Altered RAS signaling pathways drive uncontrolled cell proliferation, migration, and survival, ultimately leading to tumor development and progression Effective inhibition of RAS signaling holds the potential to benefit roughly a quarter of human cancer patients whose tumors are driven by RAS mutations. In pancreatic ductal adenocarcinoma (PDAC), the third leading cause of cancer related mortality in the US, over 90% of patient cases are driven by activating mutations in KRAS. Here we assessed two divergent approaches to target aberrant RAS signaling for PDAC treatment.In Part I we investigated the therapeutic potential of the RAS(ON) multi-selective inhibitor RMC-7977, a highly potent compound blocking the active GTP-bound forms of KRAS, HRAS, and NRAS, showing affinity for both mutant and wild type (WT) variants. We evaluated RMC-7977 in a comprehensive range of preclinical PDAC models and, following direct RAS inhibition, we observed broad and pronounced anti-tumor activity, at exposures that were well-tolerated in vivo. Pharmacological analyses revealed divergent responses to RMC-7977 in tumor versus normal tissues. Treated tumors exhibited waves of apoptosis along with sustained proliferative arrest whereas normal tissues underwent only transient decreases in proliferation, with no evidence of apoptosis. In the autochthonous KPC model, RMC-7977 treatment resulted in a profound extension of survival followed by on-treatment relapse. Analysis of relapsed tumors identified Myc copy number gain as a prevalent candidate resistance mechanism, which could be overcome by combinatorial TEAD inhibition in vitro. Together, these data established a strong preclinical rationale for the use of broad-spectrum RAS-GTP inhibition in the setting of PDAC and identified a promising candidate combination therapeutic regimen to overcome monotherapy resistance. In Part II, we investigated the therapeutic potential of combined MEK and autophagy pathways inhibition in preclinical models of PDAC. Consistent with previously published work, MEK inhibition increased the dependency of PDAC cells on autophagy, and the combination of Trametinib (a MEK inhibitor) and Hydroxychloroquine (an autophagy inhibitor) demonstrated synergistic anti-proliferative effects in murine PDAC cell lines. We added to this body of work by evaluating the combination in the clinically predictive genetically engineered mouse models (GEMMs). Trametinib/Hydroxychloroquine treatment led to significant tumor regressions, MAPK signaling inhibition, and reduced cell proliferation in the KPC mice, as well as improved survival in the KPF/FC model. Single-cell RNA sequencing analysis of treated KPC tumors revealed both treatment sensitive and resistant malignant cell populations, with the sensitive cells showing upregulated MAPK and autophagy pathway activities. Analysis of fibroblast population additionally revealed that inflammatory CAFs (iCAFs) are diminished in response to treatment, likely due to disrupted paracrine signaling mediated by IL1A secretion from malignant cells. Together, these data provided a preclinical rationale for the use of the Trametinib and Hydroxychloroquine combination in PDAC treatment. However, recent updates from ongoing clinical trials have reported limited clinical activity of this combination. The datasets generated here may help identify key discrepancies between preclinical models and clinical samples that contribute to the lack of translation, and guide the development of improved therapeutic combinations.