Abstract 4541: Utilization of K48 Poly-Ubiquitin Modulation As a Biomarker of Target Engagement for a Protein Homeostasis Inhibitor in the Clinic: Preclinical Validation with CB-5083, a First-in-class Inhibitor of the AAA ATPase P97

Abstract Background: Pharmacodynamic (PD) biomarkers are an increasingly valuable tool for decision-making and prioritization of lead compounds during preclinical and clinical studies as they link drug-target inhibition in cells with biological activity. They are of particular importance for novel, first-in-class mechanisms, where the ability of a targeted therapeutic to impact disease outcome is unknown. We recently discovered CB-5083, a novel small molecule inhibitor of p97, a protein involved in several facets of protein homeostasis, including ubiquitin-dependent protein degradation, endoplasmic reticulum-associated degradation (ERAD) and autophagy. Accumulation of K48 poly-ubiquitinated proteins is a hallmark of protein degradation inhibition, and we used this proximal biomarker to follow CB-5083 target engagement on p97 in various pre-clinical models. Results: CB-5083 is a potent inhibitor of p97, with a biochemical IC50 of 11 nM. When cancer cells are exposed to CB-5083, biological consequences linked to p97 inhibition are detected, including ERAD inhibition, ER (endoplasmic reticulum) stress, ER stress-mediated cell death and accumulation of poly-ubiquitinated proteins. In mouse models, CB-5083 is orally bio-available and causes rapid and sustained accumulation of K48 poly-ubiquitin in tumor xenografts after a single administration. Concurrent with increases in K48 poly-ubiquitin levels, activation of ER stress response pathways and induction of apoptosis markers are also observed. Accumulation of K48 poly-ubiquitin also occurs in other tissues in the body and we developed a quantitative method to detect accumulation of K48 poly-ubiquitin as a marker of target engagement in whole blood. With this method, we were able to compare the kinetics and level of K48 poly ubiquitin accumulation following CB-5083 administration in both tumor and whole blood. We also performed a dose escalation of CB-5083 in cynomolgus monkeys and defined the minimal plasma AUC and Cmax required to see accumulation of K48 poly-ubiquitin in monkey whole blood. This approach allowed us to predict human exposures that should lead to target engagement and consequent biological activity in our ongoing phase 1 studies where a flow cytometry based assay is being used to monitor K48 poly-ubiquitin levels in patient blood samples. Conclusion: K48 poly-ubiquitin is a target engagement biomarker of p97 inhibition. CB-5083, a p97 inhibitor, can induce sustained induction of K48 poly-ubiquitin, not only in tumor but also in surrogate tissues such as whole blood. K48 poly-ubiquitin accumulation is currently being assessed to follow target engagement in the blood of patients in our ongoing phase 1 dose escalation studies of CB-5083. Citation Format: Mary-Kamala MENON, Ferdie SORIANO, Steve WONG, Eduardo VALLE, Stevan Djakovic, Brajesh KUMAR, Bing YAO, Antonett MADRIAGA, Tony WU, Julie RICE, Jinhai WANG, Alessandra CESANO, Laura SHAWVER, Han-Jie ZHOU, David WUSTROW, Daniel ANDERSON, Mark ROLFE, Ronan LE MOIGNE. Utilization of K48 poly-ubiquitin modulation as a biomarker of target engagement for a protein homeostasis inhibitor in the clinic: Preclinical validation with CB-5083, a first-in-class inhibitor of the AAA ATPase p97. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 4541.