Introduction

Resistance to immune cell-mediated cytotoxicity poses a significant challenge in cancer treatment, compromising the efficacy of promising immunotherapeutic approaches, such as immune checkpoint blockade (ICB) therapy. To enhance treatment outcomes, it is crucial to elucidate interventions that synergize with ICB to overcome tumor resistance. Therefore, we need to identify the cellular mechanisms that sensitize tumors to cytotoxic CD8+ T cells. CD8+ T cells rely on cytokines such as TNFα to carry out their cytotoxicity against cancer cells. TNFα is a major mediator of cell death and immunity, and recent findings link select tumor mutations within the TNF pathway to increased T cell killing, in a manner dependent on the protein RIPK1 (Vredevoogd et al., 2019).

Key findings

Through MS-based proteomics and phosphoproteomics methods, we demonstrate that sensitized tumor cells fail to initiate inhibitory RIPK1 phosphorylation at site S25 upon T cell attack, thereby foregoing a pro-survival checkpoint early in TNF signal transduction and driving RIPK1-mediated cell death. Consequently, tumor cells experiencing a loss of TNF-induced RIPK1 S25 phosphorylation exhibit increased RIPK1 activation and fail to recruit non-canonical IKK kinases (TBK1 and IKKε) to the TNFR1 complex. Functional knockouts of TBK1 and IKKε in melanoma cells lack RIPK1 S25 phosphorylation and result in heightened sensitivity not only to CD8+T cell but also to Natural Killer cell attacks. These findings implicate TBK1 & IKKe as the checkpoint kinases responsible for pro-survival RIPK1 S25 phosphorylation and a key regulator of immune-mediated cell death in cancer cells.

MS application

To quantify phosphorylation dynamics of RIPK1 within protein signaling complexes, we combine phosphoproteomic analysis with Affinity Purification-Mass Spectrometry (AP-MS). Melanoma cell lines were stimulated with biotinylated TNFα and downstream complexes were isolated via streptavidin-coated magnetic beads. The purified complexes were digested utilizing S-TRAP microfilters, with subsequent enrichment of phosphopeptides carried out in an automated manner using the AssayMAP Bravo Platform, prior LC-MS analysis using an Orbitrap Exploris MS. In addition to our proteomics workflow, a Targeted Mass Spectrometry method was developed to quantify RIPK1 phosphorylation and validate our findings.

Impact

Our findings indicate that preventing TBK1 and IKKε recruitment to the TNF signaling complex, thereby blocking RIPK1 pro-survival phosphorylation and promoting direct RIPK1 activation, can be considered a tractable strategy to increase tumor sensitivity to immune cell killing and has the potential to benefit current immunotherapy interventions.