SLC46A3 Is Required to Transport Catabolites of Noncleavable Antibody Maytansine Conjugates from the Lysosome to the Cytoplasm.

Antibody-drug conjugates (ADC) target cytotoxic drugs to antigen-positive cells for treating cancer. After internalization, ADCs with noncleavable linkers are catabolized to amino acid-linker-warheads within the lysosome, which then enter the cytoplasm by an unknown mechanism. We hypothesized that a lysosomal transporter was responsible for delivering noncleavable ADC catabolites into the cytoplasm.

Sphingosine kinase activity is not required for tumor cell viability.

Sphingosine kinases (SPHKs) are enzymes that phosphorylate the lipid sphingosine, leading to the formation of sphingosine-1-phosphate (S1P). In addition to the well established role of extracellular S1P as a mitogen and potent chemoattractant, SPHK activity has been postulated to be an important intracellular regulator of apoptosis. According to the proposed rheostat theory, SPHK activity shifts the intracellular balance from the pro-apoptotic sphingolipids ceramide and sphingosine to the mitogenic S1P, thereby determining the susceptibility of a cell to apoptotic stress.

Measurement of Cancer Cell Growth Heterogeneity through Lentiviral Barcoding Identifies Clonal Dominance as a Characteristic of In Vivo Tumor Engraftment.

Advances in the fields of cancer initiating cells and high-throughput in vivo shRNA screens have highlighted a need to observe the growth of tumor cells in cancer models at the clonal level. While in vivo cancer cell growth heterogeneity in xenografts has been described, it has yet to be measured. Here, we tested an approach to quantify the clonal growth heterogeneity of cancer cells in subcutaneous xenograft mouse models.

STK33 kinase activity is nonessential in KRAS-dependent cancer cells.

Despite the prevalence of KRAS mutations in human cancers, there remain no targeted therapies for treatment. The serine-threonine kinase STK33 has been proposed to be required for the survival of mutant KRAS-dependent cell lines, suggesting that small molecule kinase inhibitors of STK33 may be useful to treat KRAS-dependent tumors. In this study, we investigated the role of STK33 in mutant KRAS human cancer cells using RNA interference, dominant mutant overexpression, and small molecule inhibitors.

Use of cryopreserved cell aliquots in the high-throughput screening of small interfering RNA libraries.

Screening small interfering RNA (siRNA) libraries holds the potential to elucidate gene function as well as discover new targets for the therapeutic treatment of disease. Since the inception of siRNA as a discovery tool, there have been progressive improvements in siRNA design algorithms, the transfection reagents used to deliver them, and the assay formats used to monitor phenotypic changes. These changes have helped to improve the quality of the data emerging from siRNA screens.

RNA interference screening for the discovery of oncology targets.

RNA interference (RNAi) mediated loss-of-function screens have the potential to delineate biological functions of genes and the proteins they encode. RNAi has proven to be a promising technology for identification and validation of new targets for the pharmacological treatment of many diseases including cancer. Here we review the use of high-throughput RNAi screens, examine the types of targets pursued for oncology indications, and discuss the integration of diverse datasets in both target discovery and drug discovery programs.

A functional screen for regulators of CKDN2A reveals MEOX2 as a transcriptional activator of INK4a.

The CDKN2A locus encodes two important tumor suppressors, INK4a and ARF, which respond to oncogenic stresses by inducing cellular senescence. We conducted a genome-scale cDNA overexpression screen using a reporter containing INK4a regulatory sequences to identify novel transcriptional activators of this locus. This screen revealed 285 cDNAs that putatively regulate the transcriptional activation of INK4a.

A TRAIL receptor-dependent synthetic lethal relationship between MYC activation and GSK3beta/FBW7 loss of function.

The MYC protooncogene is frequently deregulated in human cancers. Here, by screening a kinase-directed library of small inhibitory RNAs, we identify glycogen synthase kinase 3beta (GSK3beta) as a gene whose inactivation potentiates TNF-related apoptosis-inducing ligand death receptor-mediated apoptosis specifically in MYC-overexpressing cells. Small inhibitory RNA-induced silencing of GSK3beta prevents phosphorylation of MYC on T58, thereby inhibiting recognition of MYC by the E3 ubiquitin ligase component FBW7.

Synthetic lethal targeting of MYC by activation of the DR5 death receptor pathway.

The genetic concept of synthetic lethality provides a framework for identifying genotype-selective anticancer agents. In this approach, changes in cellular physiology that arise as a consequence of oncogene activation or tumor suppressor gene loss, rather than oncoproteins themselves, are targeted to achieve tumor selectivity. Here we show that agonists of the TRAIL death receptor DR5 potently induce apoptosis in human cells overexpressing the MYC oncogene, both in vitro and as tumor xenografts in vivo.

Identification of p53 regulators by genome-wide functional analysis.

The p53 tumor-suppressor protein is a critical mediator of cellular growth arrest and the induction of apoptosis. To identify proteins involved in the modulation of p53 transcriptional activity, a gain-of-function cellular screen was carried out with an arrayed matrix of approximately 20,000 cDNAs. Nine genes previously unknown to be involved in regulating p53 activity were identified.