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Figure S1. Structures of (A) PM030779 and (B) PM120306; both compounds are analogues of lurbinectedin. (C) Antiproliferative activity of PM120306 in A549 lung cancer cell line. Cell viability was analysed 72 h post lurbinectedin treatment by MTT assay. IC50 value was determined as being 49.9 nM (D) Western blot analyses of A549 cell extracts after (1h and 4h) treatment with either lurbinectedin (Lur, 30nM) or the biotinylated analog (PM120306, 30nM); NT: non-treated cells. The same antibody has been used to detect the hyper-phosphorylated (IIo) and hypo-phosphorylated (IIa) forms of the Rpb1 subunit of Pol II; β-Tubulin: Tub.

Figure S2. Lurbinectedin inhibits RNA synthesis and promotes Pol II degradation in different cancer cell lines. (A) Kinetics of RNA synthesis. Ewing sarcoma (A673), colon (HCT-116), cervix (HeLa) and breast (MDA-MB-231) cancer cells were treated with lurbinectedin or vehicle (DMSO) in normal growth medium for 0, 30, 45, 60, 90 and 120 minutes and pulsed with 5µCi/ml [3H] uridine for 30min. Data are mean {plus minus} SEM values (triplicates, n=1). (B) Western blot analyses of Pol II in the same panel of cancer cell lines after 1, 2, 3 or 4 hours of lurbinectedin (30nM) treatment.

Figure S3. NER machinery remains active in the presence of DRB. A dual incision assay (48) was performed by the co-incubation of NER (XPC, TFIIH, XPA, RPA, XPG and XPF) components with increasing amount of DRB (10, 20, 50 and 100µM).

Supplementary Figure legends

Figure S4. Proposed mechanism of action of lurbinectedin Upon administration to cancer cells, (I) lurbinectedin targets GC rich regions; (II) Pol II starts the elongation phase of transcription of activated genes after being sequentially phosphorylated by CDK7 (TFIIH) and CDK9 (pTEFb), (III) Pol II might be blocked by lurbinectedin and recruits DNA repair factors; (IV and V) the ubiquitinated Pol II is being degraded by ubiquitin proteasome machinery; (VI) the elongating Pol II stalled in front of the lesion induces DNA breaks that could have been originated from the activity of endonucleases such as XPF and/or Topo IIâ^�.

Growth inhibitory activity of lurbinectedin on different cancer cell lines.

Athletes incorporate altitude training programs into their conventional training to improve their performance. The purpose of this study was to determine the effects of an 8-week altitude training program that was supplemented with intermittent hypoxic training (IHE) on the blood biomarkers, sports performance, and safety profiles of elite athletes. In a single-blind randomized clinical trial that followed the CONSORT recommendations, 24 male athletes were randomized to an IHE group (HA, n = 12) or an intermittent normoxia group (NA, n = 12). The IHE consisted of 5-min cycles of hypoxia–normoxia with an FIO2 of between 10–13% for 90 min every day for 8 weeks. Hematological (red blood cells, hemoglobin, hematocrit, hematocrit, reticulated hemoglobin, reticulocytes, and erythropoietin), immunological (leukocytes, monocytes, and lymphocytes), and renal (urea, creatinine, glomerular filtrate, and total protein) biomarkers were assessed at the baseline (T1), day 28 (T2), and day 56 (T3). Sports performance was evaluated at T1 and T3 by measuring quadriceps strength and using three-time trials over the distances of 60, 400, and 1000 m on an athletics track. Statistically significant increases (p < 0.05) in erythropoietin, reticulocytes, hemoglobin, and reticulocyte hemoglobin were observed in the HA group at T3 with respect to T1 and the NA group. In addition, statistically significant improvements (p < 0.05) were achieved in all performance tests. No variations were observed in the immunological or renal biomarkers. The athletes who were living and training at 1065 m and were supplemented with IHE produced significant improvements in their hematological behavior and sports performance with optimal safety profiles.

Background - SCLC is the most aggressive lung cancer type and with the worst prognosis. There are four molecular subtypes based on the high expression of distinct transcription factors and with different therapeutic vulnerabilities. However, all share transcriptional addiction as pathogenic mechanism. Lurbinectedin is a novel oncogenic transcription inhibitor, approved in the United States and other countries for the treatment of adult patients with metastatic SCLC with disease progression on or after platinum-based chemotherapy. The aim of this study was to analyze the activity of lurbinectedin in the different SCLC molecular subtypes and to investigate new biomarkers of response. Methods and Results - We have characterized a panel of 20 SCLC human cell lines based on the expression of ASCL1, NEUROD1, YAP1 and POU2F3, where lurbinectedin yielded a mean IC50 value of 6.52 nM, which is considerable greater than the activity exerted by topotecan, irinotecan, carboplatin, etoposide, olaparib, alisertib and navitoclax (IC50 100 µM-100 nM). Lurbinectedin potency is high in the four molecular subtypes, with IC50 values of 4.1, 14.9, 7.2, and 0.2 nM for SCLC-A, -N, -I and -P, respectively. However, high expression of POU2F3 correlated with better responses. In fact, mean IC50 was 0.28 nM for POU2F3high cells versus 12.1 nM for all POU2F3low cells (p=0.0443). Additionally, basal SLFN11 levels were evaluated, observing that SLFN11high cells responded better to lurbinectedin, with IC50 values of 1.1 nM versus 11.8 nM for SLFN11low (p=0.05). Likewise, lurbinectedin treatment induced greater antitumor activity in SLFN11high tumor-bearing mice (H526: T/C, 18% on day 11) than in SLFN11low tumor-bearing mice (H82: T/C, 65% on day 7) after intravenous treatment at 0.18 mg/kg (on days 0, 7 and 14). Finally, SLFN11 expression was evaluated by IHC in FFPE tumor samples from SCLC patients participating in a multicenter phase II clinical trial in advanced solid tumors (NCT02454972), which allowed lurbinectedin accelerated approval in this indication by FDA. Patients with higher expression of SLFN11 had a slightly better overall survival (OS at 6 months: 68.4% ( Conclusions - Lurbinectedin is highly effective in all molecular subtypes of SCLC in vitro and in vivo, with IC50 values at least two logs more potent than for other antitumoral agents and its activity is even greater in tumors with high POU2F3 expression and/or high SLFN11 expression. Citation Format: Marta Martínez Diez, Gema Santamaría Nuñez, María José Guillén, Daniel Rueda, Eva Maria Garrido-Martin, Pablo Avilés, Carmen Cuevas. Lurbinectedin shows potent activity in all four molecular subtypes of small cell lung cancer (SCLC) and POU2F3 and SLFN11 are biomarkers for a better response. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 6247.

Background: Microtubule targeting agents have demonstrated to be very effective antitumoral drugs. The development of novel anti-tubulin agents with more efficient mechanisms of action presents several challenges due to their poor solubility, troublesome synthesis or purification, and toxicities. In this work, we present the novel anti-tubulin agent PM534, a synthetic small molecule that shows efficient antitumoral and antiangiogenic properties in vitro and in vivo. Methods and Results: PM534 exhibits a potent antitumor activity in vitro with a mean GI50 value in the low nanomolar range in several different human cancer cell lines. Washout in vitro experiments show irreversible cellular effects after 1h in contact with the compound, compelling the cells to apoptotic cell death likely due to PM534 induced disorganization of the tubulin cytoskeleton. PM534 inhibits migration and invasion of tumor cells in vitro, and arrests the cell cycle in the G2/M phase, forcing them to apoptosis. In addition, PM534 presents potent antiangiogenic effects in vitro, robustly inhibiting endothelial cells proliferation and migration, and the formation of angiotubes. The in vivo antitumor activity of PM534 was characterized in human-derived tumors xenografted in athymic nu/nu mice, namely breast (MDA-MB-231), and pancreas (Mia-Paca-2). PM534 was intravenously administered once per week for three consecutive weeks at 5.0 mg/kg. In vivo PM534-induced antitumor activity (vs. placebo) was seen in MDA-MB-231 (T/C, 0.3% on Day 28; TV, p Conclusions: Based on in vitro activity against different human tumor cell lines, in vivo activity in xenografted human tumors, as well as on its anti-angiogenic properties, the safety, pharmacology and preliminary antitumor activity of PM534 will be evaluated in a first-in-human clinical trial to be conducted in patients with advanced solid tumors. Citation Format: Marta Martínez Diez, María José Muñoz-Alonso, Gema Santamaría Nuñez, María José Guillén, María Ángela Oliva, Eva Maria Garrido-Martin, Pablo Avilés, J. Fernando Díaz, Carmen Cuevas. The novel antitubulin agent PM534 exhibits potent antitumoral and antiangiogenic properties in vitro and in vivo. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 6243.

Background - Ecubectedin (PM14) is a novel transcriptional inhibitor related to ecteinascidins family. In this work, we present its antiproliferative activity and mechanism of action. The in vitro antitumor activity obtained in cellular models has been also demonstrated in in vivo models. Methods and results - The antineoplastic activity of ecubectedin was evaluated in a panel of cell lines representative of different solid human cancers, by MTT assays. Ecubectedin exhibits potent antitumor in vitro activity with mean GI50 values in the very low nanomolar range. Electrophoretic Mobility Shift assays demonstrate that ecubectedin binds to DNA and experiments of 3H-uridine incorporation show that the drug efficiently inhibits mRNA synthesis (85% reduction after 90 min). Moreover, ecubectedin specifically inhibits transactivated transcription, as evaluated in a reporter system of the transcriptional activity mediated by NF-κB after activation with TNFα. In the presence of ecubectedin, transactivation was almost completely abolished. The mechanism of transcriptional inhibition involves the stalling and irreversible proteasomal degradation of elongating RNA Pol II. As a final event, ecubectedin induces double strand breaks in the DNA, inducing cell cycle arrest in the S-phase that can be followed through flow cytometry and triggers apoptotic death of the tumor cell as seen through Annexin V flow cytometry assays. The in vivo antitumor activity of ecubectedin was characterized in xenografted tumors of human origin in athymic nu/nu mice, namely breast (MDA-MB-231), soft tissue sarcoma (HT-1080), SCLC (H526 and H82) and prostate (22Rv1). Ecubectedin was intravenously administered (at 1.25 mg/kg) on days 0, 7 and 14. Compared to placebo, ecubectedin treatment of mice bearing xenografts resulted in a statistically significant reduction of volume in MDA-MB-231 (p≤0.0005 on days 7-35), HT-1080 (p≤0.0004 on days 2-14), H82 (p≤0.0001 on days 5-14), H526 (p≤0.0001 from day 4 to the end of the experimental period), and 22Rv1 (p≤0.0001 on days 3-14) tumors. In mice bearing MDA-MB-231, H526 and 22Rv1 tumors, complete tumor remissions were seen in 1/10 (lasting 103 days), 9/10 (lasting 220 days) and 7/10 (lasting 13 days) ecubectedin-treated animals, respectively. Conclusions - Ecubectedin is a novel transcriptional inhibitor that displays potent antiproliferative activity in different human solid cancer models in vitro and in vivo. The drug is currently in clinical development in Phase II trials. Citation Format: Gema Santamaría Nuñez, Marta Martínez Diez, María José Guillén, Eva Maria Garrido-Martin, Pablo Avilés, Carmen Cuevas. Ecubectedin is a novel transcriptional inhibitor that displays potent antitumor effects in vitro and in vivo [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 1622.

Background: Microtubule targeting compounds are a successful class of anticancer agents in the clinic. Although highly potent, the currently approved antitumor agents targeting tubulin present some drawbacks such as the development of acquired resistances, which remain an obstacle for an effective prolonged anticancer treatment. In this work, we present a novel microtubule destabilizing agent with an optimized interaction for the colchicine site and a novel mechanism of action which confers it a high affinity binding for tubulin. Methods and Results: We have determined the X-ray crystallographic structure of the complex T2R-TTL with PM534 at a resolution of 2.45 Å. We unequivocally found PM534 ligand density at the intra-dimer interface between α and β tubulin of both tubulin dimers (the colchicine domain) within the complex. The colchicine domain of tubulin can be divided into three zones: a central pocket and two accessories. Whereas other colchicine site-ligands only bind to two of the zones, we found that PM534 binds extensively along the three zones, making multiple contacts with β-tubulin N terminal domain, central hinge helix, and the intermediate domain. PM534 interaction did not affect the overall curved conformation of tubulin and its mechanism of action consists on precluding the curve-to-straight conformational transition required for tubulin to assemble intro microtubules and thus, to perform its cellular functions. In competition assays we found that the compound displaces a bona fide high-affinity colchicine-probe with a binding affinity of 5.1±0.3 × 107 M−1 at 25̊C. Moreover, PM534 shows potent antineoplastic activity in vitro, with GI50 values in the low nanomolar range, observed in different human tumor cancer cell lines. An in vivo Proof of the Concept (PoC) was performed in athymic nu/nu mice bearing H460 (NSCLC) tumors that were treated (intravenous, on days 0, 7, 14) with different doses (ranging from 0.75 mg/kg to 2.5 mg/kg) of PM534. Results demonstrated strong, dose-related PM534-induced antitumor activity in this in vivo model. Conclusions: The mechanism of action of PM534 involves an optimized interaction with the colchicine site, which is reflected in its high affinity for the substrate, being this the ultimate reason for the observed cellular activity. Additionally, PM534 presents a potent antitumor activity in vitro that was translated into a clear positive in vivo PoC that resulted in strong antitumor effect. Citation Format: Maria Ángela Oliva, Beatriz Álvarez-Bernad, Daniel Lucena-Agell, Marta Martínez Diez, María José Guillén, Gema Santamaría Nuñez, María José Muñoz-Alonso, Eva M. Garrido-Martin, Pablo Avilés, Carmen Cuevas, J. Fernando Díaz. PM534 is a novel microtubule-destabilizing agent with high affinity and potent antineoplastic properties. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 6239.

Two new bromotyrosine derivatives, anomoian B (1) and aplyzanzine B (2), were isolated, respectively, from the organic extracts of a Verongida sponge belonging to the Hexadella genus and from a two-sponge association (Jaspis sp. and Bubaris sp.), both collected off the coast of Indonesia. The planar structure of 1 and 2 was determined by 1D and 2D NMR experiments and by high-resolution mass spectrometry, while their absolute stereochemistry was assigned by comparison with optical rotation values of known bromotyrosines and by chemical degradation. Both compounds showed moderate antiproliferative activity against a panel of different cancer cell lines. Their cytotoxic activity is facilitated through the induction of apoptosis, which is mediated neither by the generation of reactive oxygen species nor by the inhibition of histone deacetylases in these cell lines.

Focusing on medieval Nepal, in this chapter Yogesh Raj argues that existing sociohistorical accounts of lynching and other extreme forms of collective cruelty are inadequate for developing “a credible historical account” due to what he considers their “narratological bias.” Raj asserts that the problematic nature of the Event Catalogues, the main methodological innovation in these accounts composed by scholars of European rioting and American lynching, “call for a radically different approach to historiography.” Using Newari medieval records of lynching, Raj argues for “employing analogy, and not argumentation, to develop a deeper historical understanding of lynching in Nepal and across societies.”

We have defined the mechanism of action of lurbinectedin, a marine-derived drug exhibiting a potent antitumor activity across several cancer cell lines and tumor xenografts. This drug, currently undergoing clinical evaluation in ovarian, breast, and small cell lung cancer patients, inhibits the transcription process through (i) its binding to CG-rich sequences, mainly located around promoters of protein-coding genes; (ii) the irreversible stalling of elongating RNA polymerase II (Pol II) on the DNA template and its specific degradation by the ubiquitin/proteasome machinery; and (iii) the generation of DNA breaks and subsequent apoptosis. The finding that inhibition of Pol II phosphorylation prevents its degradation and the formation of DNA breaks after drug treatment underscores the connection between transcription elongation and DNA repair. Our results not only help to better understand the high specificity of this drug in cancer therapy but also improve our understanding of an important transcription regulation mechanism. Mol Cancer Ther; 15(10); 2399–412. ©2016 AACR.

Purpose Trabectedin induces synthetic lethality in tumor cells carrying defects in homologous recombinant DNA repair. We evaluated the effect of concomitant inhibition of nucleotide-excision repair and poly (ADP-ribose) polymerase (PARP) activity with trabectedin and PARP inhibitors, respectively, and whether the synthetic lethality effect had the potential for a synergistic effect in breast cancer cell lines. Additionally, we investigated if this approach remained effective in BRCA1-positive breast tumor cells. Methods We have evaluated the in vitro synergistic effect of combinations of trabectedin and three different PARP inhibitors (veliparib, olaparib, and iniparib) in four breast cancer cell lines, each presenting a different BRCA1 genetic background. Antiproliferative activity, DNA damage, cell cycle perturbations and poly(ADP-ribosyl)ation were assessed by MTT assay, comet assay, flow cytometry and western blot, respectively. Results The combination of trabectedin and olaparib was synergistic in all the breast cancer cell lines tested. Our data indicated that the synergy persisted regardless of the BRCA1 status of the tumor cells. Combination treatment was associated with a strong accumulation of double-stranded DNA breaks, G2/M arrest, and apoptotic cell death. Synergistic effects were not observed when trabectedin was combined with veliparib or iniparib. Conclusion Collectively, our results indicate that the combination of trabectedin and olaparib induces an artificial synthetic lethality effect that can be used to kill breast cancer cells, independent of BRCA1 status.

Recent findings indicate that the expression of the beta-catalytic subunit of the mitochondrial H+-ATP synthase (beta-F1-ATPase) is depressed in liver, kidney and colon carcinomas, providing further a bioenergetic signature of cancer that is associated with patient survival. In the present study, we performed an analysis of mitochondrial and glycolytic protein markers in breast, gastric and prostate adenocarcinomas, and in squamous oesophageal and lung carcinomas. The expression of mitochondrial and glycolytic markers varied significantly in these carcinomas, when compared with paired normal tissues, with the exception of prostate cancer. Overall, the relative expression of beta-F1-ATPase was significantly reduced in breast and gastric adenocarcinomas, as well as in squamous oesophageal and lung carcinomas, strongly suggesting that alteration of the bioenergetic function of mitochondria is a hallmark of these types of cancer., A.I. is the recipient of a pre-doctoral fellowship from Fondo de Investigaciones Sanitarias (Ministerio de Sanidad y Consumo, Spain). M.M., G.S. and A.D.O are the recipients of pre-doctoral fellowships from Plan de Formación de Personal Investigador (Ministerio de Ciencia y Tecnología, Spain) and Programa Nacional de Formación de Profesorado (Ministerio de Educación, Cultura y Deporte, Spain) respectively. This work was supported by grants 01/0380 from Ministerio de Sanidad y Consumo and BMC2001-0710 from Ministerio de Ciencia y Tecnología to J.M.C. (Spain), 01/1519 from Ministerio de Sanidad y Consumo and AECC (Asociación Española Contra el Cancer) to P.L.F. (Spain) and from the National Institutes of Health (GM60554) to J.C.R. (U.S.A.). The CBMSO receives an institutional grant from Fundación Ramón Areces, Spain

Lurbinectedin (PM1183) is a new synthetic compound from the tetrahydroisoquinoline family, which has demonstrated a strong antiproliferative activity against a panel of human tumor models in preclinical assays and is currently being evaluated in phase III clinical trials in platinum-resistant ovarian cancer and small cell lung cancer. Lurbinectedin binds to DNA, inhibits trans-activated transcription, induces the degradation of elongating RNA Pol II and fools nucleotide excision repair to produce dsDNA breaks that need to be repaired mainly by homologous recombination (HR)1,2. Nearly 70% of patients diagnosed with ovarian cancer are in advanced stage, and the vast majority of them will eventually relapse after a primary cytoreductive surgery and several cycles of standard adjuvant chemotherapy including a platinum drug and a taxane. After a period of treatment with platinum drugs, patients will finally develop resistance, usually mediated by mechanisms such as drug detoxification or efflux and enhanced DNA repair. IRF-1 transcription factor expression has been shown to be up-regulated by cisplatin (CDDP) in ovarian cancer cells and might be limiting the response to the drug, likely by inhibiting cell proliferation3. Here we took advantage of the A2780/A2780cis human ovarian cancer cell lines, the second being a cisplatin resistant derivative, to investigate the role of IRF1 in the response of human ovarian cancer cells to cisplatin and lurbinectedin. A2780cis cells are, indeed, more resistant to cisplatin that their parental cell line but they do not differ in their resistance to lurbinectedin. Basal IRF-1 protein levels were actually higher in A2780cis cells than in their parental cell line, contributing to their resistance to cisplatin. Furthermore, cisplatin treatment induced the overexpression and nuclear localization of IRF-1 both, in A2780 and A2780cis cell lines. Contrarily, lurbinectedin did not induce the overexpression of IRF-1 neither in A2780 nor in A2780cis, explaining why this latter cell line is not resistant to the compound. Furthermore, lurbinectedin co-treatment with cisplatin reduced the expression of IRF-1 in A2780 and, more importantly, in A2780cis cells, explaining the synergism the combination has on these tumor cell lines. Thus, lurbinectedin not only did not activate the same mechanisms of resistance as cisplatin in ovarian cancer cells, but even reversed the resistance of these resistant cells to platinum drugs. 1 Santamaría Nuñez et al, 2016. Mol Cancer Ther 15(10):2399-2412 2 Romano et al, 2013. Int J Cancer. 2013 Nov;133(9):2024-33 3 Pavan et al, 2013. Eur J Cancer 49(4):964-973 Citation Format: Gema Santamaria Nuñez, Maria Jose Guillén, Juan F. Martínez-Leal, Pablo Avilés, Carlos M. Galmarini. Lurbinectedin reverses platinum dependent IRF1 overexpression and nuclear localization, partially responsible for resistance to platinum drugs in ovarian cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 1211. doi:10.1158/1538-7445.AM2017-1211

Lurbinectedin (PM1183) is a new tetrahydroisoquinoline derivative currently evaluated in phase II trials in BRCA1-mutated breast cancer and NSCLC patients. Two pivotal trials (in platinum-resistant ovarian cancer and in SCLC) will be launched in 2015. PM1183 shows antitumor activity against a wide variety of tumor cells with mean IC50 values in the low nanomolar range. In living cells, PM1183 inhibits trans-activated transcription and blocks NER-dependent DNA repair inducing the formation of dsDNA breaks and the collapse of replication forks that need to be repaired by homologous recombination (HR). As expected, PM1183 is more active against homologous recombination (HR)-deficient cell lines. In this work, we evaluated the in vitro antitumor effect of PM1183 when combined with the PARP inhibitors ABT-88 (Veliparib, PARP-1 and -2), AZD-2281 (Olaparib, PARP-1), BSI-201 (Iniparib, PARP-1) and BMN-673 (PARP-1 and -2) (Selleck Chemicals, Houston, TX, USA) in a panel of human breast carcinoma cells with different BRCA1 status, including MCF-7 (BRCA1 +/+), MDA-MB-231 (BRCA1 +/-), HCC-1937 (BRCA1 -/-) and MDA-MB-436 (BRCA1 -/-). The combination index (CI) of each different drug-drug combination was calculated by applying the Chou and Talalay method. Different degrees of synergism (CI < 1) were recorded when PM1183 was combined with AZD-2281 (Olaparib) and BMN-673, depending on the tumor cell line and the conditions tested. The highest degree of synergism with Olaparib was observed in MDA-MB-436 cells followed by MDA-MB-231, MCF7 and HCC-1937 cells. The highest degree of synergism with BMN-673 was observed in MCF-7 cells followed by MDA-MB-231 and HCC-1937 cells. In MDA-MB-231 cells, both combinations led to a synergistic induction of γ-H2AX expression and PARP-1 cleavage, as analyzed by western blot. Collectively, our results indicate that the combination of PM1183 and PARP inhibitors (Olaparib and BMN673) induces an artificial synthetic lethality that can be advantageously used to kill several breast cancer cells, independently from their BRCA1 status. Citation Format: Gema Santamaría, Sonia Avila, Victoria Moneo, Carmen Cuevas, Luis F. García-Fernández, Carlos M. Galmarini. Synergistic combination of lurbinectedin and PARP inhibitors in breast cancer tumor cell lines. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 2520. doi:10.1158/1538-7445.AM2015-2520

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