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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^�.

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.

Growth inhibitory activity of lurbinectedin on different cancer cell lines.

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.

Small-Cell Lung Cancer (SCLC) is an aggressive neuroendocrine malignancy with a poor prognosis. Here, we focus on the neuroendocrine SCLC subtypes, SCLC-A and SCLC-N, whose transcription addiction was driven by ASCL1 and NEUROD1 transcription factors which target E-box motifs to activate up to 40% of total genes, the promoters of which are maintained in a steadily open chromatin environment according to ATAC and H3K27Ac signatures. This leverage is used by the marine agent lurbinectedin, which preferentially targets the CpG islands located downstream of the transcription start site, thus arresting elongating RNAPII and promoting its degradation. This abrogates the expression of ASCL1 and NEUROD1 and of their dependent genes, such as BCL2, INSM1, MYC, and AURKA, which are responsible for relevant SCLC tumorigenic properties such as inhibition of apoptosis and cell survival, as well as for a part of its neuroendocrine features. In summary, we show how the transcription addiction of these cells becomes their Achilles's heel, and how this is effectively exploited by lurbinectedin as a novel SCLC therapeutic endeavor.

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