NER-deficient Cells Research Articles

Polymerase iota plays a key role during translesion synthesis of UV-induced lesions in the absence of polymerase eta.

Xeroderma pigmentosum (XP) variant cells are deficient in the translesion synthesis (TLS) DNA polymerase Polη (eta). This protein contributes to DNA damage tolerance, bypassing unrepaired UV photoproducts and allowing S-phase progression with minimal delay. In the absence of Polη, backup polymerases perform TLS of UV lesions. However, which polymerase plays this role in human cells remains an open question. Here, we investigated the potential role of Polι (iota) in bypassing ultraviolet (UV) induced photoproducts in the absence of Polη, using NER-deficient (XP-C) cells knocked down for Polι and/or Polη genes. Our results indicate that cells lacking either Polι or Polη have increased sensitivity to UVC radiation. The lack of both TLS polymerases led to increased cell death and defects in proliferation and migration. Loss of both polymerases induces a significant replication fork arrest and G1/S-phase blockage, compared to the lack of Polη alone. In conclusion, we propose that Polι acts as a bona fide backup for Polη in the TLS of UV-photoproducts.

Ubiquitin signaling and the proteasome drive human DNA-protein crosslink repair.

DNA-protein crosslinks (DPCs) are large cytotoxic DNA lesions that form following exposure to chemotherapeutic drugs and environmental chemicals. Nucleotide excision repair (NER) and homologous recombination (HR) promote survival following exposure to DPC-inducing agents. However, it is not known how cells recognize DPC lesions, or what mechanisms selectively target DPC lesions to these respective repair pathways. To address these questions, we examined DPC recognition and repair by transfecting a synthetic DPC lesion comprised of the human oxoguanine glycosylase (OGG1) protein crosslinked to double-stranded M13MP18 into human cells. In wild-type cells, this lesion is efficiently repaired, whereas cells deficient in NER can only repair this lesion if an un-damaged homologous donor is co-transfected. Transfected DPC is subject to rapid K63 polyubiquitination. In NER proficient cells, the DPC is subject to K48 polyubiquitination, and is removed via a proteasome-dependent mechanism. In NER-deficient cells, the DNA-conjugated protein is not subject to K48 polyubiquitination. Instead, the K63 tag remains attached, and is only lost when a homologous donor molecule is present. Taken together, these results support a model in which selective addition of polyubiquitin chains to DNA-crosslinked protein leads to selective recruitment of the proteasome and the cellular NER and recombinational DNA repair machinery.

Development of Comprehensive Ultraperformance Liquid Chromatography-High-Resolution Mass Spectrometry Assays to Quantitate Cisplatin-Induced DNA-DNA Cross-Links.

Cisplatin (CP) is a common antitumor drug that is used to treat many solid tumors. The activity of CP is attributed to the formation of DNA-DNA cross-links, which consist of 1,2-intra-, 1,3-intra-, and interstrand cross-links. To better understand how each intrastrand cross-link contributes to the activity of CP, we have developed comprehensive ultraperformance liquid chromatography-selective ion monitoring (UPLC-SIM) assays to quantify 1,2-GG-, 1,2-AG-, 1,3-GCG-, and 1,3-GTG-intrastrand cross-links. The limit of quantitation for the developed assays ranged from 5 to 50 fmol or as low as 6 cross-links per 108 nucleotides. To demonstrate the utility of the UPLC-SIM assays, we first performed in vitro cross-link formation kinetics experiments. We confirmed that the 1,2-GG-intrastrand cross-links were the most abundant intrastrand cross-link and formed at a faster rate compared to 1,2-AG- and 1,3-intrastrand cross-links. Furthermore, we investigated the repair kinetics of intrastrand cross-links in CP-treated wild-type and nucleotide excision repair (NER)-deficient U2OS cells. We observed a slow decrease of both 1,2- and 1,3-intrastrand cross-links in wild-type cells and no evidence of direct repair in the NER-deficient cells. Taken together, we have demonstrated that our assays are capable of accurately quantifying intrastrand cross-links in CP-treated samples and can be utilized to better understand the activity of CP.

Elevated glucose increases genomic instability by inhibiting nucleotide excision repair.

We investigated potential mechanisms by which elevated glucose may promote genomic instability. Gene expression studies, protein measurements, mass spectroscopic analyses, and functional assays revealed that elevated glucose inhibited the nucleotide excision repair (NER) pathway, promoted DNA strand breaks, and increased levels of the DNA glycation adduct N 2 -(1-carboxyethyl)-2'-deoxyguanosine (CEdG). Glycation stress in NER-competent cells yielded single-strand breaks accompanied by ATR activation, γH2AX induction, and enhanced non-homologous end-joining and homology-directed repair. In NER-deficient cells, glycation stress activated ATM/ATR/H2AX, consistent with double-strand break formation. Elevated glucose inhibited DNA repair by attenuating hypoxia-inducible factor-1α-mediated transcription of NER genes via enhanced 2-ketoglutarate-dependent prolyl hydroxylase (PHD) activity. PHD inhibition enhanced transcription of NER genes and facilitated CEdG repair. These results are consistent with a role for hyperglycemia in promoting genomic instability as a potential mechanism for increasing cancer risk in metabolic disease. Because of the pleiotropic functions of many NER genes beyond DNA repair, these results may have broader implications for cellular pathophysiology.

UV Radiation-induced Impairment of Cellular Morphology and Motility is Enhanced by DUSP3/VHR Loss and FAK Activation.

DUSP3 is a phosphatase expressed and active in several tissues that dephosphorylates tyrosine residues in many regulatory proteins of cellular activities such as proliferation, survival, and cell death. Recently, two new independent functions were assigned to this enzyme: dephosphorylation of focal adhesion kinase (FAK) and regulation of nucleotide-excision repair (NER) pathway. Genotoxic stress by UV radiation is known to affect cell morphology, adhesion, and migration for affecting, for example, the Rho GTPases that regulate actin cytoskeleton. This work investigated the intersection of DUSP3 function, XPA protein activity, and UV toxicity by examining cell migration, FAK, and SRC kinase phosphorylation status, in addition to cell morphology, in fibroblast cells proficient (MRC-5) or deficient (XPA) of the NER pathway. DUSP3 loss reduced cell migration of normal cells, which was stimulated by the genotoxic stress, effects evidenced in presence of serum mitogenic stimulus. However, NER-deficient cells migration response was the opposite since DUSP3 loss increased migration, especially after cells being exposed to UV stress. The levels of pFAK(Y397) peaked 15 min and 1 h after UV radiation in normal cells, but only slightly increased in repair-deficient cells. However, the DUSP3 knockdown strongly raised pFAK(Y397) levels in both cells, but especially in XPA cells as supported by the higher SRC activity. These effects impacted on the dynamics of actin-based structures formation, such as stress fibres, apparently dependent on DUSP3 and DNA-repair (NER) proficiency of the cells. Altogether our findings suggest this dual-phosphatase is bridging gaps between the complex regulation of cell morphology, motility, and genomic stability.

RHOAming Through the Nucleotide Excision Repair Pathway as a Mechanism of Cellular Response Against the Effects of UV Radiation.

Typical Rho GTPases include the enzymes RhoA, Rac1, and Cdc42 that act as molecular switches to regulate essential cellular processes in eukaryotic cells such as actomyosin dynamics, cell cycle, adhesion, death and differentiation. Recently, it has been shown that different conditions modulate the activity of these enzymes, but their functions still need to be better understood. Here we examine the interplay between RhoA and the NER (Nucleotide Excision Repair) pathway in human cells exposed to UVA, UVB or UVC radiation. The results show high levels and accumulation of UV-induced DNA lesions (strand breaks and cyclobutane pyrimidine dimers, CPDs) in different cells with RhoA loss of function (LoF), either by stable overexpression of negative dominant RhoA (RhoA-N19 mutant), by inhibition with C3 toxin or by transient silencing with siRNA. Cells under RhoA LoF showed reduced levels of γH2AX, p-Chk1 (Ser345) and p-p53 (Ser15) that reflected causally in their accumulation in G1/S phases, in low survival rates and in reduced cell proliferation, also in accordance with the energy of applied UV light. Even NER-deficient cells (XPA, XPC) or DNA translesion synthesis (TLS)-deficient cells (XPV) showed substantial hypersensitivity to UV effects when previously submitted to RhoA LoF. In contrast, analyses of apoptosis, necrosis, autophagy and senescence revealed that all cells displaying normal levels of active RhoA (RhoA-GTP) are more resistant to UV-promoted cell death. This work reaffirms the role of RhoA protein signaling in protecting cells from damage caused by UV radiation and demonstrates relevant communicating mechanisms between actin cytoskeleton and genomic stability.

Topoisomerase I-driven repair of UV-induced damage in NER-deficient cells

Nucleotide excision repair (NER) removes helix-destabilizing adducts including ultraviolet (UV) lesions, cyclobutane pyrimidine dimers (CPDs), and pyrimidine (6-4) pyrimidone photoproducts (6-4PPs). In comparison with CPDs, 6-4PPs have greater cytotoxicity and more strongly destabilizing properties of the DNA helix. It is generally believed that NER is the only DNA repair pathway that removes the UV lesions as evidenced by the previous data since no repair of UV lesions was detected in NER-deficient skin fibroblasts. Topoisomerase I (TOP1) constantly creates transient single-strand breaks (SSBs) releasing the torsional stress in genomic duplex DNA. Stalled TOP1-SSB complexes can form near DNA lesions including abasic sites and ribonucleotides embedded in chromosomal DNA. Here we show that base excision repair (BER) increases cellular tolerance to UV independently of NER in cancer cells. UV lesions irreversibly trap stable TOP1-SSB complexes near the UV damage in NER-deficient cells, and the resulting SSBs activate BER. Biochemical experiments show that 6-4PPs efficiently induce stable TOP1-SSB complexes, and the long-patch repair synthesis of BER removes 6-4PPs downstream of the SSB. Furthermore, NER-deficient cancer cell lines remove 6-4PPs within 24 h, but not CPDs, and the removal correlates with TOP1 expression. NER-deficient skin fibroblasts weakly express TOP1 and show no detectable repair of 6-4PPs. Remarkably, the ectopic expression of TOP1 in these fibroblasts led them to completely repair 6-4PPs within 24 h. In conclusion, we reveal a DNA repair pathway initiated by TOP1, which significantly contributes to cellular tolerance to UV-induced lesions particularly in malignant cancer cells overexpressing TOP1.

TGF beta promotes repair of bulky DNA damage through increased ERCC1/XPF and ERCC1/XPA interaction.

Transforming growth factor beta (TGFβ) is multifunctional cytokine that is involved in the coordination and regulation of many cellular homeostatic processes. Compromised TGFβ activity has been attributed to promotion of human cancers. Recent studies have identified a role for TGFβ in response to radiation-induced DNA damage, suggesting a link between TGFβ and the DNA damage response with implications for cancer development. In this study, the effects of TGFβ on promoting the repair of bulky DNA damage, through modulation of nucleotide excision repair (NER), were investigated. We show that treatment of cells with exogenous TGFβ leads to enhanced repair of DNA damage formed by polycyclic aromatic hydrocarbons and ultraviolet-C radiation; similarly, cells with constitutively activated endogenous TGFβ signaling show comparable responses. This effect of TGFβ is independent of the cell cycle. The response to TGFβ is decreased in cells that have compromised TGFβ signaling through RNA interference of Smad4 and is decreased in NER-deficient cells and cells with compromised NER through RNA interference of excision repair cross-complementing group 1 (ERCC1). Increased interaction and nuclear localization of ERCC1/xeroderma pigmentosum (XP) F and ERCC1/XPA proteins is observed after TGFβ treatment. Our study represents the first experimental evidence of a role for TGFβ in the repair of bulky DNA damage resulting from promotion of the interaction and localization of repair protein complexes involved in the incision step of NER.

NDRG1 disruption alleviates cisplatin/sodium glycididazole-induced DNA damage response and apoptosis in ERCC1-defective lung cancer cells.

Resistance to platinum-based chemotherapy becomes a major obstacle in lung cancer treatment. Compensatory activation of nucleotide excision repair (NER) pathway is the major mechanism accounting for cisplatin-resistance. We aimed at identifying additional regulators in NER-mediated chemoresistance in a hypoxic setting induced by sodium glycididazole (CMNa)-sensitized cisplatin chemotherapy of non-small cell lung cancer (NSCLC). We performed an RNA-sequencing (RNA-Seq) analysis to identify the genes whose expression had been differentially regulated in NER-deficient cells that had been treated by cisplatin/CMNa. DNA damage, apoptosis, and correlational analysis between the differentially expressed gene and drug sensitivity were determined by Western blots, flow cytometry and Oncomine expression analysis. The stress response gene, NDRG1 (N-Myc downstream-regulated gene 1), was among the differentially expressed genes in NER-deficient cells upon treatment of cisplatin/CMNa. Downregulation of NDRG1 by ERCC1 (excision repair cross-complementing 1) could be a prevalent mechanism for drug resistance. Furthermore, lower NDRG1 level is observed in human lung cancer cells showing chemotherapeutic drug resistance compared with the drug-sensitive cells. NDRG1 is an important modulator linking DNA damage response and hypoxia-related cellular stress response during the development of drug resistance to cisplatin/CMNa in lung cancer. Targeting both NDRG1 and ERCC1 may be a viable strategy for overcoming drug resistance in cancer therapy, and has significant clinical implications.

Repair of UV-Induced DNA Damage Independent of Nucleotide Excision Repair Is Masked by MUTYH

DNA lesions caused by UV damage are thought to berepaired solely by the nucleotide excision repair (NER) pathway in human cells. Patients carrying mutations within genes functioning in this pathwaydisplay a range of pathologies, including an increased susceptibility to cancer, premature aging, and neurological defects. There are currently no curative therapies available. Here we performed a high-throughput chemical screen for agents that could alleviate the cellular sensitivity of NER-deficient cells to UV-induced DNA damage. This led tothe identification of the clinically approved anti-diabeticdrug acetohexamide, which promoted clearance of UV-induced DNA damage without the accumulation of chromosomal aberrations, hence promoting cellular survival. Acetohexamide exerted this protective function by antagonizing expressionof the DNA glycosylase, MUTYH. Together, our data reveal the existence of an NER-independent mechanism to remove UV-induced DNA damage and prevent cell death.

An assay to detect DNA-damaging agents that induce nucleotide excision-repairable DNA lesions in living human cells

Biochemical risk assessment studies of chemicals that induce DNA lesions are important, because lesions in genomic DNA frequently result in cancer, neurodegeneration, and aging in humans. Many classes of DNA lesions induced by chemical agents are eliminated via DNA repair mechanisms, such as nucleotide excision repair (NER) and base excision repair (BER), for the maintenance of genomic integrity. Individuals with NER-defective xeroderma pigmentosum (XP), in which bulky DNA lesions are not efficiently removed, are cancer-prone and suffer neurodegeneration. For research into cancer and neurological diseases, therefore, it might be important to identify DNA damage from agents that induce NER-repairable bulky DNA lesions. However, simple and quick assays to detect such damaging agents have not been developed using human cells. Here, we report a simple, non-isotopic assay for determining DNA damaging agents that induce NER-repairable DNA lesions by visualizing gene expression from treated fluorescent protein vectors in a mammalian cell system. This assay is based on a comparison of fluorescent protein expression in NER-proficient and NER-deficient cells. When we tested UV-irradiated fluorescent protein vectors, the fluorescent protein was observed in NER-proficient cells, but not in NER-deficient cells. Similar results were obtained for vectors treated with the anticancer drug, cisplatin. In contrast, when treated with the DNA alkylating agent methyl methanesulfonate, believed to cause BER-repairable damage, no difference in gene expression between NER-proficient and NER-deficient cells was observed. These results suggest that our assay can specifically detect DNA-damaging agents that induce NER-repairable DNA lesions, and could be used to analyze chemicals with the potential to cause cancer and neurological diseases. With further validation, the assay might be also applicable to XP diagnosis.

The Nucleotide Excision Repair Pathway Limits L1 Retrotransposition.

Long interspersed elements 1 (L1) are active mobile elements that constitute almost 17% of the human genome. They amplify through a “copy-and-paste” mechanism termed retrotransposition, and de novo insertions related to these elements have been reported to cause 0.2% of genetic diseases. Our previous data demonstrated that the endonuclease complex ERCC1-XPF, which cleaves a 3′ DNA flap structure, limits L1 retrotransposition. Although the ERCC1-XPF endonuclease participates in several different DNA repair pathways, such as single-strand annealing, or in telomere maintenance, its recruitment to DNA lesions is best characterized in the nucleotide excision repair (NER) pathway. To determine if the NER pathway prevents the insertion of retroelements in the genome, we monitored the retrotransposition efficiencies of engineered L1 elements in NER-deficient cells and in their complemented versions. Core proteins of the NER pathway, XPD and XPA, and the lesion binding protein, XPC, are involved in limiting L1 retrotransposition. In addition, sequence analysis of recovered de novo L1 inserts and their genomic locations in NER-deficient cells demonstrated the presence of abnormally large duplications at the site of insertion, suggesting that NER proteins may also play a role in the normal L1 insertion process. Here, we propose new functions for the NER pathway in the maintenance of genome integrity: limitation of insertional mutations caused by retrotransposons and the prevention of potentially mutagenic large genomic duplications at the site of retrotransposon insertion events.

DNA damage and repair kinetics of the Alternaria mycotoxins alternariol, altertoxin II and stemphyltoxin III in cultured cells

The Alternaria mycotoxins alternariol (AOH) and altertoxin II (ATX II) have previously been shown to elicit mutagenic and genotoxic effects in bacterial and mammalian cells, although with vastly different activities. For example, ATX II was about 50 times more mutagenic than AOH. We now report that stemphyltoxin III (STTX III) is also highly mutagenic. The more pronounced effects of the perylene quinones ATX II and STTX III at lower concentrations compared to the dibenzo-α-pyrone AOH indicate a marked dependence of the genotoxic potential on the chemical structure and furthermore suggest that the underlying modes of action may be different. We have now further investigated the type of DNA damage induced by AOH, ATX II and STTX III, as well as the repair kinetics and their dependence on the status of nucleotide excision repair (NER).DNA double strand breaks induced by AOH due to poisoning of topoisomerase IIα were completely repaired in less than 2h. Under cell-free conditions, inhibition of topoisomerase IIα could also be measured for ATX II and STTX III at low concentrations, but the perylene quinones were catalytic inhibitors rather than topoisomerase poisons and did not induce DSBs. DNA strand breaks induced by ATX II and STTX III were more persistent and not completely repaired within 24h. A dependence of the repair rate on the NER status could only be demonstrated for STTX III, resulting in an accumulation of DNA damage in NER-deficient cells. Together with the finding that the DNA glycosylase formamidopyrimidine-DNA glycosylase (Fpg), but not T4 endonuclease V, is able to generate additional DNA strand breaks measurable by the alkaline unwinding assay, we conclude that the genotoxicity of the perylene quinones with an epoxide group is probably caused by the formation of DNA adducts which may be converted to Fpg sensitive sites.

Recombination between homologous chromosomes induced by unrepaired UV-generated DNA damage requires Mus81p and is suppressed by Mms2p.

DNA lesions caused by UV radiation are highly recombinogenic. In wild-type cells, the recombinogenic effect of UV partially reflects the processing of UV-induced pyrimidine dimers into DNA gaps or breaks by the enzymes of the nucleotide excision repair (NER) pathway. In this study, we show that unprocessed pyrimidine dimers also potently induce recombination between homologs. In NER-deficient rad14 diploid strains, we demonstrate that unexcised pyrimidine dimers stimulate crossovers, noncrossovers, and break-induced replication events. The same dose of UV is about six-fold more recombinogenic in a repair-deficient strain than in a repair-proficient strain. We also examined the roles of several genes involved in the processing of UV-induced damage in NER-deficient cells. We found that the resolvase Mus81p is required for most of the UV-induced inter-homolog recombination events. This requirement likely reflects the Mus81p-associated cleavage of dimer-blocked replication forks. The error-free post-replication repair pathway mediated by Mms2p suppresses dimer-induced recombination between homologs, possibly by channeling replication-blocking lesions into recombination between sister chromatids.

48 Trabectedin and lurbinectedin are effective against leukemic cells derived from patients affected by chronic and juvenile myelomonocytic leukemia

RNA Pol II in global NER (XPC) deficient cells, but failed to do it in TC NER (CSB, XPD and XPG) deficient cells. Importantly, these effects were confirmed to be specific for the Rpb1 subunit of RNA Pol II, since other subunits were not affected (Rpb2 and Rpb4) as well as other factors of the transcriptional machinery, such as TBP (TFIID), p62 (TFIIH), XPD or the RPA194 subunit of the RNA Pol I. Finally, it was also demonstrated that, contrary to what occurs after DNA damage with UV light, the transcription of p53 target genes important for DNA repair, including p21 or mdm2, was irreversibly inhibited after PM01183 treatment. Together, these results show the mechanism by which PM01183 inhibits trans-activated transcription process on tumor cells.

Abstract 5365: Cdt2-mediated XPG degradation promotes DNA repair synthesis following DNA damage excision in nucleotide excision repair

Abstract Xeroderma pigmentosum group G (XPG) protein is a structure-specific repair endonuclease, which cleaves DNA strands on the 3′ side of the DNA damage during nucleotide excision repair (NER). In addition, XPG plays a crucial role in initiating DNA repair synthesis through recruitment of PCNA to the repair sites. However, the fate of XPG protein subsequent to the excision of DNA damage has remained unresolved. Here, we show that XPG is degraded through proteasome-mediated proteolysis upon induction of bulky lesions from exposures to UV irradiation and cisplatin. NER process is required for XPG degradation because both UV and cisplatin treatment-induced XPG degradation is compromised in NER-deficient XP-A, XP-B, XP-C, and XP-F cells. In addition, the NER-related XPG degradation requires Cdt2, a component of an E3 ubiquitin ligase, CRL4Cdt2. Micropore local UV irradiation and in situ Proximity Ligation assays demonstrated that Cdt2 is recruited to the UV-damage sites and interacts with XPG in the presence of PCNA. Importantly, Cdt2-mediated XPG degradation is crucial to the subsequent recruitment of DNA polymerase δ and DNA repair synthesis. Collectively, our data supports the idea of PCNA recruitment to damage sites in conjunction with XPG, recognition of the PCNA-bound XPG by CRL4Cdt2 for specific ubiquitylation and protein degradation. Thus, XPG removal clears the space needed at the damage site for the subsequent recruitment of DNA pol δ and initiation of DNA synthesis. (This work was supported by grants from NIH). Note: This abstract was not presented at the meeting. Citation Format: Chunhua Han, Ran Zhao, Jiang Qian, Nidhi Sharma, Gulzar Wani, Jinshan He, Qianzheng Zhu, Qi-En Wang, Altaf A. Wani. Cdt2-mediated XPG degradation promotes DNA repair synthesis following DNA damage excision in nucleotide excision repair. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 5365. doi:10.1158/1538-7445.AM2014-5365

Abstract P3-04-08: A proteo-probe for nucleotide excision repair in breast cancers

Abstract The response of tumor cells to DNA damaging agents is often compromised, a weakness readily exploited for therapeutic purposes. Despite the general use of genotoxic treatments, the nature and extent of DNA damage in tumor cells is generally under-investigated, or remains unknown. We postulate DNA repair deficient breast tumor cells exhibit significantly more DNA damage after genotoxic treatments, compared to repair proficient cells. We present a technology to detect specific DNA damage and monitor repair as a basis to categorize cancers into homogenous groups of malignancies. We rely on the use of biochemically purified DNA damage recognition complexes as ready-to-use reagents, or “proteo-probes” for DNA damage. We developed a proteo-probe based on a purified DDB2 protein complex. DDB2 is a DNA damage recognition protein part of the nucleotide excision repair (NER) that recognizes and repairs bulky DNA adducts caused for example by UV irradiation or cisplatin. We show the DDB2 proteo-probe binds to nuclei of UV-irradiated cells. We followed the DDB2 proteo-probe signal over time, and observed a decrease concordant with the completion of NER. We then successfully distinguished repair proficient cells from genetically characterized NER-deficient cells obtained from patients. We conclude the DDB2 proteo-probe can be used to detect activation of NER by UV-induced DNA damage, and subsequent repair in situ. It has been proposed that some breast cancers carry NER deficiencies. We screened a panel of cancer cell lines using the DDB2 proteo-probe and discovered obvious NER deficient (NER-D) cell lines. In addition, we correlated NER activity with cisplatin sensitivity. We further determined whether NER deficiency in breast cancer cell lines was caused by loss-of-function of NER genes through complementation. The classification of breast cancer cell lines based on NER activity presented here suggests more investigation is needed about NER in breast cancers. Ultimately, we envision NER deficiencies could be used to select breast cancer patients for DNA-repair targeted therapy. Citation Information: Cancer Res 2013;73(24 Suppl): Abstract nr P3-04-08.

Abstract 5558: Lurbinectedin (PM01183) specifically targets RNA Pol II for degradation via the proteasome pathway in a TC-NER-dependent fashion.

Abstract Lurbinectedin (PM01183), a new synthetic tetrahydroisoquinoline alkaloid, is a minor groove specific DNA binder that interacts directly with specific factors involved in DNA repair and transcription pathways. In living cells, PM01183-DNA adducts stall replication and transcription giving rise to double strand breaks, inducing accumulation of cells in the S-phase of the cell cycle and triggering apoptosis. In both the ongoing phase II trials (pancreas and platinum-resistant ovarian cancer) the first stage was concluded with positive results in clinical activity, ensuring the continuity of the studies. Also, phase I trials in combination with doxorubicin and gemcitabine as well as in advanced acute leukemia are ongoing. Here, we examined the effects of PM01183 on the activity and stability of the RNAPol II as well as on other factors of the transcriptional machinery, including TBP (TFIID), p62 (TFIIH), XPG and XPF. Our results showed that PM01183 induced a rapid, time- and concentration-dependent degradation of RNA Pol II in a panel of different human tumor cell lines, including HCT-116 (colon), A549 (NSCLC), HeLa (cervix) and A763 (sarcoma). This degradative process was efficiently abrogated in the presence of transcriptional (DRB or Actinomycin D) or proteasome inhibitors (MG132), demonstrating that PM01183 specifically targets the transcriptionally active RNA Pol II for degradation via the proteasome pathway. In addition, it was also shown that the effect of PM01183 on the RNA Pol II was dependent on the presence of a functional TC NER repair machinery. PM01183 induced degradation of RNA Pol II in global NER (XPC) deficient cells, but failed to do it in TC NER (CSB, XPD and XPG) deficient cells. Importantly, these effects were confirmed to be specific for the RNA Pol II, since other factors of the transcriptional machinery, such as TBP (TFIID), p62 (TFIIH), XPG or XPF or the RPA194 subunit of the RNA Pol I were not affected. Together, these results show up the complex mechanism of action of the drug on tumor cells. Citation Format: Gema Santamaria, Juan F. Martinez-Leal, Carmen Cuevas, Luis F. Garcia-Fernandez, Carlos M. Galmarini. Lurbinectedin (PM01183) specifically targets RNA Pol II for degradation via the proteasome pathway in a TC-NER-dependent fashion. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 5558. doi:10.1158/1538-7445.AM2013-5558

Nucleotide excision repair activity on DNA damage induced by photoactivated methylene blue

The nucleotide excision repair (NER) mechanism is well known to be involved in the removal of UV-induced lesions. Nevertheless, the involvement of this pathway in the repair of lesions generated after DNA oxidation remains controversial. The effects of visible-light-excited methylene blue (MB), known to generate reactive oxygen species (ROS), were examined directly in xeroderma pigmentosum (XP)-A and XP-C NER-deficient human fibroblasts. Initially, MB was confirmed as being incorporated in similar amounts by the cells and that its photoexcitation induces the generation of 1O2 within cells. The analysis of cell survival indicated that NER-deficient cells were hypersensitive to photoactivated MB. This sensitivity was confirmed with cells silenced for the XPC gene and by host-cell reactivation (HCR) of plasmid exposed to the photosensitizing effects of photoexcited MB. The sensitivity detected by HCR was restored in complemented cells, confirming the participation of XPA and XPC proteins in the repair of DNA lesions induced by photosensitized MB. Furthermore, DNA damage (single- and double-strand breaks and alkali-sensitive sites) was observed in the nuclei of treated cells by alkaline comet assay, with higher frequency of lesions in NER-deficient than in NER-proficient cells. Likewise, NER-deficient cells also presented more γ-H2AX-stained nuclei and G2/M arrest after photoactivated MB treatment, probably as a consequence of DNA damage response. Notwithstanding, the kinetics of both alkali- and FPG-sensitive sites repair were similar among cells, thereby demonstrating not only that MB photoexcitation generates nuclear DNA damage, but also that the removal of these lesions is NER-independent. Therefore, this work provides further evidence that XPA and XPC proteins have specific roles in cell protection and repair/tolerance of ROS-induced DNA damage. Moreover, as XPC-deficient patients do not present neurodegeneration, premature aging, or developmental clinical symptoms, the results indicate that defects in the repair/tolerance of oxidatively generated DNA lesions are not sufficient to explain these severe clinical features of certain XP patients.

222 Lurbinectedin (PM01183): Pharmacokinetics/Pharmacodynamic (PK/PD) Properties in Pancreas, Ovarian and NSCLC Xenografts

breast and platinum-resistant/refractory ovarian) clinical evaluation. In vitro studies demonstrate that lurbinectedin causes delayed progression of S phase and cycle arrest in G2M. Also, lurbinectedin was 3−4 time less potent in NER deficient cells whereas it was 100 time more potent in HR deficient cells. The in vivo antitumor activity of lurbinectedin was evaluated in human ovarian carcinoma models, growing subcutaneously (OCX) or in the peritoenal cavity (IPX). Material and Methods: The in vivo pharmacological behavior of lurbinectedin was evaluated in M5076 (murine reticulosarcoma), MN-MCA1 (murine fibrosarcoma), 2 human OCX (HOC 18 and MNB-PTX-1) and 2 IPX (HOC8 and HOC22). Lurbinectedin was given i.v. at 0.2 mg/kg/day on days 0, 7, 14 (q7d×3). The antitumor effect was calculated either by T/C (as a percentage of the change in tumor size for treated [T] and placebo [C]) or by the increase of like span (ILS) (as 100·[(T − C)/C], where ‘T’ and ‘C’ are the median survival time of the treated and placebo); treatment-related anti-metastic activity was also evaluated. Results: In M5076 lurbinectedin was only marginally active (T/C, 73.4%) against the primary tumor whereas in MN-MCA1 demonstrated a good antitumor activity (best T/C 15%). In both tumor models lurbinectedin produced a striking antimetastatic effect (p < 0.001). Lurbinectedin treatment resulted in no antitumor activity in the MNB-PTX-1 model. In animals bearing OCX HOC18 tumors, lurbinectedin treatment induced a strong and long-lasting antitumor effect (minimal T/C, 13% Day 16). Tumor volume were reduced (P < 0.01) compared to placebo on days 10−17. Also, lurbinectedin treatment reduced tumor growth rate: all animals were sacrificed due to tumor volume on Day 63, later than placebo (Day 15). Lurbinectedin treatment of HOC8 IPX xenografts resulted in (P < 0.05) antitumor activity compared to untreated animals (ILS % of 140.0). Eight out of 9 of mice bearing HOC22 IPX tumors and treated with lurbinectedin survived 4 months after the death of the last placebo-treated animal and, then considered as tumor-free. Conclusions: The in vivo antitumor effect of lurbinectedin was demonstrated in a panel of human and murine experimental models, characterized by different behaviors and drug sensitivity.