Abstract Insulinoma-associated 1 (INSM1) is neuroendocrine (NE) transcription factor which temporal and spatial expression pattern is restricted to regions undergoing NE differentiation during embryogenesis. INSM1 is however re-expressed at high levels in small-cell lung cancer (SCLC) together with an array of other NE markers. The NE signature of SCLC is a central diagnostic tool for the disease but recent data pinpoints that this distinct signature might play a role in SCLC pathogenesis. The functional role of INSM1 in SCLC was here investigated by shRNA-mediated silencing of INSM1 in a panel of SCLC cell lines. Upon silencing of INSM1, significant reduction in cell viability was observed as measured by MTT assay. This reduction in cell viability was concomitant with decreased cell proliferation and increased apoptosis as measured by BrdU incorporation and cleaved caspase 3/7 levels, respectively. Accordingly with phenotypic growth changes it was demonstrated that INSM1 silencing caused reduced phosphorylation of members of the growth- and survival-promoting PI3K/Akt, MAPK and JAK-STAT signaling pathways and reduced levels of the inhibitors of apoptosis proteins (IAPs) members c-IAP1, XIAP and survivin. Importantly, it was shown that INSM1 is an upstream regulator of signatures previously shown to play a role in SCLC tumorigenesis, including achate-scute homolog 1 (ASCL1) and Sonic Hedgehog (SHh) pathway. Upon silencing of INSM1, a decrease in mRNA and protein levels of ASCL1 and downstream targets aldehyde dehydrogenase 1 (ALDH1) and delta-like ligand 3 (DLL3) was observed. Furthermore, mRNA levels of the SHh positive mediators Gli2, Gli3 and Smoothened was downregulated while the SHh inhibitor Patched was upregulated upon INSM1 silencing. Futhermore, overexpression of the Notch1 intracellular domain in SCLC cells resulted in a significant reduction in INSM1 expression and cell viability. This suggest that a master inhibitor of the NE signature, Notch1 signaling, could be an inhibitor of INSM1-driven tumorigenesis. In conclusion, INSM1 is a potential key driver of growth- and survival-promoting pathways in SCLC and warrants further investigation of INSM1 as a therapeutic target. Citation Format: Camilla L. Christensen, Takeshi Shimamura, Esra A. Akbay, Signe R. Michaelsen, Hans S. Poulsen, Kwok-kin Wong. Insulinoma-associated 1 is a key transcriptional regulator of growth and survival-promoting pathways in small cell lung cancer. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 3126. doi:10.1158/1538-7445.AM2013-3126