Kampmann, Martin

Abstract Hematological malignancies such as multiple myeloma (MM) have an increased reliance on the ubiquitin proteasome system (UPS) presumably as a consequence of their high protein synthetic and secretory burden. Chemical agents that target the proteasome, such as bortezomib and carfilzomib, have been successful in treating multiple myeloma; however patients treated with these drugs ultimately relapse. The AAA-ATPase p97/VCP (p97) facilitates ATP-dependent extraction and degradation of ubiquitinated proteins destined for proteasomal elimination. In addition to ubiquitin-dependent protein degradation, p97 is also closely involved in other aspects of protein homeostasis, including endoplasmic reticulum-associated degradation (ERAD) and autophagy. Pharmacologic inhibition of p97 provides a compelling therapeutic approach for hematological malignancies that rely on tight regulation of protein homeostasis as a component of their survival. CB-5083 is a novel small molecule 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 as well as an accumulation of substrates of the UPS and ERAD. CB-5083 causes a profound induction of the unfolded protein response (UPR) with consequent activation of the DR5 death receptor, caspase 8, caspase 3/7 and ultimately cell death. Induction of the UPR occurs to a greater magnitude with CB-5083 when compared to the proteasome inhibitor, bortezomib, suggesting the potential for increased efficacy in cancers with sensitivity to UPR-mediated cell death. In addition, activation of apoptosis and cell death occur more rapidly with CB-5083 than with bortezomib. Sequencing of cell lines made resistant to CB-5083 reveals missense mutations mapping to the D2 ATPase site in p97, supporting on-target association with cytotoxicity. In an expanded panel of MM cell lines there is no correlation between the cytotoxic sensitivity to CB-5083 and the cytotoxic sensitivity to proteasome inhibitors, suggesting differential mechanisms of cytotoxicity and potential activity of CB-5083 in proteasome inhibitor resistant settings. Compared to myeloma cell lines, CB-5083 has reduced cytotoxic potency in immortalized stromal cell lines and in patient-derived CD138-negative bone marrow mononuclear cells. Furthermore, unlike the reduced potency demonstrated by carfilzomib in the context of MM cell-bone marrow stromal cell (BMSC) interactions, the cyto-reductive potential of CB-5083 is unaffected in co-cultures of MM cells with patient-derived BMSCs or immortalized BMSCs from healthy donors. In vivo, CB-5083 is orally bioavailable, shows a pharmacodynamic effect in tumor tissue (as measured by poly-ubiquitin accumulation) and demonstrates robust anti-tumor activity across several MM models. CB-5083 treatment of mice bearing subcutaneous xenografts leads to tumor stasis and regression in RPMI8226 and AMO1 MM models, respectively. In advanced models of disseminated, ortho-metastatic disease, intermittent oral administration of CB-5083 demonstrates significant inhibition of myeloma burden and improves survival, with an overall efficacy profile that compares favorably to that of clinically approved proteasome inhibitors. Furthermore, in the Vk*Myc genetically engineered mouse model of MM, treatment with CB-5083 results in a significant reduction in M-spike by 55%. Combination treatment of mice bearing the RPMI8226 subcutaneous xenograft model with CB-5083, dexamethasone and lenalidomide results in tumor regression. Taken together, these data demonstrate that CB-5083 is a potent and selective inhibitor of the p97 ATPase with robust activity in vitro and in vivo in numerous MM models and strongly support clinical evaluation. Based on these observations, a phase 1 dose-escalation trial has recently been initiated and is currently underway in patients with relapsed/refractory multiple myeloma. Disclosures Anderson: Cleave Biosciences: Employment. Le Moigne:Cleave Biosciences: Employment. Djakovic:Cleave Biosciences: Employment. Rice:Cleave Biosciences: Employment. Wong:Cleave Biosciences: Employment. Kumar:Cleave Biosciences: Employment. Valle:Cleave Biosciences: Employment. Menon:Cleave Biosciences: Employment. Kiss von Soly:Cleave Biosciences: Employment. Wang:Cleave Biosciences: Employment. Yao:Cleave Biosciences: Employment. Soriano:Cleave Biosciences: Employment. Bergsagel:ONYX: Consultancy; Janssen: Consultancy; BMS: Consultancy; Novartis: Research Funding. Yakes:Cleave Biosciences: Employment. Zhou:Cleave Biosciences: Employment. Wustrow:Cleave Biosciences: Employment. Rolfe:Cleave Biosciences: Employment.

How dipeptide repeats cause pathology A repeat expansion in the chromosome 9 open reading frame 72 ( C9orf72 ) gene is the most common known cause of two neurodegenerative diseases: frontotemporal dementia and amyotrophic lateral sclerosis. This expansion leads to the abnormal production of proteins of repeating dipeptides, but their contribution to disease pathogenesis remains unclear. Zhang et al. engineered a mouse model to study the consequences of one of these dipeptides—prolinearginine dipeptide repeat protein, poly(PR)—in the brain. They found that poly(PR) caused neuron loss as well as motor and memory impairments. These detrimental effects resulted from poly(PR)-induced perturbation of heterochromatin function, a tightly packed form of DNA that represses gene expression. Science , this issue p. eaav2606