Eduardo Valle

Targeting the AAA ATPase p97 as an Approach to Treat Cancer through Disruption of Protein Homeostasis

p97 is a AAA-ATPase with multiple cellular functions, one of which is critical regulation of protein homeostasis pathways. We describe the characterization of CB-5083, a potent, selective, and orally bioavailable inhibitor of p97. Treatment of tumor cells with CB-5083 leads to accumulation of poly-ubiquitinated proteins, retention of endoplasmic reticulum-associated degradation (ERAD) substrates, and generation of irresolvable proteotoxic stress, leading to activation of the apoptotic arm of the unfolded protein response. In xenograft models, CB-5083 causes modulation of key p97-related pathways, induces apoptosis, and has antitumor activity in a broad range of both hematological and solid tumor models. Molecular determinants of CB-5083 activity include expression of genes in the ERAD pathway, providing a potential strategy for patient selection.

Discovery of a First-in-Class, Potent, Selective, and Orally Bioavailable Inhibitor of the p97 AAA ATPase (CB-5083)

The AAA-ATPase p97 plays vital roles in mechanisms of protein homeostasis, including ubiquitin-proteasome system (UPS) mediated protein degradation, endoplasmic reticulum-associated degradation (ERAD), and autophagy. Herein we describe our lead optimization efforts focused on in vitro potency, ADME, and pharmaceutical properties that led to the discovery of a potent, ATP-competitive, D2-selective, and orally bioavailable p97 inhibitor 71, CB-5083. Treatment of tumor cells with 71 leads to significant accumulation of markers associated with inhibition of UPS and ERAD functions, which induces irresolvable proteotoxic stress and cell death. In tumor bearing mice, oral administration of 71 causes rapid accumulation of markers of the unfolded protein response (UPR) and subsequently induces apoptosis leading to sustained antitumor activity in in vivo xenograft models of both solid and hematological tumors. 71 has been taken into phase 1 clinical trials in patients with multiple myeloma and solid tumors.

The p97 inhibitor CB-5083 is a unique disrupter of protein homeostasis in models of Multiple Myeloma.

Inhibition of the AAA ATPase, p97, was recently shown to be a novel method for targeting the ubiquitin proteasome system, and CB-5083, a first-in-class inhibitor of p97, has demonstrated broad antitumor activity in a range of both hematologic and solid tumor models. Here, we show that CB-5083 has robust activity against multiple myeloma cell lines and a number of in vivo multiple myeloma models. Treatment with CB-5083 is associated with accumulation of ubiquitinated proteins, induction of the unfolded protein response, and apoptosis. CB-5083 decreases viability in multiple myeloma cell lines and patient-derived multiple myeloma cells, including those with background proteasome inhibitor (PI) resistance. CB-5083 has a unique mechanism of action that combines well with PIs, which is likely owing to the p97-dependent retro-translocation of the transcription factor, Nrf1, which transcribes proteasome subunit genes following exposure to a PI. In vivo studies using clinically relevant multiple myeloma models demonstrate that single-agent CB-5083 inhibits tumor growth and combines well with multiple myeloma standard-of-care agents. Our preclinical data demonstrate the efficacy of CB-5083 in several multiple myeloma disease models and provide the rationale for clinical evaluation as monotherapy and in combination in multiple myeloma. Mol Cancer Ther; 16(11); 2375–86. ©2017 AACR.

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

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.

Abstract DDT02-01: Inhibition of the AAA-ATPase p97 with the first in class inhibitor CB-5083 as a novel approach to treat cancer

The ubiquitin-proteasome system (UPS) sustains cancer cell viability by alleviating proteotoxic stress caused by an imbalance of protein synthesis and degradation. While proteasome inhibitors and immunomodulators (IMiDs) are approved for the treatment of hematological malignancies, relapse following treatment is common. Additionally, these agents have poor efficacy in solid tumors, prompting the need for discovery of novel drug targets within the UPS which might provide a more profound anti-tumor effect. The homo-hexameric AAA-ATPase VCP/p97 functions by converting chemical energy into mechanical force to extract proteins from cell membranes and protein complexes. p97 is directly involved in several facets of protein homeostasis, including ubiquitin-dependent protein degradation, endoplasmic reticulum-associated degradation (ERAD), and autophagy. Therefore p97 inhibition could provide a novel and more potent approach than proteasome inhibitors to exploit cancer cell addiction to protein homeostatic mechanisms. Through a targeted medicinal chemistry effort we have discovered CB-5083, a novel small molecule ATP-competitive inhibitor of p97 ATPase activity with nanomolar enzymatic and cellular potency. Treatment of cancer cells with CB-5083 causes a dramatic increase in poly-ubiquitinated proteins and an accumulation of substrates of the UPS and ERAD. The blockade of ERAD causes induction of an irresolvable unfolded protein response (UPR), leading to caspase cleavage and apoptosis in a manner that is distinct from proteasome inhibitors. Indeed, ER-related stress and ERAD genes were the most significantly upregulated gene ontologies uncovered from transcriptome profiling. Additionally, knockdown of DR5 protects against CB-5083 mediated cell death, suggesting that death receptor signaling plays a key role in the apoptotic response caused by inhibition of p97 function. In animal models, our p97 inhibitor is orally bio-available and causes rapid and sustained accumulation of poly-ubiquitin and markers of the UPR and apoptosis. Furthermore, strong anti-cancer effects were observed in solid tumor and hematological murine models. We have developed a clinical-grade multiparametric flow cytometry assay to measure the accumulation of lysine-48 linked poly-ubiquitin and cPARP from human whole blood and circulating multiple myeloma cells, providing a way to monitor changes to the UPS and biological activity in real time. CB-5083 is currently being tested in ongoing phase I clinical trials for relapsed/refractory multiple myeloma and solid tumor patients. Taken together, these results indicate that CB-5083 has great promise as a novel therapeutic agent for the treatment of cancer. Citation Format: Daniel J. Anderson, Ronan Le Moigne, Stevan Djakovic, Brajesh Kumar, Julie Rice, Steve Wong, Jinhai Wang, Bing Yao, Eduardo Valle, MK Menon, Szerenke Kiss von Soly, Antonett Madriaga, Ferdie Sorlano, Mike Longhi, Alessandra Cesano, Han-Jie Zhou, David Wustrow, Mark Rolfe. Inhibition of the AAA-ATPase p97 with the first in class inhibitor CB-5083 as a novel approach to treat cancer. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr DDT02-01. doi:10.1158/1538-7445.AM2015-DDT02-01

Abstract 951: CB-5083 is a novel first in class p97 inhibitor that disrupts cellular protein homeostasis and demonstrates anti-tumor activity in solid and hematological models

Background: The AAA-ATPase p97/VCP facilitates the extraction and degradation of ubiquitinated proteins by converting chemical energy into mechanical force. p97 is closely involved in several facets of protein homeostasis, including ubiquitin-dependent protein degradation, endoplasmic reticulum-associated degradation (ERAD) and autophagy. p97 has been increasingly linked to cancer: it showed elevated protein expression in tumors, it can mediate the degradation of proteins in cancer-relevant pathways and is required for orchestrating the ubiquitin-governed DNA-damage response. In this context, p97 inhibitors may have an advantage versus other protein homeostasis inhibitors and may be active in solid tumors where 26S proteasome inhibitors, bortezomib and carfilzomib, have shown poor efficacy. We report here p97 inhibition as a novel approach to exploit cancer cell addiction to protein homeostatic mechanisms. Results: We have discovered novel small molecule inhibitors of p97 ATPase activity with nanomolar enzymatic and cellular potency. In cellular models, treatment of cancer cells with our lead compound CB-5083 causes disruptions in specific p97 functions, including ubiquitin-dependent protein degradation, ERAD, endocytosis and autophagy. In mouse models, CB-5083 is orally bio-available and causes rapid and sustained accumulation of poly-ubiquitin in tumor xenografts after a single administration. Concurrent with increases in polyubiquitin levels, activation of ER stress response pathways and induction of apoptosis markers are also observed. Sub-chronic oral administration of CB-5083 is generally well-tolerated with 50% reduction in M-spike. Additional efforts are focused on the development of translational assays to monitor p97 target engagement and antitumor efficacy in upcoming clinical trials of CB-5083. Conclusion: These data demonstrate that CB-5083 is a potent inhibitor of p97 that translates to tumor growth inhibition in multiple rodent models of human cancer. Furthermore, CB-5083 appears to exhibit greater potency over current proteasome inhibitors that further validate targeting p97 and protein homeostasis in the treatment of cancer. Citation Format: Ronan Le Moigne, Steve Wong, Ferdie Soriano, Eduardo Valle, Daniel J. Anderson, Stevan Djakovic, Mary-Kamala Menon, Bing Yao, Julie Rice, Jinhai Wang, Szerenke Kiss Von Soly, Brajesh Kumar, Marta Chesi, P. Leif Bergsagel, Han-Jie Zhou, David Wustrow, Mark Rolfe, F. Michael Yakes. CB-5083 is a novel first in class p97 inhibitor that disrupts cellular protein homeostasis and demonstrates anti-tumor activity in solid and hematological models. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 951. doi:10.1158/1538-7445.AM2014-951

Abstract C188: Novel small molecule inhibitors of p97 disrupt cellular protein homeostasis and demonstrate anti-tumor activity in solid and hematological models.

Background: The ubiquitin-proteasome system (UPS) sustains cancer cell viability by alleviating proteotoxic stress caused by an imbalance of protein synthesis and degradation. Bortezomib and carfilzomib are 26S proteasome inhibitors approved for the treatment of multiple myeloma. Unfortunately, these agents have poor efficacy in solid tumors, prompting the need for discovery of novel drugs targeting other enzymes within the UPS. The AAA-ATPase p97/VCP functions by converting chemical energy into mechanical force and is closely involved in several facets of protein homeostasis. These include ubiquitin-dependent protein degradation, endoplasmic reticulum-associated degradation (ERAD), and autophagy. p97 inhibition will provide a novel approach to exploit cancer cell addiction to protein homeostatic mechanisms. Results: Through a targeted medicinal chemistry effort, we have discovered novel small molecule inhibitors of p97 ATPase activity with nanomolar enzymatic and cellular potency. Sequencing of human cancer cell lines that have developed resistance to our p97 inhibitors has revealed mutations in p97, strongly suggesting on-target cellular activity. Treatment of normal and cancer cells with this class of p97 inhibitors causes a dramatic increase in poly-ubiquitinated proteins and an accumulation of substrates of the UPS and ERAD. These inhibitors also disrupt other specific p97 cellular functions including macroautophagy and receptor endocytosis. Inhibitor treatment ultimately leads to a blockade of NF-kB signaling and a decrease in downstream survival factors followed by the induction of caspase cleavage and apoptosis. In animal models, our p97 inhibitors are orally bio-available and cause rapid accumulation of poly-ubiquitin in tumor xenografts at levels that exceed the accumulation seen with bortezomib. Furthermore, significant tumor growth inhibition was observed in a number of solid tumor and hematological models. Conclusion: Together, these data provide novel insights into the role of p97 in cancer cell growth and the mechanism of death due to p97 inhibition. Animal model data suggests promise for our inhibitors as therapeutic agents for patients with hematological and solid tumors. Citation Information: Mol Cancer Ther 2013;12(11 Suppl):C188. Citation Format: Stevan N. Djakovic, Daniel J. Anderson, Szerenke Kiss von Soly, Ronan Le Moigne, Julie Rice, Mark Rolfe, Ferdie Soriano, Eduardo Valle, Jinhai Wang, Steve Wong, David Wustrow, F. Michael Yakes, Bing Yao, Han-Jie Zhou. Novel small molecule inhibitors of p97 disrupt cellular protein homeostasis and demonstrate anti-tumor activity in solid and hematological models. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2013 Oct 19-23; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2013;12(11 Suppl):Abstract nr C188.