KRAS, RAS

Cell line panels have proven to be an invaluable tool for investigators researching a range of topics from drug mechanism or drug sensitivity studies to disease-specific etiology. The cell lines used in these panels may range from heterogeneous tumor populations grown from primary tumor isolations to genetically engineered clonal cell lines which express specific gene isoforms. Mouse embryonic fibroblast (MEF) cells are a commonly used cell line for biological research due to their accessibility and ease of genetic manipulation...

With recent advances proving that effective inhibition of KRAS is possible, there have been significant efforts made to develop inhibitors of specific mutant alleles. Here we describe a detailed protocol that employs homogeneous time-resolved fluorescence (HTRF) to identify compounds acting on KRAS signaling in malignant cell lines. This method allows for high-throughput, cell-based screens of large compound libraries for the development of RAS-targeted therapeutics.

Fluorescence lifetime imaging performed under FRET conditions between two interacting molecules is a sensitive and robust way to quantify intermolecular interactions in cells. The fluorescence lifetime, an inherent property of the fluorophore, remains unaffected by factors such as concentration, laser intensity, and other photophysical artifacts. In the context of FLIM-FRET, the focus lies on measuring the fluorescence lifetime of the donor molecule, which diminishes upon interaction with a neighboring acceptor molecule...

Missense mutations in the RAS family of oncogenes (HRAS, KRAS, and NRAS) are present in approximately 20% of human cancers, making RAS a valuable therapeutic target (Prior et al., Cancer Res 80:2969-2974, 2020). Although decades of research efforts to develop therapeutic inhibitors of RAS were unsuccessful, there has been success in recent years with the entrance of FDA-approved KRASG12C -specific inhibitors to the clinic (Skoulidis et al., N Engl J Med 384:2371-2381, 2021; Jänne et al., N Engl J Med 387:120-131, 2022)...

Knowledge of how effectors interact with RAS GTPases is key to understanding how these switch-like proteins function in cells. Effectors bind specifically to GTP-loaded RAS using RAS association (RA) or RAS binding domains (RBDs) that show wide-ranging affinities and thermodynamic characteristics. Both normal development and RAS-induced tumorigenesis depend on multiple distinct effector proteins that are frequently co-expressed and co-localized, suggesting an antagonistic nature to signaling whereby multiple proteins compete for a limited pool of activated GTPase...

RAS is regulated by specific guanine nucleotide exchange factors, such as Son of Sevenless (SOS), that activates RAS by facilitating the exchange of inactive, GDP-bound RAS with GTP. The catalytic activity of SOS is known to be allosterically modulated by an active, GTP-bound RAS. However, it remains poorly understood how oncogenic RAS mutants interact with SOS and modulate its activity. In this chapter, we describe the application of native mass spectrometry (MS) to monitor the assembly of the catalytic domain of SOS (SOScat ) with RAS and cancer-associated mutants...

MALDI-TOF mass spectrometry enables high-throughput screening of covalent fragment libraries and SAR compound progressions of selective KRAS G12C inhibitors. Using the MALDI-TOF platform instead of the more traditional ESI-MS TOF/orbitrap instrumentation can radically shorten sample acquisition time, allowing up to 384 samples to be screened in 30 min. The typical throughput for a covalent library screen is 1152 samples per 8 h, including processing, calculation, and reporting steps. The throughput can be doubled without any significant assay modification...

Surface plasmon resonance (SPR) is an optical effect at an electron-rich surface that enables affinity measurements of biomolecules in real time. It is label free and versatile, not limited to proteins, nucleic acids, and small molecules. SPR is a widely accepted method to measure not only affinity of molecular interactions but also association and dissociation rates of such interactions. In this chapter, we describe a general method to measure the affinity of a small molecule drug, MRTX849, to GDP bound HRAS, KRAS, and NRAS...

Oncogenic mutations in KRAS typically impact the GAP-mediated and intrinsic GTP hydrolysis activity resulting in elevated levels of cellular KRAS-GTP. The development of biochemical assays for GTPase activity provides an opportunity to quantitatively measure the impact of these mutations on GTP hydrolysis. Here we describe a biochemical assay that measures the release of free phosphate upon hydrolysis of the GTP nucleotide and allows the measurement of intrinsic or GAP-stimulated GTP hydrolysis by KRAS. This assay can be used to measure GTPase activity under single turnover conditions...

RAS proteins play a vital role in regulating downstream signaling and essential cellular processes, positioning them as key players in normal cellular physiology and disease development. Among the various isoforms of RAS, KRAS stands out as one of the most frequently mutated genes in human cancer. The prevalence of RAS mutations in cancer often involves single amino acid substitutions at codons 12, 13, or 61. These mutations disrupt the RAS protein's inherent ability to transition between its active and inactive states, resulting in a constant activation signal and driving uncontrolled cell growth...

Mutant forms of the RAS genes KRAS, NRAS, and HRAS are important and common drivers of cancer. Recently, two independent teams that integrated cancer genomics with cancer epidemiology estimated that approximately 15-20% of all human cancers harbor a mutation in one of these three RAS genes. These groups also estimate KRAS mutations occur in 11-14% of all human cancers. Although these estimates are lower than many commonly encountered values, these estimates continue to rank KRAS and the ensemble of RAS oncogenes among the most common genetic drivers of cancer across all forms of malignancy...

RAS research has entered the world of translational and clinical science. Progress has been based on our appreciation of the role of RAS mutations in different types of cancer and the effects of these mutations on the biochemical, structural, and biophysical properties of the RAS proteins themselves, particularly KRAS, on which most attention has been focused. This knowledge base, while still growing, has enabled creative chemical approaches to targeting KRAS directly. Our understanding of RAS signaling pathways in normal and cancer cells plays an important role for developing RAS inhibitors but also continues to reveal new approaches to targeting RAS through disruption of signaling complexes and downstream pathways...

Small GTPases of the Ras subfamily are best known for their role as proto-oncoproteins, while their function during microbial infection has remained elusive. Here, we show that Legionella pneumophila hijacks the small GTPase NRas to the Legionella-containing vacuole (LCV) surface. A CRISPR interference screen identifies a single L. pneumophila effector, DenR (Lpg1909), required for this process. Recruitment is specific for NRas, while its homologs KRas and HRas are excluded from LCVs. The C-terminal hypervariable tail of NRas is sufficient for recruitment, and interference with either NRas farnesylation or S-acylation sites abrogates recruitment...

The YAP-TEAD protein-protein interaction mediates YAP oncogenic functions downstream of the Hippo pathway. To date, available YAP-TEAD pharmacologic agents bind into the lipid pocket of TEAD, targeting the interaction indirectly via allosteric changes. However, the consequences of a direct pharmacological disruption of the interface between YAP and TEADs remain largely unexplored. Here, we present IAG933 and its analogs as potent first-in-class and selective disruptors of the YAP-TEAD protein-protein interaction with suitable properties to enter clinical trials...

The next-generation sequencing technologies application discovers novel genetic alterations frequently in pediatric acute lymphoblastic leukemia (ALL). RAS signaling pathway mutations at the time of relapse ALL frequently appear as small subclones at the time of onset, which are considered as the drivers in ALL relapse. Whether subclones alterations in the RAS signaling pathway should be considered for risk group stratification of ALL treatment is not decided yet. In this work, we investigate the RAS signaling pathway mutation spectrum and the related prognosis in pediatric ALL...

Prognoses for TNBC remain poor due to its aggressive nature and the lack of therapies that target its "drivers". RASA1, a RAS-GAP or GTPase-activating protein whose activity inhibits RAS signaling, is downregulated in up to 77% of TNBC cases. As such, RAS proteins become hyperactive and similar in effect to mutant hyperactive RAS proteins with impaired GTPase activities. PCAIs are a novel class of agents designed to target and disrupt the activities of KRAS and other G-proteins that are hyperactive in various cancers...

A remote carcinogen exposure can predispose to breast cancer onset decades later, suggesting carcinogen-induced mutations generate long-lived premalignant clones. How subsequent events influence the progression of specific premalignant clones remains poorly understood. Here, we generated multistage mouse models of mammary carcinogenesis by combining chemical carcinogen exposure (using 7,12-dimethylbenzanthracene, DMBA) with transgenes that enable inducible expression of one of two clinically relevant mammary oncogenes: c-MYC (MYC) or PIK3CAH1047R (PIK)...

PURPOSE: Small bowel adenocarcinoma (SBA) is a rare malignancy of the gastrointestinal tract, and its unique location within the small intestine presents difficulties in obtaining tissue samples from the lesions. This limitation hinders the research and development of effective clinical treatment methods. Circulating tumor DNA (ctDNA) analysis holds promise as an alternative approach for investigating SBA and guiding treatment decisions, thereby improving the prognosis of SBA. METHODS: Between January 2017 and August 2021, a total of 336 tissue or plasma samples were obtained and the corresponding mutation status in tissue or blood was evaluated with NGS...

Circular RNAs (circRNAs) have been recognized as key components in the intricate regulatory network of the KRAS pathway across various cancers. The KRAS pathway, a central signalling cascade crucial in tumorigenesis, has gained substantial emphasis as a possible therapeutic target. CircRNAs, a subgroup of non-coding RNAs known for their closed circular arrangement, play diverse roles in gene regulation, contributing to the intricate landscape of cancer biology. This review consolidates existing knowledge on circRNAs within the framework of the KRAS pathway, emphasizing their multifaceted functions in cancer progression...

CRAF is a primary effector of RAS GTPases and plays a critical role in the tumorigenesis of several KRAS-driven cancers. In addition, CRAF is a hotspot for germline mutations, which are shown to cause the developmental RASopathy, Noonan syndrome. All RAF kinases contain multiple phosphorylation-dependent binding sites for 14-3-3 regulatory proteins. The differential binding of 14-3-3 to these sites plays essential roles in the formation of active RAF dimers at the plasma membrane under signaling conditions and in maintaining RAF autoinhibition under quiescent conditions...