Abstract Background Small cell lung cancer (SCLC) is an aggressive high grade neuroendocrine tumor (Hann et al. 2019; Bernhardt et al. 2016). Whilst MAPK mutations can be found in roughly 30% of human cancers (Schubbert et al. 2007) including non-small cell lung cancer (NSCLC), genomic and proteomic analyses have indicated suppression of MAPK pathway activity in SCLC (Cerami et al. 2012; Gao et al. 2013). Previous attempts to determine whether this might be therapeutically important (Ravi et al, 1998; Cristea et al. 2016/2020) have had conflicting conclusions. SCLC has recently been defined by the relative expression of four major transcriptional regulators (ASCL1, NEUROD1, POU2F3, YAP1) (Rudin et al., 2019). In this study we aimed to elucidate the effect of MAPK activation in these different SCLC subtypes and explore its therapeutic vulnerability. Results ASCL1-, NEUROD1- and POU2F3- driven SCLC cell lines were transduced with a doxycycline-inducible vector for expression of MEKDDS217D/S221D (MEK1). Activation through MEK1 in ASCL1-driven SCLC cell lines resulted in a significant decrease in cell growth over 9 days. This was associated with G2 cell-cycle arrest and senescence. Expression of MEK1 in the cells of other SCLC subtypes and NSCLC failed to show any appreciable changes in cell growth. Remarkably, athymic mice injected with a MEK1 expressing ASCL1-driven cell line showed significantly slower tumor formation and longer survival than when MEK1 was not expressed. Notably, we observed the opposite when MEK1 expressing NEUROD1-driven cells were injected into athymic mice. We observed strong upregulation of negative feedback regulators DUSP6 and SPRY2 upon MAPK activation which has previously been described in solid tumors and pre-B ALL (Courtois-Cox et al 2006; Shojaee et al. 2015). Interestingly, phosphokinase array demonstrated that, almost exclusively, STAT3 through phosphorylation at S727 was strongly upregulated in the ASCL1-driven subtype after MEK1 expression. This prompted us to look at other regulators of the JAK-STAT pathway and found an increase in phosphorylation of the inhibitory phosphatase PTPN6 (SHP1) in the ASCL1-driven subtype and no STAT5 phosphorylation in any of the subtypes. We next examined whether these cells were sensitive to STAT3 inhibition. Upon treatment with a STAT3 inhibitor, stattic, ASCL1-driven SCLC cells reached their IC50 after 3-5 days in comparison to 9 days for other SCLC subtypes. MEK inhibition through PD0325901 rescued growth inhibition upon MAPK activation and was further associated with a decrease in DUSP6, SPRY2 and pS727 STAT3. JAK1/2 inhibition through ruxolitinib had no effect on cell viability. Summary These findings suggest that ASCL1-driven SCLC in vitro and in vivo is sensitive to activation of MAPK signaling. Our data provides additional understanding of SCLC biology and its complex signaling networks and potential subtype-specific drug susceptibilities. Citation Format: Rebecca Caeser, Christopher Hulton, Emily Costa, Vidushi Durani, Megan Little, Xiaoping Chen, Sam E. Tischfield, Marina Asher, Faruk Erdem Kombak, Shweta S. Chavan, Nisargbhai S. Shah, Metamia Ciampricotti, Elisa de Stanchina, John T. Poirier, Charles M. Rudin, Triparna Sen. STAT3-driven MAPK activation represents a therapeutic vulnerability in ASCL1 high SCLC [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 90.