The lysate was then diluted in IP buffer containing protease and phosphatase inhibitors at ratio 1:10 and incubated on ice for 30 min

The lysate was then diluted in IP buffer containing protease and phosphatase inhibitors at ratio 1:10 and incubated on ice for 30 min. heavy-chain (transcription and CCND1 protein levels [8, 9]. Improved CCND1 levels also happen due to genomic deletions or point mutations in the 3 UTR, which results in shorter, more NU6300 stable transcripts [10, 11]. Experimental models that indicated a non-degradable CCND1 variant, in which T286 was substituted by alanine, or manifestation of an on the other hand spliced CCND1b isoform, which lacks T286, have resulted in mainly nuclear CCND1 manifestation and cellular transformation [12, 13]. In addition, aberrant activation of AKT and mTOR signaling results in down-regulation of GSK3B, also leading to reduced phosphorylation-dependent proteolysis and improved CCND1 protein levels [14]. Mantle cell lymphoma (MCL) is an incurable B-cell malignancy that regularly develops resistance to standard chemotherapy and has a prognosis having a median overall survival of approximately 1C2 years after relapse [15, 16]. Recent treatment improvements using the FDA-approved drug ibrutinib, which focuses on the B-cell antigen receptor (BCR) signaling molecule Bruton’s tyrosine kinase (BTK), have produced durable reactions in MCL [17]. However, one-third of MCL individuals are ibrutinib-resistant, and even ibrutinib-sensitive individuals eventually acquire resistance to the drug [17, 18]. The mechanisms underlying primary resistance to ibrutinib are not well understood. Recent studies have begun to provide some clues about potential mechanisms of main ibrutinib resistance, including activation of the alternative NF-kB [19], ERK1/2 or AKT signaling pathways [20]. Mechanisms of NU6300 acquired resistance to ibrutinib in patients who initially responded to the drug but then relapsed have also been reported, including recurrent mutations of the enzyme active site in BTK (C481S) or its substrate phospholipase C gamma 2 (PLCG2) [18, 21, 22]. These studies suggest that multiple mechanisms likely contribute to ibrutinib resistance in MCL. Recent large-scale genomic studies of MCL have recognized a hotspot for recurring somatic mutations in exon 1 of in 18C35% of the cases, likely arising through somatic hypermutation [23C25]. However, little is known about the functional role of these mutations in MCL. This study investigated the functional effects of mutation on protein stability and sensitivity of MCL cells to ibrutinib therapy. The three most frequent mutations (E36K, Y44D and C47S) were cloned and expressed in MCL cell lines or HEK-293T cells. CCND1 protein stability and conversation with GSK3B were evaluated by cyclohexamide treatment and immunoprecipitation, respectively. Subcellular localization of the mutant CCND1 protein was determined by cell fractionation and immunofluorescence. In addition, main MCL tumors with mutations were examined for CCND1 protein NU6300 stability and sensitivity to ibrutinib. These studies have uncovered an important role for mutations in deregulating protein turnover, and a potential role in resistance to ibrutinib in some MCL tumors. RESULTS mutations increased CCND1 protein levels through defective proteolysis To study the role of somatic mutations, the exon 1 of eight MCL cell lines was sequenced and found to have the germ-line sequence (data not shown). Therefore, site-directed mutagenesis was used to generate the three most frequent mutations, E36K, Y44D and C47S, as previously NU6300 reported (Physique ?(Figure1A)1A) [19, 23C25]. Hemagglutinin (HA)-tagged wild type (WT) or mutant cDNA was cloned into a TCEB1L retroviral vector and expressed in the MCL cell lines UPN-1, Z-138 and JEKO-1. An empty vector was used as a negative control. After establishing stably transduced cells by hygromycin selection, equal numbers of cells from each culture were harvested and mRNA and total protein lysates were prepared. Anti-HA antibody was used to assess exogenous CCND1 protein expression by immunoblot analysis. All three mutants showed increased protein expression compared to the WT counterparts in all three MCL cell NU6300 lines (Physique ?(Physique1B,1B, Supplementary Physique S1A). In Supplementary Physique S1A, JEKO-1 cells that expressed the non-degradable T286A mutant [5] were also included for comparison. Compared to WT, mutant CCND1 proteins did not impact the kinase function of CDK4, as determined by phosphorylation of the CDK4 substrate Rb in JEKO-1 cells (Supplementary Physique S1B). To determine whether increased protein expression was due to increased transcription, mRNA expressed from WT and mutant.