Stine, L. B. | bioRxiv Science

bioRxiv2025

CK2 directly controls CARD9 protein homeostasis

CARD9 is an attractive target for therapeutic intervention because the human genetic data provides strong evidence for the causal role of CARD9 in both protection and susceptibility to autoimmune disease. Expression quantitative trait loci (eQTLs) link higher CARD9 expression to increased disease risk and lower CARD9 expression to protection. Additionally, a rare allele variant leading to a C-terminal truncation of CARD9 (CARD9{Delta}11) and subsequent loss-of-function is also protective for inflammatory bowel disease (IBD). The mechanism of CARD9-driven inflammation through scaffold assembly with BCL10 and MALT1 (CBM complex) suggests a durable inflammatory signal driven by increasing levels of CARD9. Therefore, CARD9 depletion is a desired therapeutic strategy for drug discovery, yet a difficult one due to the nature of CARD9 as an adaptor protein target and the limited number of chemical tools available to engage it. Here, we uncover through a protein homeostasis screen that casein kinase 2 (CK2/CSNK2) inhibition leads to cellular CARD9 depletion. Following up with arrayed CRISPR screening, we identify key casein kinase isoforms/subunits responsible for CARD9 depletion. We find that CK2 directly binds to CARD9 and phosphorylates S424/S425 as well as S483/S484. Orthosteric CK2 inhibition prevents CK2 binding to CARD9 and leads to CARD9 destabilization. We show that the interaction between CK2 and CARD9{Delta}11 is significantly attenuated and not sensitive to CK2-mediated protein stabilization. The CK2-driven CARD9 depletion mechanism is preserved outside of immortalized cell lines and conserved between primary, differentiated mouse and human dendritic cells. Finally, we demonstrate therapeutic proof of concept in vivo using CK2 inhibition to deplete CARD9 in murine whole blood, peritoneum, and colon. Our study expands the scope of cellular consequences dealt by kinase inhibition, offers an unconventional approach for engaging a therapeutically intractable target, and identifies a novel mechanism that could contribute to disease protection conferred by the CARD9{Delta}11 allele.

immunology↗

bioRxiv2024

Orthosteric STING inhibition elucidates molecular correction of SAVI STING

STING is broadly implicated in diseases ranging from cancer, autoimmune disease, neurodegeneration to rare, monogenic diseases.1 Early drug discovery campaigns focused on STING activation as a promising platform for cancer immunotherapy yet failed in multiple clinical trials due to lack of efficacy thus far.2 Current research and development activities concentrate on STING inhibition for treating autoimmune disease and neuroinflammation. While the progression of STING activators into the clinic has been successful, the discovery and clinical progression of STING inhibitors remain elusive. Questions persist about the molecular properties needed to distinguish between a STING activator and inhibitor, particularly within SAVI disease, a monogenic autoinflammatory disease that renders STING constitutively active.3 Here we leverage an orthosteric STING activator and inhibitor from the same chemical series to discover that STING M271 is a critical residue for molecular activation. The M271CH3 NMR chemical shifts reveal a unique molecular signature for pharmacological or genetically driven activation and inhibition that is not captured by x-ray crystallography. Additionally, M271 directly interacts with the most common SAVI mutation, V155M, and using an orthosteric STING inhibitor, we show partial rescue and molecular correction of STING V155M. Finally, these data present insights into therapeutic STING molecular correction for treating SAVI patients. Our results elucidate an unappreciated structural interaction critical for STING modulation that could be utilized as a molecular diagnostic tool for drug discovery. Furthermore, we demonstrate for the first time how the therapeutic requirements of a molecular corrector differ from an orthosteric STING inhibitor, and why this is important for the SAVI disease population.

molecular biology↗