Abstract Oncogenic KRAS signaling not only promotes progression and survival of cancer cells, but it can also play a major role in suppressing anti-tumor immunity through several mechanisms, including secretion of cytokines, reduction of antigen presentation and suppression of tumor-intrinsic interferon responses. Consequently, treatment with KRASG12C inhibitors alleviates oncogenic KRAS driven immune suppression, resulting in a profound remodeling of the tumor microenvironment (TME). In immunogenic mouse models, these changes can lead to the generation of some complete responses which are dependent on the engagement of the immune system. In this project we aim to investigate whether the induction of immune responses triggered by KRASG12C inhibition can effectively target inhibitor-resistant subclones. To achieve this, we mimic the development of drug resistance by combining reporter-traced isogenic KRASG12C and KRASG12D mutant lung cancer cells, where the KRASG12D mutant cells are inherently resistant to KRASG12C inhibitors. Growth of resistant cells was monitored via in vivo luciferase imaging and end point flow cytometry. KRASG12C inhibition as monotherapy, either with the active-state KRASG12C inhibitor RMC-4998 or the inactive-state inhibitor adagrasib, led to a proliferative advantage of the drug-resistant subpopulation, suggesting that any immune response to KRASG12C inhibition in the bulk tumor population was insufficient to target the resistant cell subclones. To investigate if immune mediated elimination of resistant cells could be achieved using combinations, we assessed two strategies commonly evaluated in clinical trials with KRASG12C inhibitors: anti-PD1 blockade and SHP2 inhibition. Of note, KRASG12D mutant cells do not respond to these therapies when grown subcutaneously. Treatment of RMC-4998 in combination with either anti-PD1 or the SHP2 inhibitor RMC-4550 was able to induce complete eradication of both KRASG12C and KRASG12D mutant lung cancer cells, despite the resistant cells being insensitive to these therapies when treated in the absence of sensitive cells. Furthermore, both combinations induced immune memory towards the resistant cells. These long-term effects were dependent on the immune system as they were not observed in immune deficient mice. Immune profiling of treated tumors revealed that both combinations can result in a more profound remodeling of the TME and potentiate the immune responses. Overall, our preclinical results demonstrate that the anti-tumor responses generated by the combination of KRASG12C inhibitors with SHP2 inhibition and/or immune checkpoint blockade can result in the bystander immune mediated killing of subclones of KRASG12C inhibitor resistant cells, providing a paradigm for the development of therapeutic combinations that may be more able to counter the development of inhibitor resistance. Citation Format: Mona Tomaschko, Christopher Moore, Claire E. Pillsbury, Kangbo Ng, Sophie de Carne, Sareena Rana, Andrea de Castro, Panayiotis Anastasiou, Miriam Molina-Arcas, Julian Downward. Investigating therapeutic strategies to promote immune rejection of KRASG12C inhibitor-resistant subpopulations in lung cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 1214.