Uncovering Novel Protease Substrates and Inhibitors to Modulate Pyroptosis and Prevent Cancer Progression

Project Summary Proteases are pivotal players in cellular biology, acting as both guardians of homeostasis and mediators of stress responses. Their roles are indispensable to cellular function, ranging from protein turnover and immune responses to mitochondrial maintenance and beyond. Understanding the precise regulatory mechanisms of proteases provides unique opportunities to identify and exploit vulnerabilities in cancer progression. Aim 1 (F99 phase) focuses on identifying the physiologically relevant DPP9 inhibitor. DPP9 is a protease, which regulates inflammasomes—multiprotein complexes that detect danger signals and trigger pyroptosis, a lytic form of cell death. Under normal conditions, DPP9 represses the NLRP1 and CARD8 inflammasomes. Importantly, synthetic DPP9 inhibitors have been shown to activate these inflammasomes, triggering pyroptosis. However, the identity of an endogenous inhibitor remains elusive. Discovering this physiologically relevant inhibitor is crucial, as it will reveal the evolutionarily conserved danger signals that the NLRP1 and CARD8 inflammasomes evolved to sense, opening novel avenues for modulating inflammasome activation—a promising strategy for cancer immunotherapy. Preliminary data indicate that DPP9 interacts with the redox sensor KEAP1 through an ESGE motif, leading to mutual inhibition of both proteins. This interaction suppresses DPP9's catalytic activity and inhibits KEAP1's ability to sequester NRF2, stabilizing NRF2 and activating the antioxidant response system. Intriguingly, in DPP9’s native state, the ESGE motif is structurally incompatible with KEAP1 binding, suggesting that a conformational change in DPP9 is required for this interaction to occur. In Aim 1, this proposal seeks to (1) identify the stimulus that drives this conformational change, enabling KEAP1 binding and mutual inhibition, and (2) unbiasedly identify genetic regulators of DPP9 activity. Together, these studies aim to uncover the endogenous inhibitor of DPP9, define its role in inflammasome activation, and ultimately harness pyroptosis as a targeted cancer therapy. Aim 2 (K00 phase) focuses on identifying mitochondrial proteases critical for maintaining mitochondrial function under the metabolic and oxidative stresses of the tumor microenvironment. Unlike the cytosol, mitochondria lack a proteasomal degradation system and depend on proteases to prevent the accumulation of misfolded or damaged proteins, ensuring cellular adaptation and survival. In this Aim we will employ unbiased proteomics and CRISPR-based approaches to identify mitochondrial proteases and their cognate substrates that are essential for cancer cell survival. These studies will reveal how mitochondrial proteostasis enables cancer cell resilience and uncover therapeutic opportunities to selectively disrupt these processes, compromising cancer cell survival while sparing normal cells.