Abstract Background: Resistance arises in patients with small cell lung cancer (SCLC) following treatment with chemotherapeutic agents. Suicide gene (SG) therapy is a novel treatment strategy for cancer in which the introduced therapeutic gene encodes an enzyme capable of transforming a non-toxic prodrug into a cell poison. By coupling the SCLC specific promoter Insulinoma-associated 1 (INSM1) to the SG, it is possible to target SG expression and resulting cytotoxicity exclusive to SCLC cells. The aim of this project was to investigate the influence of chemo-resistance on transcriptional targeted SG therapy. Results: As an in vitro model of chemo-resistance we used a number of SCLC cell lines resistant to different chemotherapeutic agents used in first-line treatment of SCLC patients. Protein expression analysis showed the cells lines to vary in expression of a several proteins correlated to development of resistance. Despite of this the INSM1 promoter activity was found to be equally or increased in chemo-resistant compared to chemo-sensitive cells as measured by luciferase reporter assays and determination of INSM1 mRNA levels by RT-PCR. Additionally, in tumor xenografts stably expressing EGFP from the INSM1 promoter it was apparent that the INSM1 promoter activity is very strong and stable in vivo. Cytotoxic effects of two different SG systems driven by the INSM1 promoter were tested in the chemo-resistant cell lines by MTT assays. One consisted of the fusion SG of yeast-cytosine-deaminase (YCD) and yeast-uracil-phosphoribosyl-transferase (YUPRT) and the prodrug 5-fluorocytosin (5-FC). The other system consisted of the Nitroreductase (NTR) gene and SN27686 prodrug. While equal sensitivity was found in chemo-sensitive and -resistant cells towards YCD-YUPRT/5-FC therapy, the NTR/SN276868 system showed reduced cytotoxicity in cells resistant to alkylating agents. Importantly, equal cytotoxicity could be obtained in these chemo-resistant cells upon NTR/SN27686 therapy by exposing cells to higher prodrug doses, still not inducing off-target toxicity in NTR-negative cells. Finally, the combination of YCD-YUPRT/5-FC SG therapy with standard chemotherapy and with NTR/SN27686 SG therapy, respectively, were tested and evaluated by MTT assay. In contrast to chemo-sensitive cells, where the combination therapy had no additive effect, significantly additive cytotoxicity was achieved in chemo-resistant SCLC cells exposed to combination therapy compared to single agent therapy. Conclusion: The results demonstrate that targeted SG therapy is equally effective in chemo-resistant and -sensitive SCLC cells and that additional effect can be achieved in chemo-resistant SCLC cells when SG therapy is given in combination with chemotherapy or another SG therapy system. INSM1-driven SG therapy therefore seems potent to be effective in the treatment of SCLC patients with chemo-resistant tumors. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 832. doi:1538-7445.AM2012-832