CCND1 mutations increase protein stability and promote ibrutinib resistance in mantle cell lymphoma.

Atish Mohanty,Lan V Pham,Natalie Sandoval,Robert W Chen,Lu Chen,Raju Pillai,Manasi Das,Steven T Rosen,Guido Marcucci,Dennis D Weisenburger,Michael Wang,Vu N Ngo,Hesham M Amin

doi:10.18632/oncotarget.12434

Abstract

Mantle cell lymphoma (MCL) is characterized by the t(11;14) translocation, which leads to deregulated expression of the cell cycle regulatory protein cyclin D1 (CCND1). Genomic studies of MCL have also identified recurrent mutations in the coding region of CCND1. However, the functional consequence of these mutations is not known. Here, we showed that, compared to wild type (WT), single E36K, Y44D or C47S CCND1 mutations increased CCND1 protein levels in MCL cell lines. Mechanistically, these mutations stabilized CCND1 protein through attenuation of threonine-286 phosphorylation, which is important for proteolysis through the ubiquitin-proteasome pathway. In addition, the mutant proteins preferentially localized to the nucleus. Interestingly, forced expression of WT or mutant CCND1 increased resistance of MCL cell lines to ibrutinib, an FDA-approved Bruton tyrosine kinase inhibitor for MCL treatment. The Y44D mutant sustained the resistance to ibrutinib even at supraphysiologic concentrations (5–10 μM). Furthermore, primary MCL tumors with CCND1 mutations also expressed stable CCND1 protein and were resistant to ibrutinib. These findings uncover a new mechanism that is critical for the regulation of CCND1 protein levels, and is directly relevant to primary ibrutinib resistance in MCL.

Highlights

cyclin D1 (CCND1) is an important cell cycle regulator in many cell types
To determine whether increased protein expression was due to increased transcription, mRNA expressed from wild type (WT) and mutant CCND1 was compared by real-time quantitative PCR using primers specific for the exogenous CCND1-HA mRNA (Figure 1C)
In both UPN-1 and Z-138 cells, while more than 50% of WT CCND1 was degraded as early as 90 min after CHX, little reduction in Y44D CCND1 levels was observed during the same period (Figure 1D, 1E)

Summary

Introduction

CCND1 binds and activates cyclindependent kinase (CDK) 4, which in turn phosphorylates and inactivates retinoblastoma (Rb) protein, leading to G1/S cell cycle progression [1, 2]. Cell cycledependent proteolysis of CCND1 requires phosphorylation of threonine-286 (T286) residue, which is predominantly mediated by glycogen synthase kinase 3 beta (GSK3B) [5]. Other kinases including DYRK1B and p38 MAPK are known to phosphorylate T286, leading to ubiquitindependent degradation of CCND1 [6, 7]. Increased CCND1 levels occur due to genomic deletions or point mutations in the 3′ UTR, which results in shorter, more stable transcripts [10, 11]. Aberrant activation of AKT and mTOR signaling results in down-regulation of GSK3B, leading to reduced phosphorylation-dependent proteolysis and increased CCND1 protein levels [14]

Methods

Results

Conclusion