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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.

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.

Mutations in the XPD subunit of the transcription/DNA repair factor (TFIIH) give rise to trichothiodystrophy (TTD), a rare hereditary multisystem disorder with skin abnormalities. Here, we show that TTD primary dermal fibroblasts contain low amounts of collagen type VI alpha1 subunit (COL6A1), a fundamental component of soft connective tissues. We demonstrate that COL6A1 expression is downregulated by the sterol regulatory element-binding protein-1 (SREBP-1) whose removal from the promoter is a key step in COL6A1 transcription upregulation in response to cell confluence. We provide evidence for TFIIH being involved in transcription derepression, thus highlighting a new function of TFIIH in gene expression regulation. The lack of COL6A1 upregulation in TTD is caused by the inability of the mutated TFIIH complexes to remove SREBP-1 from COL6A1 promoter and to sustain the subsequent high rate of COL6A1 transcription. This defect might account for the pathologic features that TTD shares with hereditary disorders because of mutations in COL6A genes.

International audience; The xeroderma pigmentosum group D (XPD) helicase is a subunit of transcription/DNA repair factor, transcription factor II H (TFIIH) that catalyzes the unwinding of a damaged DNA duplex during nucleotide excision repair. Apart from two canonical helicase domains, XPD is composed of a 4Fe-S cluster domain involved in DNA damage recognition and a module of uncharacterized function termed the “ARCH domain.” By investigating the consequences of a mutation found in a patient with trichothiodystrophy, we show that the ARCH domain is critical for the recruitment of the cyclin-dependent kinase (CDK)-activating kinase (CAK) complex. Indeed, this mutation not only affects the interaction with the MAT1 CAK subunit, thereby decreasing the in vitro basal transcription activity of TFIIH itself and impeding the efficient recruitment of the transcription machinery on the promoter of an activated gene, but also impairs the DNA unwinding activity of XPD and the nucleotide excision repair activity of TFIIH. We further demonstrate the role of CAK in downregulating the XPD helicase activity within TFIIH. Taken together, our results identify the ARCH domain of XPD as a platform for the recruitment of CAK and as a potential molecular switch that might control TFIIH composition and play a key role in the conversion of TFIIH from a factor active in transcription to a factor involved in DNA repair.

SummaryTrabectedin and Zalypsis are two potent anticancer tetrahydroisoquinoline alkaloids that can form a covalent bond with the amino group of a guanine in selected triplets of DNA duplexes and eventually give rise to double-strand breaks. Using well-defined in vitro and in vivo assays, we show that the resulting DNA adducts stimulate, in a concentration-dependent manner, cleavage by the XPF/ERCC1 nuclease on the strand opposite to that bonded by the drug. They also inhibit RNA synthesis by: (1) preventing binding of transcription factors like Sp1 to DNA, and (2) arresting elongating RNA polymerase II at the same nucleotide position regardless of the strand they are located on. Structural models provide a rationale for these findings and highlight the similarity between this type of DNA modification and an interstrand crosslink.