Large collections of knockout organisms facilitate the elucidation of gene functions. Here we used retroviral insertion or homologous recombination to disrupt 472 genes encoding secreted and membrane proteins in mice, providing a resource for studying a large fraction of this important class of drug target. The knockout mice were subjected to a systematic phenotypic screen designed to uncover alterations in embryonic development, metabolism, the immune system, the nervous system and the cardiovascular system. The majority of knockout lines exhibited altered phenotypes in at least one of these therapeutic areas. To our knowledge, a comprehensive phenotypic assessment of a large number of mouse mutants generated by a gene-specific approach has not been described previously.

The echinoderm microtubule-associated protein-like 4-anaplastic lymphoma kinase (EML4-ALK) fusion gene has been identified as an oncogene in a subset of non-small cell lung cancers (NSCLC). We used profiling of cancer genomes on an exon array to develop a novel computational method for the global search of gene rearrangements. This approach led to the detection of EML4-ALK fusion in breast and colorectal carcinomas in addition to NSCLC. Screening of a large collection of patient tumor samples showed the presence of EML4-ALK fusion in 2.4% of breast (5 of 209), 2.4% of colorectal (2 of 83), and in 11.3% of NSCLC (12 of 106). Besides previously known EML4-ALK variants 1 (E13; A20) and 2 (E20; A20), a novel variant E21; A20 was found in colorectal carcinoma. The presence of an EML-ALK rearrangement was verified by identifying genomic fusion points in tumor samples representative of breast, colon, and NSCLC. EML4-ALK translocation was also confirmed by fluorescence in situ hybridization assay, which revealed its substantial heterogeneity in both primary tumors and tumor-derived cell lines. To elucidate the functional significance of EML4-ALK, we examined the growth of cell lines harboring the fusion following EML4 and ALK silencing by small interfering RNA. Significant growth inhibition was observed in some but not all cell lines, suggesting their variable dependence on ALK-mediated cell survival signaling. Collectively, these findings show the recurrence of EML4-ALK fusion in multiple solid tumors and further substantiate its role in tumorigenesis. (Mol Cancer Res 2009;7(9):1466-76)

Deregulation of apoptosis is a common occurrence in cancer, for which emerging oncology therapeutic agents designed to engage this pathway are undergoing clinical trials. With the aim of uncovering strategies to activate apoptosis in cancer cells, we used a pooled shRNA screen to interrogate death receptor signaling. This screening approach identified 16 genes that modulate the sensitivity to ligand induced apoptosis, with several genes exhibiting frequent overexpression and/or copy number gain in cancer. Interestingly, two of the top hits, EDD1 and GRHL2, are found 50 kb apart on chromosome 8q22, a region that is frequently amplified in many cancers. By using a series of silencing and overexpression studies, we show that EDD1 and GRHL2 suppress death-receptor expression, and that EDD1 expression is elevated in breast, pancreas, and lung cancer cell lines resistant to death receptor-mediated apoptosis. Supporting the relevance of EDD1 and GRHL2 as therapeutic candidates to engage apoptosis in cancer cells, silencing the expression of either gene sensitizes 8q22-amplified breast cancer cell lines to death receptor induced apoptosis. Our findings highlight a mechanism by which cancer cells may evade apoptosis, and therefore provide insight in the search for new targets and functional biomarkers for this pathway.

Here we describe the development of two complementary high‐throughput screening technologies consisting of single clone expression cloning and protein microarray platforms that permit genome‐wide screening of a protein of interest against thousands of interactions either displayed on a cell surface or on a microarray. The single clone expression cloning platform is fully automated and capable of efficient transfection, expression, and binding analysis of a bait protein to cells transfected with individual clones from the clone library. For low affinity interactions, we generated bait protein with an Fc fusion to increase the avidity. Our secreted protein microarray has over 1500 secreted or extracellular domain of single transmembrane human proteins represented to enable simultaneous systematic probing of a bait protein against proteins on the microarray. To enhance detection of low affinity interactions, we assembled bait Fc fusion proteins into multivalent complexes with protein A microbeads. As proof of concept, we evaluated well‐characterized extracellular receptor‐ligand interactions with diverse binding affinities on the single clone expression cloning and protein microarray platforms. Overall, we found that the interactions identified using both of our screening methodologies were highly specific and displayed minimal off‐target binding. Furthermore, we demonstrate the utility of both technologies for novel receptor‐ligand discovery.