A RIPK1-specific PROTAC degrader achieves potent antitumor activity by enhancing immunogenic cell death.

Receptor-interacting serine/threonine-protein kinase 1 (RIPK1) functions as a critical stress sentinel that coordinates 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-κB, 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 treatment-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.

MT1-MMP-dependent cell migration: proteolytic and non-proteolytic mechanisms.

Membrane-type 1 matrix metalloproteinase (MT1-MMP) is a type I transmembrane proteinase that belongs to the matrix metalloproteinase (MMP) family. It is a potent modifier of cellular microenvironment and promotes cell migration and invasion of a wide variety of cell types both in physiological and pathological conditions. It promotes cell migration by degrading extracellular matrix on the cell surface and creates a migration path, by modifying cell adhesion property by shedding cell adhesion molecules to increase cell motility, and by altering cellular metabolism. Thus, MT1-MMP is a multifunctional cell motility enhancer. In this review, we will discuss the current understanding of the proteolytic and non-proteolytic mechanism of MT1-MMP-dependent cell migration.

Design, synthesis and biological evaluation of bifunctional inhibitors of membrane type 1 matrix metalloproteinase (MT1-MMP).

Collagen degradation and proMMP-2 activation are major functions of MT1-MMP to promote cancer cell invasion. Since both processes require MT1-MMP homodimerization on the cell surface, herein we propose that the use of bifunctional inhibitors of this enzyme could represent an innovative approach to efficiently reduce tumor growth. A small series of symmetrical dimers derived from previously described monomeric arylsulfonamide hydroxamates was synthesized and tested in vitro on isolated MMPs. A nanomolar MT1-MMP inhibitor, compound 6, was identified and then submitted to cell-based assays on HT1080 fibrosarcoma cells. Dimer 6 reduced MT1-MMP-dependent proMMP-2 activation, collagen degradation and collagen invasion in a dose-dependent manner with better results even compared to its monomeric analogue 4. This preliminary study suggests that dimeric MT1-MMP inhibitors might be further developed and exploited as an alternative tool to reduce cancer cell invasion.

Coordination of two kinesin superfamily motor proteins, KIF3A and KIF13A, is essential for pericellular matrix degradation by membrane-type 1 matrix metalloproteinase (MT1-MMP) in cancer cells.

MT1-MMP plays a crucial role in promoting the cellular invasion of cancer cells by degrading the extracellular matrix to create a path for migration. During this process, its localization at the leading edge of migrating cells is critical, and it is achieved by targeted transport of MT1-MMP-containing vesicles along microtubules by kinesin superfamily motor proteins (KIFs). Here we identified three KIFs involved in MT1-MMP vesicle transport: KIF3A, KIF13A, and KIF9. Knockdown of KIF3A and KIF13A effectively inhibited MT1-MMP-dependent collagen degradation and invasion, while knockdown of KIF9 increased collagen degradation and invasion. Our data suggest that KIF3A/KIF13A dependent MT1-MMP vesicles transport takes over upon KIF9 knockdown. Live-cell imaging analyses have indicated that KIF3A and KIF13A coordinate to transport the same MT1-MMP-containing vesicles from the trans-Golgi to the endosomes, and KIF13A alone transports the vesicle from the endosome to the plasma membrane. Taken together, we have identified a unique interplay between three KIFs to regulate leading edge localization of MT1-MMP and MT1-MMP-dependent cancer cell invasion.

A common SNP risk variant MT1-MMP causative for Dupuytren's disease has a specific defect in collagenolytic activity.

Dupuytren's Disease (DD) is a common fibroproliferative disease of the palmar fascia. We previously identified a causal association with a non-synonymous variant (rs1042704, p.D273N) in MMP14 (encoding MT1-MMP). In this study, we investigated the functional consequences of this variant, and demonstrated that the variant MT1-MMP (MT1-N273) exhibits only 17% of cell surface collagenolytic activity compared to the ancestral enzyme (MT1-D273). Cells expressing both MT1-D273 and MT1-N273 in a 1:1 ratio, mimicking the heterozygous state, possess 38% of the collagenolytic activity compared to the cells expressing MT1-D273, suggesting that MT1-N273 acts in a dominant negative manner. Consistent with the above observation, patient-derived DD myofibroblasts with the alternate allele demonstrated around 30% of full collagenolytic activity detected in ancestral G/G genotype cells, regardless of the heterozygous (G/A) or homozygous (A/A) state. Small angle X-ray scattering analysis of purified soluble Fc-fusion enzymes allowed us to construct a 3D-molecular envelope of MT1-D273 and MT1-N273, and demonstrate altered flexibility and conformation of the ectodomains due to D273 to N substitution. Taking together, rs1042704 significantly reduces collagen catabolism in tissue, which tips the balance of homeostasis of collagen in tissue, contributing to the fibrotic phenotype of DD. Since around 30% of the worldwide population have at least one copy of the low collagenolytic alternate allele, further investigation of rs1042704 across multiple pathologies is needed.