Elucidation of the mutational landscape of human cancer has progressed rapidly and been accompanied by the development of therapeutics targeting mutant oncogenes. However, a comprehensive mapping of cancer dependencies has lagged behind and the discovery of therapeutic targets for counteracting tumor suppressor gene loss is needed. To identify vulnerabilities relevant to specific cancer subtypes, we conducted a large-scale RNAi screen in which viability effects of mRNA knockdown were assessed for 7,837 genes using an average of 20 shRNAs per gene in 398 cancer cell lines. We describe findings of this screen, outlining the classes of cancer dependency genes and their relationships to genetic, expression, and lineage features. In addition, we describe robust gene-interaction networks recapitulating both protein complexes and functional cooperation among complexes and pathways. This dataset along with a web portal is provided to the community to assist in the discovery and translation of new therapeutic approaches for cancer.

Constitutive overexpression and activation of NPM-ALK fusion protein [t(2:5)(p23;q35)] is a key oncogenic event that drives the survival and proliferation of anaplastic large-cell lymphomas (ALCLs). We have identified a highly potent and selective small-molecule ALK inhibitor, NVP-TAE684, which blocked the growth of ALCL-derived and ALK-dependent cell lines with IC 50 values between 2 and 10 nM. NVP-TAE684 treatment resulted in a rapid and sustained inhibition of phosphorylation of NPM-ALK and its downstream effectors and subsequent induction of apoptosis and cell cycle arrest. In vivo , NVP-TAE684 suppressed lymphomagenesis in two independent models of ALK-positive ALCL and induced regression of established Karpas-299 lymphomas. NVP-TAE684 also induced down-regulation of CD30 expression, suggesting that CD30 may be used as a biomarker of therapeutic NPM-ALK kinase activity inhibition.

ABL1 tyrosine-kinase inhibitors (TKI) are a front-line therapy for chronic myelogenous leukemia and represent the best known examples of targeted cancer therapeutics. However, the dynamic uptake of low molecular weight TKIs into cells and their intracellular behavior is largely unknown due to the difficulty of observing non-fluorescent small molecules at subcellular resolution. Here we report the direct label-free visualization and quantification of two TKI drugs – imatinib and nilotinib inside living cells using hyperspectral stimulated Raman scattering imaging. Both drugs were enriched over 1000-fold in lysosomes as a result of their lysosomotropic properties. In addition, low solubility appeared to contribute significantly to the surprisingly large accumulation of nilotinib. We further show that the lysosomal trapping of imatinib was reduced by more than 10-fold when using chloroquine simultaneously, suggesting that chloroquine may increase the efficacy of TKIs through lysosome mediated drug-drug interaction besides the commonly proposed autophagy inhibition mechanism.

Rapid quantitative methods for characterizing small molecules, peptides, proteins, or RNAs in a broad array of cellular assays would allow one to discover new biological activities associated with these molecules and also provide a more comprehensive profile of drug candidates early in the drug development process. Here we describe a robotic system, termed the automated compound profiler, capable of both propagating a large number of cell lines in parallel and assaying large collections of molecules simultaneously against a matrix of cellular assays in a highly reproducible manner. To illustrate its utility, we have characterized a set of 1,400 kinase inhibitors in a panel of 35 activated tyrosine-kinasedependent cellular assays in dose-response format in a single experiment. Analysis of the resulting multidimensional dataset revealed subclusters of both inhibitors and kinases with closely correlated activities. The approach also identified activities for the p38 inhibitor BIRB796 and the dual src͞abl inhibitor BMS-354825 and exposed the expected side activities for Glivec͞STI571, including cellular inhibition of c-kit and platelet-derived growth factor receptor. This methodology provides a powerful tool for unraveling the cellular biology and molecular pharmacology of both naturally occurring and synthetic chemical diversity.drug discovery ͉ high-throughput screening ͉ tyrosine kinase T he ability to simultaneously interrogate the activities of a library of molecules against a large panel of cellular assays would provide a rapid efficient means to begin to characterize and correlate the biological properties of both natural and synthetic chemical diversity. For example, libraries of noncoding RNAs, mutant growth factors, small molecule kinase inhibitors, or even existing drugs could be assayed for their potency and selectivity in pathway-based or receptor screens or toxicity and metabolic stability in diverse cell types to discover a new biological activity or optimize the pharmacological properties of a molecule (1-3). Although whole-cell systems represent an attractive milieu to characterize gene and small-molecule function, no robust and systematic method exists to correlate chemical structure and biological activity across a large number of molecules and cellular assays. To address this problem, we have developed an approach that affords rapid cost-effective broad-based cellular profiling in parallel against molecular libraries. An industrial-scale automated compound profiling (ACP) system has been designed, which consists of an automated tissue culture system for propagating cell lines, integrated with a system for automatically performing miniaturized cell-based assays in 384-or 1,536-well microplates. The ACP can rapidly test thousands of arrayed molecules, in replicates, in doseresponse format against hundreds of unique cellular assays in a single experiment.To demonstrate this capability, we focused on the problem of identifying selective small-molecule inhibitors of protein tyrosine kinases. Tyrosine ...

The Wnt signaling pathway is critical to the regulation of key cellular processes. When deregulated, it has been shown to play a crucial role in the growth and progression of multiple human cancers. The identification of small molecule modulators of Wnt signaling has proven challenging, largely due to the relative paucity of druggable nodes in this pathway. Several recent publications have identified small molecule inhibitors of the Wnt pathway, and tankyrase (TNKS) inhibition has been demonstrated to antagonize Wnt signaling via axin stabilization. Herein, we report the early hit assessment of a series of compounds previously reported to antagonize Wnt signaling. We report the biophysical, computational characterization, structure-activity relationship, and physicochemical properties of a novel series of [1,2,4]triazol-3-ylsulfanylmethyl)-3-phenyl-[1,2,4]oxadiazole inhibitors of TNKS1 and 2. Furthermore, a cocrystal structure of compound 24 complexed to TNKS1 demonstrates an alternate binding mode for PARP family member proteins that does not involve interactions with the nicotinamide binding pocket.