TheRASoncogene is both the most frequently mutated oncogene in human cancer and the first confirmed human oncogene to be discovered in 1982. After decades of research, in 2013, the Shokat lab achieved a seminal breakthrough by showing that the activated KRAS isozyme caused by the G12C mutation in theKRASgene can be directly inhibited via a newly unearthed switch II pocket. Building upon this groundbreaking discovery, sotorasib (AMG510) obtained approval by the United States Food and Drug Administration in 2021 to become the first therapy to directly target the KRAS oncoprotein in any KRAS-mutant cancers, particularly those harboring theKRAS^G12Cmutation. Adagrasib (MRTX849) and other direct KRAS^G12Cinhibitors are currently being investigated in multiple clinical trials. In this review, we delve into the path leading to the development of this novel KRAS inhibitor, starting with the discovery, structure, and function of the RAS family of oncoproteins. We then examine the clinical relevance of KRAS, especially the KRAS^G12Cmutation in human cancer, by providing an in-depth analysis of its cancer epidemiology. Finally, we review the preclinical evidence that supported the initial development of the direct KRAS^G12Cinhibitors and summarize the ongoing clinical trials of all direct KRAS^G12Cinhibitors.

Activated RAS is a common driver of cancer that was considered undruggable for decades. Recent advances have enabled the development of RAS inhibitors, but the efficacy of these inhibitors remains limited by resistance. In this study, we developed a pan-RAS inhibitor, ADT-007, (Z)-2-(5-fluoro-1-(4-hydroxy-3,5-dimethoxybenzylidene)-2-methyl-1H-inden-3-yl)-N-(furan-2-ylmethyl)acetamide, that binds nucleotide-free RAS to block GTP activation of effector interactions and MAPK/AKT signaling, resulting in mitotic arrest and apoptosis. ADT-007 potently inhibited the growth of RAS-mutant cancer cells irrespective of the RAS mutation or isozyme. Wild-type RAS (RASWT) cancer cells with GTP-activated RAS from upstream mutations were equally sensitive. Conversely, RASWT cancer cells harboring downstream BRAF mutations and normal cells were essentially insensitive to ADT-007. Sensitivity of cancer cells to ADT-007 required activated RAS and dependence on RAS for proliferation, whereas insensitivity was attributed to metabolic deactivation by UDP-glucuronosyltransferases that were expressed in RASWT and normal cells but repressed in RAS-mutant cancer cells. ADT-007 displayed unique advantages over KRAS mutant-specific, pan-KRAS, and pan-RAS inhibitors that could impact in vivo antitumor efficacy by escaping compensatory mechanisms that lead to resistance. Local administration of ADT-007 showed robust antitumor activity in syngeneic immunocompetent and xenogeneic immune-deficient mouse models of colorectal and pancreatic cancers. The antitumor activity of ADT-007 was associated with the suppression of MAPK signaling and activation of innate and adaptive immunity in the tumor immune microenvironment. Oral administration of ADT-007 prodrug also inhibited tumor growth. Thus, ADT-007 has the potential to address the complex RAS mutational landscape of many human cancers and to improve treatment of RAS-driven tumors. Significance: ADT-007, a first-in-class pan-RAS inhibitor, has unique selectivity for cancer cells with mutant RAS or activated RAS protein and the capability to circumvent resistance to suppress tumor growth, supporting further development of ADT-007 analogs.

The nonsteroidal anti-inflammatory drug (NSAID) sulindac demonstrates attractive anticancer activity, but the toxicity resulting from cyclooxygenase (COX) inhibition and the suppression of physiologically important prostaglandins precludes its long-term, high dose use in the clinic for cancer prevention or treatment. While inflammation is a known tumorigenic driver, evidence suggests that sulindac’s antineoplastic activity is partially or fully independent of its COX inhibitory activity. One COX-independent target proposed for sulindac is cyclic guanosine monophosphate phosphodiesterase (cGMP PDE) isozymes. Sulindac metabolites, i.e., sulfide and sulfone, inhibit cGMP PDE enzymatic activity at concentrations comparable with those associated with cancer cell growth inhibitory activity. Additionally, the cGMP PDE isozymes PDE5 and PDE10 are overexpressed during the early stages of carcinogenesis and appear essential for cancer cell proliferation and survival based on gene silencing experiments. Here, we describe a novel amide derivative of sulindac, sulindac sulfide amide (SSA), which was rationally designed to eliminate COX-inhibitory activity while enhancing cGMP PDE inhibitory activity. SSA was 68-fold and 10-fold less potent than sulindac sulfide (SS) in inhibiting COX-1 and COX-2, respectively, but 10-fold more potent in inhibiting growth and inducing apoptosis in breast cancer cells. The pro-apoptotic activity of SSA was associated with inhibition of cGMP PDE activity, elevation of intracellular cGMP levels, and activation of cGMP-dependent protein kinase (PKG) signaling, as well as the inhibition of β-catenin/Tcf transcriptional activity. SSA displayed promising in vivo anticancer activity, resulting in a 57% reduction in the incidence and a 62% reduction in the multiplicity of tumors in the N-methyl-N-nitrosourea (MNU)-induced model of breast carcinogenesis. These findings provide strong evidence for cGMP/PKG signaling as a target for breast cancer prevention or treatment and the COX-independent anticancer properties of sulindac. Furthermore, this study validates the approach of optimizing off-target effects by reducing the COX-inhibitory activity of sulindac for future targeted drug discovery efforts to enhance both safety and efficacy.

Approximately 45% of colorectal cancers harbor mutations in the KRAS gene, resulting in constitutive activation of RAS signaling through effector proteins, primarily RAF and PI3K. Members of the RAS family are GTPases that function as a molecular switch, cycling between inactive (GDP-bound) and active (GTP-bound) states in cells to regulate proliferation and survival. Constitutive signaling from mutant RAS drives tumorigenesis, in part, by deregulation of the cell cycle, resulting in increased proliferation (mitosis) and decreased apoptosis of tumor cells. From an extensive medicinal chemistry/screening campaign, we identified a novel compound class that potently and selectively inhibits the growth of tumor cell lines harboring constitutively activated RAS by a mechanism involving the disruption of RAS nucleotide loading, blockage of effector activation, and consequent inhibition of signaling. A prototype of the class, MCI-062, inhibits the growth of KRAS-driven (HCT-116) colon tumor cells with low nanomolar IC50 values, while RAS-independent (HT-29) colon tumor cells harboring the BRAF V600E mutation, are essentially insensitive. A strong correlation was measured among a larger panel of tumor cell lines between potency to inhibit tumor cell growth and levels of activated RAS. The growth inhibitory effects of MCI-062 were sustained and irreversible as demonstrated by colony formation and apoptosis assays. MCI-062 treatment of HCT-116 colon tumor cells reduced levels of activated RAS and RAS-mediated signaling as measured by GST-RBD pulldown assays and phospho-specific immunoblotting. Within the same concentration range, MCI-062 induced mitotic arrest as measured by cell cycle analysis of DNA content and phospho-histone H3B immunofluorescence. Further analysis revealed that MCI-062 interfered with localization of the mitosis-inducing protein, PLK1 to kinetochores and decreased nuclear localization of its substrate, Cdc25C, a downstream target of RAS-RAF signaling involved in both mitotic entry and exit checkpoints. In vivo testing of MCI-062 in a syngeneic mouse model of KRAS-driven colon cancer (CT-26) demonstrated that MCI-062 engages its molecular target, depleting GTP-RAS and suppressing activation of the MAPK signal transduction pathway, and inhibiting tumor growth. MCI-062 also suppresses PD-L1 expression and activates anti-tumor immunity, which may contribute to its antitumor activity and suggests potential benefits of combining with immunotherapy. From these studies, we have identified a novel class of RAS inhibitors that potently and selectively inhibits RAS-driven tumor growth by disrupting downstream signaling, leading to cell cycle arrest and apoptosis. These findings support further development of MCI-062 for treatment of RAS-driven colorectal and other cancers. Funding provided by NCI grants R01CA131378, R01CA148817, R01CA197147, and R01CA155638. Citation Format: Adam B. Keeton, Antonio Ward, Xi Chen, Jacob Valiyaveettil, Bing Zhu, Veronica Ramirez-Alcantara, Yulia Maxuitenko, Kristy Berry, Tyler E. Mattox, Michael R. Boyd, Gary A. Piazza. A novel RAS inhibitor, MCI-062, inhibits colon tumor growth in vivo and activates antitumor immunity [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 2707.