Ribosome Profiling Reveals Translational Reprogramming and mTOR Activation in Omacetaxine Resistant Multiple Myeloma
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PROVIDER: PRJNA1099202 | ENA |
REPOSITORIES: ENA
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Project description:Protein homeostasis is critical to the survival of multiple myeloma (MM) cells. While this is targeted with proteasome inhibitors, mRNA translation inhibition has not entered trials. Recent work illustrates broad sensitivity MM cells to translation inhibitor omacetaxine. We hypothesized that understanding how MM cells become omacetaxine resistant will lead to the development of drug combinations to prevent or delay relapse. We generated omacetaxine resistance in H929 and MM1S MM cell lines and compared them to their parental lines. Resistant lines displayed decreased sensitivity to omacetaxine, with EC50 > 100 nM, compared to parental line sensitivity of 24-54 nM. To adapt to omacetaxine, H929 and MM1S exhibited an increased percentage of multi-nucleated polyaneuploid cells that led to distinct molecular mechanisms of resistance Interestingly, both resistant lines showed a defect in oncologic potential via extended survival in a MM xenograft model. Since omacetaxine inhibits protein synthesis, we performed both RNA-sequencing and ribosome profiling (Ribo-seq) to identify shared and unique regulatory strategies of resistance. Transcripts encoding translation factors and containing TOP motifs in their 5’ UTR were translationally upregulated in both resistant cell lines. The mTOR pathway promotes the translation of TOP motif containing mRNAs. Indeed, mTOR inhibition restored partial sensitivity to omacetaxine in both MM1S and H929 cells. Primary MM cells from patient samples were sensitive to combinations of omacetaxine and mTOR inhibitors rapamycin and Torin 1. These results provide a rational approach for omacetaxine-based combination in patients with multiple myeloma, which have historically shown better responses to multi-agent regimens.
2025-01-27 | GSE263767 | GEO
Project description:The combination of bortezomib and dexamethasone should become the reference induction treatment for multiple myeloma patients younger than 65 years. Pharmacogenomic profiles of genes involved in response to treatment may help to understand resistance. We performed gene expression profiling in 9 myeloma cell lines, incubated or not with bortezomib and dexamethasone for 6 hours. Supervised analysis identified significantly up regulated genes involved in stress responses. We focused on REDD1 a gene known to be rapidly induced by a wide variety of stress conditions and DNA damages. REDD1 expression was early and highly induced after bortezomib exposure. REDD1 induction was associated with the dephosphorylation of P70 S6 ribosomal kinase (P70S6K), a key substrate of mTOR. These effects were dependent upon cell line. REDD1 was overexpressed within two hours in bortezomib resistant cell lines in association with a cell size decrease. In sensitive cell lines, neither REDD1 induction nor morphological changes occurred. RNA interference mediated inhibition of REDD1 induction abrogates P70S6K dephosphorylation, early transitory cell size reduction and enhances sensitivity to bortezomib - dexamethasone. Our results suggest that mTOR regulation could be a resistance mechanism mediated by REDD1 expression in myeloma cells. Nine cell lines of multiple myeloma studied, incubated or not with bortezomib and dexamethasone, examined with spotted cDNA nylon membrane. Cell line and Code : JJN3 = Vel_1x; L363 = Vel_2x; LP1 = Vel_3x; MDN = Vel_4x; NCI = Vel_5x; RPMI = Vel_6x; SBN = Vel_7x; U266 = Vel_8x; XG1 = Vel_9x.
2010-02-14 | E-GEOD-11866 | biostudies-arrayexpress
Project description:The combination of bortezomib and dexamethasone should become the reference induction treatment for multiple myeloma patients younger than 65 years. Pharmacogenomic profiles of genes involved in response to treatment may help to understand resistance. We performed gene expression profiling in 9 myeloma cell lines, incubated or not with bortezomib and dexamethasone for 6 hours. Supervised analysis identified significantly up regulated genes involved in stress responses. We focused on REDD1 a gene known to be rapidly induced by a wide variety of stress conditions and DNA damages. REDD1 expression was early and highly induced after bortezomib exposure. REDD1 induction was associated with the dephosphorylation of P70 S6 ribosomal kinase (P70S6K), a key substrate of mTOR. These effects were dependent upon cell line. REDD1 was overexpressed within two hours in bortezomib resistant cell lines in association with a cell size decrease. In sensitive cell lines, neither REDD1 induction nor morphological changes occurred. RNA interference mediated inhibition of REDD1 induction abrogates P70S6K dephosphorylation, early transitory cell size reduction and enhances sensitivity to bortezomib - dexamethasone. Our results suggest that mTOR regulation could be a resistance mechanism mediated by REDD1 expression in myeloma cells.
2010-02-15 | GSE11866 | GEO
Project description:The rate of protein synthesis varies according to the mRNA sequence in ways that affect gene expression. Global analysis of translational pausing is now possible with ribosome profiling. Here, we revisit an earlier report that Shine-Dalgarno sequences are the major determinant of translational pausing in bacteria. Using refinements in the profiling method as well as biochemical assays, we find that SD motifs have little (if any) effect on elongation rates. We argue that earlier evidence of pausing arose from two factors. First, in previous analyses, pauses at Gly codons were difficult to distinguish from pauses at SD motifs. Second, and more importantly, the initial study preferentially isolated long ribosome-protected mRNA fragments that are enriched in SD motifs. These findings clarify the landscape of translational pausing in bacteria as observed by ribosome profiling. Ribosome profiling (three replicates) and RNAseq (two replicates) of E. coli MG1655
2016-01-13 | E-GEOD-72899 | biostudies-arrayexpress
Project description:Translational control is a widespread mode of gene regulation in organisms ranging from bacteria to mammals. Computational models posit that translational control of protein expression during elongation is exerted through a traffic jam of multiple ribosomes at ribosome pause sites on mRNAs. Yet neither the in vivo frequency of ribosome traffic jams nor the contribution of such traffic jams to protein expression has been measured in any organism. Here we show that upon starvation for single amino acids in the bacterium Escherichia coli, ribosome traffic jams are pervasive across the transcriptome, but they occur at only a subset of codons cognate to the limiting amino acid, and their severity is determined by the translation efficiency of mRNAs. Surprisingly, a computational model based on the observed traffic jams at ribosome pause sites is quantitatively inconsistent with measured protein synthesis rates. By comparison, a model incorporating abortion of protein synthesis at ribosome pause sites in addition to ribosome traffic jams predicts protein synthesis rate with higher accuracy. Consistent with the latter model, a significant fraction of the nascent polypeptides at ribosome pause sites is degraded through the activity of the transfer-messenger RNA during amino acid starvation in E. coli. Our work provides a minimal, experimentally-constrained model for predicting protein expression from ribosome dynamics, and it suggests the existence of a trade-off between the cellular translational capacity and the processivity of protein synthesis in vivo. 6 samples for ribosome profiling and 5 samples for total mRNA profiling
2014-05-15 | E-GEOD-51052 | biostudies-arrayexpress
Project description:Pancreatic ductal adenocarcinoma (PDAC) relies on hyper-activated protein synthesis. Consistently, human and mouse PDAC lose expression of the translational repressor and mTOR target 4E-BP1. Using genome-wide polysome-profiling, we here explore mRNAs whose translational efficiencies depend on the mTOR/4E-BP1 axis in Miapaca-2 cells. This was performed by isolating cytoplasmic and efficiently translated (heavy polysome-associated) mRNAs from MiaPaca-2 cells upon PP242-mediated mTOR inhibition
2019-09-18 | GSE137553 | GEO
Project description:The mammalian target of rapamycin (mTOR) kinase is a master regulator of protein synthesis that couples nutrient sensing to cell growth and cancer. However, the downstream translationally regulated nodes of gene expression that may direct cancer development are poorly characterized. Employing ribosome profiling we uncover specialized translation of the prostate cancer genome by oncogenic mTOR signaling, revealing a remarkably specific repertoire of genes involved in cell proliferation, metabolism, and invasion. We extend these findings by functionally characterizing a class of translationally controlled pro-invasion mRNAs that we show direct prostate cancer invasion and metastasis downstream of oncogenic mTOR signaling. We further develop a clinically relevant ATP site inhibitor of mTOR, INK128, which reprograms this gene expression signature with therapeutic benefit for prostate cancer metastasis, for which there is presently no cure. Together, these findings significantly extend our understanding of how the M-bM-^@M-^\cancerousM-bM-^@M-^] translation machinery steers specific cancer cell behaviors and may be therapeutically targeted. Examination of mRNA translation in human prostate cancer upon differential inhibition of the mTOR signaling pathway.
2012-02-28 | E-GEOD-35469 | biostudies-arrayexpress
Project description:Ribo-seq (Ribosome profiling) analyses in human multiple myeloma cells
| PRJNA656190 | ENA
Project description:Purpose: The aim is to analyze the translational kinetics of the basal and LPS-stimulated conditions of RAW264 macrophages focusing on ribosome density Method: RAW264 macrophages were cultured at 3.0 x 10^5 cells/ml in media (DMEM, 2mM Glutamine, 10% FBS, 100 units penicillin and 100µg streptomycin/mL) 24 hours prior to harvest. It was confirmed that the confluency of cells never surpassed 80 ~ 90%. Cell were harvested in their basal state or LPS stimulated condition (100 ng/ml for 30 min) and ribosome profiling (Ribo-Seq) and high-throughput mRNA sequencing (mRNA-Seq) were conducted. Ribosome protected fragments (RPF) were sequenced and the density was normalized by mRNA abundance. Result and conclusion: We discovered where in the ribosome translational arrest ocurs and how this happens. Translational stall was striking at A-site and P-site of ribosomal complex. The other arrest site was observed 3 to 5 residues away from the peptidyl transfer center of the exit tunnel, in which ribosomal density and hydrophobicity showed a significant negative correlation. mRNA and RPF of RAW264 macrophages were deep-sequenced with two independent biological replicates by Ion PGM sequencer
2015-11-01 | E-GEOD-60930 | biostudies-arrayexpress
Project description:Proteasome inhibitor (PI) resistance remains a central challenge in multiple myeloma. To identify pathways mediating resistance, we first mapped proteasome-associated genetic co-dependencies. We identified cytosolic heat shock protein 70 (HSP70) chaperones as potential targets, consistent with proposed mechanisms of myeloma tumor cells overcoming PI-induced stress. These results led us to explore allosteric HSP70 inhibitors (JG compounds) as myeloma therapeutics. We showed these compounds exhibit increased efficacy against acquired and intrinsic PI-resistant myeloma models, unlike HSP90 inhibition. Surprisingly, shotgun and pulsed-SILAC mass spectrometry found that JGs have the most pronounced effect on the proteome not through inhibiting cytosolic HSP70s but instead through mitochondrial-localized HSP70, HSPA9/mortalin, destabilizing the 55S mitoribosome. Analysis of myeloma patient data further supports strong effects of global proteostasis capacity, and particularly HSPA9 expression, on PI response. Our results characterize myeloma proteostasis networks under therapeutic pressure while motivating further investigation of HSPA9 as a specific vulnerability in PI-resistant disease.
2022-05-27 | PXD032030 | Pride