HSPA8 inhibitors augment cancer chemotherapeutic effectiveness via potentiating necroptosis.

Our recent work has uncovered a novel function of HSPA8 as an amyloidase, capable of dismantling the RHIM-containing protein fibrils to suppress necroptosis. However, the impact of HSPA8 inhibitors on cancer regression via necroptosis remains unexplored. In this study, we conducted a comprehensive investigation to assess the potential of HSPA8 inhibitors in enhancing necroptosis both in vitro and in vivo. Our findings indicate that pharmacologic inhibition of HSPA8, achieved either through VER (VER-155008) targeting the nucleotide binding domain or pifithrin-µ targeting the substrate binding domain of HSPA8, significantly potentiates necroptosis induced by diverse treatments in cellular assays. These inhibitors effectively disrupt the binding of HSPA8 to the RHIM protein, impeding its regulatory function on RHIM amyloid formation. Importantly, HSPA8 inhibitors significantly enhanced cancer cell sensitivity to microtubule-targeting agents (MTAs) in vitro, while reversing chemoresistance and facilitating tumor regression by augmenting necroptosis in vivo. Our findings suggest a promising therapeutic approach to cancer through necroptosis modulation via HSPA8 targeting, particularly in combination with MTA drugs for enhanced treatment efficacy.

HSPA8 acts as an amyloidase to suppress necroptosis by inhibiting and reversing functional amyloid formation.

Ultra-stable fibrous structure is a hallmark of amyloids. In contrast to canonical disease-related amyloids, emerging research indicates that a significant number of cellular amyloids, termed 'functional amyloids', contribute to signal transduction as temporal signaling hubs in humans. However, it is unclear how these functional amyloids are effectively disassembled to terminate signal transduction. RHIM motif-containing amyloids, the largest functional amyloid family discovered thus far, play an important role in mediating necroptosis signal transduction in mammalian cells. Here, we identify heat shock protein family A member 8 (HSPA8) as a new type of enzyme - which we name as 'amyloidase' - that directly disassembles RHIM-amyloids to inhibit necroptosis signaling in cells and mice. Different from its role in chaperone-mediated autophagy where it selects substrates containing a KFERQ-like motif, HSPA8 specifically recognizes RHIM-containing proteins through a hydrophobic hexapeptide motif N(X1)φ(X3). The SBD domain of HSPA8 interacts with RHIM-containing proteins, preventing proximate RHIM monomers from stacking into functional fibrils; furthermore, with the NBD domain supplying energy via ATP hydrolysis, HSPA8 breaks down pre-formed RHIM-amyloids into non-functional monomers. Notably, HSPA8's amyloidase activity in disassembling functional RHIM-amyloids does not require its co-chaperone system. Using this amyloidase activity, HSPA8 reverses the initiator RHIM-amyloids (formed by RIP1, ZBP1, and TRIF) to prevent necroptosis initiation, and reverses RIP3-amyloid to prevent necroptosis execution, thus eliminating multi-level RHIM-amyloids to effectively prevent spontaneous necroptosis activation. The discovery that HSPA8 acts as an amyloidase dismantling functional amyloids provides a fundamental understanding of the reversibility nature of functional amyloids, a property distinguishing them from disease-related amyloids that are unbreakable in vivo.