SPOP mediates apoptosis and protects against necroptosis by regulating ubiquitination of RIPK1 and RIPK3.

Apoptosis and necroptosis are 2 distinct destinies of cells stimulated with TNF-α; however, it remains unclear how apoptosis and necroptosis are differentially regulated. This study validates the key regulatory role of speckle-type POZ protein (SPOP) in balancing apoptosis and necroptosis. SPOP promotes the polyubiquitination and degradation of receptor-interacting serine/threonine-protein kinase 3 (RIPK3), thereby inhibiting necrosome formation and decreasing cellular sensitivity to necroptosis. Conversely, SPOP interacted with RIPK1 independently of its E3 ubiquitin ligase activity, protecting it from ubiquitination and degradation, thereby enhancing RIPK1 expression and cellular sensitivity to apoptosis. Inhibiting RIPK1 kinase activity with 7-Cl-O-Nec-1 impeded both SPOP-mediated apoptosis and SPOP deficiency-mediated necroptosis. Besides, inhibition or loss of RIPK3 rescued SPOP deficiency-mediated necroptosis. Pancancer analyses indicated that the SPOP/RIPK1/RIPK3 axis is dysfunctional in a variety of tumors. In 3 representative tumor types with high expression of SPOP and RIPK1, kidney renal clear cell carcinoma, liver hepatocellular carcinoma, and breast invasive carcinoma, this regulatory mechanism remains applicable. Based on these findings, a combination therapy using the second mitochondria-derived activator of caspases (Smac) mimetic SM164 and sunitinib was developed, demonstrating a more pronounced efficacy than sunitinib monotherapy, and this sensitizing effect was dependent on the expression level of RIPK1. These results suggest that the combination of Smac mimetics with tyrosine kinase inhibitors holds potential clinical value for tumors with dysregulated SPOP/RIPK1/RIPK3 signaling.