Gene expression profiles of NCI-H929 cells after knockdown of RRM1 and RRM2
ABSTRACT: Purpose: Overexpression or polymorphisms of ribonucleotide reductase (RR) are described in many solid tumors, and its expression is highly correlated with survival. However, the biologic significance in multiple myeloma (MM) has not yet been elucidated. Here, we identify the role of RR in MM pathogenesis. Experimental Design: Here we studied the potential utility of RRM1 knockdown effect in multitple myeloma in vitro and in vivo. Results: Knockdown of RRM1 (large subunit) triggered significant growth inhibition, associated with apoptotic cells death, in MM cells, regardless of the existence of bone marrow microcnrivonment. To validate the role of RRM1 of xenograft model, tumor growth was significantly reduced in RRM1-knocked-down MM cells versus control MM cells. Gene expression profiling showed that p53 regulated genes were upregulated after RRM1 knockdown. Immunoblot and QRT-PCR validated that p53 pathways were acviated as well. Clofarabine (CLO), a purine nucleoside analog which inhibits both DNA polymerases and RRM1, is a potential therapeutic agent in MM. CLO induced growth arrest in p53 wild-type cell lines, but not in p53 mutant or null cells. Moreover, CLO treatment with combined with DNA damaging agents triggered synergetic cell death even in p53 mutant MM cells. Conclusions: Our results therefore demonstrate that RRM1 is a novel therapeutic target in patients with MM, and provide the basis for clinical evaluation of RRM1 inhibitor, alone or in combination with DNA damaging agents, to improve patient outcome. Overall design: Myeloma cell line (NCI-H929) was transduced with either siRNAs targeting RRM1, RRM2 or scramble (control) in duplicate. The gene expression profiles of RRM1 and RRM2 knockdown cells were compared with that of control cells. A total of 6 RNA samples (2 RRM1 knockdown, 2 RRM2 knockdown, and 2 control) were analyzed.
INSTRUMENT(S): [HG-U133A_2] Affymetrix Human Genome U133A 2.0 Array
Project description:RBM10 is an RNA-binding protein that plays an essential role in development and is frequently mutated in the context of human disease. RBM10 recognizes a diverse set of RNA motifs in introns and exons and regulates alternative splicing. However, the molecular mechanisms underlying this seemingly relaxed sequence specificity are not understood and functional studies have focused on 3’ intronic sites only. Here we dissect the RNA code recognized by RBM10 and relate it to the splicing regulatory function of this protein. We show that a two-domain RRM1-ZnF unit recognises a GGA-centred motif enriched in RBM10 exonic sites with high affinity and specificity and test that the interaction with these exonic sequences promotes exon skipping. Importantly, a second RRM domain (RRM2) of RBM10 recognises a C-rich sequence, which explains its known interaction with the intronic 3’ site of NUMB exon 9 contributing to regulation of the Notch pathway in cancer. Together, these findings explain RBM10’s broad RNA specificity and suggest that RBM10 functions as a splicing regulator using two RNA-binding units with different specificities to promote exon skipping. Overall design: In the RNAcompete assay, purified GST-tagged RBM10 proteins (RRM1-ZnF and RRM1-ZnF-RRM2) are incubated with an excess of RNA pool and bound RNA from individual pulldown experiments are directly labeled and hybridized to a custom Agilent 244K microarray.
Project description:To understand how mutations in Matrin 3 (MATR3) cause amyotrophic lateral sclerosis (ALS) and distal myopathy, we used transcriptome and interactome analysis. We found over-expression of wild-type (WT) or F115C mutant MATR3 had little impact on gene expression in neuroglia cells. We identified ~123 proteins that bound MATR3, with proteins associated with stress granules and RNA processing/splicing being prominent. The interactome of myopathic S85C and ALS-variant F115C MATR3 were virtually identical to WT protein. Deletion of RNA recognition motif (RRM1) or Zn finger motifs (ZnF1 or ZnF2) diminished the binding of a subset of MATR3 interacting proteins. Remarkably, deletion of the RRM2 motif caused enhanced binding of >100 hundred proteins. In live cells, MATR3 lacking RRM2 (ΔRRM2) formed intranuclear spherical structures that fused over time into large structures. Our findings in the cell models used here suggest that MATR3 with disease-causing mutations is not dramatically different from WT protein in modulating gene regulation or in binding to normal interacting partners. The intra-nuclear localization and interaction network of MATR3 is strongly modulated by its RRM2 domain.
Project description:Previous study demonstrated that HDAC3 has a critical role in MM proliferation; however, the underlying mechanism has not yet been elucidated. We identify that HDAC3 inhibition targets DNMT1 through dual regulations. We demonstrate that knockdown of DNMT1 leads to apoptosis and significant growth inhibition in myeloma cells. HDAC3 inhibition by gene silencing or HDAC3 selective inhibitor BG45 downregulates an oncoprotein c-Myc through its acetylation. c-Myc directly regulates DNMT1 expression at its enhancer region. Furthermore, HDAC3 directly regulates the stability of DNMT1 protein through its acetylation. Pharmaceutical inhibition of HDAC3 and DNMT1 synergistically induce MM growth inhibition in in vitro and in vivo settings. The goal of this analysis is to identify genes whose expression changes after shRNA-mediated knockdown of HDAC3 or DNMT1 using the human U133 plus 2.0 Affymetrix GeneChip in myeloma cell line (MM.1S). Overall design: Myeloma cell line (MM.1S) was transduced with either shRNAs targeting HDAC3 (duplicate hairpins), DNMT1 (duplicate hairpins) or luciferase (control) in duplicate. The gene expression profiles of HDAC3 or DNMT1 knockdown cells were compared with that of control cells. A total of 10 RNA samples (4 HDAC3 knockdown, 4 DNMT1 knockdown, and 2 control) were analyzed.
Project description:To understand how mutations in Matrin 3 (MATR3) cause amyotrophic lateral sclerosis (ALS) and distal myopathy, we used transcriptome and interactome analysis, coupled with microscopy. Over-expression of wild-type (WT) or F115C mutant MATR3 had little impact on gene expression in neuroglia cells. Only 23 genes, expressed at levels of >100 transcripts showed ≥1.6-fold changes in expression by transfection with WT or mutant MATR3:YFP vectors. We identified ~123 proteins that bound MATR3, with proteins associated with stress granules and RNA processing/splicing being prominent. The interactome of myopathic S85C and ALS-variant F115C MATR3 were virtually identical to WT protein. Deletion of RNA recognition motif (RRM1) or Zn finger motifs (ZnF1 or ZnF2) diminished the binding of a subset of MATR3 interacting proteins. Remarkably, deletion of the RRM2 motif caused enhanced binding of >100 hundred proteins. In live cells, MATR3 lacking RRM2 (ΔRRM2) formed intranuclear spherical structures that fused over time into large structures. Our findings in the cell models used here suggest that MATR3 with disease-causing mutations is not dramatically different from WT protein in modulating gene regulation or in binding to normal interacting partners. The intra-nuclear localization and interaction network of MATR3 is strongly modulated by its RRM2 domain. Overall design: RNA-seq of untransfected cells or cells with overexpression of YFP, WT MATR3, or ALS mutation MATR3
Project description:The Human antigen R protein (HuR) is a RNA-binding protein that recognizes U/AU-rich elements in diverse RNAs through two RNA-recognition motifs, RRM1 and RRM2, and post-transcriptionally regulates the fate of target RNAs. The natural product Dihydrotanshinone-I (DHTS) prevents the association of HuR and target RNAs in vitro and in cultured cells by interfering with the binding of HuR to RNA. Here, we report the structural determinants of the interaction between DHTS and HuR and the impact of DHTS on HuR binding to target mRNAs transcriptome-wide. NMR titration and Molecular Dynamic simulation identified the residues within RRM1 and RRM2 responsible for the interaction between DHTS and HuR. RNA Electromobility Shifts and Alpha Screen Assays showed that DHTS interacts with HuR through the same binding regions as target RNAs, stabilizing HuR in a locked conformation that hampers RNAs binding competitively. HuR ribonucleoprotein immunoprecipitation followed by microarray (RIP-chip) analysis showed that DHTS treatment of HeLa cells paradoxically enriched HuR binding to mRNAs with longer 3’UTR and with higher density of U/AU-rich elements, suggesting that DHTS inhibits the association of HuR to weaker target mRNAs. In vivo, DHTS potently inhibited xenograft tumor growth in a HuR-dependent model without systemic toxicity. Overall design: The influence of DHTS on the interaction of HuR with endogenous mRNAs was analyzed in HeLa cells exposed to either 1 micromolar (1 uM) DHTS or DMSO vehicle for 3 hours, followed by immunoprecipitation (IP) of endogenous ribonucleoprotein complexes, and extraction of RNA from the IP samples for cDNA microarray analysis or q-RT-PCR validation.
Project description:p53 wildtype or complementing DNA binding cooperativity versions (EE, RR, EE/RR) were overexpressed in SAOS cells and 18h later harvested to perform ChIP with an p53-specific antibody. The enriched DNA fragments were purified and identified by high throughput sequencing.
Project description:The introduction of novel therapeutic agents has considerably improved the median survival of patients with multiple myeloma (MM). However, the natural history of the disease is characterized by continuous relapses over time. As a consequence, the development of new drugs is still required to treat MM recurrence. Here, we report for the first time the potent anti-myeloma activity of amiloride, an old potassium-sparing diuretic approved for the treatment of hypertension and edema due to heart failure. Amilorideinduced apoptosis was observed in a broad panel of MM cell lines and in xenograft mouse models. Moreover, amiloride also had a synergistic effect when combined with dexamethasone and melphalan. RNA-seq experiments showed that amiloride not only significantly altered the level of transcript isoforms and alternative splicing events, but also deregulated the spliceosomal machinery. Additionally, disruption of the splicing machinery in immunofluorescence studies was associated with the inhibition of myeloma cell viability after amiloride exposure. Although amiloride was able to induce apoptosis in myeloma cells lacking p53 expression, activation of p53 signaling was observed in wild-type and mutated TP53 cells after amiloride exposure. On the other hand, the manageable toxicity profile of amiloride is well known and we did not find a significant systemic toxicity in mice treated with amiloride. Overall, our results provide a mechanistic rationale for the use of amiloride as an alternative treatment option for relapsed MM patients. Overall design: Poly A+ RNA from KMS12-BM and JJN3 cells untreated or treated with amiloride or TG003 (0.1 mM, 0.4 mM, and 0.4 mM respectively) for 24 h was isolated and prepared for RNA-seq.
Project description:We developed genetically engineered mice to generate brain tumors with especific genetic lessions. The animals were split in three groups: NRAS, P53 knockdown, IDH1-R132H and ATRX knock down (NPAID); NRAS, P53 knockdown, IDH1-R132H (NPI); NRAS, P53 knockdown, (NshP53). From these tumor we obtain and culture tumor cells growth like neurospheres and attached cells.
Project description:Targeting p53 by the small molecule PRIMA-1Met/APR-246 has shown promising preclinical activity in various cancer types. However, the mechanism of PRIMA-1Met-induced apoptosis is not completely understood and its effect on multiple myeloma (MM) cells is unknown. In this study we evaluated anti-tumor effect of PRIMA-1Met alone or combined with current anti-myeloma agents in MM cell lines, patient samples, and a mouse xenograft model. Results of our study showed that PRIMA-1Met decreased the viability of MM cells irrespective of p53 status with limited cytotoxicity toward normal hematopoietic cells. PRIMA-1Met restored wild type conformation of mutant p53 and induced activation of p73 up-regulating Noxa and down-regulating Mcl-1 without significant modulation of p53 level. Importantly, PRIMA-1Met delayed tumor growth and prolonged survival of mice bearing MM tumor. To identify the potential targets of PRIMA-1Met, we performed gene expression profiling (GEP) by microarray in three different cell lines harboring wild type, mutant or null p53 and analysed differential expression of target genes between PRIMA-1Met treated and non-treated samples. Based on our we conclude that treatment of MM cells with PRIMA-1Met lead to induction of p73-mediated apoptosis by up-regulating Noxa and down-regulating Mcl-1 irrespective of p53 status. MM.1S, U266, and 8266R5 cells were treated with 20, 40, and 40 µM PRIMA-1Met, respectively for 8 hrs and total RNA was isolated. Gene expression was analyzed with Illumina RNA analysis Beadchips (Illumina Inc. San Diego, CA) representing 48,000 human genes (Human HT12). Array data analysis was carried out with Bead Studio software. Genes showing at least a 2.0-fold difference in expression levels between control and PRIMA-1Met-treated cells were considered to be modulated by PRIMA-1Met.