Transcription-coupled Nucleotide Excision Repair Pathway Research Articles

Global genome and transcription-coupled nucleotide excision repair pathway in prokaryotes

Global genome and transcription-coupled nucleotide excision repair pathway in prokaryotes

Abstract P4-01-06: LP-184, an acylfulvene class small molecule therapeutic, is synthetically lethal in HR deficient and PARP inhibitor resistant triple negative breast cancer

Abstract Triple-negative breast cancer (TNBC) is the most aggressive subtype of breast cancer accounting for approximately 15% to 20% of all newly diagnosed breast cancer cases. TNBCs are defined by lack of expression of estrogen receptor (ER), progesterone receptor (PR) and human epidermal growth factor receptor 2 (HER-2).Due to its special molecular phenotype, it is not sensitive to endocrine therapy or targeted therapy. Therefore, chemotherapy such as anthracyclines or taxanes is the main systemic treatment, but the efficacy of conventional postoperative adjuvant chemoradiotherapy is poor. ~35% of TNBC tumors show abnormalities in the HR pathway, making them sensitive to poly (ADP-ribose) polymerase (PARP) inhibitors (PARPi) and DNA-damaging agents. Although more than 40% BRCA1/2-deficient patients fail to respond to PARPi and a substantial proportion of patients acquire PARPi resistance over time. TNBCs heterogeneous nature, poor prognosis and limited treatment options presents the urgent need of development of novel agents targeting tumor specific alterations. Lantern Pharma is advancing LP-184, an acylfulvene-derived prodrug that is specifically activated in tumors that overexpress the oxidoreductase enzyme Prostaglandin Reductase 1 (PTGR1), for the treatment of solid tumor indications including TNBC. LP-184 has multiple mechanisms of action (MOA). While It is synthetically lethal in tumors harboring DNA damage repair defects including Homologous Recombination (HR) deficiencies, it can also interrupt transcription. LP-184 efficacy was tested in a panel of breast cancer cell lines and patient derived TNBC xenografts models, both sensitive as well as resistant to PARP inhibitors and anthracyclines. LP-184 demonstrated nanomolar potency in six NCI-60 breast cancer cell lines (median IC50 = 327 nM). Subcutaneous patient-derived TNBC xenograft mouse models were used to determine tumor volume responses to LP-184 treatment in vivo. Xenograft tumors were derived from 10 treatment-naive HR deficient (HRD score > 50) primary TNBC patients with known BRCA1/2 loss of heterozygosity (LOH), 7 of which subsequently progressed on PARP inhibitor Olaparib. LP-184 (4mg/kg i.v., (q2d x 5 then 7 days off)x2) led to complete and durable regression in all 10 TNBC HRD-PDX models tested as compared to control (p < 0.0001). A tumor growth inhibition range of 107-141% was achieved across all the 10 models. For LP-184, T/C at the control group end day was 0% in 10/10 models, whereas across the same models for Olaparib, T/C was 0% in 2/10 models and ranged from 15 - 90% in 8/10 models. Terminal body weight change was only a transient weight loss < 4% with LP-184 treatment across all models. LP-184 exhibited superior potency than olaparib in TNBC PDX models that carry HRD mutations including PARP resistant models and was well tolerated in mice. As acylfulvene-induced damage is primarily repaired by transcription-coupled nucleotide-excision repair (TC-NER) and HR pathways, response to LP-184 is influenced by tumor DNA damage repair pathway status. Recent data highlight an important role of super enhancer driven core transcription regulatory circuits in the pathogenesis of TNBCs. Our results support the superior efficacy of LP-184 in TNBCs, likely linked to the multiple MOAs, and establish LP-184 as a promising new agent for future clinical testing in TNBC patients. We finally propose that LP-184 may be broadly efficacious in solid tumors with HR and/or TC-NER pathway defects, such as pancreatic, prostate, ovarian and bladder cancers. Citation Format: Aditya Kulkarni, Neha Biyani, Laura Brullé-Soumaré, Stefano Cairo, Panna Sharma, Kishor Bhatia. LP-184, an acylfulvene class small molecule therapeutic, is synthetically lethal in HR deficient and PARP inhibitor resistant triple negative breast cancer [abstract]. In: Proceedings of the 2022 San Antonio Breast Cancer Symposium; 2022 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2023;83(5 Suppl):Abstract nr P4-01-06.

Next-Generation Sequencing-Based Analysis of the Effects of N1- and N6-Methyldeoxyadenosine Adducts on DNA Transcription.

DNA methylation can occur naturally or be induced by various environmental and chemotherapeutic agents. The regioisomeric N1- and N6-methyldeoxyadenosine (1mdA and 6mdA, respectively) represent an important class of methylated DNA adducts. In this study, we developed a shuttle vector- and next-generation sequencing-based assay to quantitatively assess the effects of 1mdA and 6mdA on the accuracy and efficiency of DNA transcription. Our results revealed that 1mdA can induce multiple types of mutant transcripts and strongly inhibit DNA transcription, whereas 6mdA is a nonmutagenic DNA adduct that can exhibit a subtle but significant inhibitory effect on DNA transcription in vitro and in human cells. Moreover, our results demonstrated that the transcription-coupled nucleotide excision repair pathway is dispensable for the removal of 1mdA and 6mdA from the template DNA strand in human cells. These findings provided new important insights into the functional interplay between DNA methylation modifications and transcription in mammalian cells.

Set2 histone methyltransferase regulates transcription coupled-nucleotide excision repair in yeast.

Helix-distorting DNA lesions, including ultraviolet (UV) light-induced damage, are repaired by the global genomic-nucleotide excision repair (GG-NER) and transcription coupled-nucleotide excision repair (TC-NER) pathways. Previous studies have shown that histone post-translational modifications (PTMs) such as histone acetylation and methylation can promote GG-NER in chromatin. Whether histone PTMs also regulate the repair of DNA lesions by the TC-NER pathway in transcribed DNA is unknown. Here, we report that histone H3 K36 methylation (H3K36me) by the Set2 histone methyltransferase in yeast regulates TC-NER. Mutations in Set2 or H3K36 result in UV sensitivity that is epistatic with Rad26, the primary TC-NER factor in yeast, and cause a defect in the repair of UV damage across the yeast genome. We further show that mutations in Set2 or H3K36 in a GG-NER deficient strain (i.e., rad16Δ) partially rescue its UV sensitivity. Our data indicate that deletion of SET2 rescues UV sensitivity in a GG-NER deficient strain by activating cryptic antisense transcription, so that the non-transcribed strand (NTS) of yeast genes is repaired by TC-NER. These findings indicate that Set2 methylation of H3K36 establishes transcriptional asymmetry in repair by promoting canonical TC-NER of the transcribed strand (TS) and suppressing cryptic TC-NER of the NTS.

The Positive Enantiomer of a Novel Chiral DNA Alkylating Agent Exhibits Nanomolar Potency in Hematologic Cancers

LP-184, or (-)-hydroxyurea methylacylfulvene, is a potent DNA alkylating agent that effectively kills solid tumors. It belongs to the acylfulvene compound family known to induce DNA lesions repaired by the Transcription-Coupled Nucleotide Excision Repair (TC-NER) pathway. Here, we show that LP-284, the synthetic positive enantiomer of LP-184, exhibited the greatest and broadest hematologic cancer antiproliferative activities among the 6 acylfulvenes, including illudin S, illudin M, Irofulven (LP-100), the semisynthetic racemic LP-184, the synthetic negative enantiomer LP-184, and LP-284. The distinct pharmacological activities of LP-284 may be due to differences in metabolic activation, transport, or affinity to cellular macromolecules. To determine whether metabolic activation plays a role, we compared the correlation between the expression of Prostaglandin Reductase 1 (PTGR1), the NADPH-dependent oxidoreductase known to convert Irofulven into its active metabolite, and the IC50 of LP-184, Irofulven, and LP-284. We found that the expression level of PTGR1 is highly correlated with LP-184 (r=0.88, p=8.4e-20) and Irofulven (r=0.71, p=4.7e-10) sensitivity, but not with LP-284 (r=-0.01, p=0.93). We also found that the average expression level of PTGR1 is significantly lower in hematologic cancer cell lines (n=180) than in solid tumor cell lines (n=856), indicating the existence of an alternative LP-284 activator in hematologic cells. Next, we checked mutation status, RNA expression, protein expression, and DNA methylation of 489 oxidoreductases, but none of the enzymes was highly correlated with LP-284 activity.To further explore the potential clinical application of LP-284 in hematologic cancers, we conducted cell viability assays in 18 hematologic cancer cell lines and found that LP-284 exhibited nanomolar potency in acute lymphocytic leukemia (average IC50: 351 nM), chronic myeloid leukemia (average IC50: 360 nM), B-cell lymphoma (average IC50: 366 nM), and Multiple Myeloma (MM, IC50: 334 nM). We also investigated the therapeutic potential of LP-284 in combination with spironolactone in treating MM. Spironolactone, an FDA approved drug for hypertension, degrades one of the key TC-NER players ERCC3 in MM, which in turn makes cells more vulnerable to helix-distorting DNA lesions likely caused by LP-284. While Spironolactone alone didn't cause cytotoxicity to the MM cell line RPMI8226, it reduced LP-284 IC50 by 2.4 fold.Taken together, we have demonstrated the importance of stereochemistry in acylfulvene activity. LP-284, likely to be activated through a different route, is a unique and potent acylfulvene for hematologic cancers. Additionally, pharmacological inhibition of the TC-NER pathway greatly promoted LP-284 cytotoxicity. We hypothesize that LP-284 induces DNA lesions, which may be lethal to TC-NER deficient cells and may block transcription of short-lived fusion genes that are essential for cancer cell survival until repaired. Therefore, our discovery of the novel enantiomer LP-284 may provide a targeted therapy option for hematologic cancers with compromised DNA repair. DisclosuresZhou: Lantern Pharma: Current Employment. Biyani: Lantern Pharma: Current Employment. Kathad: Lantern Pharma: Current Employment, Current equity holder in publicly-traded company. Kulkarni: Lantern Pharma: Current Employment. McDermott: Lantern Pharma: Current Employment. Bhatia: Lantern Pharma: Current Employment.

Unraveling the antitrypanosomal mechanism of benznidazole and related 2-nitroimidazoles: From prodrug activation to DNA damage.

Nitroheterocycles represent an important class of compound used to treat trypanosomiasis. They often function as prodrugs and can undergo type I nitroreductase (NTR1)-mediated activation before promoting their antiparasitic activities although the nature of these downstream effects has yet to be determined. Here, we show that in an NTR1-dependent process, benznidazole promotes DNA damage in the nuclear genome of Trypanosoma brucei, providing the first direct link between activation of this prodrug and a downstream trypanocidal mechanism. Phenotypic and protein expression studies revealed that components of the trypanosome's homologous recombination (HR) repair pathway (TbMRE11, γH2A, TbRAD51) cooperate to resolve the benznidazole-induced damage, indicating that the prodrug-induced lesions are most likely double stand DNA breaks, while the sequence/recruitment kinetics of these factors parallels that in other eukaryotes HR systems. When extended to other NTR1-activated 2-nitroimidazoles, some were shown to promote DNA damage. Intriguingly, the lesions induced by these required TbMRE11 and TbCSB activities to fix leading us to postulate that TbCSB may operate in systems other than the transcription-coupled nucleotide excision repair pathway. Understanding how existing trypanosomal drugs work will aid future drug design and help unlock novel reactions/pathways that could be exploited as targets for therapeutic intervention.

Transcription-coupled nucleotide excision repair: New insights revealed by genomic approaches

Elongation of RNA polymerase II (Pol II) is affected by many factors including DNA damage. Bulky damage, such as lesions caused by ultraviolet (UV) radiation, arrests Pol II and inhibits gene transcription, and may lead to genome instability and cell death. Cells activate transcription-coupled nucleotide excision repair (TC-NER) to remove Pol II-impeding damage and allow transcription resumption. TC-NER initiation in humans is mediated by Cockayne syndrome group B (CSB) protein, which binds to the stalled Pol II and promotes assembly of the repair machinery. Given the complex nature of the TC-NER pathway and its unique function at the interface between transcription and repair, new approaches are required to gain in-depth understanding of the mechanism. Advances in genomic approaches provide an important opportunity to investigate how TC-NER is initiated upon damage-induced Pol II stalling and what factors are involved in this process. In this Review, we discuss new mechanisms of TC-NER revealed by genome-wide DNA damage mapping and new TC-NER factors identified by high-throughput screening. As TC-NER conducts strand-specific repair of mutagenic damage, we also discuss how this repair pathway causes mutational strand asymmetry in the cancer genome.

FACT subunit Spt16 controls UVSSA recruitment to lesion-stalled RNA Pol II and stimulates TC-NER.

Transcription-coupled nucleotide excision repair (TC-NER) is a dedicated DNA repair pathway that removes transcription-blocking DNA lesions (TBLs). TC-NER is initiated by the recognition of lesion-stalled RNA Polymerase II by the joint action of the TC-NER factors Cockayne Syndrome protein A (CSA), Cockayne Syndrome protein B (CSB) and UV-Stimulated Scaffold Protein A (UVSSA). However, the exact recruitment mechanism of these factors toward TBLs remains elusive. Here, we study the recruitment mechanism of UVSSA using live-cell imaging and show that UVSSA accumulates at TBLs independent of CSA and CSB. Furthermore, using UVSSA deletion mutants, we could separate the CSA interaction function of UVSSA from its DNA damage recruitment activity, which is mediated by the UVSSA VHS and DUF2043 domains, respectively. Quantitative interaction proteomics showed that the Spt16 subunit of the histone chaperone FACT interacts with UVSSA, which is mediated by the DUF2043 domain. Spt16 is recruited to TBLs, independently of UVSSA, to stimulate UVSSA recruitment and TC-NER-mediated repair. Spt16 specifically affects UVSSA, as Spt16 depletion did not affect CSB recruitment, highlighting that different chromatin-modulating factors regulate different reaction steps of the highly orchestrated TC-NER pathway.

Epistatic SNP interaction of ERCC6 with ERCC8 and their joint protein expression contribute to gastric cancer/atrophic gastritis risk.

Excision repair cross-complementing group 6 and 8 (ERCC6 and ERCC8) are two indispensable genes for the initiation of transcription-coupled nucleotide excision repair pathway. This study aimed to evaluate the interactions between single nucleotide polymorphisms of ERCC6 (rs1917799) and ERCC8 (rs158572 and rs158916) in gastric cancer and its precancerous diseases. Besides, protein level analysis were performed to compare ERCC6 and ERCC8 expression in different stages of gastric diseases, and to correlate SNPs jointly with gene expression. Sequenom MassARRAY platform method was used to detect polymorphisms of ERCC6 and ERCC8 in 1916 subjects. In situ ERCC6 and ERCC8 protein expression were detected by immunohistochemistry in 109 chronic superficial gastritis, 109 chronic atrophic gastritis and 109 gastric cancer cases. Our results demonstrated pairwise epistatic interactions between ERCC6 and ERCC8 SNPs that ERCC6 rs1917799-ERCC8 rs158572 combination was associated with decreased risk of chronic atrophic gastritis and increased risk of gastric cancer. ERCC6 rs1917799 also showed a significant interaction with ERCC8 rs158916 to reduce gastric cancer risk. The expressions of ERCC6, ERCC8 and ERCC6-ERCC8 combination have similarities that higher positivity was observed in chronic superficial gastritis compared with chronic atrophic gastritis and gastric cancer. As for the effects of ERCC6 and ERCC8 SNPs on the protein expression, single SNP had no correlation with corresponding gene expression, whereas the ERCC6 rs1917799–ERCC8 rs158572 pair had significant influence on ERCC6 and ERCC6-ERCC8 expression. In conclusion, ERCC6 rs1917799, ERCC8 rs158572 and rs158916 demonstrated pairwise epistatic interactions to associate with chronic atrophic gastritis and gastric cancer risk. The ERCC6 rs1917799–ERCC8 rs158572 pair significantly influence ERCC6 and ERCC6-ERCC8 expression.

Abstract C55: Nedd8-activating enzyme inhibitor pevonedistat synergizes with cisplatin and carboplatin through interference with nucleotide excision repair and interstrand cross-link repair mechanisms in non-small cell lung cancer

Abstract Platinum-based therapies, both cisplatin and carboplatin, are utilized as part of first-line standard of care regimens for advanced non-small cell lung cancer (NSCLC), but overall response rate and overall survival remain limited, with a 5-year survival rate of 18%. Therefore, agents that can improve responses and survival are needed. Pevonedistat (MLN4924) is an investigational small molecule inhibitor of the Nedd8-activating enzyme (NAE) currently in Phase Ib clinical trials. Nedd8 is a small ubiquitin-like protein whose activation of the cullin family of E3 ubiquitin ligases is dependent on NAE activity. Importantly, pevonedistat synergized with carboplatin in a cell viability assay for 6 of 20 NSCLC cell lines tested in vitro, and synergy was also detected in the triple combination of carboplatin, pevonedistat, and paclitaxel. Order of addition experiments in the A549 NSCLC cell line demonstrated a benefit of simultaneous treatment or addition of pevonedistat prior to platinum treatment, while a reduced combination benefit was observed when cisplatin was added 24-48 hrs before pevonedistat. Levels of NAE pathway inhibition were similar in pevonedistat and pevonedistat+cisplatin treated cells, indicating that a drug-drug interaction was not likely the cause of the synergy. Using patient-derived xenograft (PDX) models a combination benefit of carboplatin and pevonedistat was demonstrated in a carboplatin-insensitive model but a similar improvement in response was not observed in a carboplatin-sensitive model, adding to evidence that pevonedistat might target platinum resistance mechanisms. To evaluate the mechanism of synergy between pevonedistat and platinum, in vitro experiments with RNAi were performed in 4 cell lines. These studies identified 2 mechanisms of synergy between pevonedistat and platinums in vitro. Depletion of genes within the TC-NER (transcription-coupled nucleotide excision repair) and ICR (interstrand crosslink repair) pathways reduced the synergy between pevonedistat and platinum, with the contribution of each pathway varying by cell line. Because these pathways are also implicated in the response to single agent platinums, the results suggest pevonedistat could delay completion of platinum-induced DNA repair and therefore result in enhanced cell death. Inhibition of neddylation of the E3 ubiquitin ligase CUL4-RBX1-DDB1-ERCC8 by pevonedistat may delay the TC-NER pathway, providing a direct mechanism of resistance reversal by pevonedistat. The combination of pevonedistat with carboplatin and paclitaxel is under evaluation as part of a phase 1b trial in adult patients with solid tumors (NCT01862328). Citation Format: David C. Bouck, Khristofer Garcia, Jonathan L. Blank, Xiaozhen J. Liu, Hugues Bernard, Allison J. Berger, Mike Kuranda, Erik Koenig, Eric S. Lightcap. Nedd8-activating enzyme inhibitor pevonedistat synergizes with cisplatin and carboplatin through interference with nucleotide excision repair and interstrand cross-link repair mechanisms in non-small cell lung cancer. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2015 Nov 5-9; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2015;14(12 Suppl 2):Abstract nr C55.

Rad26, the Transcription-Coupled Repair Factor in Yeast, Is Required for Removal of Stalled RNA Polymerase-II following UV Irradiation

Transcription coupled nucleotide excision repair (TCR) is a major pathway responsible for removal of helix distorting DNA lesions from transcriptionally active regions of the genome. Rad26, a key factor of the TCR pathway, is known to play a role during early steps of TCR. Here, we show that Rad26-mediated TCR is not absolutely dependent on active transcription elongation in budding yeast. As per our results, RAD26-deleted cells show enhanced UV sensitivity compared to wild type cells under conditions where transcription elongation is inhibited. The increased UV sensitivity observed in RAD26-deleted cells, however, is not due to reduced expression of the major NER-responsive genes. Interestingly, transcription of the constitutively expressed RPB2 gene is adversely affected in RAD26-deleted cells during UV-induced DNA damage repair. In consonance, chromatin immunoprecipitation analysis showed that unlike in wild type, in RAD26-deleted cells no significant reduction in RNA polymerase II occupancy occurs during nucleotide excision repair in the transcriptionally active loci like, RPB2, PYK1 and RPL2B. These results collectively indicate that removal of RNAPII during DNA damage repair following UV irradiation is dependent on Rad26.

Nucleotide Excision Repair, Mismatch Repair, and R-Loops Modulate Convergent Transcription-Induced Cell Death and Repeat Instability

Expansion of CAG•CTG tracts located in specific genes is responsible for 13 human neurodegenerative disorders, the pathogenic mechanisms of which are not yet well defined. These disease genes are ubiquitously expressed in human tissues, and transcription has been identified as one of the major pathways destabilizing the repeats. Transcription-induced repeat instability depends on transcription-coupled nucleotide excision repair (TC-NER), the mismatch repair (MMR) recognition component MSH2/MSH3, and RNA/DNA hybrids (R-loops). Recently, we reported that simultaneous sense and antisense transcription–convergent transcription–through a CAG repeat not only promotes repeat instability, but also induces a cell stress response, which arrests the cell cycle and eventually leads to massive cell death via apoptosis. Here, we use siRNA knockdowns to investigate whether NER, MMR, and R-loops also modulate convergent-transcription-induced cell death and repeat instability. We find that siRNA-mediated depletion of TC-NER components increases convergent transcription-induced cell death, as does the simultaneous depletion of RNase H1 and RNase H2A. In contrast, depletion of MSH2 decreases cell death. These results identify TC-NER, MMR recognition, and R-loops as modulators of convergent transcription-induced cell death and shed light on the molecular mechanism involved. We also find that the TC-NER pathway, MSH2, and R-loops modulate convergent transcription-induced repeat instability. These observations link the mechanisms of convergent transcription-induced repeat instability and convergent transcription-induced cell death, suggesting that a common structure may trigger both outcomes.

Alkyltransferase-like Proteins: Brokers Dealing with Alkylated DNA Bases

A new pathway for the repair of DNA alkylation damage is described in this issue of Molecular Cell (Latypov et al., 2012). Alkyltransferase-like enzymes mark O(6)-alkylguanine lesions and, depending on adduct size, channel them into global genome or transcription-coupled nucleotide excision repair pathways.

Sapacitabine, the prodrug of CNDAC, is a nucleoside analog with a unique action mechanism of inducing DNA strand breaks.

Sapacitabine is an orally bioavailable prodrug of the nucleoside analog 2′-C-cyano-2′-deoxy-1-β-D-arabino-pentofuranosylcytosine (CNDAC). Both the prodrug and active metabolite are in clinical trials for hematologic malignancies and/or solid tumors. CNDAC has a unique mechanism of action: after incorporation into DNA, it induces single-strand breaks (SSBs) that are converted into double-strand breaks (DSBs) when cells go through a second S phase. In our previous studies, we demonstrated that CNDAC-induced SSBs can be repaired by the transcription-coupled nucleotide excision repair pathway, whereas lethal DSBs are mainly repaired through homologous recombination. In the current work, we used clonogenic assays to compare the DNA damage repair mechanism of CNDAC with two other deoxycytidine analogs: cytarabine, which is used in hematologic malignacies, and gemcitabine, which shows activity in solid tumors. Deficiency in two Rad51 paralogs, Rad51D and XRCC3, greatly sensitized cells to CNDAC, but not to cytarabine or gemcitabine, indicating that homologous recombination is not a major mechanism for repairing damage caused by the latter two analogs. This study further suggests clinical activity and application of sapacitabine that is distinct from that of cytarabine or gemcitabine.

1096 Molecular signatures of disseminated tumour cells in metastatic breast cancer patients

Gemcitabine (Gemzar; Eli Lilly and Company, Indianapolis, IN) and cisplatin are commonly used in the treatment of many solid tumors, although the impact of chemotherapy is limited in metastatic non-small cell lung cancer. However, in clinical practice, there is a minority of patients who can attain long-term survival. Upregulation of mRNA transcripts has been linked to chemoresistance, and in some instances, mRNA expression has been correlated with polymorphisms. Cisplatin resistance is directly linked to the nucleotide excision repair system, specifically to the transcription-coupled nucleotide excision repair pathway that involves genes that are deficient in rare inborn disorders such as Cockayne syndrome and xeroderma pigmentosum. Overexpression of ERCC1 correlates with poor survival in gemcitabine/cisplatin-treated non-small cell lung cancer patients. At the preclinical level, ERCC1 and XPD mRNA expression correlate with each other, and overexpression of XPD causes selective cisplatin resistance in human tumor cell lines. XPD polymorphisms have been associated with lower DNA repair capacity. In our experience, time to progression is significantly higher in gemcitabine/cisplatin-treated patients with the Lys751Gln genotype (9.6 months) than in those with the Lys751Lys genotype (4.2 months; P = .03). Other polymorphisms involved in parallel DNA repair systems may well provide the same information, indicating a high degree of biologic redundancy. The overexpression of the subunit M1 of ribonucleotide reductase (RRM1) has been linked to gemcitabine resistance in our retrospective assessment. Preliminary findings that a subset of gemcitabine/cisplatin-treated patients with low ERCC1 and RRM1 mRNA levels show a significantly longer survival and highlight the possibilities of individually tailored chemotherapy.

Diverse effects of individual mismatch repair components on transcription-induced CAG repeat instability in human cells

Several neurodegerative diseases are caused by expansion of a trinucleotide repeat tract in a critical gene. The mechanism of repeat instability is not yet defined, but in mice it requires MutSβ, a complex of MSH2 and MSH3. We showed previously that transcription through a CAG repeat tract induces repeat instability in human cells via a pathway that requires the mismatch repair (MMR) components, MSH2 and MSH3, and the entire transcription-coupled nucleotide excision repair pathway [Y. Lin, V. Dion, J.H. Wilson, Transcription promotes contraction of CAG repeat tracts in human cells, Nat. Struct. Mol. Biol. 13 (2006) 179–180; Y. Lin, J.H. Wilson, Transcription-induced CAG repeat contraction in human cells is mediated in part by transcription-coupled nucleotide excision repair, Mol. Cell Biol. 27 (2007) 6209–6217]. Here, we examine the role of downstream MMR processing components on transcription-induced CAG instability, using our selection assay for repeat contraction. In contrast to knockdowns of MSH2 or MSH3, which reduce repeat contractions, we show that siRNA-mediated depletion of MLH1 or PMS2 increases contraction frequency. Knockdown of DNMT1, which has been identified as an MMR factor in genetic studies, also elevates the frequency of contraction. Simultaneous knockdowns of MLH1 or DNMT1 along with MSH2, XPA, or BRCA1, whose individual knockdowns each decrease CAG contraction, yield intermediate frequencies. In sharp contrast, double knockdown of MLH1 and DNMT1 additively increases the frequency of CAG contraction. These results show that MMR components can alter repeat stability in diverse ways, either enhancing or suppressing CAG contraction, and they provide insight into the influence of MMR components on transcription-induced CAG repeat instability.

The Involvement of Mismatch Repair in Transcription Coupled Nucleotide Excision Repair

Nucleotide excision repair (NER) is a versatile repair pathway to remove a variety of DNA distorting lesions. NER operate via two subpathways, that are global genome repair (GGR) and transcription coupled nucleotide excision repair (TCR). GGR removes DNA damage from the genome over all, whilst TCR is selectively directed to DNA lesions in the transcribed strand of expressed genes. The damage recognition step in GGR and TCR is also different. In GGR, the XPC-HR23B complex is an essential factor to recruit proteins for subsequent process. In TCR, a stalled RNA polymerase II is a presumed trigger to initiate TCR machinery in concert with Cockayne syndrome (CS) proteins. Mismatch repair (MMR) keeps fidelity of DNA replication through correcting replication errors. A distinctive feature of MMR pathway is that this repair is directed exclusively to the newly synthesized strand. This characteristic contributes to mediation of cytotoxity by methylating agents, and MMR deficient cells are more resistant to methylating agents than MMR proficient cells. The interaction between MMR and NER has been reported by several investigators. However, the most controversial problem is the role of MMR in TCR TCR in E. coli requires the participation of the MutS and MutL MMR proteins. On the contrary, TCR in yeast is independent of the yeast MutS and MutL homologues. To date, in mammalian cells, there are conflicting evidences for the association of MMR with TCR pathway. The aim of this article is to provide a brief overview of the recent literature on this subject.

Targeted therapy in combination with gemcitabine in non-small cell lung cancer

Gemcitabine (Gemzar; Eli Lilly and Company, Indianapolis, IN) and cisplatin are commonly used in the treatment of many solid tumors, although the impact of chemotherapy is limited in metastatic non-small cell lung cancer. However, in clinical practice, there is a minority of patients who can attain long-term survival. Upregulation of mRNA transcripts has been linked to chemoresistance, and in some instances, mRNA expression has been correlated with polymorphisms. Cisplatin resistance is directly linked to the nucleotide excision repair system, specifically to the transcription-coupled nucleotide excision repair pathway that involves genes that are deficient in rare inborn disorders such as Cockayne syndrome and xeroderma pigmentosum. Overexpression of ERCC1 correlates with poor survival in gemcitabine/cisplatin-treated non-small cell lung cancer patients. At the preclinical level, ERCC1 and XPD mRNA expression correlate with each other, and overexpression of XPD causes selective cisplatin resistance in human tumor cell lines. XPD polymorphisms have been associated with lower DNA repair capacity. In our experience, time to progression is significantly higher in gemcitabine/cisplatin-treated patients with the Lys751Gln genotype (9.6 months) than in those with the Lys751Lys genotype (4.2 months; P = .03). Other polymorphisms involved in parallel DNA repair systems may well provide the same information, indicating a high degree of biologic redundancy. The overexpression of the subunit M1 of ribonucleotide reductase (RRM1) has been linked to gemcitabine resistance in our retrospective assessment. Preliminary findings that a subset of gemcitabine/cisplatin-treated patients with low ERCC1 and RRM1 mRNA levels show a significantly longer survival and highlight the possibilities of individually tailored chemotherapy.

Transcription-coupled repair removes both cyclobutane pyrimidine dimers and 6-4 photoproducts with equal efficiency and in a sequential way from transcribed DNA in xeroderma pigmentosum group C fibroblasts.

We investigated the contribution of the global and the transcription-coupled nucleotide excision repair pathway to the removal of structurally different DNA lesions. The repair kinetics of UV-induced cyclobutane pyrimidine dimers (CPDs) and pyrimidine (6-4) pyrimidone photoproducts (6-4PPs) were determined in an active and inactive gene in normal human fibroblasts and in xeroderma pigmentosum group C (XP-C) fibroblasts. Previously we have shown that in normal human cells exposed to a UV dose of 10 J/m2 repair of CPDs takes place via two pathways: global repair and transcription-coupled repair, the latter being responsible for accelerated repair of CPDs in the transcribed strand of active genes. So far, no clear evidence for transcription-coupled repair of 6-4PPs has been presented. Here we demonstrate that 6-4PPs really form a target for transcription-coupled repair. In XP-C cells, exposed to 30 J/m2 and only capable of performing transcription-coupled repair, CPDs as well as 6-4PPs are removed selectively and with similar kinetics from the transcribed strand of the adenosine deaminase (ADA) gene. The non-transcribed strand of the ADA gene and the inactive 754 gene are hardly repaired. In contrast to XP-C cells, normal cells exposed to 30 J/m2 lack strand-specific repair of both 6-4PPs and CPDs, suggesting that transcription-coupled repair is overruled by global repair, probably due to severe inhibition of transcription at this high UV dose. The much more rapid repair of 6-4PPs compared with CPDs in normal cells may be related to higher affinity of the global repair system for the former lesion. In XP-C cells the similarity of the rate of repair of both 6-4PPs and CPDs in the transcribed strand at 30 J/m2 indicates that transcription-coupled repair of photolesions takes place in a sequential way. Our results strongly suggest that the significance of transcription-coupled repair for removal of lesions depends on the type of lesion and on the dose employed.