Calpain 9 functions in TNF receptor mediated apoptosis

Evasion of apoptosis is a hallmark of cancer development. The Inhibitor of Apoptosis Proteins, IAPs, block Caspase activity and cell death. Release of the Second Mitochondria-Derived Activator of Caspases, Smac, from the mitochondria relieves IAP Caspase inhibition, activating apoptosis. Our lab has developed a small molecule Smac mimetic. Surprisingly, approximately 25% of cell lines show single agent Smac mimetic sensitivity through activation of autocrine TNF-a secretion and TNF dependent apoptosis. Using Smac mimetic sensitivity as a model system, I performed a genome-wide high-throughput siRNA screen and identified Calpain 9, CAPN9, as a novel component of TNF-alpha induced apoptosis. CAPN9 knockdown does not affect TNF-a secretion, yet is essential for downstream activation. Two splice variants are reported for CAPN9. The smaller splice, CAPN9-SP2, is required for effective TNF-a induced apoptosis. CAPN9 is essential for RIPK1 recruitment and ubiquitination at the TNFR1 upon activation with TNF-alpha. CAPN9 knockdown demonstrates previously unreported association of ubiquitinated proteins, and actin binding proteins with TNFR1 in the absence of stimulus. This interaction is CAPN9 dependent and correlates with CAPN9 regulation of TNF-a induced apoptosis. [Keywords: Calpain, SMAC, IAR, TNF, ubiquitin, RJPK1]