Harold E. Varmus

Acquired Resistance of Lung Adenocarcinomas to Gefitinib or Erlotinib Is Associated with a Second Mutation in the EGFR Kinase Domain

Background Lung adenocarcinomas from patients who respond to the tyrosine kinase inhibitors gefitinib (Iressa) or erlotinib (Tarceva) usually harbor somatic gain-of-function mutations in exons encoding the kinase domain of the epidermal growth factor receptor (EGFR). Despite initial responses, patients eventually progress by unknown mechanisms of “acquired” resistance. Methods and Findings We show that in two of five patients with acquired resistance to gefitinib or erlotinib, progressing tumors contain, in addition to a primary drug-sensitive mutation in EGFR, a secondary mutation in exon 20, which leads to substitution of methionine for threonine at position 790 (T790M) in the kinase domain. Tumor cells from a sixth patient with a drug-sensitive EGFR mutation whose tumor progressed on adjuvant gefitinib after complete resection also contained the T790M mutation. This mutation was not detected in untreated tumor samples. Moreover, no tumors with acquired resistance had KRAS mutations, which have been associated with primary resistance to these drugs. Biochemical analyses of transfected cells and growth inhibition studies with lung cancer cell lines demonstrate that the T790M mutation confers resistance to EGFR mutants usually sensitive to either gefitinib or erlotinib. Interestingly, a mutation analogous to T790M has been observed in other kinases with acquired resistance to another kinase inhibitor, imatinib (Gleevec). Conclusion In patients with tumors bearing gefitinib- or erlotinib-sensitive EGFR mutations, resistant subclones containing an additional EGFR mutation emerge in the presence of drug. This observation should help guide the search for more effective therapy against a specific subset of lung cancers.

Somatic mutations affect key pathways in lung adenocarcinoma

Determining the genetic basis of cancer requires comprehensive analyses of large collections of histopathologically well-classified primary tumours. Here we report the results of a collaborative study to discover somatic mutations in 188 human lung adenocarcinomas. DNA sequencing of 623 genes with known or potential relationships to cancer revealed more than 1,000 somatic mutations across the samples. Our analysis identified 26 genes that are mutated at significantly high frequencies and thus are probably involved in carcinogenesis. The frequently mutated genes include tyrosine kinases, among them the EGFR homologue ERBB4; multiple ephrin receptor genes, notably EPHA3; vascular endothelial growth factor receptor KDR; and NTRK genes. These data provide evidence of somatic mutations in primary lung adenocarcinoma for several tumour suppressor genes involved in other cancers—including NF1, APC, RB1 and ATM—and for sequence changes in PTPRD as well as the frequently deleted gene LRP1B. The observed mutational profiles correlate with clinical features, smoking status and DNA repair defects. These results are reinforced by data integration including single nucleotide polymorphism array and gene expression array. Our findings shed further light on several important signalling pathways involved in lung adenocarcinoma, and suggest new molecular targets for treatment.

A New Initiative on Precision Medicine

President Obama has announced a research initiative that aims to accelerate progress toward a new era of precision medicine, with a near-term focus on cancers and a longer-term aim to generate knowledge applicable to the whole range of health and disease.

Many tumors induced by the mouse mammary tumor virus contain a provirus integrated in the same region of the host genome.

We have asked whether oncogenesis by the mouse mammary tumor virus (MMTV), a slowly oncogenic retrovirus, involves integration of viral DNA within a certain region of the host genome. We first identified a C3H mouse mammary tumor bearing a single new MMTV provirus and cloned a 19 kilobase (kb) DNA restriction fragment containing a junction of viral and host sequences. Host sequences from this clone were used to retrieve 25 kb of the uninterrupted locus (termed MMTV int1) from a bacteriophage library of normal mouse DNA. Hybridization with subcloned DNA fragments of MMTV int1 detected abnormal restriction fragments in digests of DNA from 18 of 26 C3H mammary tumors. The rearrangements all appeared to be due to the insertion of an MMTV provirus, and the integration sites were located in at least five clusters over a total distance of 19 kb. A polyadenylated 2.6 kb RNA species transcribed from int1 was found in the few tumors tested, but not in lactating mammary glands from C3H mice. Of 12 tested viral oncogenes, none exhibited homology with cloned DNA from this locus. We propose that tumorigenesis by MMTV is strongly favored by proviral insertion within the int1 locus, perhaps as a consequence of enhanced expression of a novel cellular oncogene.

Characterizing the cancer genome in lung adenocarcinoma

Somatic alterations in cellular DNA underlie almost all human cancers. The prospect of targeted therapies and the development of high-resolution, genome-wide approaches are now spurring systematic efforts to characterize cancer genomes. Here we report a large-scale project to characterize copy-number alterations in primary lung adenocarcinomas. By analysis of a large collection of tumours (n = 371) using dense single nucleotide polymorphism arrays, we identify a total of 57 significantly recurrent events. We find that 26 of 39 autosomal chromosome arms show consistent large-scale copy-number gain or loss, of which only a handful have been linked to a specific gene. We also identify 31 recurrent focal events, including 24 amplifications and 7 homozygous deletions. Only six of these focal events are currently associated with known mutations in lung carcinomas. The most common event, amplification of chromosome 14q13.3, is found in ∼12% of samples. On the basis of genomic and functional analyses, we identify NKX2-1 (NK2 homeobox 1, also called TITF1), which lies in the minimal 14q13.3 amplification interval and encodes a lineage-specific transcription factor, as a novel candidate proto-oncogene involved in a significant fraction of lung adenocarcinomas. More generally, our results indicate that many of the genes that are involved in lung adenocarcinoma remain to be discovered.

Structure and variability of human chromosome ends.

Mammalian telomeres are thought to be composed of a tandem array of TTAGGG repeats. To further define the type and arrangement of sequences at the ends of human chromosomes, we developed a direct cloning strategy for telomere-associated DNA. The method involves a telomere enrichment procedure based on the relative lack of restriction endonuclease cutting sites near the ends of human chromosomes. Nineteen (TTAGGG)n-bearing plasmids were isolated, two of which contain additional human sequences proximal to the telomeric repeats. These telomere-flanking sequences detect BAL 31-sensitive loci and thus are located close to chromosome ends. One of the flanking regions is part of a subtelomeric repeat that is present at 10 to 25% of the chromosome ends in the human genome. This sequence is not conserved in rodent DNA and therefore should be a helpful tool for physical characterization of human chromosomes in human-rodent hybrid cell lines; some of the chromosomes that may be analyzed in this manner have been identified, i.e., 7, 16, 17, and 21. The minimal size of the subtelomeric repeat is 4 kilobases (kb); it shows a high frequency of restriction fragment length polymorphisms and undergoes extensive de novo methylation in somatic cells. Distal to the subtelomeric repeat, the chromosomes terminate in a long region (up to 14 kb) that may be entirely composed of TTAGGG repeats. This terminal segment is unusually variable. Although sperm telomeres are 10 to 14 kb long, telomeres in somatic cells are several kilobase pairs shorter and very heterogeneous in length. Additional telomere reduction occurs in primary tumors, indicating that somatic telomeres are unstable and may continuously lose sequences from their termini.

The Knockout Mouse Project

Mouse knockout technology provides a powerful means of elucidating gene function in vivo, and a publicly available genome-wide collection of mouse knockouts would be significantly enabling for biomedical discovery. To date, published knockouts exist for only about 10% of mouse genes. Furthermore, many of these are limited in utility because they have not been made or phenotyped in standardized ways, and many are not freely available to researchers. It is time to harness new technologies and efficiencies of production to mount a high-throughput international effort to produce and phenotype knockouts for all mouse genes, and place these resources into the public domain.Mouse knockout technology provides a powerful means of elucidating gene function in vivo, and a publicly available genome-wide collection of mouse knockouts would be significantly enabling for biomedical discovery. To date, published knockouts exist for only about 10% of mouse genes. Furthermore, many of these are limited in utility because they have not been made or phenotyped in standardized ways, and many are not freely available to researchers. It is time to harness new technologies and efficiencies of production to mount a high-throughput international effort to produce and phenotype knockouts for all mouse genes, and place these resources into the public domain.

Evidence that transgenes encoding components of the Wnt signaling pathway preferentially induce mammary cancers from progenitor cells

Breast cancer is a genetically and clinically heterogeneous disease, and the contributions of different target cells and different oncogenic mutations to this heterogeneity are not well understood. Here we report that mammary tumors induced by components of the Wnt signaling pathway contain heterogeneous cell types and express early developmental markers, in contrast to tumors induced by other signaling elements. Expression of the Wnt-1 protooncogene in mammary glands of transgenic mice expands a population of epithelial cells expressing progenitor cell markers, keratin 6 and Sca-1; subsequent tumors express these markers and contain luminal epithelial and myoepithelial tumor cells that share a secondary mutation, loss of Pten, implying that they arose from a common progenitor. Mammary tumors arising in transgenic mice expressing β-catenin and c-Myc, downstream components of the canonical Wnt signaling pathway, also contain a significant proportion of myoepithelial cells and cells expressing keratin 6. Progenitor cell markers and myoepithelial cells, however, are lacking in mammary tumors from transgenic mice expressing Neu, H-Ras, or polyoma middle T antigen. These results suggest that mammary stem cells and/or progenitors to mammary luminal epithelial and myoepithelial cells may be the targets for oncogenesis by Wnt-1 signaling elements. Thus, the developmental heterogeneity of different breast cancers is in part a consequence of differential effects of oncogenes on distinct cell types in the breast.

Signals for ribosomal frameshifting in the rous sarcoma virus gag-pol region

Abstract The gag-pol protein of Rous sarcoma virus (RSV), the precursor to the enzymes responsible for reverse transcription and integration, is expressed from two genes that lie in different translational reading frames by ribosomal frameshifting. Here, we localize the site of frameshifting and show that the frameshifting reaction is mediated by slippage of two adjacent tRNAs by a single nucleotide in the 5′ direction. The gag terminator, which immediately follows the frameshift site, is not required for frameshifting. Other suspected retroviral frameshift sites mediate frameshifting when placed at the end of RSV gag. Mutations in RSV pol also affect synthesis of the gag-pol protein in vitro. The effects of these mutations best correlate with the potential to form an RNA stem-loop structure adjacent to the frameshift site. A short sequence of RSV RNA, 147 nucleotides in length, containing the frameshift site and stem-loop structure, is sufficient to direct frameshifting in a novel genetic context.

Evidence that the transforming gene of avian sarcoma virus encodes a protein kinase associated with a phosphoprotein.

Avian sarcoma virus (ASV) induces sarcomas in animals and transforms fibroblasts to a neoplastic state in cell culture. A single viral gene (src) is responsible for both the induction and maintenance of neoplastic transformation. Recent work has identified a protein with a molecular weight of 60,000 daltons that is apparently encoded in src and may be the effector molecule for the gene (Brugge and Erikson, 1977; Purchio et al, 1978). The putative product of src can be immunoprecipitated by antisera obtained from rabbits bearing tumors induced by ASV. We have used this approach to isolate the protein to characterize further its genetic origins and possible function. Our rabbit tumor antisera precipitated a protein with a molecular weight of 60,000 daltons; according to serological, biochemical and genetic criteria, this protein is encoded in src. We found that this protein is phosphorylated and therefore denoted it pp60. Phosphorylation of pp60 could be accomplished in vitro with extracts of ASV-infected cells. A temperature-sensitive conditional mutation in src had no demonstrable effect on either the production or stability of pp60 in the infected cell, but phosphorylation of the protein was temperature-sensitive. Since the mutant src is not expressed at the restrictive temperature, our findings raise the possibility that phosphorylation of pp60 is required for its function as the putative effector of src. Immunoprecipitates prepared with extracts of ASV-infected cells and the rabbit tumor antisera contained a protein kinase activity that catalyzed phosphorylation of the heavy chains of immunoglobulin molecules, using either ATP or GTP as phosphate donor. The kinase activity immunoprecipitated in parallel with pp60 was obtained only from cells that contained a functioning product of src and could not be precipitated with antisera directed against structural proteins of ASV. A temperature-sensitive conditional mutation in src caused the kinase activity to be thermally inactivated in vitro far more rapidly than the activity from cells infected with wild-type virus. We conclude that both the protein kinase and pp60 are encoded in src, and that the enzymatic activity may be an intrinsic property of pp60. Phosphorylation of pp60 in cellular extracts was inhibited by calcium ion, whereas the immunoprecipitable kinase activity was not, suggesting that the kinase responsible for pp60 phosphorylation may be distinct from that encoded in src. Collett and Erikson (1978) have also identified a protein kinase activity associated with pp60. These findings raise the possibility that phosphorylation of specific cellular targets might account for transformation of the host cell by src.