We report here the purification of the third protein factor, Apaf-3, that participates in caspase-3 activation in vitro. Apaf-3 was identified as a member of the caspase family, caspase-9. Caspase-9 and Apaf-1 bind to each other via their respective NH2-terminal CED-3 homologous domains in the presence of cytochrome c and dATP, an event that leads to caspase-9 activation. Activated caspase-9 in turn cleaves and activates caspase-3. Depletion of caspase-9 from S-100 extracts diminished caspase-3 activation. Mutation of the active site of caspase-9 attenuated the activation of caspase-3 and cellular apoptotic response in vivo, indicating that caspase-9 is the most upstream member of the apoptotic protease cascade that is triggered by cytochrome c and dATP.

Summary Wnt/β-catenin signaling plays a key role in the pathogenesis of colon and other cancers; emerging evidence indicates that oncogenic β-catenin regulates several biological processes essential for cancer initiation and progression. To decipher the role of β-catenin in transformation, we classified β-catenin activity in 85 cancer cell lines in which we performed genome scale loss-of-function screens and found that β-catenin active cancers are dependent on a signaling pathway involving the transcriptional regulator YAP1. Specifically, we found that YAP1 and the transcription factor TBX5 form a complex with β-catenin. Phosphorylation of YAP1 by the tyrosine kinase YES1 leads to localization of this complex to the promoters of anti-apoptotic genes including BCL2L1 and BIRC5. A small molecule inhibitor of YES1 impeded the proliferation of β-catenin-dependent cancers in both cell lines and animal models. These observations define a β-catenin-YAP1-TBX5 complex essential to the transformation and survival of β-catenin-driven cancers.

Ultraviolet (UV) irradiation of

Indisulam is an aryl sulfonamide drug with selective anticancer activity. Its mechanism of action and the basis for its selectivity have so far been unknown. Here we show that indisulam promotes the recruitment of RBM39 (RNA binding motif protein 39) to the CUL4-DCAF15 E3 ubiquitin ligase, leading to RBM39 polyubiquitination and proteasomal degradation. Mutations in RBM39 that prevent its recruitment to CUL4-DCAF15 increase RBM39 stability and confer resistance to indisulam's cytotoxicity. RBM39 associates with precursor messenger RNA (pre-mRNA) splicing factors, and inactivation of RBM39 by indisulam causes aberrant pre-mRNA splicing. Many cancer cell lines derived from hematopoietic and lymphoid lineages are sensitive to indisulam, and their sensitivity correlates with DCAF15 expression levels. Two other clinically tested sulfonamides, tasisulam and chloroquinoxaline sulfonamide, share the same mechanism of action as indisulam. We propose that DCAF15 expression may be a useful biomarker to guide clinical trials of this class of drugs, which we refer to as SPLAMs (splicing inhibitor sulfonamides).