Abstract RAS mutations are highly prevalent in tumors, especially those of the lung, colon, skin and pancreas. The tissues in which these tumors arise are enriched for specific RAS isoform and codon preferences. For example, mutations in KRAS are prevalent in lung, colon and pancreas, while NRAS mutations are predominantly found in melanoma. Additionally, mutations in KRAS predominantly occur at the hotspot codons G12 or G13 and NRAS mutations predominantly occur at the hotspot codon Q61. Independent of the RAS isoform, the mutated codon and specifically the amino acid substitution at that codon contribute to distinct biochemical differences. We aim to address if different amino acid substitutions at hotspot codons induce differential transformation, i.e., KRAS G12C versus KRAS G12D. Further, we aim to address if different codons are disposed to differential transformation capabilities and if these are isoform specific, for example, if KRAS Q61 mutations are similar to KRAS G12 mutations and if KRAS Q61 mutations are similar to NRAS Q61 mutations. Additionally, we aim to characterize the transforming capacity of RAS alterations outside of hotspot codons. Hundreds of sporadic insertions and point mutations in RAS have been observed clinically, yet most of these alterations have not been experimentally investigated. To address these questions, we created a pooled library of 184 RAS (KRAS, NRAS and HRAS) alterations identified in 29,000 tumors from a panel of ~100,000 pan-cancer tumors. These alterations include missense, insertion and deletion mutations irrespective of the incidence in which they occur. The library contains the greatest representation of KRAS alterations, 104. Using this pooled library, we measured the relative proliferation effect of these RAS variants using Ba/F3 cells in a competition assay. In interleukin 3 (IL-3)-dependent murine pro B-cells, Ba/F3, expression of well-characterized RAS hotspot mutations led to IL-3-independent proliferation. Due to the robust Ba/F3 cellular transformation we observed upon expression of RAS variants, we sought to use this assay for the pooled-library screen. The Ba/F3 system enables us to interrogate the transformation capacity of the RAS alterations and to identify the relative strength of these mutations while assaying independent of tissue type. Citation Format: Amanda R. Moore, Jean-Philippe Fortin, Michael Costa, Marinella Callow, Benjamin Haley, Gerard Manning, Ethan S. Sokol, Shiva Malek. A pan-cancer RAS mutant library elucidates the transformation potential of RAS variants [abstract]. In: Proceedings of the AACR Special Conference on Targeting RAS-Driven Cancers; 2018 Dec 9-12; San Diego, CA. Philadelphia (PA): AACR; Mol Cancer Res 2020;18(5_Suppl):Abstract nr A46.