Biochemical Characterization of a Novel KRAS Insertion Mutation from a Human Leukemia

Bollag, Gideon; Adler, Felix; elMasry, Nadia; McCabe, Peter C.; Conner, Edward; Thompson, Patrick; McCormick, Frank; Shannon, Kevin

doi:10.1074/jbc.271.51.32491

Cited by 55 publications

(48 citation statements)

References 24 publications

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“…S5 A). Similar molecular alterations had been previously found in animal and human tumors (26,27). Biochemical analysis demonstrated that this insertion strongly activates KRAS by permanently switching the corresponding mutated protein into the GTP-bound active state (Fig.…”

Section: Knock-in Cells Display Drug Responses Resembling Those Of Tusupporting

confidence: 57%

Replacement of normal with mutant alleles in the genome of normal human cells unveils mutation-specific drug responses

Nicolantonio

Arena

Gallicchio

et al. 2008

Proc. Natl. Acad. Sci. U.S.A.

124

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Mutations in oncogenes and tumor suppressor genes are responsible for tumorigenesis and represent favored therapeutic targets in oncology. We exploited homologous recombination to knock-in individual cancer mutations in the genome of nontransformed human cells. Sequential introduction of multiple mutations was also achieved, demonstrating the potential of this strategy to construct tumor progression models. Knock-in cells displayed allele-specific activation of signaling pathways and mutation-specific phenotypes different from those obtainable by ectopic oncogene expression. Profiling of a library of pharmacological agents on the mutated cells showed striking sensitivity or resistance phenotypes to pathway-targeted drugs, often matching those of tumor cells carrying equivalent cancer mutations. Thus, knock-in of single or multiple cancer alleles provides a pharmacogenomic platform for the rational design of targeted therapies.cancer mutation ͉ oncogene addiction ͉ pharmacogenomic ͉ targeted therapies ͉ tumor progression model T he construction of model systems that accurately recapitulate the genetic alterations present in human cancer is a prerequisite to understand the cellular properties imparted by the mutated alleles and to identify genotype and tumor-specific pharmacological responses. In this regard, mammalian cell lines have been widely used as model systems to functionally characterize cancer alleles carrying point mutations and to develop and validate anticancer drugs. These models typically involve the ectopic expression (by means of plasmid transfection or viral infection) of mutated cDNAs in human or mouse cells (1). Although these approaches have yielded remarkable results, they are typically hampered by at least two caveats. First, the expression is achieved by transient or stable transfection of cDNAs, often resulting in over-expression of the target allele at levels that do not recapitulate what occurs in human cancers. Second, the expression of the mutated cDNA is achieved under the control of nonendogenous viral promoters. As a result, the mutated alleles cannot be appropriately (endogenously) modulated in the target cells. While such systems in which mutated oncogenes are ectopically expressed under exogenous promoters have been instrumental in dissecting their oncogenic properties, they have also led to controversial results. For example, studies focused on oncogene-mediated transformation and senescence have generated conflicting data depending on whether the cancer alleles were ectopically expressed or permanently introduced in the genome of mouse or human cells (2-5). To address the limitation of current models, we have used targeted homologous recombination to introduce (knock-in, KI) a panel of cancer alleles in human somatic cells. Specifically, we focused on EGFR, KRAS, BRAF, and PIK3CA mutated alleles that are found in multiple cancer types. Mutant cells have then been used to study the biochemical and transforming potential of common cancer alleles and to identify genotype-specific ...

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Section: Knock-in Cells Display Drug Responses Resembling Those Of Tusupporting

confidence: 57%

Replacement of normal with mutant alleles in the genome of normal human cells unveils mutation-specific drug responses

Nicolantonio

Arena

Gallicchio

et al. 2008

Proc. Natl. Acad. Sci. U.S.A.

124

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“…Multiple studies have shown that alterations across KRAS exons 2, 3, and 4 similarly predict cetuximab and panitumumab resistance, and the National Comprehensive Cancer Network (NCCN) and American Society for Clinical Oncology (ASCO) guidelines have expanded to recommend extended KRAS testing, as well as testing for NRAS and BRAF mutations [6,9,15,16,52]. Additionally, KRAS insertions within or adjacent to hotspot regions have been reported; these mutations have been characterized as activating and oncogenic, and may similarly predict resistance, although this has not yet been established [53][54][55][56].…”

Section: Discussionmentioning

confidence: 99%

Broad Detection of Alterations Predicted to Confer Lack of Benefit From EGFR Antibodies or Sensitivity to Targeted Therapy in Advanced Colorectal Cancer

Rankin¹,

Klempner

Erlich³

et al. 2016

The Oncologist

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Introduction. A KRAS mutation represented the first genomic biomarker to predict lack of benefit from anti‐epidermal growth factor receptor (EGFR) antibody therapy in advanced colorectal cancer (CRC). Expanded RAS testing has further refined the treatment approach, but understanding of genomic alterations underlying primary and acquired resistance is limited and further study is needed. Materials and Methods. We prospectively analyzed 4,422 clinical samples from patients with advanced CRC, using hybrid‐capture based comprehensive genomic profiling (CGP) at the request of the individual treating physicians. Comparison with prior molecular testing results, when available, was performed to assess concordance. Results. We identified a RAS/RAF pathway mutation or amplification in 62% of cases, including samples harboring KRAS mutations outside of the codon 12/13 hotspot region in 6.4% of cases. Among cases with KRAS non‐codon 12/13 alterations for which prior test results were available, 79 of 90 (88%) were not identified by focused testing. Of 1,644 RAS/RAF wild‐type cases analyzed by CGP, 31% harbored a genomic alteration (GA) associated with resistance to anti‐EGFR therapy in advanced CRC including mutations in PIK3CA, PTEN, EGFR, and ERBB2. We also identified other targetable GA, including novel kinase fusions, receptor tyrosine kinase amplification, activating point mutations, as well as microsatellite instability. Conclusion. Extended genomic profiling reliably detects alterations associated with lack of benefit to anti‐EGFR therapy in advanced CRC, while simultaneously identifying alterations potentially important in guiding treatment. The use of CGP during the course of clinical care allows for the refined selection of appropriate targeted therapies and clinical trials, increasing the chance of clinical benefit and avoiding therapeutic futility. Implications for Practice: Comprehensive genomic profiling (CGP) detects diverse genomic alterations associated with lack of benefit to anti‐epidermal growth factor receptor therapy in advanced colorectal cancer (CRC), as well as targetable alterations in many other genes. This includes detection of a broad spectrum of activating KRAS alterations frequently missed by focused molecular hotspot testing, as well as other RAS/RAF pathway alterations, mutations shown to disrupt antibody binding, RTK activating point mutations, amplifications, and rearrangements, and activating alterations in downstream effectors including PI3K and MEK1. The use of CGP in clinical practice is critical to guide appropriate selection of targeted therapies for patients with advanced CRC.

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“…Translocations involving this region were also shown to be important for ALL leukemogenesis [44][45][46]. Similarly, KRAS2 protein with a genetic insertion was shown to play a role in malignant transformation by activating the RAS-activated signaling pathway in acute myeloid leukemia (AML) cells [47]. Additionally, some cases of AML were caused by alterations in CEBPA and NPM proteins via mutations [48,49].…”

Section: Exploitation Of Protein Profiling In Leukemia Researchmentioning

confidence: 99%

The importance of protein profiling in the diagnosis and treatment of hematologic malignancies

Şanlı-Mohamed¹,

Turan²,

Ekiz³

et al. 2011

Turk J Hematol

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Proteins are important targets in cancer research because malignancy is associated with defects in cell protein machinery. Protein profiling is an emerging independent subspecialty of proteomics that is rapidly expanding and providing unprecedented insight into biological events. Quantitative assessment of protein levels in hematologic malignancies seeks a comprehensive understanding of leukemiaassociated protein patterns for use in aiding diagnosis, follow-up treatment, and the prediction of clinical outcomes. Many recently developed high-throughput proteomic methods can be applied to protein profiling. Herein the importance of protein profiling, its exploitation in leukemia research, and its clinical usefulness in the treatment and diagnosis of various cancer types, and techniques for determining changes in protein profiling are reviewed. (Turk J Hematol 2011; 28: 1-14) Key words: Hematologic malignancies, leukemia, proteomics, protein profiling Received: November 24, 2010 Accepted: January 19, 2011 ÖzetMalignitelerde proteinlerin hücresel mekanizmalarında meydana gelen bozukluklardan dolayı, proteinler kanser araştırmaları için önemli hedeflerdir. Proteomiksin alt uzmanlık dalı olarak ortaya çıkan ve bağımsız bir alan olan protein profilleme biyolojik olaylara farklı bir bakış açısı sağlamak amacıyla hızla gelişmektedir. Hematolojik malignitelerdeki protein düzeylerinin kantitatif olarak değerlendiril-mesi, teşhise yardımcı olması, tedavinin izlenmesi ve klinik sonuçların tahmininde mükemmel bir yaklaşım olması nedeni ile lösemi ile ilgili protein modellerinin kapsamlı bir şekilde incelenmesini amaçlamaktadır. Son dönemlerde geliştirilen yüksek verimli yöntemler protein profillemede kullanıla-bilir. Bu makalede, protein profillemenin önemi, lösemi araştırmalarındaki rolü, çeşitli kanser tiplerinin tanısı ve tedavisi için klinik kullanımları ve protein profilindeki değişikliklerin belirlenmesinde kullanılan teknikler değerlendirilmiştir.

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Biochemical Characterization of a Novel KRAS Insertion Mutation from a Human Leukemia

Cited by 55 publications

References 24 publications

Replacement of normal with mutant alleles in the genome of normal human cells unveils mutation-specific drug responses

Replacement of normal with mutant alleles in the genome of normal human cells unveils mutation-specific drug responses

Broad Detection of Alterations Predicted to Confer Lack of Benefit From EGFR Antibodies or Sensitivity to Targeted Therapy in Advanced Colorectal Cancer

The importance of protein profiling in the diagnosis and treatment of hematologic malignancies

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