Cullin-1 Controls The Clinical and Cellular response to Bortezomib Through NF-Kb Pathway Activation In Myeloma

The Ubiquitin (Ub)+Proteasome system (UPS) is a highly complex network that maintains cellular homeostasis through the selective turnover of targeted proteins. The proteasome serves as the catalytic core of the UPS to execute the efficient removal of ubiquitin-conjugated proteins. Pharmacologic inhibitors that exploit the pivotal role of the proteasome in cellular metabolism promote tumor cytotoxicity and yield durable clinical responses that have significantly improved survival of patients diagnosed with the invariably fatal plasma cell malignancy multiple myeloma (MM). However, while functional blockade of the proteasome has emerged as a successful anti-cancer strategy, drug resistance inevitably emerges through mechanisms that remain elusive. Since E3 Ub ligases target biologically-relevant proteins for proteasomal degradation and are frequently overexpressed in cancers, we hypothesized that altered E3 Ub ligase expression served as a mechanism of resistance to proteasome inhibitors (PIs). To address the role of individual E3s in myelomagenegesis, gene expression profiles of MM patient samples obtained prior to treatment were analyzed. Results indicated that Cullin-1 was overexpressed in MM patient tumor samples compared to normal or MGUS samples. Cullin-1 is an essential scaffolding component of multiple Skp1-Cullin-1-F-box protein E3 complexes that mediate the ubiquitination of proteins involved in cell cycle progression. Next. bone marrow-derived CD138⁺ plasma cells were obtained from MM patients that were then treated with the PI bortezomib. Samples were similarly probed to identify differences in E3 expression and to identify features that dictate therapeutic response. Again, Cullin-1 was overexpressed in samples from MM patients that did not respond to bortezomib but Cullin-1 was not overexpressed in samples from those patients that responded to bortezomib. Cullin-1 levels were also higher in bortezomib-resistant myeloma cell lines and drug-resistant cells were re-sensitized to bortezomib after short-hairpin-RNA-mediated Cul-1 knockdown. Cells overexpressing Cullin-1 displayed increased NF-Kappa B activity and a reduced sensitivity to bortezomib-induced apoptosis as demonstrated by staining for annexin-positivity. The effect of Cullin-1 expression level on the sensitivity to bortezomib treatment was determined using MM xenografts in athymic SCID mice. We have used a biologically-supervised, microarray-based approach to identify Cul-1 overexpression in a subset of myeloma patients and correlated expression with clinical resistance to bortezomib. Functional studies demonstrated that Cullin-1 sustains activation of the NF-Kappa B pathway and decreases cellular response to bortezomib in vitro in human cell lines. We conclude that Cullin-1 attenuates bortezomib anti-MM activity by maintaining NF-Kappa B signaling and hence promoting tumor survival. Engineered overexpression or shRNA-mediated inactivation of Cullin-1 modulated the cytotoxic effect of bortezomib to further recapitulate the premise that tumor genomics dictate therapeutic response. Cullin-1 is a novel effector within the UPS that offers promise as an oncologic target either alone or in synergistic combination with proteasome inhibitors.