Biochemical and Structural Analysis of Common Cancer-Associated KRAS Mutations

Hunter, John C.; Manandhar, Anuj; Carrasco, Martin A.; Gurbani, Deepak; Gondi, Sudershan; Westover, Kenneth D.

doi:10.1158/1541-7786.mcr-15-0203

Cited by 581 publications

(674 citation statements)

References 67 publications

(79 reference statements)

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“…Specifically, Q61 mutant tumors exhibit less MAPK activity compared with other mutants and are associated with better prognosis (Witkiewicz et al 2015), a finding consistent with the notion that different KRAS mutations drive diverse signaling outputs with distinct biological consequences (Ihle et al 2012;Hunter et al 2015). The complexity of KRAS in PDAC is also manifest by genomic studies identifying the coexistence of multiple different KRAS mutations in the same tumor (Murphy et al 2013).…”

Section: Complexity Of Kras Oncogene Mutations As Pdac Driverssupporting

confidence: 58%

Genetics and biology of pancreatic ductal adenocarcinoma

et al. 2016

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With 5-year survival rates remaining constant at 6% and rising incidences associated with an epidemic in obesity and metabolic syndrome, pancreatic ductal adenocarcinoma (PDAC) is on track to become the second most common cause of cancer-related deaths by 2030. The high mortality rate of PDAC stems primarily from the lack of early diagnosis and ineffective treatment for advanced tumors. During the past decade, the comprehensive atlas of genomic alterations, the prominence of specific pathways, the preclinical validation of such emerging targets, sophisticated preclinical model systems, and the molecular classification of PDAC into specific disease subtypes have all converged to illuminate drug discovery programs with clearer clinical path hypotheses. A deeper understanding of cancer cell biology, particularly altered cancer cell metabolism and impaired DNA repair processes, is providing novel therapeutic strategies that show strong preclinical activity. Elucidation of tumor biology principles, most notably a deeper understanding of the complexity of immune regulation in the tumor microenvironment, has provided an exciting framework to reawaken the immune system to attack PDAC cancer cells. While the long road of translation lies ahead, the path to meaningful clinical progress has never been clearer to improve PDAC patient survival. Pancreatic ductal adenocarcinoma (PDAC) pathogenesisOn gross inspection, PDAC presents as a poorly demarcated, firm white-yellow mass, with the surrounding nonmalignant pancreas typically showing atrophy, fibrosis, and dilated ducts due to the obstructive effects of expanding carcinoma. Microscopically, these neoplasms vary from well-differentiated gland-forming carcinomas to poorly differentiated "sarcomatoid" carcinomas diagnosed only upon immunolabeling due to predominant mesenchymal features (Hruban et al. 2007). PDAC evolves from well-defined precursor lesions that, in the context of their genetic features, define the genetic progression model of pancreatic carcinogenesis (Yachida et al. 2010). Early disease histology manifests as several distinct types of precursor lesions-the most common are microscopic pancreatic intraepithelial neoplasia (PanIN), followed by the macroscopic cysts; namely, the intraductal papillary mucinous neoplasm (IPMN) and mucinous cystic neoplasm (MCN) (Matthaei et al. 2011). Since most PDAC cases become clinically apparent at advanced stages, major opportunities to reduce mortality rest with diagnostics and treatments that would enable the reliable identification and elimination of high-risk precursor lesions. Thus, the identification of these precursor lesions has provided an essential framework to define the genomic features that drive progression to advanced disease and develop effective screening and targeted therapeutics for earlier stage disease (Eser et al. 2011).The noninvasive PanIN lesions were formerly classified into three grades according to the extent of cytological and architectural atypia: PanIN1A (flat lesion) and PanIN1B (micropapillary...

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Section: Complexity Of Kras Oncogene Mutations As Pdac Driverssupporting

confidence: 58%

Genetics and biology of pancreatic ductal adenocarcinoma

et al. 2016

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“…These factors are likely to include allele-specific attributes of oncogenic mutations in genes such as KRAS 36 and BRAF [36][37][38] ; the cell lineage in which the cancer has arisen 22,37,39 ; the levels of expression of mutant cancer genes 32,38,40,41 ; the co-existence of certain additional mutations 42 ;…”

Section: Discussionmentioning

confidence: 99%

Hyperactivation of extracellular signal-regulated kinase (ERK) by RAS-mediated signaling or inhibition of dual specificity phosphatase 6 (DUSP6) is associated with toxicity in lung adenocarcinoma cells with mutations in KRAS or EGFR

Harbourne

et al. 2017

Preprint

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We recently described the synthetic lethality that results when mutant KRAS and mutant EGFR are coexpressed in human lung adenocarcinoma (LUAD) cells, revealing the biological basis for the mutual exclusivity of KRAS and EGFR mutations in lung cancers. We have now further defined the biochemical events responsible for the toxic effects of signaling through the RAS pathway. By combining pharmacological and genetic approaches, we have developed multiple lines of evidence that signaling through extracellular signalregulated kinases (ERK1/2) mediates the toxicity. These findings imply that tumors with mutant oncogenes that drive signaling through the RAS pathway must restrain the activity of ERK1/2 to avoid cell toxicities and enable tumor growth. In particular, a dual specificity phosphatase, DUSP6, regulates phosphorylated (P)-ERK levels in lung adenocarcinoma cells, providing negative feedback to the RAS signaling pathway. Accordingly, inhibition of DUSP6 is cytotoxic in LUAD cells driven by either mutant KRAS or mutant EGFR, phenocopying the effects of co-expression of mutant KRAS and EGFR. Together, these data suggest that targeting DUSP6 or other feedback regulators of the EGFR-KRAS-ERK pathway may offer a strategy for treating certain cancers by exceeding an upper threshold of RAS-mediated signaling.

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“…Furthermore, different mutations may differentially impact the affinity of RIT1 for the BRAF RBD probe used to assess activation in the cell, akin to the previously reported effects of the H-RAS G12V mutation on ARAF and RGL1 RBD binding (33). It is important to note that the binding affinity for RAF-RBD is reduced by most of the germ-line and cancer-associated K-RAS mutations that have been studied, including those that are distal from the RBD-binding site (28,31). The activation state of such mutants would be underestimated in RBD pulldown assays.…”

Section: Ar S Ygi P F I E T S Aktmentioning

confidence: 93%

“…4, C and D) increases the basal activation state from 30 to 70 -99% at 100 mM NaCl. The G13D substitution introduces a negative charge in the phosphate-binding site that renders binding of nucleotide less favorable (28). Affinity for nucleotide can also be reduced through an engineered H-RAS mutation (F28L) that disrupts a hydrophobic interaction with the base (29).…”

Section: Ar S Ygi P F I E T S Aktmentioning

confidence: 99%

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Biochemical Classification of Disease-associated Mutants of RAS-like Protein Expressed in Many Tissues (RIT1)

Fang

Marshall

Yin³

et al. 2016

Journal of Biological Chemistry

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RAS-like protein expressed in many tissues 1 (RIT1) is a disease-associated RAS subfamily small guanosine triphosphatase (GTPase). Recent studies revealed that germ-line and somatic RIT1 mutations can cause Noonan syndrome (NS), and drive proliferation of lung adenocarcinomas, respectively, akin to RAS mutations in these diseases. However, the locations of these RIT1 mutations differ significantly from those found in RAS, and do not affect the three mutational "hot spots" of RAS. Moreover, few studies have characterized the GTPase cycle of RIT1 and its disease-associated mutants. Here we developed a realtime NMR-based GTPase assay for RIT1 and investigated the effect of disease-associated mutations on GTPase cycle. RIT1 exhibits an intrinsic GTP hydrolysis rate similar to that of H-RAS, but its intrinsic nucleotide exchange rate is ϳ4-fold faster, likely as a result of divergent residues near the nucleotide binding site. All of the disease-associated mutations investigated increased the GTP-loaded, activated state of RIT1 in vitro, but they could be classified into two groups with different intrinsic GTPase properties. The S35T, A57G, and Y89H mutants exhibited more rapid nucleotide exchange, whereas F82V and T83P impaired GTP hydrolysis. A RAS-binding domain pulldown assay indicated that RIT1 A57G and Y89H were highly activated in HEK293T cells, whereas T83P and F82V exhibited more modest activation. All five mutations are associated with NS, whereas two (A57G and F82V) have also been identified in urinary tract cancers and myeloid malignancies. Characterization of the effects on the GTPase cycle of RIT1 disease-associated mutations should enable better understanding of their role in disease processes.Small GTPase proteins regulate signal transduction through a conserved "switch" mechanism (1). The GTP-bound state of these proteins adopts an activated conformation that can bind to and activate downstream effector proteins, thus driving signal transduction. Hydrolysis of GTP to GDP then terminates the signaling event (Fig. 1A). GTPase proteins have the ability to hydrolyze GTP (intrinsic hydrolysis), and to become reactivated by releasing GDP and binding a new molecule of GTP (intrinsic exchange), although both of these processes occur slowly. Most GTPases can be rapidly inactivated by GTPaseactivating proteins (GAPs), 6 which catalyze nucleotide hydrolysis, whereas guanine nucleotide exchange factors (GEFs) promote their reactivation by accelerating the exchange of GDP for GTP. Mutations in small GTPases or their GAPs and GEFs can disrupt this GTPase cycle and result in diseases such as cancer and developmental disorders (2, 3).Recent genetic analyses of Noonan syndrome (NS) patients have shown that mutations in RIT1 can cause this condition (Fig. 1B and Table 1) (4 -11). NS is a "RASopathy" characterized by short stature, distinctive facial features, and heart defects, caused by mutations in several RAS signaling pathway genes, including PTPN11, SOS1/2, RAF1, KRAS, NRAS,. RIT1 belongs to the RAS subfamily, sha...

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Biochemical and Structural Analysis of Common Cancer-Associated KRAS Mutations

Cited by 581 publications

References 67 publications

Genetics and biology of pancreatic ductal adenocarcinoma

Genetics and biology of pancreatic ductal adenocarcinoma

Hyperactivation of extracellular signal-regulated kinase (ERK) by RAS-mediated signaling or inhibition of dual specificity phosphatase 6 (DUSP6) is associated with toxicity in lung adenocarcinoma cells with mutations in KRAS or EGFR

Biochemical Classification of Disease-associated Mutants of RAS-like Protein Expressed in Many Tissues (RIT1)

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