Key Publications
A Panel of Isogenic Human Cancer Cells Suggests a Therapeutic Approach for Cancers with Inactivated p53
www.pnas.org/cgi/doi/10.1073/pnas.0813333106
Through targeted homologous recombination, we developed a panel of matched colorectal cancer cell lines that differ only with respect to their endogenous TP53 status. We then used these lines to define the genes whose expression was altered after DNA damage induced by ionizing radiation. Transcriptome analyses revealed a consistent upregulation of polo-like kinase 1 (PLK1) as well as other genes controlling the G2/M transition in the cells whose TP53 genes were inactivated compared with those with WT TP53 genes. This led to the hypothesis that the viability of stressed cells without WT TP53 depended on PLK1. This hypothesis was validated by demonstrating that stressed cancer cells without WT TP53 alleles were highly sensitive to PLK1 inhibitors, both in vivo and in vitro.
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PIK3CA Mutations in Colorectal Cancer Are Associated with Clinical Resistance to EGFR-Targeted Monoclonal Antibodies
Cancer Res 2009; 69: (5). March 1, 2009
The monoclonal antibodies (moAb) panitumumab and cetuximab target the epidermal growth factor receptor (EGFR) and have proven valuable for the treatment of metastatic colorectal cancer (mCRC).EGFR -mediated signaling involves two main intracellular cascades: on one side KRAS activates BRAF, which in turn triggers the mitogen-activated protein kinases.On the other, membrane localization of the lipid kinase PIK3CA counteracts PTEN and promotes AKT1 phosphorylation, thereby activating a parallel intracellular axis. Constitutive activation of KRAS bypasses the corresponding signaling cascade and, accordingly, patients with mCRC bearing KRAS mutations are clinically resistant to therapy with panitumumab or cetuximab.W e hypothesized that mutations activating PIK3CA could also preclude responsiveness to EGFR-targeted moAbs through a similar mechanism. Here, we present the mutational analysis of PIK3CA and KRAS and evaluation of the PTEN protein status in a cohort of 110 patients with mCRC treated with anti-EGFR moAbs. We observed 15 (13.6%) PIK3CA and 32 (29.0%) KRAS mutations. PIK3CA mutations were significantly associated with clinical resistance to panitumumab or cetuximab; none of the mutated patients achieved objective response (P = 0.038). When only KRAS wild-type tumors were analyzed, the statistical correlation was stronger (P = 0.016). Patients with PIK3CA mutations displayed a worse clinical outcome also in terms of progression-free survival (P = 0.035). Our data indicate that PIK3CA mutations can independently hamper the therapeutic response to panitumumab or cetuximab in mCRC.When the molecular status of the PIK3CA/PTEN and KRAS pathways are concomitantly ascertained, up to 70% of mCRC patients unlikely to respond to EGFR moAbs can be identified.
Knock-in of Mutant PI3KCA Activates Multiple Oncogenic Pathways
www.pnas.org/cgi/doi/10.1073/pnas/0813351106
The phosphatidylinositol 3-kinase subunit PIK3CA is frequently mutated in human cancers. Here we used gene targeting to ‘‘knock in’’ PIK3CA mutations into human breast epithelial cells to identify new therapeutic targets associated with oncogenic PIK3CA. Mutant PIK3CA knock-in cells were capable of epidermal growth factor and mTOR-independent cell proliferation that was associated with AKT, ERK, and GSK3beta phosphorylation. Paradoxically, the GSK3beta inhibitors lithium chloride and SB216763 selectively decreased the proliferation of human breast and colorectal cancer cell lines with oncogenic PIK3CA mutations and led to a decrease in the GSK3beta target gene CYCLIN D1. Oral treatment with lithium preferentially inhibited the growth of nude mouse xenografts of HCT-116 colon cancer cells with mutant PIK3CA compared with isogenic HCT-116 knockout cells containing only wild-type PIK3CA. Our findings suggest GSK3beta is an important effector of mutant PIK3CA, and that lithium, an FDA-approved therapy for bipolar disorders, has selective antineoplastic properties against cancers that harbor these mutations.
Replacement of Normal with Mutant Alleles in the Genome of Normal Human Cells Unveils Mutation-Specific Drug Responses
PNAS December 30, 2008 vol. 105, no. 52
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 lso 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.
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Wild-Type BRAF Is Required for Response to Panitumumab and Cetuximab in Metastatic Colorectal Cancer
10.1200/JCO.2008.18.0786
Cetuximab or panitumumab are effective in 10% to 20% unselected metastatic colorectal cancer (CRC) patients. KRAS mutations account for approximately 30% to 40% patients who are not responsive. The serine-threonine kinase BRAF is the principal effector of KRAS. KRAS mutations were present in 30% of the patients and were associated with resistance to cetuximab or panitumum ired the therapeutic effect of etuximab or panitumumab. Treatment with the BRAF inhibitor sorafenib restored sensitivity to panitumumab or cetuximab of CRC cells carrying the V600E allele. BRAF wild-type is required for response to panitumumab or cetuximab and could be used to select patients who are eligible for the treatment. Double-hit therapies aimed at simultaneous inhibition of epidermal growth factor receptor and BRAF warrant exploration in CRC patients carrying the V600E oncogenic mutation
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EDITORIAL | Genomic landscapes of cancers: prospects for targeted therapies
Tumor genotyping is helping clinicians to individualize therapies by matching patients with the best treatment for their tumors.
Knock-in of Mutant K-ras in Nontumorigenic Human Epithelial Cells as a New Model for Studying K-ras–Mediated Transformation
Cancer Res 2007; 67: (18). September 15, 2007
The oncogenic function of mutant ras in mammalian cells has been extensively investigated using multiple human and animal models. These systems include overexpression of exogenous mutant ras transgenes, conditionally expressed knock-in mouse models, and somatic cell knockout of mutant and wild-type ras genes in human cancer cell lines. However, phenotypic discrepancies between knock-in mice and transgenic mutant ras overexpression prompted us to evaluate the consequences of targeted knock-in of an oncogenic K-ras mutation in the nontumorigenic human breast epithelial cell line MCF-10A and hTERT- immortalized human mammary epithelial cells. Our results show several significant differences between mutant K-ras knock-in cells versus their transgene counterparts, including limited phosphorylation of the downstream olecules extracellular signal-regulated kinase and KT, minor proliferative capacity in the absence of an exogenous growth factor, and the inability to form colonies in semisolid medium. Analysis of 16 cancer cell lines carrying mutant K-ras genes indicated that 50% of cancer cells harbor nonoverexpressed heterozygous K-ras mutations similar to the expression seen in our knock-in cell lines. Thus, this system serves as a new model for elucidating the oncogenic contribution of mutant K-ras as expressed in a large fractionof human cancer cells.
Genetic targeting of the kinase activity of the Met receptor in cancer cells.
Proc Natl Acad Sci U S A. 2007 Jul 3;104(27):11412-7.
The development of kinase inhibitors is revolutionizing cancer treatment. Assessing the oncogenic potential of individual kinase activities and ensuring that a drug of interest acts by direct inhibition of its putative target kinase are clear priorities. We developed a genetic strategy to selectively inactivate the catalytic activity of kinases. This approach generates isogenic cells in which a given kinase gene is expressed but is devoid of enzymatic activity. As a model to test this approach, we chose the MET receptor, which is involved in multiple cancers and is the focus of several therapeutic efforts. The exon encoding the ATP-binding site of MET was deleted from the genome of colorectal, bladder, and endometrial cancer cells. The derivative isogenic cells expressed a kinase-inactive Met (MET-KD) and were completely unresponsive to its ligand hepatocyte growth factor (HGF), indicating the exclusivity of this ligand-receptor axis. The in vivo tumorigenic potential of MET-KD cells was reduced but could be partially restored by HGF, suggesting that concomitant targeting of the receptor and its ligand should be therapeutically exploited. A reportedly selective Met-kinase inhibitor (SU-11274) markedly affected the growth of MET-KD cancer cells, indicating this compound exerts its effects not only through the intended target. The genetic strategy presented here is not limited to kinase genes but could be broadly applicable to any drug/protein combination in which the target enzymatic domain is known.
COMMENT | Targeting how targeted therapies work
Proc Natl Acad Sci U S A. 2007 Jul 3;104(27):11122.
Oncogenic activation of the RAS/RAF signaling pathway impairs the response of metastatic colorectal cancers to anti-epidermal growth factor receptor antibody therapies.
Cancer Research 2007 Mar 15;67(6):2643-8.
Monoclonal antibodies (mAbs) against the extracellular domain of the epidermal growth factor receptor (EGFR) have been introduced for the treatment of metastatic colorectal cancer (mCRC). We have reported recently that increased copy number of the EGFR can predict response to anti-EGFR mAbs and that patients might be selected for treatment based on EGFR copy number. Here, we show that mutations activating the RAS/RAF signaling pathway are also predictive and prognostic indicators in mCRC patients, being inversely correlated with response to anti-EGFR mAbs. In cellular models of CRCs, activation of the RAS signaling pathway by introduction of an activated K-RAS allele (Gly(12)Val) impairs the therapeutic effect of anti-EGFR mAbs. In cancer cells carrying constitutively active RAS, the pharmacologic inhibition of the mitogen-activated protein kinase (MAPK) signaling cascade improves anti-EGFR treatment based on mAbs. These results have implications for the identification of patients who are likely to respond to anti-EGFR treatment. They also provide the rationale for combination therapies, targeted simultaneously to the EGFR and RAS/RAF/MAPK signaling pathways in CRC patients.
Knock-in of oncogenic KRAS does not transform mouse somatic cells but triggers a transcriptional response that classifies human cancers
Cancer Research 2007 Sep 15;67(18):8468-76.
KRAS mutations are present at a high frequency in human cancers. The development of therapies targeting mutated KRAS requires cellular and animal preclinical models. We exploited adeno-associated virus-mediated homologous recombination to insert the Kras G12D allele in the genome of mouse somatic cells. Heterozygous mutant cells displayed a constitutively active Kras protein, marked morphologic changes, increased proliferation and motility but were not transformed. On the contrary, mouse cells in which we overexpressed the corresponding Kras cDNA were readily transformed. The levels of Kras activation in knock-in cells were comparable with those present in human cancer cells carrying the corresponding mutation. Kras-mutated cells were compared with their wild-type counterparts by gene expression profiling, leading to the definition of a “mutated Kras-KI signature” of 345 genes. This signature was capable of classifying mouse and human cancers according to their KRAS mutational status, with an accuracy similar to or better than published Ras signatures. The isogenic cells that we have developed recapitulate the oncogenic activation of KRAS occurring in cancer and represent new models for studying Kras-mediated transformation. Our results have implications for the identification of human tumors in which the oncogenic KRAS transcriptional response is activated and suggest new strategies to build mouse models of tumor progression.
Novel Somatic and Germline Mutations in Cancer Candidate Genes in Glioblastoma, Melanoma, and Pancreatic Carcinoma
Cancer Research 2007;67(8):3545–50
A recent systematic sequence analysis of well-annotated human protein coding genes or consensus coding sequences led to the identification of 189 genes displaying somatic mutations in breast and colorectal cancers. Based on their mutation prevalence, a subset of these genes was identified as cancer candidate (CAN) genes as they could be potentially involved in cancer. We evaluated the mutational profiles of 19 CAN genes in the highly aggressive tumors: glioblastoma, melanoma, and pancreatic carcinoma. Among other changes, we found novel somatic mutations in EPHA3, MLL3, TECTA, FBXW7, and OBSCN, affecting amino acids not previously found to be mutated in human cancers. Interestingly, we also found a germline nucleotide variant of OBSCN that was previously reported as a somatic mutation. Our results identify specific genetic lesions in glioblastoma, melanoma, and pancreatic cancers and indicate that CAN genes and their mutational profiles are tumor specific. Some of the mutated genes, such as the tyrosine kinase EPHA3, are clearly amenable to pharmacologic intervention and could represent novel therapeutic targets for these incurable cancers. We also speculate that similar to other oncogenes and tumor suppressor genes, mutations affecting OBSCN could be involved in cancer predisposition.
REVIEW | Kinase mutations in cancer: chinks in the Enemy’s armour?
Current Opinion in Oncology 2006, 17:000 – 000
Many oncogenes are mutated kinase genes. In most cases, the mutations result in the constitutive activation of the affected kinase that can be pharmacologically inhibited. Unfortunately, upon treatment with kinase inhibitors, resistant clones develop rapidly, impairing their therapeutic effect. Strategies to overcome resistance are discussed as well as the possibility to target kinases regulating cancer stem cells.
Gene copy number for epidermal growth factor receptor (EGFR) and clinical response to antiEGFR treatment in colorectal cancer: a cohort study.
Lancet Oncology, 2005 May;6(5):279-86.
BACKGROUND: The antiepidermal growth factor receptor (antiEGFR) monoclonal antibodies cetuximab and panitumumab have good clinical activity in about 10% of patients with metastatic colorectal cancer that is resistant to chemotherapy. The molecular mechanisms underlying clinical response or resistance to these agents are unknown. METHODS: Tumours from 31 patients with metastatic colorectal cancer who had either an objective response (n=10) or stable disease or progressive disease (n=21) after treatment with cetuximab or panitumumab were screened for genetic changes in EGFR or its immediate intracellular effectors. Specifically, we assessed the EGFR copy number and the mutation profile of the EGFR catalytic domain and of selected exons in KRAS, BRAF, and PIK3CA. RESULTS: Eight of nine of patients with objective responses who were assessable by fluorescence in-situ hybridisation (FISH) had an increased EGFR copy number. By contrast, one of 21 non-responders assessable by FISH had an increased EGFR copy number (p<0.0001 for responders vs non-responders, Fisher’s exact test). The mutation status of the EGFR catalytic domain and its immediate downstream effectors PIK3CA, KRAS, and BRAF did not correlate with disease response. In colorectal-cancer cell lines, the concentration of cetuximab that completely inhibited proliferation of cells with amplified EGFR copy number did not affect proliferation of cells with unamplified EGFR. INTERPRETATION: We propose that the response to antiEGFR treatment has a genetic basis and suggest that patients might be selected for treatment on the basis of EGFR copy number.
Comment | Who will benefit from treatment against EGFR?
Lancet Oncology, 2005 May;6(5):257-8.
Use of isogenic human cancer cells for high-throughput screening and drug discovery
Nature Biotechnology. 2001 Oct;19(10):940-5.
Cell-based screening for novel tumor-specific drugs has been compromised by the lack of appropriate control cells. We describe a strategy for drug screening based on isogenic human cancer cell lines in which key tumorigenic genes have been deleted by targeted homologous recombination.As a test case, a yellow fluo-rescent protein (YFP) expression vector was introduced into the colon cancer cell line DLD-1, and a blue fluo-rescent protein (BFP) expression vector was introduced into an isogenic derivative in which the mutant K-Ras allele had been deleted. Co-culture of both cell lines allowed facile screening for compounds with selective toxicity toward the mutant Ras genotype.Among 30,000 compounds screened, a novel cytidine nucleoside analog was identified that displayed selective activity in vitro and inhibited tumor xenografts containing mutant Ras. The present data demonstrate a broadly applicable approach for mining therapeutic agents targeted to the specific genetic alterations responsible for cancer development.
News & Views: Knockout Drug Screens
Nature Biotechnology. 2001 Oct;19(10):919-20.
Cell lines that differ by a single genetic change show promise in drug screens to identify compounds with gene-selective properties.