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KRAS is the most frequently mutated oncogene in human cancer, particularly in cancers with a high mortality rate such as pancreatic, colorectal, and non-small cell lung cancer (NSCLC) (Ryan and Corcoran, 2018). While effective therapies directly targeting KRAS-mutant tumors have yet to be fully validated, recent clinical trials show positive progress for patients with the KRAS(G12C) mutation (Canon et al. 2019). Moreover, sequencing data has allowed for better understanding of how secondary mutations synergize with oncogenic KRAS to drive tumor progression. For example, activating mutations in KRAS frequently occur with loss-of-function mutations in the gene STK11, which encodes the tumor suppressor liver kinase B1 (LKB1), resulting in decreased patient survival, de novo resistance to targeted treatments and immunotherapies, and increased likelihood of tumor recurrence (Cancer Genome Atlas Research Network 2014, Skoulidis et al. 2018, Caiola et al. 2018). Additionally, previous work from genetically engineered mouse models (GEMMs) suggests loss of Lkb1 is sufficient to promote the progression and metastasis of nascent Kras-driven lung adenocarcinoma (Ji et al. 2007). Therefore, we sought to determine whether knockdown of Lkb1 by RNAi could cooperate with activating mutations in Ras to drive tissue overgrowth in wing imaginal discs of the genetically tractable model organism Drosophila melanogaster. To address this question, we obtained transgenic Drosophila expressing oncogenic RasV12, which on its own causes hyperplastic growth balanced by non-autonomous cell death in imaginal tissues (Karim and Rubin 1998). To knockdown Lkb1 we obtained an RNAi fly stock (Lkb1RNAi) developed by the Transgenic RNAi Project (TRiP) (Dietzl et al. 2007) and validated through the Harvard Medical School RNAi Stock Validation and Phenotypes (RSVP) resource (Perkins et al. 2015). Of note, the Lkb1RNAi stock was determined to have approximately 68% knockdown efficiency when used with the MTD-Gal4 driver (Sopko et al. 2014). Additional validation using the Updated Targets of RNAi Reagents (UP-TORR) Fly resource confirmed no off-target effects with this RNAi sequence (Hu et al. 2013). We generated a combined RasV12/Lkb1RNAi fly line, and crossed our double mutant (along with single transgenes as controls) to the MS1096-Gal4, UAS-GFP wing pouch driver. In order to precisely measure effects on overall organ size, we used confocal microscopy to acquire z-sections through the entire wing disc, followed by 3D reconstruction and volume measurements using IMARIS software. We determined that total wing disc volume was significantly larger in MS1096-Gal4; RasV12/Lkb1RNAi tissues compared to control genotypes. (A-B). Previous investigations have shown that Lkb1 can exert a non-autonomous role in tumor suppression (Katajisto et al. 2008; Tanwar et al. 2012; Ollila et al. 2018). Therefore, we investigated whether the increase in organ size was due to autonomous vs. non-autonomous effects on growth. To do this we measured individual volumes of GFP-positive and GFP-negative tissue across genotypes. Expression of RasV12/Lkb1RNAi led to significant autonomous overgrowth in the GFP-positive MS1096 expression domain, while the GFP-negative (non-autonomous) tissue compartment remained unchanged (C-D). Changes in organ size control can result from any number of combinations of cell growth, proliferation, and cell death phenotypes. To investigate the compartmental effects on cell proliferation and cell death in RasV12/Lkb1RNAi tissues, we used the MS1096-Gal4 driver to express Lkb1RNAi, RasV12, or RasV12/Lkb1RNAi in developing wing pouches. Tissues were either labeled with BrdU or an anti-Death Caspase-1 (DCP-1) antibody (E-F, G-H). Knockdown of Lkb1 alone resulted in no change in the absolute levels of BrdU or DCP-1 relative to control discs (F, H). Expression of RasV12 alone resulted in a mild increase in the amount of autonomous BrdU and non-autonomous DCP-1 (F, H). Alternatively, co-expression of RasV12/Lkb1RNAi led to a dramatic shift in cellular phenotypes with a large increase in autonomous and non- autonomous BrdU – and a rescue of the non-autonomous cell death observed in cells expressing RasV12 alone. Therefore, knockdown of Lkb1 in the context of oncogenic Ras in the Drosophila wing pouch can exert both non-autonomous and autonomous effects that override organ size control. Future studies will focus on the signaling pathways responsible for both phenotypes which could represent novel, targetable pathways for the thousands of cancer patients in the U.S. with LKB1 mutations.