination at sites of platinum-induced damage [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 2576.

Anticancer activity in patients with advanced ovarian and biliary tract cancers treated with NUC-1031 and a platinum agent.

3030 Background: The inhibition of cellular nucleotide metabolism to promote apoptosis is a key principle of cancer therapy. This, in combination with platinum-induced DNA-damage, is key to promoting anti-cancer activity in a variety of tumors, including ovarian, biliary tract, lung, breast and bladder. NUC-1031, a phosphoramidate transformation of gemcitabine is designed to overcome resistance mechanisms that limit the efficacy of this nucleoside analog. NUC-1031 has shown broad clinical activity across multiple solid tumors as both a single agent and in combination with platinum agents. We show potential synergism between NUC-1031 and a platinum agent in advanced ovarian (OC) and biliary tract (BTC) cancers. Methods: PRO-002 was a phase Ib study; 25 patients (pts) with recurrent OC who had exhausted all other therapy options received NUC-1031 + carboplatin. 17 pts were considered platinum resistant (10) or platinum refractory (7). ABC-08 is a phase Ib study, 14 pts with advanced BTC treated in the first-line setting with NUC-1031 + cisplatin. Results: In PRO-002, strong efficacy signals were observed in non-platinum-responsive patients. Of the 17 response-evaluable platinum-resistant or refractory pts, 5 partial responses (PRs) and 11 stable diseases (SDs) were achieved, resulting in an ORR of 29% and a DCR of 94%. NUC-1031 + carboplatin was well-tolerated with no unexpected AEs; DLTs were myelosuppression and fatigue. Encouraging response rates were also observed in ABC-08 compared to historical standard of care (ABC-02). One CR (7%), 6 PRs (43%) and 1 SD (7%) were observed, resulting in an ORR of 50%. NUC-1031 + cisplatin was well-tolerated, with no unexpected AEs or DLTs. Complementary in vitro evidence suggests that the beneficial interaction occurs whereby platinum treatment sensitizes cells to NUC-1031. Conclusions: Increasing evidence suggests that NUC-1031 in combination with a platinum agent may have synergistic properties, leading to enhanced anti-cancer activity. In both OC and BTC, durable tumor shrinkage was observed. This was particularly encouraging in a platinum resistant/refractory OC population. Future studies utilizing both NUC-1031 plus a platinum agent will further elucidate the potential of this therapeutic combination.

ERCC1-XPF deficiency is a predictor of olaparib induced synthetic lethality and platinum sensitivity in epithelial ovarian cancers

PurposePARP inhibitor maintenance therapy in platinum sensitive sporadic ovarian cancers improves progression free survival. However, biomarker for synthetic lethality in platinum sensitive sporadic disease is yet to be defined. ERCC1-XPF heterodimer is a key player in nucleotide excision repair (NER) involved in the repair of platinum induced DNA damage. In the current study, we tested whether ERCC1-XPF deficiency would predict synthetic lethality to the PARP inhibitor Olaparib and platinum sensitivity in ovarian cancers. MethodsERCC1, XPF and PARP1 protein expression was evaluated in tumors from a cohort of 331 patients treated at Nottingham University Hospitals and correlated to clinicopathological features and survival. Pre-clinically, ERCC1 and XPF was depleted in A2780 (platinum sensitive) and A2780cis (platinum resistant) ovarian cancer cell lines and tested for platinum sensitivity as well as for Olaparib induced synthetic lethality. ResultsLow ERCC1 was significantly associated with improved progression free survival (PFS) in patients with ovarian cancers in univariate (p = 0.001) and multivariate (p = 0.002) analysis. In addition, low ERCC1/low XPF (p = 0.003) or low ERCC1/low PARP1 (p = 0.0001) tumors was also linked to better PFS compared to high ERCC1/high XPF or high ERCC1/high PARP1 tumors. Pre-clinically, ERCC1 or XPF depletion not only increased platinum sensitivity but also increased toxicity to Olaparib therapy. Increased sensitivity was associated with DNA double strand breaks (DSBs) accumulation, cell cycle arrest and increased apoptosis. ConclusionThe data provide evidence that low ERCC1 is not only a predictor of platinum sensitivity but is also a promising biomarker for Olaparib induced synthetic lethality in ovarian cancers.

Single nucleotide polymorphisms in miRNA binding sites of nucleotide excision repair-related genes to predict clinical benefit of oxaliplatin in FOLFOXIRI plus bevacizumab in TRIBE trial.

663 Background: Nucleotide excision repair (NER) is known as part of the DNA damage response (DDR) against platinum-induced DNA damage. Polymorphisms in miRNA binding sites of replication protein A ( RPA2) and General Transcription Factor IIH Subunit 1 ( GTF2H1) genes in NER pathway have shown to correlate with colorectal cancer risk. We tested whether the NER-related single nucleotide polymorphisms (SNPs) predict oxaliplatin efficacy in FOLFOXIRI regimen in patients (pts) with metastatic colorectal cancer (mCRC). Methods: Genomic DNA was extracted from 457 blood samples of mCRC pts enrolled in the TRIBE trial: 230 pts received first-line (1L) FOLFOXIRI plus bevacizumab (discovery cohort: median age, 60 yrs; median follow-up, 46.6 mos); and 227 pts received 1L FOLFIRI plus bevacizumab (control cohort: median age, 60 yrs; median follow-up, 49.0 mos). SNPs were analyzed by PCR-based direct sequencing. Progression-free survival (PFS) and overall survival (OS) were analyzed using Kaplan-Meier curves, log-rank test, and Cox proportional hazards regression. Results: In KRAS wild-type population among the discovery cohort, the C/C variant in RPA2 rs7356 was associated with shorter PFS and OS than any T allele (9.1 vs 13.3 mos, HR 2.32, 95%CI: 1.07-5.03, P= 0.020; 20.8 vs 37.1 mos, HR 1.94, 95%CI: 0.94-3.99, P= 0.059). Any C allele in GTF2H1 rs4596 was associated with shorter PFS than the G/G variant (10.8 vs 13.3 mos, HR1.64, 95%CI: 0.95-2.81, P= 0.045). In multivariable analysis, RPA2 rs7356 remained significant for PFS and OS (HR 2.97, 95%CI: 1.27-6.94, P= 0.012; HR 2.58, 95%CI: 1.19-5.58, P= 0.016). In all population of the discovery cohort, pts with any GTF2H1 rs4596 C allele had remarkably shorter PFS and OS than those with the G/G variant (11.3 vs 13.2 mos, HR 1.32, P= 0.087; 28.6 vs 34.3 mos, HR 1.36, P= 0.076). The findings remained in multivariable analysis. These findings were not confirmed in the control cohort. Conclusions: We suggest that miRNA binding sites of NER-related genes regulate DDR for oxaliplatin-related DNA damage. Germline RPA2 and GTF2H1 polymorphisms may predict oxaliplatin efficacy in mCRC pts.

ERCC1 as predictive biomarker to platinum-based chemotherapy in adrenocortical carcinomas.

Platinum-based chemotherapy (PBC) is the most effective cytotoxic treatment for advanced adrenocortical carcinoma (ACC). Excision repair cross complementing group 1 (ERCC1) plays a critical role in the repair of platinum-induced DNA damage. Two studies investigating the role of ERCC1 immunostaining as a predictive marker for the response to PBC in ACC had reported conflicting results. Both studies used the ERCC1-antibody clone 8F1 that later turned out to be not specific. The aim of this study was to evaluate the predictive role of ERCC1 with a new specific antibody in a larger series of ACC. 146 ACC patients with available FFPE slides were investigated. All patients underwent PBC (median cycles = 6), including cisplatin (n = 131) or carboplatin (n = 15), in most cases combined with etoposide (n = 144), doxorubicin (n = 131) and mitotane (n = 131). Immunostaining was performed with the novel ERCC1-antibody clone 4F9. The relationship between ERCC1 expression and clinicopathological parameters, as well as best objective response to therapy and progression-free survival (PFS) during PBC was evaluated. High ERCC1 expression was observed in 66% of ACC samples. During PBC, 43 patients experienced objective response (29.5%), 49 stable disease (33.6%), 8 mixed response (5.5%) and 46 progressive disease (31.5%) without any relationship with the ERCC1 immunostaining. No significant correlation was also found between ERCC1 expression and progression-free survival (median 6.5 vs 6 months, P = 0.33, HR = 1.23, 95% CI = 0.82-2.0). ERCC1 expression is not directly associated with sensitivity to PBC in ACC. Thus, other predictive biomarkers are required to support treatment decisions in patients with ACC.

RETRACTED ARTICLE: The melanocortin signaling cAMP axis accelerates repair and reduces mutagenesis of platinum-induced DNA damage

Using primary melanocytes and HEK293 cells, we found that cAMP signaling accelerates repair of bi- and mono-functional platinum-induced DNA damage. Elevating cAMP signaling either by the agonistic MC1R ligand melanocyte stimulating hormone (MSH) or by pharmacologic cAMP induction by forskolin enhanced clearance of intrastrand cisplatin-adducts in melanocytes or MC1R-transfected HEK293 cells. MC1R antagonists human beta-defensin 3 and agouti signaling protein blocked MSH- but not forskolin-mediated enhancement of platinum-induced DNA damage. cAMP-enhanced repair of cisplatin-induced DNA damage was dependent on PKA-mediated phosphorylation of ATR on S435 which promoted ATR’s interaction with the key NER factor xeroderma pigmentosum A (XPA) and facilitated recruitment of an XPA-ATR-pS435 complex to sites of cisplatin DNA damage. Moreover, we developed an oligonucleotide retrieval immunoprecipitation (ORiP) assay using a novel platinated-DNA substrate to establish kinetics of ATR-pS435 and XPA’s associations with cisplatin-damaged DNA. Expression of a non-phosphorylatable ATR-S435A construct or deletion of A kinase-anchoring protein 12 (AKAP12) impeded platinum adduct clearance and prevented cAMP-mediated enhancement of ATR and XPA’s associations with cisplatin-damaged DNA, indicating that ATR phosphorylation at S435 is necessary for cAMP-enhanced repair of platinum-induced damage and protection against cisplatin-induced mutagenesis. These data implicate cAMP signaling as a critical regulator of genomic stability against platinum-induced mutagenesis.

Genetic variants in ERCC1 and XPC predict survival outcome of non-small cell lung cancer patients treated with platinum-based therapy

Nucleotide excision repair (NER) plays a vital role in platinum-induced DNA damage during chemotherapy. We hypothesize that regulatory single nucleotide polymorphisms (rSNPs) of the core NER genes modulate clinical outcome of patients with advanced non-small cell lung cancer (NSCLC) treated with platinum-based chemotherapy (PBS). We investigated associations of 25 rSNPs in eight NER genes with progression free survival (PFS) and overall survival (OS) in 710 NSCLC patients. We found that ERCC1 rs3212924 AG/GG and XPC rs2229090 GC/CC genotypes were associated with patients’ PFS (HRadj = 1.21, 95% CI = 1.03–1.43, Padj = 0.021 for ERCC1 and HRadj = 0.80, 95% CI = 0.68–0.94, Padj = 0.007 for XPC), compared with the AA and GG genotypes, respectively. The association of XPC rs2229090 was more apparent in adenocarcinoma than in squamous cell carcinoma patients. Additionally, ERCC4 rs1799798 GA/AA genotypes were associated with poorer OS (HRadj = 1.32, 95% CI = 1.04–1.69, Padj = 0.026), compared with the GG genotype. The expression quantitative trait loci analysis revealed that ERCC1 rs3212924 and XPC rs2229090 might regulate transcription of their genes, which is consistent with their associations with survival. Larger studies are needed to validate our findings with further functional studies to elucidate the mechanisms underlying these observed associations.

Abstract 2918: Analysis of matched pre and post cisplatin-treated muscle-invasive bladder cancer reveals a candidate cisplatin mutational signature

Abstract Background: Mutational signatures of exposure to DNA damaging agents such as UV irradiation, tobacco, and temozolomide have been described; however a cisplatin mutational signature has not been identified. We analyzed mutational changes in matched pre- and post-cisplatin based neoadjuvant chemotherapy (NAC)-treated muscle-invasive bladder cancer (MIBC) samples to identify a cisplatin mutational signature. Methods: Whole exome sequencing and mutation calling was performed on matched germline and pre- and post-cisplatin based NAC (MVAC and GC) tumor samples from 46 MIBC patients with gross residual disease (≥ pT2) at cystectomy. After quality control, samples from 30 patients were analyzed. For each tumor, we characterized single nucleotide mutations (C>A, C>T, C>G, T>A, T>C, T>G) within a tri-nucleotide context, and used an optimized non-negative matrix factorization (NMF) to discover signatures of mutational processes in pre-NAC tumors, post-NAC tumors, and among mutations unique to the post-NAC samples. We compared our discovered signatures to previously described human mutational signatures and to a signature of cisplatin exposure in a DT40 (chicken lymphoblast) cell line (normalized for a human exome context). We also performed strand asymmetry analysis to search for evidence of transcription-coupled repair. Results: In both pre and post-NAC tumors we identified mutational signatures matching those attributed to APOBEC activity and nucleotide excision repair pathway defects, consistent with past studies in MIBC. In post-NAC tumors we identified an additional novel mutational signature, with modest overall cosine similarity (0.61) to the DT40 cisplatin signature, but similar C>A and T>A motifs. There was a strong correlation in inferred mutational activity (Pearson R = 0.98) when we replaced the novel signature with the DT40 signature in the post-NAC tumors, which was highly unlikely to be due to chance (p < 0.001, empiric null distribution). Further, we found evidence of transcriptional strand bias in C>A (p = 0.00025) and T>A (p = 4.2e-06) motifs with depletion of coding strand mutations, consistent with transcription coupled repair of platinum crosslinks at GpG and ApG motifs in the non-coding strand. Finally, we were able to rediscover the novel signature when limiting analyses to mutations unique to post-NAC tumors, consistent with mutational activity during chemotherapy. Conclusions: Analysis of matched pre- and post- cisplatin treated MIBC identified a novel signature in post-cisplatin treated samples that (1) has mutational activity similar to a preclinical cisplatin mutational signature; (2) has a transcription strand bias consistent with known repair characteristics of platinum-induced DNA damage; and (3) arises in tumors following cisplatin chemotherapy. This may represent a cisplatin-induced mutational signature in human tumors. Citation Format: David Liu, Daniel Keliher, Philip Abbosh, Kent Mouw, Diana Miao, Mariel Boyd, Jean Hoffman-Censits, Gopa Iyer, Sara Tolaney, Jaegil Kim, Gad Getz, Scott Carter, Joaquim Bellmunt, Elizabeth R. Plimack, Jonathan E. Rosenberg, Eliezer M. Van Allen. Analysis of matched pre and post cisplatin-treated muscle-invasive bladder cancer reveals a candidate cisplatin mutational signature [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 2918. doi:10.1158/1538-7445.AM2017-2918

Phase ιι (window) preoperative study of olaparib with cisplatin or with durvalumab or alone or no treatment in patients with histologically proven head and neck squamous cell carcinoma who are candidates for surgery (OPHELIA).

TPS6096 Background: Novel agents are often investigated in unselected end-stage cancer patients and their efficacy is evaluated by the classical RECIST criteria making unlikely to fully exploit the antitumor potential of these targeted agents. Olaparib (O) is a potent inhibitor of PARP especially active in tumors that have homologous recombination DNA repair pathway deficiencies. Durvalumab (D) is a selective, high-affinity human IgG1 monoclonal antibody that blocks PD-L1 binding to PD-1 and CD80, overcoming PD-L1-mediated inhibition of T-cell activation. There is substantial evidence that tumor cells use PARP to repair platinum-induced DNA damage and thus escape apoptosis. In addition, O may complement the antitumor activity of D by increasing DNA damage through repair inhibition. Methods: OPHELIA is an open-label randomized multicenter phase II (window) trial in patients (pts) with head and neck squamous cell carcinoma (HNSCC). Treatment-naive HNSCC pts selected for primary curative study are randomized 3:3:3:1 in 4 neoadjuvant treatment groups: D 1500 mg on day 1 followed by O 600mg daily for 21-28 days (12 pts), cisplatin 60 mg/m2 on day 1 followed by O 75mg daily for 5 days (12 pts), monotherapy with O 600mg daily for 21-28 days (12 pts) and no treatment (5 pts). Preoperative therapy is discontinued 24 to 36 hours before surgery. Tumor biopsies, CT scans, PET and blood specimens are obtained at diagnosis and at surgery. Primary endpoint is the change in the tumor Ki-67 before and after treatment. Secondary endpoints are objective response rate according to RECIST 1.1 criteria, pathologic complete response rate and metabolic response rate assessed by FDG-PET/CT scan. Exploratory endpoints will include tumor and blood biomarkers. Translational correlates will be tested in tumor tissue, plasma and germline DNA and will include mutations in genes associated with DNA repair assessed by next generation sequencing and circulating tumor cells (CTCs) evaluated for DNA repair biomarkers and PD-L1. Trial is open to enrollment. Clinical trial information: NCT02882308.

Smoking History Predicts Sensitivity to PARP Inhibitor Veliparib in Patients with Advanced Non–Small Cell Lung Cancer

IntroductionTobacco-related NSCLC is associated with reduced survival and greater genomic instability. Veliparib, a potent poly(adenosine diphosphate–ribose) polymerase inhibitor, augments platinum-induced DNA damage. A phase 2 trial of untreated advanced NSCLC showed a trend for improved outcomes (hazard ratio [HR] = 0.80, 95% confidence interval: 0.54–1.18, p = 0.27 for overall survival and HR = 0.72, 95% CI: 0.45–1.15, p = 0.17 for progression-free survival) when veliparib was added to carboplatin/paclitaxel. Here we report an exploratory analysis by smoking history. MethodsPatients were randomized 2:1 to receive carboplatin/paclitaxel with veliparib, 120 mg (n = 105), or placebo (n = 53). Patients were stratified by histologic subtype and smoking history (recent smokers [n = 95], former smokers [n = 42], and never-smokers [n = 21]). Plasma cotinine level was measured as a chemical index of smoking. Mutation status was assessed by whole exome sequencing (n = 38). ResultsSmoking history, histologic subtype, age, Eastern Cooperative Oncology Group performance status, sex, and geographic region predicted veliparib benefit in univariate analyses. In multivariate analysis, history of recent smoking was most predictive for veliparib benefit. Recent smokers treated with veliparib derived significantly greater progression-free survival and overall survival benefits (HR = 0.38 [p < 0.01] and HR = 0.43 [p < 0.01]) than former smokers (HR = 2.098 [p = 0 0208] and HR = 1.62 [p = 0.236]) and never-smokers (HR = 1.025 [p = 0.971] and HR = 1.33 [p = 0.638]). Sequencing data revealed that mutational burden was not associated with veliparib benefit. The rate of grade 3 or 4 adverse events was higher in recent smokers with veliparib treatment; all-grade and serious adverse events were similar in both treatment arms. ConclusionsSmoking history predicted for efficacy with a veliparib-chemotherapy combination; toxicity was acceptable regardless of smoking history. A prespecified analysis of recent smokers is planned for ongoing phase 3 studies of veliparib in NSCLC.

ERCC2/XPD Lys751Gln alter DNA repair efficiency of platinum-induced DNA damage through P53 pathway

Platinum-based treatment causes Pt-DNA adducts which lead to cell death. The platinum-induced DNA damage is recognized and repaired by the nucleotide excision repair (NER) system of which ERCC2/XPD is a critical enzyme. Single nucleotide polymorphisms in ERCC2/XPD have been found to be associated with platinum resistance. The aim of the present study was to investigate whether ERCC2/XPD Lys751Gln (rs13181) polymorphism is causally related to DNA repair capacity of platinum-induced DNA damage. First, cDNA clones expressing different genotypes of the polymorphism was transfected to an ERCC2/XPD defective CHO cell line (UV5). Second, all cells were treated with cisplatin. Cellular survival rate were investigated by MTT growth inhibition assay, DNA damage levels were investigated by comet assay and RAD51 staining. The distribution of cell cycle and the change of apoptosis rates were detected by a flow cytometric method (FCM). Finally, P53mRNA and phospho-P53 protein levels were further investigated in order to explore a possible explanation. As expected, there was a significantly increased in viability of UV5ERCC2 (AA) as compared to UV5ERCC2 (CC) after cisplatin treatment. The DNA damage level of UV5ERCC2 (AA) was significant decreased compared to UV5ERCC2 (CC) at 24 h of treatment. Mutation of ERCC2rs13181 AA to CC causes a prolonged S phase in cell cycle. UV5ERCC2 (AA) alleviated the apoptosis compared to UV5ERCC2 (CC), meanwhile P53mRNA levels in UVERCC2 (AA) was also lower when compared UV5ERCC2 (CC). It co-incides with a prolonged high expression of phospho-P53, which is relevant for cell cycle regulation, apoptosis, and the DNA damage response (DDR). We concluded that ERCC2/XPD rs13181 polymorphism is possibly related to the DNA repair capacity of platinum-induced DNA damage. This functional study provides some clues to clarify the relationship between cisplatin resistance and ERCC2/XPDrs13181 polymorphism.