Background: Non-small cell lung cancer (NSCLC) is the leading cause of cancer-related death worldwide. It is a very heterogeneous disease where some frequent mutations remain untargetable. Among them there are those affecting STK11/LKB1, the third most commonly mutated gene in NSCLC adenocarcinomas. Patients harboring LKB1-mutated tumors often have a poor prognosis due to the aggressiveness of this type of cancer and the lack of specific and efficacious therapies. Previous studies in our laboratory demonstrated a peculiar in vitro and in vivo activity of ERK inhibitors (ERKi) on LKB1-mutated NSCLC preclinical models. However, due to the huge heterogeneity of this type of tumor, it is likely that ERKi alone will have limited clinical application. In this scenario, the aim of this project is to find ERKi synthetic lethal partners in order to enlarge NSCLC patient population amenable to this therapy. Methods: We performed high-throughput screenings of a FDA-approved drug library on two different cell line systems (LU99 and H358) each composed by the LKB1 wild-type (wt) parental cell line and the CRISPR-CAS9-derived LKB1 deleted clone. In both the screenings, we treated the cell lines with the FDA-approved library alone or in combination with a subtoxic concentration of the ERKi. Results: According to our aim, for each screening, we analyzed the results selecting as hits those combinations preferentially active on the LKB1-deleted clones compared to their LKB1-wt cell lines. We verified that the FDA-approved compounds were subtoxic when used as single treatment in both the parental cell lines and their clones. We highlighted 28 and 42 hit combinations for LU99 and H358 isogenic systems, respectively. Some hits were common between the two screenings and the most represented FDA-approved drug’s classes of the hit combinations were PI3K/Akt/mTOR inhibitors, tyrosine kinase receptor inhibitors, MAPK inhibitors and compounds involved in anti-inflammatory pathways neuronal signaling, metabolism and DNA damage. Among the hits, we have cross-validated 4 ERKi-based combinations (a tyrosin kinase inhibitor, a MAPK inhibitor, a serine-threonine selective protein inhibitor and a farnesyl transferase inhibitor) in four different isogenic systems and we are now performing further validation on a panel of NSCLC cell lines with different LKB1-status as well as on NSCLC organoids. Conclusions: From the FDA-approved drug library screenings, different ERKi-based combinations showed promising results worthy to be deeply studied. Further analyses are ongoing to verify their efficacy on more complex models as NSCLC 3D organoids and to clarify the mechanism of action at the basis of the hit combinations. Positive results could give the chance to develop effective and specific therapies for patient with LKB1-mutated NSCLCs. Citation Format: Marika Colombo, Matteo Demetrio Tripodi, Gabriel Caso, Elisa Perin, Mirko Marabese, Massimo Broggini, Elisa Caiola. Identification of ERK inhibitor-based combinations targeting LKB1-mutated NSCLC [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 6346.