Project description:RNA was extracted from myeloma cell lines that were either drug-naïve or resistant to bortezomib or carfilzomib and the transcriptome was characterised using RNA sequencing.
Project description:LP-1 cells were exposed to stepwise increasing concentrations of carfilzomib over a period of 18 weeks: cells adapted to growth in 4 nM carfilzomib by 4 weeks, in 6 nM in another 6 weeks and in 12 nM after a further 8 weeks. The resulting cell culture, denoted LP-1/Cfz, retained resistance to carfilzomib even when tested after removal of selective pressure for approximately 8 weeks.
Project description:The clinical efficacy of bortezomib in multiple myeloma (MM) is limited due to secondary drug resistance driven by clonal evolution and currently available pre-clinical models are inadequate for comprehensive understanding of mechanisms underlying drug resistance. In the present study, we have established and characterized bortezomib-resistant cell lines BR1 and BR2, and identified upregulation of proteasome pathway as the potential mechanism underlying bortezomib resistance. Furthermore, both the cell lines retained sensitivity towards imatinib suggesting the lack of general resistance towards broad classes of inhibitors. Notably, BR1 and BR2 showed increased sensitivity than the parental cell line towards the next-generation proteasome inhibitor carfilzomib, suggesting its utility in treating patients who relapse upon bortezomib treatment. When compared to the wild type cell line, BR1 and BR2 displayed similar sensitivity towards sorafenib and vorinostat, but enhanced sensitivity towards geldanamycin treatment. Taken together, our study identified carfilzomib and HSP90 inhibition as promising therapeutic strategies to overcome bortezomib resistance.
Project description:Resistance to proteasome inhibitors (PIs) is a ubiquitous clinical concern in multiple myeloma (MM). We proposed that signaling-level responses after PI would reveal new means to enhance efficacy. Unbiased phosphoproteomics after the PI carfilzomib surprisingly demonstrated the most prominent phosphorylation changes on spliceosome components. Spliceosome modulation was invisible to RNA or protein abundance alone. Transcriptome analysis demonstrated broad-scale intron retention suggestive of PI-specific splicing interference. Direct spliceosome inhibition synergized with carfilzomib and showed potent anti-myeloma activity. Functional genomics and exome sequencing further supported the spliceosome as a specific vulnerabilityin myeloma. Our results propose splicing interference as an unrecognized modality of PI mechanism, reveal additional modes of spliceosome modulation, and suggest spliceosome targeting as a promising therapeutic strategy in myeloma.
Project description:Resistance to proteasome inhibitors (PIs) is a ubiquitous clinical concern in multiple myeloma. We proposed that signaling-level responses after PI would reveal new means to enhance efficacy. Unbiased phosphoproteomics after the PI carfilzomib surprisingly demonstrated the most prominent phosphorylation changes on spliceosome components. Spliceosome modulation was invisible to RNA or protein abundance alone. Transcriptome analysis demonstrated broad-scale intron retention suggestive of PI-specific splicing interference. Direct spliceosome inhibition synergized with carfilzomib and showed potent anti-myeloma activity. Functional genomics and exome sequencing further supported the spliceosome as a specific vulnerability in myeloma. Our results propose splicing interference as an unrecognized modality of PI mechanism, reveal additional modes of spliceosome modulation, and suggest spliceosome targeting as a promising therapeutic strategy in myeloma.
Project description:To study the oncogenic potential of cyclin D1b in the context of mature B cells we generated several cell clones derived from LP-1 MM cell line expressing either cyclin D1b, Myc or cyclin K oncogenes. Transcriptomic analysis allowed us to describe several mechanisms of cyclin D1b- and K-mediated oncogenesis. Three-condition experiment: LP-1cl1, LP-1K and LP-1D1b. Multiple myeloma LP-1 cell lines. Biological replicates: 4 control (LP-1cl1), 4 transfected with cyclin K (Lp-1K) and 4 transfected with cyclin D1b (LP-1D1b) independently grown and harvested. One replicate per array.
