Myxoid Liposarcomas (MLS), characterized by the expression of FUS-CHOP fusion gene are clinically very sensitive to the DNA binding antitumor agent, trabectedin. However, resistance eventually occurs, preventing disease eradication. To investigate the mechanisms of resistance, a trabectedin resistant cell line, 402-91/ET, was developed. The resistance to trabectedin was not related to the expression of MDR related proteins, uptake/efflux of trabectedin or GSH levels that were similar in parental and resistant cells. The 402-91/ET cells were hypersensitive to UV light because of a nucleotide excision repair defect: XPG complementation decreased sensitivity to UV rays, but only partially to trabectedin. 402-91/ET cells showed collateral sensitivity to temozolomide due to the lack of O 6 -methylguanine-DNA-methyltransferase (MGMT) activity, related to the hypermethylation of MGMT promoter. In 402-91 cells chromatin immunoprecipitation (ChIP) assays showed that FUS-CHOP was bound to the PTX3 and FN1 gene promoters, as previously described, and trabectedin caused FUS-CHOP detachment from DNA. Here we report that, in contrast, in 402-91/ET cells, FUS-CHOP was not bound to these promoters. Differences in the modulation of transcription of genes involved in different pathways including signal transduction, apoptosis and stress response between the two cell lines were found. Trabectedin activates the transcription of genes involved in the adipogenicprogram such as c/EBPa and b, in 402-91 but not in 402-91/ET cell lines. The collateral sensitivity of 402-91/ET to temozolomide provides the rationale to investigate the potential use of methylating agents in MLS patients resistant to trabectedin.Trabectedin is a marine alkaloid isolated from the tunicate Ecteinascidia turbinata that has shown striking antitumor activity in a variety of preclinical models, including some that are insensitive to conventional chemotherapeutics. [1][2][3][4][5] Clinical investigations have shown that trabectedin is effective in soft tissue sarcomas, 6-12 and in several other human malignancies, and it was approved by EMEA in 2007 for second line therapy of soft tissue sarcoma and in 2009 for second line therapy of ovarian cancer patients. [13][14][15][16][17] Trabectedin binds to the N 2 of guanine in the minor groove of DNA, causing bending of the minor groove towards the major groove. [18][19][20][21] Trabectedin affects transcription regulation in a promoter and gene-specific manner, 22,23 but the mechanisms underlining these effects are not elucidated. A peculiar aspect of trabectedin's mode of action is related to the fact that cells that are defective in transcription-coupled nucleotide excision repair (TC-NER), and are hypersensitive to UV light, are partially resistant to the drug. [24][25][26][27][28] It has therefore been hypothesized that in the presence of trabectedin, NER does not lead restoration of normal DNA function, but instead