Abstract B126: Targeting USP36 as a therapeutic strategy for high-MYC expressing pancreatic ductal adenocarcinoma

Abstract

Pancreatic ductal adenocarcinoma (PDAC) remains one of the most lethal malignancies, characterized by poor therapeutic response and aggressive disease progression driven heavily by the c-MYC oncoprotein. Ubiquitin-specific protease 36 (USP36) is a nucleolar deubiquitinating enzyme that enhances the stability of critical nucleolar proteins, including c-MYC. Consequently, targeting USP36 represents a compelling therapeutic vulnerability in high-MYC-expressing PDAC tumors that are resistant to standard-of-care therapy. To identify novel therapeutic agents, a target-based high-throughput screening of a library containing 1,430 FDA-approved compounds was conducted utilizing a Ub-AMP fluorescence assay. In this biochemical assay, active USP36 cleaves ubiquitin from the substrate to generate a fluorescent signal; effective inhibitors are identified by a corresponding decrease in fluorescence intensity. From this screen, napabucasin, originally characterized as a cancer stemness inhibitor that blocks STAT3 phosphorylation and subsequent transcription of tumor-survival genes, was identified as a top candidate USP36 inhibitor. To validate this finding, well-established human PDAC cell lines (MIA PaCa-2 and Capan) were treated with napabucasin, demonstrating a robust time- and dose-dependent downregulation of c-MYC expression in both lines. Furthermore, in vitro cell viability assays demonstrated that napabucasin inhibits the proliferation of KMC murine PDAC cells, derived from tumors initiated through deregulated constitutive expression of MYC and mutant KRAS in adult pancreatic acinar cells, in a dose-dependent manner. Notably, cells with reduced MYC expression (MYC knockdown) exhibited significantly less sensitivity to napabucasin compared to shMYC control cells, supporting a MYC-dependent mechanism of action. To further validate these findings in patient-derived models, napabucasin was evaluated in patient-derived cell lines (PDCLs) established from primary human PDAC tumors. PDCLs possessing a high-MYC gene signature, as determined by bulk RNA-sequencing analysis, demonstrated heightened sensitivity to USP36 inhibition compared to cell lines with low-MYC signatures. Finally, to evaluate in vivo efficacy, napabucasin was administered in an orthotopic KMC PDAC mouse model. Tumors from this KMC model histologically and molecularly recapitulate the heterogeneity observed in human PDAC. Mice treated with napabucasin exhibited a significant reduction in average tumor volume compared to vehicle-treated controls. Furthermore, combining napabucasin with the pan-KRAS inhibitor RMC-6236 demonstrated superior efficacy in suppressing tumor growth compared to RMC-6236 monotherapy. Collectively, these data demonstrate that USP36 inhibition by napabucasin effectively impairs PDAC tumor growth in a MYC-dependent manner. These findings underscore the potential of USP36-targeted therapies, particularly when paired with pan-KRAS inhibition, as a promising strategy to overcome therapeutic resistance in MYC-driven pancreatic cancer.