The nuclear enzyme poly(ADP-ribose) polymerase (PARP) facilitates the repair of DNA strand breaks and is implicated in the resistance of cancer cells to certain DNA-damaging agents. Inhibitors of PARP have clinical potential as resistance-modifying agents capable of potentiating radiotherapy and the cytotoxicity of some forms of cancer chemotherapy. The preclinical development of 2-aryl-1H-benzimidazole-4-carboxamides as resistance-modifying agents in cancer chemotherapy is described. 1H-Benzimidazole-4-carboxamides, particularly 2-aryl derivatives, are identified as a class of potent PARP inhibitors. Derivatives of 2-phenyl-1H-benzimidazole-4-carboxamide (23, Ki = 15 nM), in which the phenyl ring contains substituents, have been synthesized. Many of these derivatives exhibit Ki values for PARP inhibition < 10 nM, with 2-(4-hydroxymethylphenyl)-1H-benzimidazole-4-carboxamide (78, Ki = 1.6 nM) being one of the most potent. Insight into structure−activity relationships (SAR) for 2-aryl-1H-benzimidazole-4-carboxamides has been enhanced by studying the complex formed between 2-(3-methoxyphenyl)-1H-benzimidazole-4-carboxamide (44, Ki = 6 nM) and the catalytic domain of chicken PARP. Important hydrogen-bonding and hydrophobic interactions with the protein have been identified for this inhibitor. 2-(4-Hydroxyphenyl)-1H-benzimidazole-4-carboxamide (45, Ki = 6 nM) potentiates the cytotoxicity of both temozolomide and topotecan against A2780 cells in vitro (by 2.8- and 2.9-fold, respectively).
Defective DNA damage response (DDR) pathways are enabling characteristics of cancers that not only can be exploited to specifically target cancer cells but also can predict chemotherapy response. Defective Homologous Recombination Repair (HRR) function, e.g., due to BRCA1/2 loss, is a determinant of response to platinum agents and PARP inhibitors in ovarian cancers. Most chemotherapies function by either inducing DNA damage or impacting on its repair but are generally used in the clinic unselectively. The significance of HRR and other DDR pathways in determining response to several other chemotherapy drugs is not well understood. In this study, the genomic, transcriptomic and functional analysis of DDR pathways in a panel of 14 ovarian cancer cell lines identified that defects in DDR pathways could determine response to several chemotherapy drugs. Carboplatin, rucaparib, and topotecan sensitivity were associated with functional loss of HRR (validated in 10 patient-derived primary cultures) and mismatch repair. Two DDR gene expression clusters correlating with treatment response were identified, with PARP10 identified as a novel marker of platinum response, which was confirmed in The Cancer Genome Atlas (TCGA) ovarian cancer cohort. Reduced non-homologous end-joining function correlated with increased sensitivity to doxorubicin, while cells with high intrinsic oxidative stress showed sensitivity to gemcitabine. In this era of personalised medicine, molecular/functional characterisation of DDR pathways could guide chemotherapy choices in the clinic allowing specific targeting of ovarian cancers.
Abstract DNA double-strand breaks (DSB) are the most cytotoxic lesions induced by ionizing radiation and topoisomerase II poisons, such as etoposide and doxorubicin. A major pathway for the repair of DSB is nonhomologous end joining, which requires DNA-dependent protein kinase (DNA-PK) activity. We investigated the therapeutic use of a potent, specific DNA-PK inhibitor (NU7441) in models of human cancer. We measured chemosensitization by NU7441 of topoisomerase II poisons and radiosensitization in cells deficient and proficient in DNA-PKCS (V3 and V3-YAC) and p53 wild type (LoVo) and p53 mutant (SW620) human colon cancer cell lines by clonogenic survival assay. Effects of NU7441 on DSB repair and cell cycle arrest were measured by γH2AX foci and flow cytometry. Tissue distribution of NU7441 and potentiation of etoposide activity were determined in mice bearing SW620 tumors. NU7441 increased the cytotoxicity of ionizing radiation and etoposide in SW620, LoVo, and V3-YAC cells but not in V3 cells, confirming that potentiation was due to DNA-PK inhibition. NU7441 substantially retarded the repair of ionizing radiation–induced and etoposide-induced DSB. NU7441 appreciably increased G2-M accumulation induced by ionizing radiation, etoposide, and doxorubicin in both SW620 and LoVo cells. In mice bearing SW620 xenografts, NU7441 concentrations in the tumor necessary for chemopotentiation in vitro were maintained for at least 4 hours at nontoxic doses. NU7441 increased etoposide-induced tumor growth delay 2-fold without exacerbating etoposide toxicity to unacceptable levels. In conclusion, NU7441 shows sufficient proof of principle through in vitro and in vivo chemosensitization and radiosensitization to justify further development of DNA-PK inhibitors for clinical use. (Cancer Res 2006; 66(10): 5354-62)
Epithelial ovarian cancer is recognized to be heterogeneous but is currently treated with a single treatment strategy. Successful patient stratification of emerging chemotherapy agents is dependent upon the availability of reliable biomarkers indicative of the entire tumor.The aim of this study was to evaluate intertumor and intratumor heterogeneity within a series of epithelial ovarian cancer using homologous recombination (HR) DNA repair status.Primary cultures generated from ascites and solid tumor from multiple intra-abdominal sites were characterized by their morphology and expression of protein markers. Results were compared with Formalin fixed paraffin embedded tissue pathology.Homologous recombination function was determined by quantification of nuclear Rad51 foci. Growth inhibition (sulforhodamine B) assays were used to calculate the GI50 for cisplatin and rucaparib.Ascites with matched solid tumor were cultured from 25 patients.Concordance in functional HR status between ascites and solid tumor subcultures was seen in only 13 (52%) of 25 patients. Heterogeneity in HR status was seen even in patients with homogeneous histological subtype. Homologous recombination defective cultures were significantly more sensitive to cisplatin and rucaparib.Additionally, intertumor and intratumor heterogeneity was seen between the expression of epithelial and ovarian markers (EpCAM, cytokeratin, CA125, MOC-31, and vimentin). There was no relationship between heterogeneity of HR functional status and antigen expression.Intertumor and intratumor functional HR heterogeneity exists that cannot be detected using histological classification. This has implications for biomarker-directed treatment.
Half of all cancer patients receive radiotherapy, which makes a substantial contribution to their long-term disease control/cure. There are significant inter-patient differences in response, both in terms of efficacy and toxicity (frequently delayed onset) which are difficult to predict. With the introduction of technological improvements (e.g. stereotactic body radiotherapy and proton therapy) and development of combination therapies (e.g. radiotherapy and immune checkpoint inhibition), predictive biomarkers are needed even more. Whilst genomic studies have contributed significantly to predictions of response to anticancer therapy, there is no doubt that more information can be gathered from patient tissue samples. Patients are willing to donate their tissues to biobanks and wish them to be used as widely as possible for high-quality research. We report here a survey of the current practices in the UK from several groups collecting material from patients in radiotherapy trials and have identified barriers to collecting and sharing data and samples. We believe the current situation represents a significant missed opportunity to improve the personalisation of radiotherapy. We propose a greater involvement of patients and/or their advocates, a standardisation of the patient information leaflet, consent form content and data set, with easy linkage to clinical data, which would facilitate widespread sample and data discovery and availability to other researchers. The greater sharing of data and samples, nationally and internationally, would facilitate robust multicentre studies and avoid duplication of effort.
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