There is an increase in the incidence of early onset colorectal carcinoma (EOCRC). To better understand if there is any difference in molecular pathogenesis of EOCRC and late onset colorectal carcinoma (LOCRC), we compared the clinical, histological, transcriptome, and methylome profile of paired CRC and healthy colonic tissue from 67 EOCRC and 98 LOCRC patients. The frequency of stage 3 CRC, lymph node involvement, lymphovascular invasion, and perineural invasion was higher in the EOCRC group. Many of the cancer related pathways were differentially expressed in CRC tissue in both EOCRC and LOCRC patients. However, the magnitude of differential expression for some groups of genes, such as DNA damage repair genes and replication stress genes, were significantly less pronounced in the EOCRC group, suggesting less efficient DNA damage repair to be associated with EOCRC. A more marked methylation of \"growth factor receptor\" genes in LOCRC correlated with a more pronounced down-regulation of those genes in that group. From a therapeutic point of view, more over-expression of fatty acid synthase (FASN) among the LOCRC patients may suggest a better response of FASN targeted therapy in that group. The age of onset of CRC did not appear to modify the response of cis-platin or certain immune checkpoint inhibitors. We found some differences in the molecular pathogenesis in EOCRC and LOCRC that may have some biological and therapeutic significance.

Several proteins are involved in DNA repair mechanisms attempting to repair damages to the DNA continuously. One such protein is Xeroderma Pigmentosum Complementation Group G (XPG), a significant component in the Nucleotide Excision Repair (NER) pathway. XPG is accountable for making the 3\' incision in the NER, while XPF-ERCC4 joins ERCC1 to form the XPF-ERCC1 complex. This complex makes a 5\' incision to eliminate bulky DNA lesions. XPG is also known to function as a cofactor in the Base Excision Repair (BER) pathway by increasing hNth1 activity, apart from its crucial involvement in the NER. Reports suggest that XPG also plays a non-catalytic role in the Homologous Recombination Repair (HRR) pathway by forming higher-order complexes with BRCA1, BRCA2, Rad51, and PALB2, further influencing the activity of these molecules. Studies show that, apart from its vital role in repairing DNA damages, XPG is also responsible for R-loop formation, which facilitates exhibiting phenotypes of Werner Syndrome. Though XPG has a role in several DNA repair pathways and molecular mechanisms, it is primarily a NER protein. Unrepaired and prolonged DNA damage leads to genomic instability and facilitates neurological disorders, aging, pigmentation, and cancer susceptibility. This review explores the vital role of XPG in different DNA repair mechanisms which are continuously involved in repairing these damaged sites and its failure leading to XP-G, XP-G/CS complex phenotypes, and cancer progression.

The 5\',8-cyclo-2\'-deoxypurines (cdPus) affect the DNA structure. When these bulky structures are a part of clustered DNA lesions (CDL), they affect the repair of the other lesions within the cluster. Mitochondria are crucial for cell survival and have their own genome, hence, are highly interesting in the context of CDL repair. However, no studies are exploring this topic. Here, the initial stages of mitochondrial base excision repair (mtBER) were considered-the strand incision and elongation. The repair of a single lesion (apurinic site (AP site)) accompanying the cdPu within the double-stranded CDL has been investigated for the first time. The type of cdPu, its diastereomeric form, and the interlesion distance were taken into consideration. For these studies, the established experimental model of short oligonucleotides (containing AP sites located ≤7 base pairs to the cdPu in both directions) and mitochondrial extracts of the xrs5 cells were used. The obtained results have shown that the presence of cdPus influenced the processing of an AP site within the CDL. Levels of strand incision and elongation were higher for oligos containing RcdA and ScdG than for those with ScdA and RcdG. Investigated stages of mtBER were more efficient for DNA containing AP sites located on 5\'-end side of cdPu than on its 3\'-end side. In conclusion, the presence of cdPus in mtDNA structure may affect mtBER (processing the second mutagenic lesion within the CDL). As impaired repair processes may lead to serious biological consequences, further studies concerning the mitochondrial repair of CDL are highly demanded.

Uracil-DNA glycosylase (UDG) is one of the most important base excision repair (BER) enzymes involved in the repair of uracil-induced DNA lesion by removing uracil from the damaged DNA. Uracil in DNA may occur due to cytosine deamination or deoxy uridine monophosphate (dUMP) residue misincorporation during DNA synthesis. Medical evidences show that an abnormal expression of UDG is related to different types of cancer, including colorectal cancer, lung cancer, and liver cancer. Therefore, the research of UDG is crucial in cancer treatment and prevention as well as other clinical activities. Here we applied multiple computational methods to study UDG in several perspectives: Understanding the stability of the UDG enzyme in different pH conditions; studying the differences in charge distribution between the pocket side and non-pocket side of UDG; analyzing the field line distribution at the interfacial area between UDG and DNA; and performing electrostatic binding force analyses of the special region of UDG (pocket area) and the target DNA base (uracil) as well as investigating the charged residues on the UDG binding pocket and binding interface. Our results show that the whole UDG binding interface, and not the UDG binding pocket area alone, provides the binding attractive force to the damaged DNA at the uracil base.

BACKGROUND: TRC102 inhibits base excision repair by binding abasic sites and preventing AP endonuclease processing; it potentiates the activity of alkylating agents, including temozolomide, in murine models. In published xenograft studies, TRC102 enhanced the antitumor effect of temozolomide regardless of cell line genetic characteristics, e.g., O6-methylguanine DNA methyltransferase (MGMT), mismatch repair (MMR), or p53 status.
METHODS: We conducted a phase 1 trial of TRC102 with temozolomide given orally on days 1-5 of 28-day cycles in adult patients with refractory solid tumors that had progressed on standard therapy. Tumor induction of nuclear biomarkers of DNA damage response (DDR) γH2AX, pNBs1, and Rad51 was assessed in the context of MGMT and MMR protein expression for expansion cohort patients.
RESULTS: Fifty-two patients were enrolled (37 escalation, 15 expansion) with 51 evaluable for response. The recommended phase 2 dose was 125 mg TRC102, 150 mg/m2 temozolomide QDx5. Common adverse events (grade 3/4) included anemia (19%), lymphopenia (12%), and neutropenia (10%). Four patients achieved partial responses (1 non-small cell lung cancer, 2 granulosa cell ovarian cancer, and 1 colon cancer) and 13 patients had a best response of stable disease. Retrospective analysis of 15 expansion cohort patients did not demonstrate a correlation between low tumor MGMT expression and patient response, but treatment induced nuclear Rad51 responses in 6 of 12 patients.
CONCLUSIONS: The combination of TRC 102 with temozolomide is active, with 4 of 51 patients experiencing a partial response and 13 of 51 experiencing stable disease, and the side effect profile is manageable.

Mechanistic and functional studies by gene disruption or editing approaches often suffer from confounding effects like compensatory cellular adaptations generated by clonal selection. These issues become particularly relevant when studying factors directly involved in genetic or epigenetic maintenance. To provide a genetic tool for functional and mechanistic investigation of DNA-repair mediated active DNA demethylation, we generated experimental models in mice and murine embryonic stem cells (ESCs) based on a minigene of the thymine-DNA glycosylase (TDG). The loxP-flanked miniTdg is rapidly and reliably excised in mice and ESCs by tamoxifen-induced Cre activation, depleting TDG to undetectable levels within 24 hours. We describe the functionality of the engineered miniTdg in mouse and ESCs (TDGiKO ESCs) and validate the pluripotency and differentiation potential of TDGiKO ESCs as well as the phenotype of induced TDG depletion. The controlled and rapid depletion of TDG allows for a precise manipulation at any point in time of multistep experimental procedures as presented here for neuronal differentiation in vitro. Thus, we provide a tested and well-controlled genetic tool for the functional and mechanistic investigation of TDG in active DNA (de)methylation and/or DNA repair with minimal interference from adaptive effects and clonal selection.

Nuclear poly(ADP-ribose) polymerases 1 and 2 (PARP1 and PARP2) catalyze the synthesis of poly(ADP-ribose) (PAR) and use NAD+ as a substrate for the polymer synthesis. Both PARP1 and PARP2 are involved in DNA damage response pathways and function as sensors of DNA breaks, including temporary single-strand breaks formed during DNA repair. Consistently, with a role in DNA repair, PARP activation requires its binding to a damaged DNA site, which initiates PAR synthesis. Here we use atomic force microscopy to characterize at the single-molecule level the interaction of PARP1 and PARP2 with long DNA substrates containing a single damage site and representing intermediates of the short-patch base excision repair (BER) pathway. We demonstrated that PARP1 has higher affinity for early intermediates of BER than PARP2, whereas both PARPs efficiently interact with the nick and may contribute to regulation of the final ligation step. The binding of a DNA repair intermediate by PARPs involved a PARP monomer or dimer depending on the type of DNA damage. PARP dimerization influences the affinity of these proteins to DNA and affects their enzymatic activity: the dimeric form is more effective in PAR synthesis in the case of PARP2 but is less effective in the case of PARP1. PARP2 suppresses PAR synthesis catalyzed by PARP1 after single-strand breaks formation. Our study suggests that the functions of PARP1 and PARP2 overlap in BER after a site cleavage and provides evidence for a role of PARP2 in the regulation of PARP1 activity.

BACKGROUND: It is frequently asked whether chemotherapy can still play a role in metastatic melanoma considering the effectiveness of the available drugs today, including antiCTLA4/antiPD1 immunotherapy and antiBRAF/antiMEK inhibitors. However, only approximately half of patients respond to these drugs, and the majority progress after 6-11 months. Therefore, a need for other therapeutic options is still very much apparent. We report the first large trial of a sequential full dose of fotemustine (FM) preceded by a low dose of temozolomide (TMZ) as a chemo-modulator in order to inactivate the DNA repair action of O(6)-methylguanine DNA-methyltransferase (MGMT). Primary endpoints were overall response and safety. We also evaluated specific biological parameters aiming to tailor these chemotherapies to selected patients.
METHODS: A total of 69 consecutive patients were enrolled. The main features included a median age of 60 years (21-81) and M1c stage, observed in 74% of the patients, with brain metastases in 15% and high LDH levels in 42% of the patients. The following schedule was used: oral TMZ 100 mg/m2 on days 1 and 2 and FM iv 100 mg/m2 on day 2, 4 h after TMZ; A translational study aiming to analyse MGMT methylation status and base-excision repair (BER) gene expression was performed in a subset of 14 patients.
RESULTS: We reported an overall response rate of 30.3% with 3 complete responses and a disease control rate of 50.5%. The related toxicity rate was low and mainly of haematological types. Although our population had a very poor prognosis, we observed a PFS of 6 months and an OS of 10 months. A non-significant correlation with response was found with the mean expression level of the three genes involved in the BER pathway (APE1, XRCC1 and PARP1), whereas no association was found with MGMT methylation status.
CONCLUSIONS: This schedule could represent a good alternative for patients who are not eligible for immune or targeted therapy or whose previous therapies have failed.
BACKGROUND: EUDRACT 2009-016487-36l ; date of registration 23 June 2010.

OBJECTIVE: We determined the safety, pharmacokinetics, pharmacodynamics and recommended phase II dose of the base excision repair blocker methoxyamine combined with fludarabine.
METHODS: This was a phase I study with intravenous fludarabine (25 mg/m2, days 1-5), and methoxyamine (15 mg/m2-120 mg/m2, once). A maximum of six cycles were given. Adult patients with relapsed/refractory hematologic malignancies, excluding acute myeloid leukemia, were eligible.
RESULTS: Twenty patients were treated; diagnoses included CLL/SLL (n = 10), follicular lymphoma (n = 3), DLBCL (n = 3), mantle cell lymphoma (n = 1), anaplastic large cell lymphoma (n = 1) and plasma cell myeloma (n = 2). No DLTs were observed and dose escalation reached the maximum planned dose. Hematologic toxicity was frequent; most common grade 3-4 toxicities were lymphopenia (70%), neutropenia (60%), leukopenia (50%) and anemia (40%). Four patients achieved a partial remission and 8 achieved stable disease. The drug combination resulted in increased DNA damage measured with the Comet assay.
CONCLUSIONS: Methoxyamine combined with fludarabine was safe and well tolerated. Hematologic toxicity was comparable to single agent fludarabine. Activity appears to correlate with increased levels of DNA damage. Further studies will examine use of this combination of as part conditioning regimens of stem cell transplant and use of methoxyamine as fludarabine dose-sparing agent.

BACKGROUND: Smoking has been considered to be the major cause of lung cancer. However, only a fraction of cigarette smokers develop this disease. This suggests the importance of genetic constitution in predicting the individual\'s susceptibility towards lung cancer. This genetic susceptibility may result from inherited polymorphisms in genes controlling carcinogen metabolism and repair of damaged deoxyribonucleic acid (DNA). These repair systems are fundamental to the maintenance of genomic integrity. X-ray repair cross complimenting group I (XRCC1), a major DNA repair gene in the base excision repair (BER) pathway. It is involved in repair by interacting with components of DNA at the site of damage. Inconsistent results have been reported regarding the associations between the Arg399Gln polymorphism of XRCC1. This study demonstrates the importance of recognition of this relationship of lung carcinoma and genetic constitution of the person which will help guide clinicians on the optimal screening of this disease.
OBJECTIVE: To assess the role of XRCC1 gene polymorphism (Arg399Gln) directly on the variation in susceptibility to development of lung cancer in North Indian subjects.
METHODS: One hundred males with diagnosed cases of lung cancer were recruited from Delhi State Cancer Institute (DSCI). Hundred healthy volunteers were taken as controls. DNA isolation was done and Polymerase chain reaction-Restriction Fragment Length Polymorphism (PCR-RFLP) procedure undertaken to amplify the region containing Arg/Gln substitution at codon 399 (in exon 10).
RESULTS: XRCC1 gene polymorphism is associated with increased risk of lung cancer when the Arg/Arg genotype was used as the reference group. The Arg/Gln and Gln/Gln was associated with statistically increased risk for cancer.
CONCLUSIONS: Arg399Gln polymorphism in XRCC1 gene polymorphism is associated with lung cancer in North Indian subjects and screening for this polymorphism will help in targeting predisposed individuals and its prevention.