Identification of small molecule activators of Type I interferon signaling for cancer treatment

In the past decade, immunotherapies have revolutionized the way many cancers are treated. By activating the immune system, these treatments enable the body's own defense mechanisms to attack cancer cells and halt tumor growth. Unfortunately, many cancers are refractory to these new therapeutics and there continues to be a desperate need for drugs with novel mechanisms of action to enhance the long-term effectiveness of cancer treatment. Type I interferons (IFN-Is) are attractive candidate drugs to fill this role since these cytokines “interfere” with the growth of tumors; indeed, manufactured IFN-Is have been used in the clinic to treat more than 10 types of cancers but the outcomes were somewhat disappointing. Due to negative feedback regulation of IFN signaling, most interferon stimulated genes (ISGs) are transiently expressed, and these feedback mechanisms ultimately suppress the anticancer effectiveness of administered IFN-Is over time. This process also limits the signaling from intrinsically produced IFN-Is, critical to the success of chemo-, radiation, and immuno-therapy. In pioneering work over many years, we unraveled the details of the negative feedback pathway and identified the ubiquitin protease family member USP18 as the central regulator driving feedback suppression of the IFN-I response. By single-cell RNA-seq analysis, we revealed that cancer stem cells are especially sensitive to USP18 depletion- triggered cell death. These novel and exciting discoveries demonstrate that targeting USP18 represents an effective and promising, yet to-date underutilized, therapeutic option for treating cancer by directly promoting immunogenic cancer stem cell death and by increasing both innate and adaptive immune responses against cancer. With the goal to discover small molecule inhibitors of the USP18-mediated IFN-I feedback loop, we conceptualized an innovative high-throughput screening (HTS) assay based on our novel IFN-I signaling biosensor cell line with CRISPR/Cas9 inserted fluorescent reporters and validated it in a pilot screen of known compounds. Since it is currently unclear which specific mechanisms for USP18 inhibition are druggable by small molecules and provide the best therapeutic window, we opted for a phenotypic discovery approach combined with target deconvolution assays. Additionally, we developed a pipeline of secondary and mechanistic assays that validates the hit compounds and provides initial insight into the targeted components of the feedback pathway. Here we propose to 1) identify inhibitors of the USP18-mediated feedback pathway through a large phenotypic HTS, 2) validate the hits in secondary assays and map their effects to the specific pathway components, and 3) evaluate the therapeutic anti-cancer potential of the final chemical probes. Successful completion of these studies will identify and validate small molecule inhibitors of USP18 that can be used to probe the therapeutic potential of the different mechanisms for inhibiting the USP18-mediated negative feedback regulation of IFN signaling. Additionally, such immunomodulators could be further development toward a new class of cancer therapeutics. Project Number: 1R01CA299139-01A1 | Fiscal Year: 2026 | NIH Institute/Center: National Cancer Institute (NCI) | Principal Investigator: DONG-ER ZHANG (+1 co-PI) | Institution: UNIVERSITY OF CALIFORNIA, SAN DIEGO, LA JOLLA, CA | Award Amount: $771,811 | Activity Code: R01 | Study Section: Special Emphasis Panel[ZRG1 CTH-T (80)] View on NIH RePORTER: https://reporter.nih.gov/project-details/11297664