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RNA-DNA hybrids immunofluorescence after trabectedin or lurbinectedin treatment

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.

Supplementary Figure 2 from Trabectedin and Its C Subunit Modified Analogue PM01183 Attenuate Nucleotide Excision Repair and Show Activity toward Platinum-Resistant Cells

DNA damage foci and colony survival after trabectedin treatment in RNase H1-overexpressing cells

FANCD2 foci accumulation in neocarzinostatin-treated cells

Supplementary Figure 1 from Trabectedin and Its C Subunit Modified Analogue PM01183 Attenuate Nucleotide Excision Repair and Show Activity toward Platinum-Resistant Cells

Supplementary Figure 3 from Trabectedin and Its C Subunit Modified Analogue PM01183 Attenuate Nucleotide Excision Repair and Show Activity toward Platinum-Resistant Cells

Supplementary Figure 4 from Trabectedin and Its C Subunit Modified Analogue PM01183 Attenuate Nucleotide Excision Repair and Show Activity toward Platinum-Resistant Cells

EdU incorporation in trabectedin-treated cells after transcription inhibition

Supplementary information contains the supplementary material and methods and the legends of the supplemetary figures.

53BP1 foci after trabectedin treatment in RNase H1-overexpressing cells

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 β-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.

Description of synthetic method for PM120160 Supplementary Figure 1. Analysis of MI130004. A: Analytical size exclusion chromatography analysis performed on a Superdex-100 10/300 column running an isocratic method with PBS at 1 mL/min. B: HIC analysis of trastuzumab (dotted red line) and MI130004 (solid blue line) run as described under Materials and Methods. The dotted black line shows the ammonium sulfate gradient. C: UV-Vis chromatogram at 280 nm resulting from the elution of trastuzumab digested with IdeS and then reduced with DTT. D: UV-Vis chromatogram at 280 nm resulting from the elution of MI130004 digested with IdeS and then reduced with DTT. Supplementary Figure 2. Deconvoluted mass spectra of peaks common to trastuzumab and MI130004 in Suppl. Fig. 1. A: Analysis of peak eluted at 12.7 min. B: Analysis of peak eluted at 15.2 min. C: Analysis of peak eluted at 19.0 min. Supplementary Figure 3. Deconvoluted mass spectra of peaks exclusive for MI130004 in Suppl. Fig. 1. A: Analysis of peak eluted at 20.82 min. B: Analysis of peak eluted at 23.88 min. Supplementary Figure 4. Antiproliferative assay showing the in vitro potency of MI130004. The assay was performed as described under Materials and Methods. A: Effect on breast tumor cells: JIMT-1 (HER2 positive, solid circles), BT-474 (HER2 positive, solid squares), MDA-MB-231 (HER2 negative, hollow circles) and MCF7 (HER2 negative, hollow squares) cell lines. B: Effect on gastric tumor cells: HGC-27 (HER2 positive, solid circles) and Hs 746T (HER2 negative, hollow circles) cell lines. Supplementary Figure 5. Biological effects of PM050489 in tumor cell lines of bbreast and gastric origins. A: Fluorescence microscopy analysis of cells treated with 1% (v/v) DMSO ("control") or with 1 nM PM050489 in 1% DMSO. Cells were stained for α-tubulin (green), γ-tubulin (red) and nuclei (blue).. B: Effect of PM050489 in cell cycle as determined by flow cytometry. Figures denote the percentage of cells arrested in G2/M according to the area of the peaks pointed by the arrows. Supplementary Figure 6. Biological effects of MI130004 in tumor cell lines of breast and gastric origin. Fluorescence microscopy analysis of cells treated with MI130004 at 0.1 µg/mL. Cells were stained for α-tubulin (green), γ-tubulin (red) and nuclei (blue). Left hand column, control untreated cells. Right hand column, cells treated with MI130004. Supplementary Figure 7. Immunohistochemistry analysis of tumors from mice xenografts. Twenty-four hours after MI130004 first administration, HER2 levels were determined by immunohistochemistry, as described under Materials and Methods, in samples from MI130004-treated animals bearing breast JIMT-1, BT-474 (both HER2 positive), MDA-MB-231 (HER2 negative), gastric N87, Gastric-008 (bth HER2 positive), Hs 746T (HER2 negative) and ovarian SK-OV-3 and A2780cis (HER2 positive) tumor cells. Magnification is 20x. Supplementary Table 1: Supplementary Table 1. Mass assignments of species eluted in the UV chromatograms shown in Suppl. Fig. 1 Supplementary Table 2: Supplementary Table 2. Antiproliferative activity of PM050489 in HER2 positive and negative cell lines.

PM01183 is a novel marine-derived covalent DNA binder in clinical development. PM01183 is structurally similar to trabectedin (yondelis, ecteinascidin-743) except for the C subunit, and this modification is accompanied by different pharmacokinetics in cancer patients. We here characterize the interaction of PM01183 with the nucleotide excision repair (NER) pathway in comparison with trabectedin. Our results show for the first time that although neither PM01183 nor trabectedin is repaired by NER, both compounds are able to interfere with the NER machinery thereby attenuating the repair of specific NER substrates. We further show that the NER activity is increased in 3 of 4 cellular models with acquired resistance to cisplatin or oxaliplatin, confirming the involvement of NER in the resistance to platinum derivatives. Importantly, both PM01183 and trabectedin show unchanged or even enhanced activity toward all 4 cisplatin- and oxaliplatin-resistant cell lines. We finally show that combinations of PM01183 and cisplatin were mostly synergistic toward both parental and cisplatin-resistant ovarian carcinoma cells as indicated by Chou and Talalay analysis. These data show that the C subunit of trabectedin can be subjected to at least some structural modifications without loss of activity or NER interaction. While PM01183 and trabectedin appear functionally similar in cellular models, it is likely that the differences in pharmacokinetics may allow different dosing and scheduling of PM01183 in the clinic that could lead to novel and/or increased antitumor activity. Taken together, our results provide a mechanistic basis to support clinical trials of PM01183 alone or in combination with cisplatin. Mol Cancer Ther; 10(8); 1481–9. ©2011 AACR.

Supplementary Fig. from Polymeric nanogels containing the triphosphate form of cytotoxic nucleoside analogues show antitumor activity against breast and colorectal cancer cell lines

Supplementary Table 1 from Class III β-Tubulin Isotype Predicts Response in Advanced Breast Cancer Patients Randomly Treated Either with Single-Agent Doxorubicin or Docetaxel

The therapeutic efficiency of anticancer nucleoside analogues (NA) strongly depends on their intracellular accumulation and conversion into 5′-triphosphates. Because active NATP cannot be directly administrated due to instability, we present here a strategy of nanoencapsulation of these active drugs for efficient delivery to tumors. Stable lyophilized formulations of 5′-triphosphates of cytarabine (araCTP), gemcitabine (dFdCTP), and floxuridine (FdUTP) encapsulated in biodegradable PEG-cl-PEI or F127-cl-PEI nanogel networks (NGC and NGM, respectively) were prepared by a self-assembly procedure. Cellular penetration, in vitro cytotoxicity, and drug-induced cell cycle perturbations of these nanoformulations were analyzed in breast and colorectal cancer cell lines. Cellular accumulation and NATP release from nanogel was studied by confocal microscopy and direct high-performance liquid chromatography analysis of cellular lysates. Antiproliferative effect of dFdCTP nanoformulations was evaluated in human breast carcinoma MCF7 xenograft animal model. Nanoencapsulated araCTP, dFdCTP, and FdUTP showed similar to NA cytotoxicity and cell cycle perturbations. Nanogels without drugs showed very low cytotoxicity, although NGM was more toxic than NGC. Treatment by NATP nanoformulations induced fast increase of free intracellular drug concentration. In human breast carcinoma MCF7 xenograft animal model, i.v. dFdCTP-nanogel was equally effective in inhibiting tumor growth at four times lower administered drug dose compared with free gemcitabine. Active triphosphates of NA encapsulated in nanogels exhibit similar cytotoxicity and cell cycle perturbations in vitro and faster cell accumulation and equal tumor growth-inhibitory activity in vivo at much lower dose compared with parental drugs, illustrating their therapeutic potential for cancer chemotherapy. [Mol Cancer Ther 2008;7(10):3373–80]

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.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.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.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.