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A RIPK1-specific PROTAC degrader achieves potent antitumor activity by enhancing immunogeniccell death

ABSTRACT: Receptor-interacting serine/threonine-protein kinase 1 (RIPK1) functions as a critical stress sentinel that co- ordinates cell survival, inflammation, and immunogenic cell death (ICD). Although the catalytic function of RIPK1 is required to trigger cell death, its non-catalytic scaffold function mediates strong pro-survival signaling. Accordingly, cancer cells can hijack RIPK1 to block necroptosis and evade immune detection. We generated a small-molecule proteolysis-targeting chimera (PROTAC) that selectively degraded human and murine RIPK1. PROTAC-mediated depletion of RIPK1 deregulated TNFR1 and TLR3/4 signaling hubs, accentuating the output of NF-kB, MAPK, and IFN signaling. Additionally, RIPK1 degradation simultaneously promoted RIPK3 activation and necroptosis induction. We further demonstrated that RIPK1 degradation enhanced the immunostimulatory effects of radio- and immunotherapy by sensitizing cancer cells to treat- ment-induced TNF and interferons. This promoted ICD, antitumor immunity, and durable treatment responses. Consequently, targeting RIPK1 by PROTACs emerges as a promising approach to overcome radio- or immunotherapy resistance and enhance anticancer therapies.

INSTRUMENT(S): Orbitrap Fusion Lumos

ORGANISM(S): Homo Sapiens (human)

SUBMITTER: Graeme Benstead-Hume

LAB HEAD: Jyoti Choudhary

PROVIDER: PXD043560 | Pride | 2024-05-23

REPOSITORIES: Pride

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			Action	DRS
	20210528_SU_Meier_lab_LMS03_set01_01.raw	Raw
	20210528_SU_Meier_lab_LMS03_set01_02.raw	Raw
	20210528_SU_Meier_lab_LMS03_set01_03.raw	Raw
	20210528_SU_Meier_lab_LMS03_set01_04.raw	Raw
	20210528_SU_Meier_lab_LMS03_set01_05.raw	Raw

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Project description:Background: Phospholamban (PLN) is a tail-anchored sarco-endoplasmic reticulum (SER) membrane protein that regulates cardiac contraction/relaxation by reversibly inhibiting the SERCA2a Ca2+ 36 -reuptake pump. The R14Δ-PLN mutation causes severe cardiomyopathy that is resistant to conventional treatment. Multi-protein complexes and higher-order supercomplexes, such as intercalated disk components and SER Ca+2 -cycling domains, underlie many critical cardiac functions, a subset of which may be disrupted by R14∆-PLN. Complexome profiling (CP) is a systems biology approach to characterize high molecular weight (MW) assemblies in biological samples. Most current CP data analysis strategies rely on external protein42 complex databases that are predominantly curated from immortalized or cancerous cell lines; thus, assemblies unique to specialized cell types such as cardiomyocytes, as well as higher-order supercomplexes that may have fluid stoichiometry/composition are significantly underrepresented. The requirement for computer programming expertise also presents a barrier to generalized adoption. Methods: We developed and applied a novel CP workflow to identify protein-complexes/supercomplexes disrupted in presymptomatic 28wk-old R14Δ/+ knockin mice hearts. Cardiac tissues were homogenized under non-denaturing conditions, fractionated by size-exclusion chromatography (SEC) and subjected to quantitative data-independent acquisition mass spectrometry (DIA-MS)-based proteomics analysis. As expected, traditional database-dependent analysis offered only limited insights due to underrepresentation of cardiac-specific assemblies and higher-order supercomplexes. We thus developed a novel data analysis strategy (PERCOM) that does not rely upon external protein-complex databases and can, furthermore, be implemented on widely available spreadsheet software. Results: SEC-DIA-MS coupled with PERCOM analysis identified 296 proteins with disrupted MW-distribution profiles. Hits were significantly enriched for mitochondrial and intercalated disk (ICD) components. Mitochondrial alterations were confirmed at the level of global protein expression and metabolic function. Disruption of mitochondrial and ICD supercomplexes was confirmed in mice as young as 9wks of age. Conclusion: We present a novel CP workflow that is highly suited to the analysis of very high MW protein-complexes and supercomplexes in specialized tissue types. It includes a data analysis strategy that removes a computer programming resource barrier to general application. Our findings are among the first to show altered mitochondrial function as a hallmark of early-stage R14Δ-PLN, and identify disruption of the intercalated disk as a novel potential disease pathway. These molecular-level alterations target key machinery underlying cardiac energy generation and electromechanical connectivity, and may significantly contribute to the R14∆-PLN pathogenesis.

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A RIPK1-specific PROTAC degrader achieves potent antitumor activity by enhancing immunogeniccell death

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