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Directly targeting RAS is a desirable approach for treating RAS-mutated tumors because direct RAS inhibitors block all RAS effector pathways simultaneously. Recently, several groups have developed agents that directly target KRAS by covalently modifying KRASG12C. KRAS is commonly altered in adult malignancies such as lung, pancreatic, and colorectal adenocarcinoma, but is less commonly altered in pediatric cancer. However, rare pediatric tumors harboring K-, H-, or NRASG12C are observed. The efficacy of KRASG12C inhibitors in pediatric malignancies is currently unknown, and the ability of these drugs to modify H- and NRASG12C has been incompletely characterized. Here we show that the KRASG12C inhibitors AMG 510, MRTX849, and ARS-1620 slow the release of GDP from H-, N-, and KRASG12C in vitro. Using RASless MEFs, we show that the activity of AMG 510 is specific for H-, N- or KRASG12C, while MRTX849 has both G12C and RAS-independent effects on cell viability. The KRASG12C inhibitors decreased cell viability in KRAS-mutant cell lines derived from patients with the pediatric solid tumor neuroblastoma. MRTX849 was more potent than AMG 510 in neuroblastoma cell lines but was less efficacious and less G12C-specific. The KRASG12C inhibitors also decreased cell viability in an NRASG12C-mutant T-cell ALL cell line derived from an adolescent patient but only MTRX849 decreased cell viability in an HRASG12C-mutant rhabdomyosarcoma cell line. These results suggest that KRASG12C inhibitors may be a treatment option for pediatric patients with H-, N- or KRASG12C mutant solid tumors or leukemia. Future studies will be aimed at testing the efficacy of these inhibitors in xenograft models of pediatric cancer and identifying non-RAS targets of MRTX849. Citation Format: Hannah Price, Stacey Stauffer, Katie Powell, Andrew Baker, Andrew Perciaccante, Edjay Ralph Hernandez, Patience Odeniyide, Christine Pratilas, William Burgan, Lisa Jenkins, Kent L. Rossman, Marielle Yohe. Preclinical efficacy of KRASG12C inhibitors in models of pediatric cancer [abstract]. In: Proceedings of the AACR Special Conference: Targeting RAS; 2023 Mar 5-8; Philadelphia, PA. Philadelphia (PA): AACR; Mol Cancer Res 2023;21(5_Suppl):Abstract nr B038.

Shortly after the formation of the NCI Ras Initiative, a H/N/KRAS-less mouse embryonic fibroblast (MEF) cell line panel was generated, validated, and shared internationally to aid the research community in its reinvigorated effort to apply new technical advancements to understanding the structure and function of Ras and to ultimately design inhibitors. While this panel has been beneficial to the Ras research community, an inherent variability due in part to the ploidy of the parental pool from which the panel was derived complicated comparisons between isolates and alleles. We have minimized this variation by utilizing a diploid clone derived from the DU1473 MEF; Hras-/-; Nras-/-; Kraslox/lox; RERTert/ert - parental cell line. A diploid cell population was sorted, cloned and clones were cultured continuously for up to 20 passages to identify those that retained a diploid signature. These clones were further screened for p53 mutations and mouse exome sequenced from which a final clone was selected to develop the improved cell line panel. The selected clone was treated with 4-hydroxytamoxifen (4-OHT) to activate the Cre-recombinase which excised a portion of the endogenous Kras located between two loxP sites effectively arresting cells in a senescent G1 state. Molecular verification of Kras removal was performed, and the cells were then transduced with a third generation VSV-G pseudotyped lentiviral vector which delivered different versions of the human Ras transgene as well as an antibiotic selection marker. This transduction restored the proliferative ability of the cells allowing for expansion and selection of the transduced pool. The pools were single cell cloned and subsequent clones underwent a systematic, rigorous quality control evaluation culminating with another round of exome sequencing before clones were selected to be part of the MEF 2.0 panel. Clones contained in this new panel were confirmed to be diploid, had fewer integrations than the initial panel, and performed similarly on proliferation assays as well as epidermal growth factor stimulation signaling assays. The new panel has successfully recapitulated the initial panel, but in a far more stable and reproducible cellular background. Instructions to obtain the cell line panel will be presented on the Ras Initiative website at www.cancer.gov and these cells are scheduled to become available to the Ras research community late in the spring of 2023. Citation Format: William E. Burgan, Nicole Fer, Katie Powell, Bingfang Xu, Mackenzie Meyer, Scott Eury, Abigail Neish, Daniel Soppet, Dominic Esposito, Frank McCormick, Dwight V. Nissley. An isogenic H/N/KRAS-less mouse embryonic fibroblast cell line panel derived from a size sorted diploid clonal parent [abstract]. In: Proceedings of the AACR Special Conference: Targeting RAS; 2023 Mar 5-8; Philadelphia, PA. Philadelphia (PA): AACR; Mol Cancer Res 2023;21(5_Suppl):Abstract nr A028.

The RAS family of GTPases (HRAS, KRAS4A, KRAS4B, NRAS) provide a formidable challenge to standard proteomic workflows. Linking key N-terminal mutations (e.g., G12C, G13D) with C-terminal post-translational modifications (PTMs) on a given RAS isoform by proteolytic digestion and peptide-based analysis is further complicated by their high sequence identity, low endogenous abundance, and labile PTMs. Conversely, top-down mass spectrometry (TDMS), which measures intact and modified protein forms (proteoforms), can precisely identify and confidently localize mutations and PTMs within each RAS isoform sequence. Moreover, by combining immunoprecipitation with TDMS (IP-TDMS), endogenous RAS proteoforms can be isolated to near-homogeneity from malignant cell lines, detected at high signal-to-noise ratios via selected ion monitoring, and targeted for optimized MS2-based characterization to provide molecular detail simply unachievable by other proteomic methods. The NCI RAS Initiative has developed an optimized RAS proteoform assay incorporating IP-TDMS analysis on an Orbitrap Fusion Lumos mass spectrometer. We initially employed recombinant versions of each RAS isoform to determine the optimal targeted MS2 fragmentation (tMS2) parameters for sequence characterization and localization of endogenous PTMs. We next isolated FLAG-tagged KRAS4B -WT, -G12D, and -G12C expression constructs from HeLa cells to refine our liquid chromatography and tMS2 parameters while targeting stoichiometrically abundant KRAS4B proteoforms within each population. We then isolated FLAG-tagged HRAS-, KRAS4A-, KRAS4B-, and NRAS-G12V proteins from HeLa or Panc1 cells for further refinement of isoform-specific tMS2 parameters with emphasis on capturing unusual or labile PTMs (e.g. palmitoylation). This allowed us to visualize and directly compare the most abundant RAS isoform-specific proteoform populations within these cellular contexts. Complementary peptide-based analyses were performed on all samples to validate RAS isoform-specific PTM landscapes and facilitate detection of their respective endogenous versions. Finally, we applied our optimized IP-TDMS workflow to a panel of well-characterized malignant and RAS mutant cell lines with the goal of expanding our knowledge of the endogenous RAS proteoform landscape. In doing so, we identified a wealth of novel HRAS, KRAS4A, KRAS4B, and NRAS proteoforms bearing stoichiometrically abundant PTMs differing markedly from the established literature. We also performed direct comparisons between isoform- and mutation-specific RAS proteoforms, revealing clear indications of context dependence. These initial results further underscore the potential for top-down proteomics to broaden our understanding of RAS-dependent signaling within a range of cancer contexts. They also reveal that extensive investigations remain to be performed to elucidate the roles of RAS PTMs and proteoforms in normal and oncogenic signaling. Citation Format: Caroline J. DeHart, Robert A. D'Ippolito, Kanika Sharma, Nicole Fer, Brian Smith, Mackenzie Meyer, Scott Eury, Abigail Neish, Katie Powell, Vanessa Wall, William Burgan, Dominic Esposito, Anna E. Maciag, Frank McCormick, Dwight V. Nissley. Expanded RAS proteoform landscape in malignant cell lines revealed by top-down mass spectrometry [abstract]. In: Proceedings of the AACR Special Conference: Targeting RAS; 2023 Mar 5-8; Philadelphia, PA. Philadelphia (PA): AACR; Mol Cancer Res 2023;21(5_Suppl):Abstract nr A016.