Targeting Neddylation to Overcome Drug Resistance in Multiple Myeloma

Background: The overexpression of CKS1B resulting from chromosomal amplification of the 1q21 region identifies a subset of multiple myeloma (MM) patients with poor clinical outcomes. Bortezomib (Btz) is an FDA-approved first-in-class proteasome inhibitor that has greatly improved clinical outcomes in MM. However, toxicities affiliated with pan-proteasomal inhibition and resistant populations remain a problem, highlighting the need for improved therapies. We have previously demonstrated that elevated expression of CKS1B results in the destabilization of p21 and contributes to insensitivity to bortezomib. MLN4924 is an inhibitor of the NEDD8-Activating Enzyme E1 (NAE1) and prevents the neddylation and subsequent activation of Cullin-1, a crucial component in the activation of SCF-driven ubiquitin-mediated degradation. We therefore explored if MLN4924 might serve as an alternative inhibitor to proteasomal degradation in MM.

Materials and Methods: CKS1B was overexpressed or knocked down in myeloma cell lines using lentiviral vectors and shRNA. Cell proliferation and viability were assessed by cell counts and using PrestoBlue reagent. Clonogenicity was assessed by colony formation in soft agar. SA-b-galactosidase staining was used to assess senescence. Gene expression profiling was performed using the publicly available databases Total Therapy 2 (TT2) and APEX trials.

Results: To examine the efficacy of Btz and MLN4924 in elevated CKS1B expression environments, we compared treatment with the MLN4924 to Btz in CKS1B OE cells in vitro. Cells with basal levels of CKS1B were sensitive to treatment with either Btz or MLN4924, with treatment leading to decreased proliferation and cellular viability.  In the CKS1B OE background, we found cells were resistant to Btz repression of proliferation but sensitive to MLN4924. We also found that MLN4924 could more potently reduce colony formation in soft agar and more potently induce senescence in CKS1B OE cells compared to treatment with Btz. Immunoblot analyses confirmed a correlation between CKS1B expression and Cullin‑1 neddylation. Further immunoblotting of known SCF-mediated ubiquitin targets in cells with and without CKS1B OE demonstrated a stabilization of p21 in all cells upon MLN4924 treatment that was not exhibited upon Btz treatment. To investigate the role of p21 in CKS1BOE cell sensitivity to MLN4924, we stably knocked down expression of p21. We found the knockdown of p21 partially abrogated the sensitivity of CKS1B OE cells to MLN4924, increasing cell viability, increasing colony formation in soft agar, and decreasing senescence induction.

The importance of neddylation in the clinic was confirmed using GEP. We found that expression of the neddylation-related genes (i.e. NAE1, UBA3, and UBC12) is significantly upregulated (p<0.05) in MM patients relative to patients in MGUS or to healthy donors. Expression of NEDD8 was also significantly elevated in patients unresponsive to either Btz treatment or treatment with the combination of Btz and dexamethasone compared to responsive patients. The elevated expression of CKS1B highlights a population of patients with poor clinical outcomes. Considering the elevated expression of neddylation-related genes in MM progression, we performed Kaplan Meier survival analyses looking at patients segregated on two axes by CKS1B expression levels (high/low) and neddylation-related gene expression levels (either UBA3 or UBC12).  We found patients with high/high expression of CKS1B and neddylation-related genes had significantly decreased survival relative to patients with mixed high/low expression from either axis. Together, these data suggest the importance of neddylation contributions to MM progression and clinical outcomes.

Conclusions: These results shed light on the subtle differences in the blockade of proteasomal inhibition, illustrating the distinct effects of pan inhibition from inhibition of SCF-driven ubiquitin-mediated degradation. Mechanistically, we demonstrate at least a partial role for p21 in mediating cell sensitivity to MLN4924 in CKS1B overexpressing cells. Our findings highlight the important contributions of neddylation to MM disease progression and suggest the utility of targeting neddylation as a means of overcoming drug resistance in MM.