Analysis of transcriptomic responses to SARS-CoV-2 reveals plausible defective pathways responsible for increased susceptibility to infection and complications and helps to develop fast-track repositioning of drugs against COVID-19.

BACKGROUND: To understand the transcriptomic response to SARS-CoV-2 infection, is of the utmost importance to design diagnostic tools predicting the severity of the infection. METHODS: We have performed a deep sampling analysis of the viral transcriptomic data oriented towards drug repositioning. Using different samplers, the basic principle of this methodology the biological invariance, which means that the pathways altered by the disease, should be independent on the algorithm used to unravel them. RESULTS: The transcriptomic analysis of the altered pathways, reveals a distinctive inflammatory response and potential side effects of infection. The virus replication causes, in some cases, acute respiratory distress syndrome in the lungs, and affects other organs such as heart, brain, and kidneys. Therefore, the repositioned drugs to fight COVID-19 should, not only target the interferon signalling pathway and the control of the inflammation, but also the altered genetic pathways related to the side effects of infection. We also show via Principal Component Analysis that the transcriptome signatures are different from influenza and RSV. The gene COL1A1, which controls collagen production, seems to play a key/vital role in the regulation of the immune system. Additionally, other small-scale signature genes appear to be involved in the development of other COVID-19 comorbidities. CONCLUSIONS: Transcriptome-based drug repositioning offers possible fast-track antiviral therapy for COVID-19 patients. It calls for additional clinical studies using FDA approved drugs for patients with increased susceptibility to infection and with serious medical complications.

Why we do what we do. A brief analysis of cancer therapies.

The goal of all medical activity is to preserve health in fit people, and to restore the sick into a state of complete physical, mental and social wellbeing. In an effort to determine whether we are achieving this last goal in oncology, herein we review the biological and clinical framework that has led to the foundations of the current anticancer treatment paradigm. Currently, cancer therapy is still based on the ancient axiom that states that the complete eradication of the tumor burden is the only way to achieve a cure. This strategy has led to a substantial improvement in survival rates as cancer mortality rates have dropped in an unprecedented way. Despite this progress, more than 9 million people still die from cancer every year, indicating that the current treatment strategy is not leading to a cancer cure, but to a cancer remission, that is "the temporary absence of manifestations of a particular disease"; after months or years of remission, in most patients, cancer will inevitably recur. Our critical analysis indicates that it is time to discuss about the new key challenges and future directions in clinical oncology. We need to generate novel treatment strategies more suited to the current clinical reality.

Deep Neural Frameworks Improve the Accuracy of General Practitioners in the Classification of Pigmented Skin Lesions.

This study evaluated whether deep learning frameworks trained in large datasets can help non-dermatologist physicians improve their accuracy in categorizing the seven most common pigmented skin lesions. Open-source skin images were downloaded from the International Skin Imaging Collaboration (ISIC) archive. Different deep neural networks (DNNs) (n = 8) were trained based on a random dataset constituted of 8015 images. A test set of 2003 images was used to assess the classifiers' performance at low (300 × 224 RGB) and high (600 × 450 RGB) image resolution and aggregated data (age, sex and lesion localization). We also organized two different contests to compare the DNN performance to that of general practitioners by means of unassisted image observation. Both at low and high image resolution, the DNN framework differentiated dermatological images with appreciable performance. In all cases, the accuracy was improved when adding clinical data to the framework. Finally, the least accurate DNN outperformed general practitioners. The physician's accuracy was statistically improved when allowed to use the output of this algorithmic framework as guidance. DNNs are proven to be high performers as skin lesion classifiers and can improve general practitioner diagnosis accuracy in a routine clinical scenario.

Lessons from Hippocrates: Time to Change the Cancer Paradigm.

The ultimate goal of all medical activity is to restore patients to a state of complete physical, mental, and social wellbeing. In cancer, it is assumed that this can only be obtained through the complete eradication of the tumor burden. So far, this strategy has led to a substantial improvement in cancer survival rates. Despite this, more than 9 million people die from cancer every year. Therefore, we need to accept that our current cancer treatment paradigm is obsolete and must be changed. The new paradigm should reflect that cancer is a systemic disease, which affects an individual patient living in a particular social reality, rather than an invading organism or a mere cluster of mutated cells that need to be eradicated. This Hippocratic holistic view will ultimately lead to an improvement in health and wellbeing in cancer patients. They deserve nothing less.

ATLANTIS: a Phase III study of lurbinectedin/doxorubicin versus topotecan or cyclophosphamide/doxorubicin/vincristine in patients with small-cell lung cancer who have failed one prior platinum-containing line.

Lurbinectedin is an inhibitor of active transcription of protein-coding genes, causing DNA-break accumulation, apoptosis and modulation of the tumor microenvironment. Early-phase clinical trials indicate promising activity of lurbinectedin in small-cell lung cancer. Here, we describe the rationale and design of ATLANTIS (NCT02566993), an open-label, randomized, multicenter Phase III study to compare the efficacy of lurbinectedin and doxorubicin combination with standard-of-care chemotherapy, investigator's choice of cyclophosphamide/doxorubicin/vincristine or topotecan, in patients with small-cell lung cancer that has progressed following one line of platinum-based chemotherapy. Patients are randomized in a 1:1 ratio. The primary end point is overall survival and key secondary end points include progression-free survival, best tumor response and duration of response, each assessed by independent review committee.

The Antitumor Drugs Trabectedin and Lurbinectedin Induce Transcription-Dependent Replication Stress and Genome Instability.

R-loops are a major source of replication stress, DNA damage, and genome instability, which are major hallmarks of cancer cells. Accordingly, growing evidence suggests that R-loops may also be related to cancer. Here we show that R-loops play an important role in the cellular response to trabectedin (ET743), an anticancer drug from marine origin and its derivative lurbinectedin (PM01183). Trabectedin and lurbinectedin induced RNA-DNA hybrid-dependent DNA damage in HeLa cells, causing replication impairment and genome instability. We also show that high levels of R-loops increase cell sensitivity to trabectedin. In addition, trabectedin led to transcription-dependent FANCD2 foci accumulation, which was suppressed by RNase H1 overexpression. In yeast, trabectedin and lurbinectedin increased the presence of Rad52 foci, a marker of DNA damage, in an R-loop-dependent manner. In addition to providing new insights into the mechanisms of action of these drugs, our study reveals that R-loops could be targeted by anticancer agents. Given the increasing evidence that R-loops occur all over the genome, the ability of lurbinectedin and trabectedin to act on them may contribute to enhance their efficacy, opening the possibility that R-loops might be a feature shared by specific cancers. IMPLICATIONS: The data presented in this study provide the new concept that R-loops are important cellular factors that contribute to trabectedin and lurbinectedin anticancer activity.

Binding of eEF1A2 to the RNA-dependent protein kinase PKR modulates its activity and promotes tumour cell survival.

BACKGROUND: Through several not-fully-characterised moonlighting functions, translation elongation factor eEF1A2 is known to provide a fitness boost to cancer cells. Furthermore, eEF1A2 has been demonstrated to confer neoplastic characteristics on preneoplastic, nontumourigenic precursor cells. We have previously shown that eEF1A2 is the target of plitidepsin, a marine drug currently in development for cancer treatment. Herein, we characterised a new signalling pathway through which eEF1A2 promotes tumour cell survival. METHODS: Previously unknown binding partners of eEF1A2 were identified through co-immunoprecipitation, high-performance liquid chromatography-mass spectrometry and proximity ligation assay. Using plitidepsin to release eEF1A2 from those protein complexes, their effects on cancer cell survival were analysed in vitro. RESULTS: We uncovered that double-stranded RNA-activated protein kinase (PKR) is a novel eEF1A2-interacting partner whose pro-apoptotic effect is hindered by the translation factor, most likely through sequestration and inhibition of its kinase activity. Targeting eEF1A2 with plitidepsin releases PKR from the complex, facilitating its activation and triggering a mitogen-activated protein kinase signalling cascade together with a nuclear factor-κB-dependent activation of the extrinsic apoptotic pathway, which lead to tumour cell death. CONCLUSIONS: Through its binding to PKR, eEF1A2 provides a survival boost to cancer cells, constituting an Achilles heel that can be exploited in anticancer therapy.

Multicenter Phase II Study of Lurbinectedin in BRCA-Mutated and Unselected Metastatic Advanced Breast Cancer and Biomarker Assessment Substudy.

PURPOSE: This multicenter phase II trial evaluated lurbinectedin (PM01183), a selective inhibitor of active transcription of protein-coding genes, in patients with metastatic breast cancer. A unicenter translational substudy assessed potential mechanisms of lurbinectedin resistance. PATIENTS AND METHODS: Two arms were evaluated according to germline BRCA1/2 status: BRCA1/2 mutated (arm A; n = 54) and unselected ( BRCA1/2 wild-type or unknown status; arm B; n = 35). Lurbinectedin starting dose was a 7-mg flat dose and later, 3.5 mg/m2 in arm A. The primary end point was objective response rate (ORR) per Response Evaluation Criteria in Solid Tumors (RECIST). The translational substudy of resistance mechanisms included exome sequencing (n = 13) and in vivo experiments with patient-derived xenografts (n = 11) from BRCA1/2-mutated tumors. RESULTS: ORR was 41% (95% CI, 28% to 55%) in arm A and 9% (95% CI, 2% to 24%) in arm B. In arm A, median progression-free survival was 4.6 months (95% CI, 3.0 to 6.0 months), and median overall survival was 20.0 months (95% CI, 11.8 to 26.6 months). Patients with BRCA2 mutations showed an ORR of 61%, median progression-free survival of 5.9 months, and median overall survival of 26.6 months. The safety profile improved with lurbinectedin dose adjustment to body surface area. The most common nonhematologic adverse events seen at 3.5 mg/m2 were nausea (74%; grade 3, 5%) and fatigue (74%; grade 3, 21%). Neutropenia was the most common severe hematologic adverse event (grade 3, 47%; grade 4, 10%). Exome sequencing showed mutations in genes related to the nucleotide excision repair pathway in four of seven tumors at primary or acquired resistance and in one patient with short-term stable disease. In vivo, sensitivity to cisplatin and lurbinectedin was evidenced in lurbinectedin-resistant (one of two) and cisplatin-resistant (two of three) patient-derived xenografts. CONCLUSION: Lurbinectedin showed noteworthy activity in patients with BRCA1/2 mutations. Response and survival was notable in those with BRCA2 mutations. Additional clinical development in this subset of patients with metastatic breast cancer is warranted.

MI130004, a Novel Antibody-Drug Conjugate Combining Trastuzumab with a Molecule of Marine Origin, Shows Outstanding In Vivo Activity against HER2-Expressing Tumors.

In the search for novel payloads to design new antibody-drug conjugates (ADC), marine compounds represent an interesting opportunity given their unique chemical features. PM050489 is a marine compound that binds ß-tubulin at a new site and disrupts the microtubule network, hence leading to mitotic aberrations and cell death. PM050489 has been conjugated to trastuzumab via Cys residues through a noncleavable linker, and the resulting ADC, named MI130004, has been studied. Analysis of MI130004 delivered data consistent with the presence of two molecules of PM050489 per antibody molecule, likely bound to both sides of the intermolecular disulfide bond connecting the antibody light and heavy chains. The antitumor activity of MI130004 was analyzed in vitro and in vivo in different cell lines of diverse tumor origin (breast, ovary, and gastric cancer) expressing different levels of HER2. MI130004 showed very high in vitro potency and good selectivity for tumor cells that overexpressed HER2. At the cellular level, MI130004 impaired tubulin polymerization, causing disorganization and disintegration of the microtubule network, which ultimately led to mitotic failure, mirroring the effect of its payload. Treatment with MI130004 in mice carrying histologically diverse tumors expressing HER2 induced a long-lasting antitumor effect with statistically significant inhibition of tumor growth coupled with increases in median survival time compared with vehicle or trastuzumab. These results strongly suggest that MI130004 is endowed with remarkable anticancer activity and confirm the extraordinary potential of marine compounds for the design of new ADCs. Mol Cancer Ther; 17(4); 786-94. ©2018 AACR.

Plocabulin, a novel tubulin-binding agent, inhibits angiogenesis by modulation of microtubule dynamics in endothelial cells.

BACKGROUND: Vascular supply of tumors is one of the main targets for cancer therapy. Here, we investigated if plocabulin (PM060184), a novel marine-derived microtubule-binding agent, presents antiangiogenic and vascular-disrupting activities. METHODS: The effects of plocabulin on microtubule network and dynamics were studied on HUVEC endothelial cells. We have also studied its effects on capillary tube structures formation or destabilization in three-dimensional collagen matrices. In vivo experiments were performed on different tumor cell lines. RESULTS: In vitro studies show that, at picomolar concentrations, plocabulin inhibits microtubule dynamics in endothelial cells. This subsequently disturbs the microtubule network inducing changes in endothelial cell morphology and causing the collapse of angiogenic vessels, or the suppression of the angiogenic process by inhibiting the migration and invasion abilities of endothelial cells. This rapid collapse of the endothelial tubular network in vitro occurs in a concentration-dependent manner and is observed at concentrations lower than that affecting cell survival. The in vitro findings were confirmed in tumor xenografts where plocabulin treatment induced a large reduction in vascular volume and induction of extensive necrosis in tumors, consistent with antivascular effects. CONCLUSIONS: Altogether, these data suggest that an antivascular mechanism is contributing to the antitumor activities of plocabulin.

Dynamic cellular maps of molecular species: Application to drug-target interactions.

The design of living cell studies aimed at deciphering the mechanism of action of drugs targeting proteins with multiple functions, expressed in a wide range of concentrations and cellular locations, is a real challenge. We recently showed that the antitumor drug plitidepsin (APL) localizes sufficiently close to the elongation factor eEF1A2 so as to suggest the formation of drug-protein complexes in living cells. Here we present an extension of our previous micro-spectroscopy study, that combines Generalized Polarization (GP) images, with the phasor approach and fluorescence lifetime imaging microscopy (FLIM), using a 7-aminocoumarin drug analog (APL*) as fluorescence tracer. Using the proposed methodology, we were able to follow in real time the formation and relative distribution of two sets of APL-target complexes in live cells, revealing two distinct patterns of behavior for HeLa-wt and APL resistant HeLa-APL-R cells. The information obtained may complement and facilitate the design of new experiments and the global interpretation of the results obtained with other biochemical and cell biology methods, as well as possibly opening new avenues of study to decipher the mechanism of action of new drugs.

Lurbinectedin reduces tumour-associated macrophages and the inflammatory tumour microenvironment in preclinical models.

BACKGROUND: Lurbinectedin is a novel anticancer agent currently undergoing late-stage (Phase II /III) clinical evaluation in platinum-resistant ovarian, BRCA1/2-mutated breast and small-cell lung cancer. Lurbinectedin is structurally related to trabectedin and it inhibits active transcription and the DNA repair machinery in tumour cells. METHODS: In this study we investigated whether lurbinectedin has the ability to modulate the inflammatory microenvironment and the viability of myeloid cells in tumour-bearing mice. RESULTS: Administration of lurbinectedin significantly and selectively decreased the number of circulating monocytes and, in tumour tissues, that of macrophages and vessels. Similar findings were observed when a lurbinectedin-resistant tumour variant was used, indicating a direct effect of lurbinectedin on the tumour microenviroment. In vitro, lurbinectedin induced caspase-8-dependent apoptosis of human purified monocytes, whereas at low doses it significantly inhibited the production of inflammatory/growth factors (CCL2, CXCL8 and VEGF) and dramatically impaired monocyte adhesion and migration ability. These findings were supported by the strong inhibition of genes of the Rho-GTPase family in lurbinectedin-treated monocytes. CONCLUSIONS: The results illustrate that lurbinectedin affects at multiple levels the inflammatory microenvironment by acting on the viability and functional activity of mononuclear phagocytes. These peculiar effects, combined with its intrinsic activity against cancer cells, make lurbinectedin a compound of particular interest in oncology.

Antitumour activity of trabectedin in myelodysplastic/myeloproliferative neoplasms.

BACKGROUND: Juvenile myelomonocytic leukaemia (JMML) and chronic myelomonocytic leukaemia (CMML) are myelodysplastic myeloproliferative (MDS/MPN) neoplasms with unfavourable prognosis and without effective chemotherapy treatment. Trabectedin is a DNA minor groove binder acting as a modulator of transcription and interfering with DNA repair mechanisms; it causes selective depletion of cells of the myelomonocytic lineage. We hypothesised that trabectedin might have an antitumour effect on MDS/MPN. METHODS: Malignant CD14+ monocytes and CD34+ haematopoietic progenitor cells were isolated from peripheral blood/bone marrow mononuclear cells. The inhibition of CFU-GM colonies and the apoptotic effect on CD14+ and CD34+ induced by trabectedin were evaluated. Trabectedin's effects were also investigated in vitro on THP-1, and in vitro and in vivo on MV-4-11 cell lines. RESULTS: On CMML/JMML cells, obtained from 20 patients with CMML and 13 patients with JMML, trabectedin - at concentration pharmacologically reasonable, 1-5 nM - strongly induced apoptosis and inhibition of growth of haematopoietic progenitors (CFU-GM). In these leukaemic cells, trabectedin downregulated the expression of genes belonging to the Rho GTPases pathway (RAS superfamily) having a critical role in cell growth and cytoskeletal dynamics. Its selective activity on myelomonocytic malignant cells was confirmed also on in vitro THP-1 cell line and on in vitro and in vivo MV-4-11 cell line models. CONCLUSIONS: Trabectedin could be good candidate for clinical studies in JMML/CMML patients.

Translation Elongation Factor eEF1A2 is a Novel Anticancer Target for the Marine Natural Product Plitidepsin.

eEF1A2 is one of the isoforms of the alpha subunit of the eukaryotic Elongation Factor 1. It is overexpressed in human tumors and is endowed with oncogenic properties, favoring tumor cell proliferation while inhibiting apoptosis. We demonstrate that plitidepsin, an antitumor agent of marine origin that has successfully completed a phase-III clinical trial for multiple myeloma, exerts its antitumor activity by targeting eEF1A2. The drug interacts with eEF1A2 with a KD of 80 nM and a target residence time of circa 9 min. This protein was also identified as capable of binding [14C]-plitidepsin in a cell lysate from K-562 tumor cells. A molecular modelling approach was used to identify a favorable binding site for plitidepsin at the interface between domains 1 and 2 of eEF1A2 in the GTP conformation. Three tumor cell lines selected for at least 100-fold more resistance to plitidepsin than their respective parental cells showed reduced levels of eEF1A2 protein. Ectopic expression of eEF1A2 in resistant cells restored the sensitivity to plitidepsin. FLIM-phasor FRET experiments demonstrated that plitidepsin localizes in tumor cells sufficiently close to eEF1A2 as to suggest the formation of drug-protein complexes in living cells. Altogether, our results strongly suggest that eEF1A2 is the primary target of plitidepsin.

Dual inhibition of ATR and ATM potentiates the activity of trabectedin and lurbinectedin by perturbing the DNA damage response and homologous recombination repair.

Trabectedin (Yondelis®, ecteinascidin-743, ET-743) is a marine-derived natural product approved for treatment of advanced soft tissue sarcoma and relapsed platinum-sensitive ovarian cancer. Lurbinectedin is a novel anticancer agent structurally related to trabectedin. Both ecteinascidins generate DNA double-strand breaks that are processed through homologous recombination repair (HRR), thereby rendering HRR-deficient cells particularly sensitive. We here characterize the DNA damage response (DDR) to trabectedin and lurbinectedin in HeLa cells. Our results show that both compounds activate the ATM/Chk2 (ataxia-telangiectasia mutated/checkpoint kinase 2) and ATR/Chk1 (ATM and RAD3-related/checkpoint kinase 1) pathways. Interestingly, pharmacological inhibition of Chk1/2, ATR or ATM is not accompanied by any significant improvement of the cytotoxic activity of the ecteinascidins while dual inhibition of ATM and ATR strongly potentiates it. Accordingly, concomitant inhibition of both ATR and ATM is an absolute requirement to efficiently block the formation of γ-H2AX, MDC1, BRCA1 and Rad51 foci following exposure to the ecteinascidins. These results are not restricted to HeLa cells, but are shared by cisplatin-sensitive and -resistant ovarian carcinoma cells. Together, our data identify ATR and ATM as central coordinators of the DDR to ecteinascidins and provide a mechanistic rationale for combining these compounds with ATR and ATM inhibitors.

Unique features of trabectedin mechanism of action.

Trabectedin (Yondelis®, ET-743) is a marine-derived natural product that was initially isolated from the marine ascidian Ecteinascidia turbinata and is currently prepared synthetically. Trabectedin is used as a single agent for the treatment of patients with soft tissue sarcoma after failure of doxorubicin or ifosfamide or who are unsuited to receive these agents, and in patients with relapsed, platinum-sensitive ovarian cancer in combination with pegylated liposomal doxorubicin. Trabectedin presents a complex mechanism of action affecting key cell biology processes in tumor cells as well as in the tumor microenvironment. The inhibition of trans-activated transcription and the interaction with DNA repair proteins appear as a hallmark of the antiproliferative activity of trabectedin. Inhibition of active transcription is achieved by an initial direct mechanism that involves interaction with RNA polymerase II, thereby inducing its ubiquitination and degradation by the proteasome. This subsequently modulates the production of cytokines and chemokines by tumor and tumor-associated macrophages. Another interesting effect on activated transcription is mediated by the displacement of oncogenic transcription factors from their target promoters, thereby affecting oncogenic signaling addiction. In addition, it is well established that DNA repair systems including transcription-coupled nucleotide excision repair and homologous recombination play a role in the antitumor activity of trabectedin. Ongoing studies are currently addressing how to exploit these unique mechanistic features of trabectedin to combine this agent either with immunological or microenvironmental modulators or with classical chemotherapeutic agents in a more rational manner.

Elisidepsin Interacts Directly with Glycosylceramides in the Plasma Membrane of Tumor Cells to Induce Necrotic Cell Death.

Plasma membrane integrity is essential for cell life. Any major break on it immediately induces the death of the affected cell. Different molecules were described as disrupting this cell structure and thus showing antitumor activity. We have previously defined that elisidepsin (Irvalec®, PM02734) inserts and self-organizes in the plasma membrane of tumor cells, inducing a rapid loss of membrane integrity, cell permeabilization and necrotic death. Here we show that, in sensitive HCT-116 colorectal cells, all these effects are consequence of the interaction of elisidepsin with glycosylceramides in the cell membrane. Of note, an elisidepsin-resistant subline (HCT-116-Irv) presented reduced levels of glycosylceramides and no accumulation of elisidepsin in the plasma membrane. Consequently, drug treatment did not induce the characteristic necrotic cell death. Furthermore, GM95, a mutant derivative from B16 mouse melanoma cells lacking ceramide glucosyltransferase (UGCG) activity and thus the synthesis of glycosylceramides, was also resistant to elisidepsin. Over-expression of UGCG gene in these deficient cells restored glycosylceramides synthesis, rendering them sensitive to elisidepsin, at a similar level than parental B16 cells. These results indicate that glycosylceramides act as membrane targets of elisidepsin, facilitating its insertion in the plasma membrane and the subsequent membrane permeabilization that leads to drug-induced cell death. They also indicate that cell membrane lipids are a plausible target for antineoplastic therapy.

Survivorship in untreated breast cancer patients.

In any disease, the knowledge of the natural history of untreated cases provides a real background against which the real advantages of a new treatment itself are judged. Fortunately, in the present days, there are scant data on outcomes in patients with untreated breast cancer. In an attempt to provide this background against which the virtues of current curative and palliative treatments can be more accurately assessed, we have reviewed the literature regarding published untreated breast cancer series. Taking into consideration all the difficulties of analyzing reports written on the last half of the nineteenth century or on the first half of the twentieth century, in most reports, patients survived almost 3-4 years without any type of treatment. Worth mentioning, approximately 5-10 % of untreated patients lived longer than 10 years. Thus, the spectrum of clinical aggressiveness of untreated breast cancer varies between virulence and chronic disease. These facts should be taken into account when considering the value of current treatments for early-stage disease.

Synergistic Effect of Trabectedin and Olaparib Combination Regimen in Breast Cancer Cell Lines.

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.

PM060184, a new tubulin binding agent with potent antitumor activity including P-glycoprotein over-expressing tumors.

PM060184 belongs to a new family of tubulin-binding agents originally isolated from the marine sponge Lithoplocamia lithistoides. This compound is currently produced by total synthesis and is under evaluation in clinical studies in patients with advanced cancer diseases. It was recently published that PM060184 presents the highest known affinities among tubulin-binding agents, and that it targets tubulin dimers at a new binding site. Here, we show that PM060184 has a potent antitumor activity in a panel of different tumor xenograft models. Moreover, PM060184 is able to overcome P-gp mediated resistance in vivo, an effect that could be related to its high binding affinity for tubulin. To gain insight into the mechanism responsible of the observed antitumor activity, we have characterized its molecular and cellular effects. We have observed that PM060184 is an inhibitor of tubulin polymerization that reduces microtubule dynamicity in cells by 59%. Interestingly, PM060184 suppresses microtubule shortening and growing at a similar extent. This action affects cells in interphase and mitosis. In the first case, the compound induces a disorganization and fragmentation of the microtubule network and the inhibition of cell migration. In the second case, it induces the appearance of multipolar mitosis and lagging chromosomes at the metaphase plate. These effects correlate with prometaphase arrest and induction of caspase-dependent apoptosis or appearance of cells in a multinucleated interphase-like state unrelated to classical apoptosis pathways. Taken together, these results indicate that PM060184 represents a new tubulin binding agent with promising potential as an anticancer agent.