Apoptosis can be mediated through the mitochondria (intrinsic) or cell surface death receptors (extrinsic). In response to a variety of apoptotic cues, initiator caspases are activated from their pro-forms, which then activate downstream executor caspases that cleave protein substrates to commit apoptosis. The detailed biochemical mechanisms that activation of caspases from pro-forms have been well elucidated. In striking contrast, mechanisms that regulate apoptosis remain poorly understood. In extrinsic apoptosis, death ligand induction triggers formation of the death-inducing signaling complex (DISC), which mediates activation of initiator caspase-8 to transduce extrinsic apoptosis. We recently discovered that ubiquitination and deubiquitination death receptors function as a novel regulatory mechanism of DISC formation. Deubiquitinase USP35 antagonizes death receptor ubiquitination and suppresses extrinsic apoptosis. Thus, USP35 is a strong suppressor of extrinsic apoptosis. In this proposal, we aim to validate USP35 as a promising anticancer drug target using experiments based on cell culture and xenograft human cancer models (Aim 1); and to develop small chemical inhibitors of USP35 using high throughput screening and medicinal chemistry optimization (Aim 2).