RESUMO
Therapeutic innovation to address sickle cell disease (SCD) is at a historical apex, characterized by a drug discovery, development, and commercialization landscape that includes potentially curative gene therapies. Given the wide geographic distribution of SCD, with a major presence in Africa, it is imperative that new medicines are designed to meet the specific needs of persons with SCD everywhere. Target product profiles (TPPs) detail the desired attributes of new medicines and serve as a guide for drug developers. To support research efforts for curative treatments for SCD, we mobilized a large multi-disciplinary expert group to generate consensus-driven TPPs for ex vivo and in vivo SCD gene therapies, utilizing a modified Delphi methodology supplemented with virtual workshops. The main findings are TPPs that describe 20 minimal and optimal criteria for novel gene therapy products in categories of scope (3 criteria), performance/safety (11 criteria), manufacturing (4 criteria), and administration (2 criteria). TPPs for ex vivo and in vivo products differed in some performance/safety criteria and all criteria pertaining to manufacturing and administration. These outputs will ideally support development of durable treatments that are safe, efficacious, and practical for persons with SCD in global settings.
RESUMO
Vascular endothelial growth factor (VEGF) is produced either as a pro-angiogenic or anti-angiogenic protein depending upon splice site choice in the terminal, eighth exon. Proximal splice site selection (PSS) in exon 8 generates pro-angiogenic isoforms such as VEGF(165), and distal splice site selection (DSS) results in anti-angiogenic isoforms such as VEGF(165)b. Cellular decisions on splice site selection depend upon the activity of RNA-binding splice factors, such as ASF/SF2, which have previously been shown to regulate VEGF splice site choice. To determine the mechanism by which the pro-angiogenic splice site choice is mediated, we investigated the effect of inhibition of ASF/SF2 phosphorylation by SR protein kinases (SRPK1/2) on splice site choice in epithelial cells and in in vivo angiogenesis models. Epithelial cells treated with insulin-like growth factor-1 (IGF-1) increased PSS and produced more VEGF(165) and less VEGF(165)b. This down-regulation of DSS and increased PSS was blocked by protein kinase C inhibition and SRPK1/2 inhibition. IGF-1 treatment resulted in nuclear localization of ASF/SF2, which was blocked by SPRK1/2 inhibition. Pull-down assay and RNA immunoprecipitation using VEGF mRNA sequences identified an 11-nucleotide sequence required for ASF/SF2 binding. Injection of an SRPK1/2 inhibitor reduced angiogenesis in a mouse model of retinal neovascularization, suggesting that regulation of alternative splicing could be a potential therapeutic strategy in angiogenic pathologies.
Assuntos
Processamento Alternativo , Inibidores da Angiogênese/biossíntese , RNA Mensageiro/biossíntese , Neovascularização Retiniana/metabolismo , Fator A de Crescimento do Endotélio Vascular/biossíntese , Inibidores da Angiogênese/genética , Animais , Linhagem Celular Transformada , Modelos Animais de Doenças , Inibidores Enzimáticos/farmacologia , Humanos , Fator de Crescimento Insulin-Like I/genética , Fator de Crescimento Insulin-Like I/metabolismo , Camundongos , Proteínas Nucleares/antagonistas & inibidores , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Isoformas de Proteínas/biossíntese , Isoformas de Proteínas/genética , Proteínas Serina-Treonina Quinases/antagonistas & inibidores , Proteínas Serina-Treonina Quinases/genética , Proteínas Serina-Treonina Quinases/metabolismo , RNA Mensageiro/genética , Proteínas de Ligação a RNA , Neovascularização Retiniana/genética , Neovascularização Retiniana/terapia , Fatores de Processamento de Serina-Arginina , Fator A de Crescimento do Endotélio Vascular/genéticaRESUMO
Large-scale, genome-wide studies report that RNA binding proteins are altered in cancers, but it is unclear how these proteins control tumor progression. We found that the RNA-editing protein ADAR (adenosine deaminase acting on double-stranded RNA) acted as a facilitator of lung adenocarcinoma (LUAD) progression through its ability to stabilize transcripts encoding focal adhesion kinase (FAK). In samples from 802 stage I LUAD patients, increased abundance of ADAR at both the mRNA and protein level correlated with tumor recurrence. Knocking down ADAR in LUAD cells suppressed their mesenchymal properties, migration, and invasion in culture. Analysis of gene expression patterns in LUAD cells identified ADAR-associated enrichment of a subset of genes involved in cell migration pathways; among these, FAK is the most notable gene whose expression was increased in the presence of ADAR. Molecular analyses revealed that ADAR posttranscriptionally increased FAK protein abundance by binding to the FAK transcript and editing a specific intronic site that resulted in the increased stabilization of FAK mRNA. Pharmacological inhibition of FAK blocked ADAR-induced invasiveness of LUAD cells, suggesting a potential therapeutic application for LUAD that has a high abundance of ADAR.
Assuntos
Adenocarcinoma/enzimologia , Adenosina Desaminase/metabolismo , Movimento Celular , Quinase 1 de Adesão Focal/metabolismo , Neoplasias Pulmonares/enzimologia , Proteínas de Neoplasias/metabolismo , Proteínas de Ligação a RNA/metabolismo , Adenocarcinoma/tratamento farmacológico , Adenocarcinoma/genética , Adenocarcinoma/patologia , Adenocarcinoma de Pulmão , Adenosina Desaminase/genética , Linhagem Celular , Estabilidade Enzimática , Quinase 1 de Adesão Focal/antagonistas & inibidores , Quinase 1 de Adesão Focal/genética , Humanos , Neoplasias Pulmonares/tratamento farmacológico , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/patologia , Proteínas de Neoplasias/antagonistas & inibidores , Proteínas de Neoplasias/genética , Proteínas de Ligação a RNA/genéticaRESUMO
The bone marrow microenvironment influences malignant hematopoiesis, but how it promotes leukemogenesis has not been elucidated. In addition, the role of the bone marrow stroma in regulating clinical responses to DNA methyltransferase inhibitors (DNMTi) is also poorly understood. In this study, we conducted a DNA methylome analysis of bone marrow-derived stromal cells from myelodysplastic syndrome (MDS) patients and observed widespread aberrant cytosine hypermethylation occurring preferentially outside CpG islands. Stroma derived from 5-azacytidine-treated patients lacked aberrant methylation and DNMTi treatment of primary MDS stroma enhanced its ability to support erythroid differentiation. An integrative expression analysis revealed that the WNT pathway antagonist FRZB was aberrantly hypermethylated and underexpressed in MDS stroma. This result was confirmed in an independent set of sorted, primary MDS-derived mesenchymal cells. We documented a WNT/ß-catenin activation signature in CD34+ cells from advanced cases of MDS, where it associated with adverse prognosis. Constitutive activation of ß-catenin in hematopoietic cells yielded lethal myeloid disease in a NUP98-HOXD13 mouse model of MDS, confirming its role in disease progression. Our results define novel epigenetic changes in the bone marrow microenvironment, which lead to ß-catenin activation and disease progression of MDS. Cancer Res; 77(18); 4846-57. ©2017 AACR.
Assuntos
Epigênese Genética , Células-Tronco Mesenquimais/patologia , Síndromes Mielodisplásicas/patologia , Proteínas Wnt/metabolismo , beta Catenina/metabolismo , Animais , Apoptose , Diferenciação Celular , Proliferação de Células , Ilhas de CpG , Metilação de DNA , Células-Tronco Hematopoéticas/metabolismo , Células-Tronco Hematopoéticas/patologia , Humanos , Células-Tronco Mesenquimais/metabolismo , Camundongos , Camundongos Transgênicos , Síndromes Mielodisplásicas/genética , Síndromes Mielodisplásicas/metabolismo , Proteínas de Fusão Oncogênica/genética , Células Tumorais CultivadasRESUMO
The identity of the RNA-binding proteins (RBPs) that govern cancer stem cells remains poorly characterized. The MSI2 RBP is a central regulator of translation of cancer stem cell programs. Through proteomic analysis of the MSI2-interacting RBP network and functional shRNA screening, we identified 24 genes required for in vivo leukemia. Syncrip was the most differentially required gene between normal and myeloid leukemia cells. SYNCRIP depletion increased apoptosis and differentiation while delaying leukemogenesis. Gene expression profiling of SYNCRIP-depleted cells demonstrated a loss of the MLL and HOXA9 leukemia stem cell program. SYNCRIP and MSI2 interact indirectly though shared mRNA targets. SYNCRIP maintains HOXA9 translation, and MSI2 or HOXA9 overexpression rescued the effects of SYNCRIP depletion. Altogether, our data identify SYNCRIP as a new RBP that controls the myeloid leukemia stem cell program. We propose that targeting these RBP complexes might provide a novel therapeutic strategy in leukemia.
Assuntos
Regulação Leucêmica da Expressão Gênica , Ribonucleoproteínas Nucleares Heterogêneas/genética , Leucemia Mieloide/genética , Proteínas de Ligação a RNA/metabolismo , Animais , Sobrevivência Celular , Feminino , Hematopoese/genética , Ribonucleoproteínas Nucleares Heterogêneas/metabolismo , Proteínas de Homeodomínio/genética , Humanos , Leucemia Aguda Bifenotípica/genética , Leucemia Aguda Bifenotípica/patologia , Leucemia Mieloide/patologia , Camundongos Endogâmicos C57BL , Camundongos Knockout , Células Progenitoras Mieloides/metabolismo , Células Progenitoras Mieloides/patologia , RNA Interferente Pequeno , Proteínas de Ligação a RNA/genética , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Breast cancer metastasis suppressor 1 (BRMS1) is decreased in non-small cell lung cancer (NSCLC) and other solid tumors, and its loss correlates with increased metastases. We show that BRMS1 is posttranslationally regulated by TNF-induced casein kinase 2 catalytic subunit (CK2α') phosphorylation of nuclear BRMS1 on serine 30 (S30), resulting in 14-3-3ε-mediated nuclear exportation, increased BRMS1 cytosolic expression, and ubiquitin-proteasome-induced BRMS1 degradation. Using our in vivo orthotopic mouse model of lung cancer metastases, we found that mutation of S30 in BRMS1 or the use of the CK2-specific small-molecule inhibitor CX4945 abrogates CK2α'-induced cell migration and invasion and decreases NSCLC metastasis by 60-fold. Analysis of 160 human NSCLC specimens confirmed that tumor CK2α' and cytoplasmic BRMS1 expression levels are associated with increased tumor recurrence, metastatic foci, and reduced disease-free survival. Collectively, we identify a therapeutically exploitable posttranslational mechanism by which CK2α-mediated degradation of BRMS1 promotes metastases in lung cancer. Cancer Res; 76(9); 2675-86. ©2016 AACR.
Assuntos
Carcinoma Pulmonar de Células não Pequenas/patologia , Neoplasias Pulmonares/patologia , Invasividade Neoplásica/patologia , Proteínas Repressoras/metabolismo , Transporte Ativo do Núcleo Celular/fisiologia , Animais , Western Blotting , Carcinoma Pulmonar de Células não Pequenas/metabolismo , Caseína Quinase II/metabolismo , Modelos Animais de Doenças , Intervalo Livre de Doença , Imunofluorescência , Xenoenxertos , Imuno-Histoquímica , Imunoprecipitação , Estimativa de Kaplan-Meier , Neoplasias Pulmonares/metabolismo , Camundongos , Análise Serial de TecidosRESUMO
Myelodysplastic syndromes (MDS) are driven by complex genetic and epigenetic alterations. The MSI2 RNA-binding protein has been demonstrated to have a role in acute myeloid leukaemia and stem cell function, but its role in MDS is unknown. Here, we demonstrate that elevated MSI2 expression correlates with poor survival in MDS. Conditional deletion of Msi2 in a mouse model of MDS results in a rapid loss of MDS haematopoietic stem and progenitor cells (HSPCs) and reverses the clinical features of MDS. Inversely, inducible overexpression of MSI2 drives myeloid disease progression. The MDS HSPCs remain dependent on MSI2 expression after disease initiation. Furthermore, MSI2 expression expands and maintains a more activated (G1) MDS HSPC. Gene expression profiling of HSPCs from the MSI2 MDS mice identifies a signature that correlates with poor survival in MDS patients. Overall, we identify a role for MSI2 in MDS representing a therapeutic target in this disease.
Assuntos
Síndromes Mielodisplásicas/metabolismo , Proteínas de Ligação a RNA/metabolismo , Idoso , Animais , Estudos de Casos e Controles , Modelos Animais de Doenças , Feminino , Células-Tronco Hematopoéticas/fisiologia , Humanos , Leucemia Mieloide Aguda/etiologia , Leucemia Mieloide Aguda/metabolismo , Masculino , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Síndromes Mielodisplásicas/etiologia , Síndromes Mielodisplásicas/mortalidadeRESUMO
Breast cancer metastasis suppressor 1 (BRMS1) is downregulated in non-small cell lung cancer (NSCLC), and its reduction correlates with disease progression. Herein, we investigate the mechanisms through which loss of the BRMS1 gene contributes to epithelial-to-mesenchymal transition (EMT). Using a short hairpin RNA (shRNA) system, we show that loss of BRMS1 promotes basal and transforming growth factor beta-induced EMT in NSCLC cells. NSCLC cells expressing BRMS1 shRNAs (BRMS1 knockdown [BRMS1(KD)]) display mesenchymal characteristics, including enhanced cell migration and differential regulation of the EMT markers. Mesenchymal phenotypes observed in BRMS1(KD) cells are dependent on RelA/p65, the transcriptionally active subunit of nuclear factor kappa B (NF-κB). In addition, chromatin immunoprecipitation analysis demonstrates that loss of BRMS1 increases Twist1 promoter occupancy of RelA/p65 K310-a key histone modification associated with increased transcription. Knockdown of Twist1 results in reversal of BRMS1(KD)-mediated EMT phenotypic changes. Moreover, in our animal model, BRMS1(KD)/Twist1(KD) double knockdown cells were less efficient in establishing lung tumors than BRMS1(KD) cells. Collectively, this study demonstrates that loss of BRMS1 promotes malignant phenotypes that are dependent on NF-κB-dependent regulation of Twist1. These observations offer fresh insight into the mechanisms through which BRMS1 regulates the development of metastases in NSCLC.
Assuntos
Regulação Neoplásica da Expressão Gênica , Neoplasias Pulmonares/metabolismo , Subunidade p50 de NF-kappa B/metabolismo , Proteínas Nucleares/metabolismo , Proteínas Repressoras/metabolismo , Proteína 1 Relacionada a Twist/metabolismo , Animais , Carcinoma Pulmonar de Células não Pequenas/metabolismo , Linhagem Celular Tumoral , Sobrevivência Celular , Transição Epitelial-Mesenquimal , Genes Supressores de Tumor , Histonas/metabolismo , Humanos , Metástase Linfática , Camundongos , Camundongos Nus , Metástase Neoplásica , Transplante de Neoplasias , Proteínas Nucleares/genética , Fenótipo , RNA Interferente Pequeno/metabolismo , Proteínas Repressoras/genética , Fator de Crescimento Transformador beta/metabolismo , Proteína 1 Relacionada a Twist/genética , CicatrizaçãoRESUMO
The angiogenic capability of colorectal carcinomas (CRC), and their susceptibility to anti-angiogenic therapy, is determined by expression of vascular endothelial growth factor (VEGF) isoforms. The intracellular protein T-cell Intracellular Antigen (TIA-1) alters post-transcriptional RNA processing and binds VEGF-A mRNA. We therefore tested the hypothesis that TIA-1 could regulate VEGF-A isoform expression in colorectal cancers. TIA-1 and VEGF-A isoform expression was measured in colorectal cancers and cell lines. We discovered that an endogenous splice variant of TIA-1 encoding a truncated protein, short TIA-1 (sTIA-1) was expressed in CRC tissues and invasive K-Ras mutant colon cancer cells and tissues but not in adenoma cell lines. sTIA-1 was more highly expressed in CRC than in normal tissues and increased with tumour stage. Knockdown of sTIA-1 or over-expression of full length TIA-1 (flTIA-1) induced expression of the anti-angiogenic VEGF isoform VEGF-A165b. Whereas flTIA-1 selectively bound VEGF-A165 mRNA and increased translation of VEGF-A165b, sTIA-1 prevented this binding. In nude mice, xenografted colon cancer cells over-expressing flTIA-1 formed smaller, less vascular tumours than those expressing sTIA-1, but flTIA-1 expression inhibited the effect of anti-VEGF antibodies. These results indicate that alternative splicing of an RNA binding protein can regulate isoform specific expression of VEGF providing an added layer of complexity to the angiogenic profile of colorectal cancer and their resistance to anti-angiogenic therapy.
Assuntos
Processamento Alternativo , Bevacizumab , Neoplasias do Colo/metabolismo , Resistencia a Medicamentos Antineoplásicos , Regulação Neoplásica da Expressão Gênica , Proteínas de Neoplasias/biossíntese , Neovascularização Patológica/metabolismo , Proteínas de Ligação a Poli(A)/biossíntese , Fator A de Crescimento do Endotélio Vascular/biossíntese , Animais , Neoplasias do Colo/irrigação sanguínea , Neoplasias do Colo/genética , Neoplasias do Colo/patologia , Células HEK293 , Células HeLa , Xenoenxertos , Humanos , Camundongos Nus , Proteínas de Neoplasias/genética , Transplante de Neoplasias , Neovascularização Patológica/genética , Neovascularização Patológica/patologia , Proteínas de Ligação a Poli(A)/genética , Isoformas de Proteínas/biossíntese , Isoformas de Proteínas/genética , Antígeno-1 Intracelular de Células T , Fator A de Crescimento do Endotélio Vascular/genéticaRESUMO
Angiogenesis is regulated by the balance of proangiogenic VEGF(165) and antiangiogenic VEGF(165)b splice isoforms. Mutations in WT1, the Wilms' tumor suppressor gene, suppress VEGF(165)b and cause abnormal gonadogenesis, renal failure, and Wilms' tumors. In WT1 mutant cells, reduced VEGF(165)b was due to lack of WT1-mediated transcriptional repression of the splicing-factor kinase SRPK1. WT1 bound to the SRPK1 promoter, and repressed expression through a specific WT1 binding site. In WT1 mutant cells SRPK1-mediated hyperphosphorylation of the oncogenic RNA binding protein SRSF1 regulated splicing of VEGF and rendered WT1 mutant cells proangiogenic. Altered VEGF splicing was reversed by wild-type WT1, knockdown of SRSF1, or SRPK1 and inhibition of SRPK1, which prevented in vitro and in vivo angiogenesis and associated tumor growth.
Assuntos
Neovascularização Patológica/genética , Proteínas Serina-Treonina Quinases/metabolismo , Fator B de Crescimento do Endotélio Vascular/genética , Proteínas WT1/genética , Animais , Linhagem Celular Tumoral , Núcleo Celular/metabolismo , Neovascularização de Coroide/genética , Neovascularização de Coroide/metabolismo , Neovascularização de Coroide/patologia , Síndrome de Denys-Drash/genética , Síndrome de Denys-Drash/metabolismo , Síndrome de Denys-Drash/patologia , Expressão Gênica , Regulação Neoplásica da Expressão Gênica , Genes Reporter , Humanos , Luciferases de Renilla/biossíntese , Luciferases de Renilla/genética , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Nus , Transplante de Neoplasias , Neoplasias/irrigação sanguínea , Proteínas Nucleares/metabolismo , Podócitos/metabolismo , Regiões Promotoras Genéticas , Ligação Proteica , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Inibidores de Proteínas Quinases/farmacologia , Proteínas Serina-Treonina Quinases/antagonistas & inibidores , Proteínas Serina-Treonina Quinases/genética , Transporte Proteico , Interferência de RNA , Splicing de RNA/genética , Proteínas de Ligação a RNA/metabolismo , Fatores de Processamento de Serina-Arginina , Fator B de Crescimento do Endotélio Vascular/metabolismo , Receptor 2 de Fatores de Crescimento do Endotélio Vascular/metabolismoRESUMO
Vascular endothelial growth factor A (VEGFA; hereafter referred to as VEGF) is a key regulator of physiological and pathological angiogenesis. Two families of VEGF isoforms are generated by alternate splice-site selection in the terminal exon. Proximal splice-site selection (PSS) in exon 8 results in pro-angiogenic VEGFxxx isoforms (xxx is the number of amino acids), whereas distal splice-site selection (DSS) results in anti-angiogenic VEGFxxxb isoforms. To investigate control of PSS and DSS, we investigated the regulation of isoform expression by extracellular growth factor administration and intracellular splicing factors. In primary epithelial cells VEGFxxxb formed the majority of VEGF isoforms (74%). IGF1, and TNFalpha treatment favoured PSS (increasing VEGFxxx) whereas TGFbeta1 favoured DSS, increasing VEGFxxxb levels. TGFbeta1 induced DSS selection was prevented by inhibition of p38 MAPK and the Clk/sty (CDC-like kinase, CLK1) splicing factor kinase family, but not ERK1/2. Clk phosphorylates SR protein splicing factors ASF/SF2, SRp40 and SRp55. To determine whether SR splicing factors alter VEGF splicing, they were overexpressed in epithelial cells, and VEGF isoform production assessed. ASF/SF2, and SRp40 both favoured PSS, whereas SRp55 upregulated VEGFxxxb (DSS) isoforms relative to VEGFxxx. SRp55 knockdown reduced expression of VEGF165b. Moreover, SRp55 bound to a 35 nucleotide region of the 3'UTR immediately downstream of the stop codon in exon 8b. These results identify regulation of splicing by growth and splice factors as a key event in determining the relative pro-versus anti-angiogenic expression of VEGF isoforms, and suggest that p38 MAPK-Clk/sty kinases are responsible for the TGFbeta1-induced DSS selection, and identify SRp55 as a key regulatory splice factor.