We have generated a molecular taxonomy of lung carcinoma, the leading cause of cancer death in the United States and worldwide. Using oligonucleotide microarrays, we analyzed mRNA expression levels corresponding to 12,600 transcript sequences in 186 lung tumor samples, including 139 adenocarcinomas resected from the lung. Hierarchical and probabilistic clustering of expression data defined distinct subclasses of lung adenocarcinoma. Among these were tumors with high relative expression of neuroendocrine genes and of type II pneumocyte genes, respectively. Retrospective analysis revealed a less favorable outcome for the adenocarcinomas with neuroendocrine gene expression. The diagnostic potential of expression profiling is emphasized by its ability to discriminate primary lung adenocarcinomas from metastases of extra-pulmonary origin. These results suggest that integration of expression profile data with clinical parameters could aid in diagnosis of lung cancer patients.

Acute lymphoblastic leukemias carrying a chromosomal translocation involving the mixed-lineage leukemia gene (MLL, ALL1, HRX) have a particularly poor prognosis. Here we show that they have a characteristic, highly distinct gene expression profile that is consistent with an early hematopoietic progenitor expressing select multilineage markers and individual HOX genes. Clustering algorithms reveal that lymphoblastic leukemias with MLL translocations can clearly be separated from conventional acute lymphoblastic and acute myelogenous leukemias. We propose that they constitute a distinct disease, denoted here as MLL, and show that the differences in gene expression are robust enough to classify leukemias correctly as MLL, acute lymphoblastic leukemia or acute myelogenous leukemia. Establishing that MLL is a unique entity is critical, as it mandates the examination of selectively expressed genes for urgently needed molecular targets.

Human T cell leukemias can arise from oncogenes activated by specific chromosomal translocations involving the T cell receptor genes. Here we show that five different T cell oncogenes (HOX11, TAL1, LYL1, LMO1, and LMO2) are often aberrantly expressed in the absence of chromosomal abnormalities. Using oligonucleotide microarrays, we identified several gene expression signatures that were indicative of leukemic arrest at specific stages of normal thymocyte development: LYL1+ signature (pro-T), HOX11+ (early cortical thymocyte), and TAL1+ (late cortical thymocyte). Hierarchical clustering analysis of gene expression signatures grouped samples according to their shared oncogenic pathways and identified HOX11L2 activation as a novel event in T cell leukemogenesis. These findings have clinical importance, since HOX11 activation is significantly associated with a favorable prognosis, while expression of TAL1, LYL1, or, surprisingly, HOX11L2 confers a much worse response to treatment. Our results illustrate the power of gene expression profiles to elucidate transformation pathways relevant to human leukemia.

Prevailing drug discovery approaches focus on compounds with molecular selectivity, inhibiting disease-relevant targets over others in vitro. However in vivo, many such agents are not therapeutically selective, either because of undesirable activity at effective doses or because the biological system responds to compensate. In theory, drug combinations should permit increased control of such complex biology, but there is a common concern that therapeutic synergy will generally be mirrored by synergistic side-effects. Here we provide evidence, from 94,110 multi-dose combination experiments representing diverse disease areas and large scale flux balance simulations of inhibited bacterial metabolism, that multi-target synergies are more specific than single agent activities to particular cellular contexts. Using an anti-inflammatory combination, we show how multi-target synergy can achieve therapeutic selectivity in animals through differential target expression. Synergistic combinations can increase the number of selective therapies using the current pharmacopeia, and offer opportunities for more precise control of biological systems.

In an effort to develop a genomics-based approach to the prediction of drug response, we have developed an algorithm for classification of cell line chemosensitivity based on gene expression profiles alone. Using oligonucleotide microarrays, the expression levels of 6,817 genes were measured in a panel of 60 human cancer cell lines (the NCI-60) for which the chemosensitivity profiles of thousands of chemical compounds have been determined. We sought to determine whether the gene expression signatures of untreated cells were sufficient for the prediction of chemosensitivity. Gene expression-based classifiers of sensitivity or resistance for 232 compounds were generated and then evaluated on independent sets of data. The classifiers were designed to be independent of the cells' tissue of origin. The accuracy of chemosensitivity prediction was considerably better than would be expected by chance. Eighty-eight of 232 expression-based classifiers performed accurately (with P < 0.05) on an independent test set, whereas only 12 of the 232 would be expected to do so by chance. These results suggest that at least for a subset of compounds genomic approaches to chemosensitivity prediction are feasible.

Rab molecules regulate vesicular trafficking in many different exocytic and endocytic transport pathways in eukaryotic cells. In neurons, rab3 has been proposed to play a crucial role in regulating synaptic vesicle release. To elucidate the role of rab3 in synaptic transmission, we isolated and characterized Caenorhabditis elegans rab-3 mutants. Similar to the mouse rab3A mutants, these mutants survived and exhibited only mild behavioral abnormalities. In contrast to the mouse mutants, synaptic transmission was perturbed in these animals. Extracellular electrophysiological recordings revealed that synaptic transmission in the pharyngeal nervous system was impaired. Furthermore, rab-3 animals were resistant to the acetylcholinesterase inhibitor aldicarb, suggesting that cholinergic transmission was generally depressed. Last, synaptic vesicle populations were redistributed in rab-3 mutants. In motor neurons, vesicle populations at synapses were depleted to 40% of normal levels, whereas in intersynaptic regions of the axon, vesicle populations were elevated. On the basis of the morphological defects at neuromuscular junctions, we postulate that RAB-3 may regulate recruitment of vesicles to the active zone or sequestration of vesicles near release sites.

C. elegans aex-3 mutations cause pleiotropic behavioral defects that are suggestive of reduced synaptic transmission. aex-3 mutations also show strong genetic interactions with mutations in unc-31 and unc-64, two other genes implicated in synaptic transmission. Physiological and pharmacological studies indicate that aex-3 defects are presynaptic. In aex-3 mutants, the synaptic vesicle-associated RAB-3 protein aberrantly accumulates in neuronal cell bodies and is reduced in synapse-rich axons. This localization defect is specific to RAB-3, since other synaptic proteins are localized normally in aex-3 mutants. aex-3 encodes a 1409 amino acid protein with strong homology to DENN, a human protein of unknown function. In C. elegans, aex-3 is expressed in all or nearly all neurons. These results suggest that AEX-3 is a novel regulator of presynaptic activity that interacts with RAB-3 to regulate synaptic vesicle release.

Rabphilin, a putative rab effector, interacts specifically with the GTP-bound form of the synaptic vesicle-associated protein rab3a. In this study, we define in vivo functions for rabphilin through the characterization of mutants that disrupt the Caenorhabditis elegans rabphilin homolog. The mutants do not display the general synaptic defects associated with rab3 lesions, as assayed at the pharmacological, physiological, and ultrastructural level. However, rabphilin mutants exhibit severe lethargy in the absence of mechanical stimulation. Furthermore, rabphilin mutations display strong synergistic interactions with hypomorphic lesions in the syntaxin, synaptosomal-associated protein of 25 kDa, and synaptobrevin soluble N-ethylmaleimide sensitive factor attachment protein receptor (SNARE) genes; double mutants were nonresponsive to mechanical stimulation. These synergistic interactions were independent of rab3 function and were not observed in rab3-SNARE double mutants. Our data reveal rab3-independent functions for rabphilin in the potentiation of SNARE function.