Structural dataset for the fast-exchanging KRAS G13D.
ABSTRACT: Cancers bearing the KRAS G13D mutation are notable for their distinct clinical behavior relative to other oncogenic KRAS mutations. We hypothesized that primary biochemical or biophysical properties of KRAS G13D might contribute to these clinical observations and as part of our study undertook structural studies using x-ray crystallography. In this data article we discuss several x-ray diffraction datasets that yielded structures of oncogenic KRAS mutants including a high resolution (1.13 Å) structure of KRAS G13D. The datasets are typical for high resolution x-ray diffraction data and allow the construction of atomic resolution, three dimensional structural models with high confidence. This data can be correlated with biochemical information such as defects in substrate binding kinetics, GTPase activities and interactions with the RAS effector RAF kinase.
Project description:KRAS mutations occur in ?35% of colorectal cancers and promote tumor growth by constitutively activating the mitogen-activated protein kinase (MAPK) pathway. KRAS mutations at codons 12, 13, or 61 are thought to prevent GAP protein-stimulated GTP hydrolysis and render KRAS-mutated colorectal cancers unresponsive to epidermal growth factor receptor (EGFR) inhibitors. We report here that KRAS G13-mutated cancer cells are frequently comutated with NF1 GAP but NF1 is rarely mutated in cancers with KRAS codon 12 or 61 mutations. Neurofibromin protein (encoded by the NF1 gene) hydrolyzes GTP directly in complex with KRAS G13D, and KRAS G13D-mutated cells can respond to EGFR inhibitors in a neurofibromin-dependent manner. Structures of the wild type and G13D mutant of KRAS in complex with neurofibromin (RasGAP domain) provide the structural basis for neurofibromin-mediated GTP hydrolysis. These results reveal that KRAS G13D is responsive to neurofibromin-stimulated hydrolysis and suggest that a subset of KRAS G13-mutated colorectal cancers that are neurofibromin-competent may respond to EGFR therapies.
Project description:KRAS is one of the most frequently mutated genes across all cancer subtypes. Two of the most frequent oncogenic KRAS mutations observed in patients result in glycine to aspartic acid substitution at either codon 12 (G12D) or 13 (G13D). Although the biochemical differences between these two predominant mutations are not fully understood, distinct clinical features of the resulting tumors suggest involvement of disparate signaling mechanisms. When we compared the global and phosphotyrosine proteomic profiles of isogenic colorectal cancer cell lines bearing either G12D or G13D KRAS mutation, we observed both shared as well as unique signaling events induced by the two KRAS mutations.
Project description:Ras GTPases are mutated at codons 12, 13, and 61, with different frequencies in KRas, HRas, and NRas and in a cancer-specific manner. The G13D mutant appears in 25% of KRas-driven colorectal cancers, while observed only rarely in HRas or NRas. Structures of Ras G13D in the three isoforms show an open active site, with adjustments to the D13 backbone torsion angles and with disconnected switch regions. KRas G13D has unique features that destabilize the nucleotide-binding pocket. In KRas G13D bound to GDP, A59 is placed in the Mg2+ binding site, as in the HRas-SOS complex. Structure and biochemistry are consistent with an intermediate level of KRas G13D bound to GTP, relative to wild-type and KRas G12D, observed in genetically engineered mouse models. The results explain in part the elevated frequency of the G13D mutant in KRas over the other isoforms of Ras.
Project description:<h4>Background</h4>The treatment of metastatic colorectal cancer (mCRC) includes drugs targeting the epidermal growth factor receptor (EGFR). Mutation in codon 12 or 13 in the Kirsten rat sarcoma viral oncogene homolog (KRAS) gene, downstream of the EGFR, evokes constitutive activation of the RAS/RAF/MAPK signaling pathway and correlates with resistance to anti-EGFR monoclonal antibody (mAb) therapies. However, a retrospective study reported that a proportion of patients with the KRAS G13D mutation may respond to cetuximab. A similar analysis for panitumumab was not as conclusive. We sought to determine the sensitivity of CRC cell lines to cetuximab or panitumumab treatment and to investigate the correlation of the KRAS mutational status of the CRC cell lines to the responsiveness to cetuximab or panitumumab.<h4>Methods</h4>To determine the responsiveness of CRC cell lines to cetuximab or panitumumab, cell lines were treated with an optimized concentration of each mAb, and proliferation assays were conducted.<h4>Results</h4>After treatment with cetuximab or panitumumab, at the optimum concentration of 8 ?g/well, the KRAS G13D mutant cell lines HCT-116, LoVo, and T84 showed intermediate sensitivity to both treatments, between the resistant KRAS G12V mutant cell line SW480 and the sensitive KRAS wild-type cell line LIM1215. One of the G13D cell lines was significantly more sensitive to panitumumab than to cetuximab (P = .02).<h4>Conclusion</h4>The specific KRAS mutation determines the responsiveness to anti-EGFR monoclonal antibody treatment, corresponding to reported clinical observations.
Project description:BACKGROUND:KRAS mutations have been characterized as the major predictive biomarkers for resistance to cetuximab treatment. However, studies indicate that not all KRAS mutations are associated with equivalent treatment outcomes. KRAS G13D mutations were observed to account for approximately 16% of all KRAS mutations in advanced colorectal cancer patients, and whether these patients can benefit from cetuximab has not been determined. METHODS:An established KRAS G13D mutant colorectal cancer (CRC) patient-derived xenograft (PDX) model was treated with cetuximab. After repeated use of cetuximab, treatment-resistant PDX models were established. Tissue samples were collected before and during treatment, and multiomics data were subsequently sequenced and processed, including whole-exome, mRNA and miRNA data, to explore potential dynamic changes. RESULTS:Cetuximab treatment initially slowed tumor growth, but resistance developed not long after treatment. WES (whole-exome sequencing) and RNA sequencing found that 145 genes had low P values (<?0.01) when analyzed between the locus genotype and its related gene expression level. Among these genes, SWAP70 was believed to be a probable cause of acquired resistance. JAK2, PRKAA1, FGFR2 and RALBP1, as well as 10 filtered immune-related genes, also exhibited dynamic changes during the treatment. CONCLUSIONS:Cetuximab may be effective in KRAS G13D mutation patients. Dynamic changes in transcription, as determined by WES and RNA sequencing, occurred after repeated drug exposure, and these changes were believed to be the most likely cause of drug resistance.
Project description:Circulating tumour DNA (ctDNA) is widely used in liquid biopsies due to having a presence in the blood that is typically in proportion to the stage of the cancer and because it may present a quick and practical method of capturing tumour heterogeneity. This paper outlines a simple electrochemical technique adapted towards point-of-care cancer detection and treatment monitoring from biofluids using a label-free detection strategy. The mutations used for analysis were the KRAS G12D and G13D mutations, which are both important in the initiation, progression and drug resistance of many human cancers, leading to a high mortality rate. A low-cost DNA sensor was developed to specifically investigate these common circulating tumour markers. Initially, we report on some developments made in carbon surface pre-treatment and the electrochemical detection scheme which ensure the most sensitive measurement technique is employed. Following pre-treatment of the sensor to ensure homogeneity, DNA probes developed specifically for detection of the KRAS G12D and G13D mutations were immobilized onto low-cost screen printed carbon electrodes using diazonium chemistry and 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride/N-hydroxysuccinimide coupling. Prior to electrochemical detection, the sensor was functionalised with target DNA amplified by standard and specialist PCR methodologies (6.3% increase). Assay development steps and DNA detection experiments were performed using standard voltammetry techniques. Sensitivity (as low as 0.58 ng/?L) and specificity (>300%) was achieved by detecting mutant KRAS G13D PCR amplicons against a background of wild-type KRAS DNA from the representative cancer sample and our findings give rise to the basis of a simple and very low-cost system for measuring ctDNA biomarkers in patient samples. The current time to receive results from the system was 3.5 h with appreciable scope for optimisation, thus far comparing favourably to the UK National Health Service biopsy service where patients can wait for weeks for biopsy results.
Project description:KRAS-mutated colorectal cancers (CRCs) are resistant to cetuximab treatment. The multifunctional Y-box binding protein 1 (YB-1) is overexpressed in CRC and is associated with chemoresistance. In this study, the effects of oncogenic mutated KRAS(G12V) and KRAS(G13D) on YB-1 phosphorylation were investigated in CRC cells. The effects of the inhibition of p90 ribosomal S6 kinase (RSK) on YB-1 phosphorylation, cell proliferation and survival were tested with and without treatment with 5-fluorouracil using pharmacological inhibitors and siRNA. YB-1 phosphorylation status and subcellular distribution in CRC patient tissues were determined by immunofluorescence staining and confocal microscopy. Endogenous expression of mutated KRAS(G13D) and conditional expression of KRAS(G12V) significantly stimulated YB-1 phosphorylation via RSK and were associated with cetuximab resistance. Inhibition of YB-1 by targeting RSK stimulated the Akt signaling pathway, and this stimulation occurred independently of KRAS mutational status. Akt activation interfered with the antiproliferative effect of the RSK inhibitor. Consequently, dual targeting of RSK and Akt efficiently inhibited cell proliferation in KRAS(G13D)-mutated HCT116 and KRAS wild-type SW48 cells. Treatment with 5-fluorouracil (5-FU) significantly enhanced YB-1 phosphorylation in KRAS(G13D)-mutated HCT116 cells but not in KRAS wild-type SW48 cells. Dual targeting of Akt and RSK sensitized HCT116 cells to 5-FU by stimulating 5-FU-induced apoptosis and inhibiting repair of 5-FU-induced DNA damage. YB-1 was highly phosphorylated in CRC patient tumor tissues and was mainly localized in the nucleus. Together, dual targeting of RSK and Akt may be an alternative molecular targeting approach to cetuximab for treating CRC in which YB-1 is highly phosphorylated.
Project description:KRAS mutations occur in one third of human cancers and cluster in several hotspots, with codons 12 and 13 being most commonly affected. It has been suggested that the position and type of amino acid exchange influence the transforming capacity of mutant KRAS proteins. We used MCF10A human mammary epithelial cells to establish isogenic cell lines that express different cancer-associated KRAS mutations (G12C, G12D, G12V, G13C, G13D, A18D, Q61H, K117N) at physiological or elevated levels, and investigated the biochemical and functional consequences of the different variants. The overall effects of low-expressing mutants were moderate compared to overexpressed variants, but allowed delineation of biological functions that were related to specific alleles rather than KRAS expression level. None of the mutations induced morphological changes, migratory abilities, or increased phosphorylation of ERK, PDK1, and AKT. KRAS-G12D, G12V, G13D, and K117N mediated EGF-independent proliferation, whereas anchorage-independent growth was primarily induced by K117N and Q61H. Both codon 13 mutations were associated with increased EGFR expression. Finally, global gene expression analysis of MCF10A-G13D versus MCF10A-G12D revealed distinct transcriptional changes. Together, we describe a useful resource for investigating the function of multiple KRAS mutations and provide insights into the differential effects of these variants in MCF10A cells.
Project description:Oncogenic mutations of Ras at codons 12, 13, or 61, that render the protein constitutively active, are found in ? 16% of all cancer cases. Among the three major Ras isoforms, KRAS is the most frequently mutated isoform in cancer. Each Ras isoform and tumor type displays a distinct pattern of codon-specific mutations. In colon cancer, KRAS is typically mutated at codon 12, but a significant fraction of patients have mutations at codon 13. Clinical data suggest different outcomes and responsiveness to treatment between these two groups. To investigate the differential effects upon cell status associated with KRAS mutations we performed a quantitative analysis of the proteome and phosphoproteome of isogenic SW48 colon cancer cell lines in which one allele of the endogenous gene has been edited to harbor specific KRAS mutations (G12V, G12D, or G13D). Each mutation generates a distinct signature, with the most variability seen between G13D and the codon 12 KRAS mutants. One notable example of specific up-regulation in KRAS codon 12 mutant SW48 cells is provided by the short form of the colon cancer stem cell marker doublecortin-like Kinase 1 (DCLK1) that can be reversed by suppression of KRAS.
Project description:Cetuximab is a standard of care for treating EGFR-expressing metastatic colorectal carcinoma (mCRC) exclusive of those with KRAS mutations at codons 12/13. However, retrospective analysis has recently suggested that KRAS-G13D patients can still benefit, while only a fraction of KRAS wild-type patients can benefit, from the treatment. We set out to test this contradicting issue experimentally in an independent cohort of patient derived xenograft (PDX) diseases. We conducted a mouse clinical trial (MCT) enrolling a random cohort of 27 transcriptome sequenced CRC-PDXs to evaluate cetuximab activity. The treatment responses were analyzed against the KRAS 12/13 mutation alleles, as well as several other well-known oncogenic alleles. If the response is defined by >80% tumor growth inhibition, 8/27 PDXs (~30%) are responders versus 19/27 non-/partial responders (~70%). We found that indeed there are no significantly fewer KRAS-12/13-allele responders (4/8 or 50%) than non-/partial responders (7/19, or 37%). In particular, there are actually no fewer G13D responders (4/8, or 50%) than in non-/partial responders (2/19 or 10.5%) statistically. Furthermore, majority of the non-/partial responders tend to have certain activating oncogenic alleles (one or more of the following common ones: K/N-RAS-G12V/D, -A146T, -Q61H/R, BRAF-V600E, AKT1-L52R and PIK3CA-E545G/K). Our data on an independent cohort support the recent clinical observation, but against the current practiced patient stratification in the cetuximab CRC treatment. Meanwhile, our data seem to suggest that a set of the six-oncogenic alleles may be of better predictive value than the current practiced stratification, justifying a new prospective clinical investigation on an independent cohort for confirmation.