RESUMO
Haploinsufficiency of ribosomal proteins (RPs) and upregulation of the tumour suppressor TP53 have been shown to be the common basis for the anaemia observed in Diamond Blackfan anaemia and 5q- myelodysplastic syndrome. We previously demonstrated that treatment with L-Leucine resulted in a marked improvement in anaemia in disease models. To determine if the L-Leucine effect was Tp53-dependent, we used antisense MOs to rps19 and rps14 in zebrafish; expression of tp53 and its downstream target cdkn1a remained elevated following L-leucine treatment. We confirmed this observation in human CD34+ cells. L-Leucine thus alleviates anaemia in RP-deficient cells in a TP53-independent manner.
Assuntos
Anemia de Diamond-Blackfan/tratamento farmacológico , Anemia Macrocítica/tratamento farmacológico , Proteína Supressora de Tumor p53/metabolismo , Proteínas de Peixe-Zebra/metabolismo , Anemia de Diamond-Blackfan/genética , Anemia de Diamond-Blackfan/metabolismo , Anemia de Diamond-Blackfan/patologia , Anemia Macrocítica/genética , Anemia Macrocítica/metabolismo , Anemia Macrocítica/patologia , Animais , Deleção Cromossômica , Cromossomos Humanos Par 5/genética , Cromossomos Humanos Par 5/metabolismo , Inibidor de Quinase Dependente de Ciclina p21/genética , Inibidor de Quinase Dependente de Ciclina p21/metabolismo , Modelos Animais de Doenças , Humanos , Leucina , Proteínas Ribossômicas/genética , Proteínas Ribossômicas/metabolismo , Proteína Supressora de Tumor p53/genética , Proteínas de Peixe-Zebra/genéticaRESUMO
NPM1 is a ubiquitously expressed nucleolar phosphoprotein, the gene for which maps to chromosome 5q35 in close proximity to a commonly deleted region associated with (del)5q, a type of myelodysplastic syndrome (MDS). This region is also a frequent target of deletions in de novo and therapy-related MDS/acute myeloid leukemia. Previous studies have shown that Npm1(+/-) mice develop an MDS-like disease that transforms to acute myeloid leukemia over time. To better understand the mechanism by which NPM1 haploinsufficiency causes an MDS phenotype, we generated factor-dependent myeloid cell lines from the bone marrow of Npm1(+/+) and Npm1(+/-) mice and demonstrated compromised neutrophil-specific gene expression in the MNPM1(+/-) cells. We attribute these observations to increased levels of the shorter, dominant negative leukemogenic isoform (p30) of CCAAT enhancer-binding protein α (C/EBPα). We show that this increase is caused, in part, by elevated levels of the activated translation initiation factor eIF4E, overexpression of which also increases translation of C/EBPαp30 in HEK293 cells. In a positive feedback loop, eIF4E expression is further elevated both at the mRNA and protein levels by C/EBPαp30 but not by the full-length C/EBPαp42. Re-expression of C/EBPαp42 or NPM1 but not C/EBPαp30 in MNPM1(+/-) cells partially rescues the myeloid phenotype. Our observations suggest that the aberrant feed-forward pathway that keeps eIF4E and C/EBPαp30 elevated in NPM1(+/-) cells contributes to the MDS phenotype associated with NPM1 deficiency.
Assuntos
Proteínas Estimuladoras de Ligação a CCAAT/metabolismo , Fator de Iniciação 4E em Eucariotos/biossíntese , Regulação Leucêmica da Expressão Gênica , Haploinsuficiência , Leucemia Mieloide Aguda/metabolismo , Síndromes Mielodisplásicas/metabolismo , Proteínas de Neoplasias/metabolismo , Proteínas Nucleares/metabolismo , Regulação para Cima , Animais , Proteínas Estimuladoras de Ligação a CCAAT/genética , Fator de Iniciação 4E em Eucariotos/genética , Células HEK293 , Humanos , Leucemia Mieloide Aguda/genética , Leucemia Mieloide Aguda/patologia , Camundongos , Camundongos Mutantes , Síndromes Mielodisplásicas/genética , Síndromes Mielodisplásicas/patologia , Proteínas de Neoplasias/genética , Proteínas Nucleares/genética , Nucleofosmina , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismoRESUMO
Haploinsufficiency for ribosomal protein genes has been implicated in the pathophysiology of Diamond-Blackfan anemia (DBA) and the 5q-syndrome, a subtype of myelodysplastic syndrome. The p53 pathway is activated by ribosome dysfunction, but the molecular basis for selective impairment of the erythroid lineage in disorders of ribosome function has not been determined. We found that p53 accumulates selectively in the erythroid lineage in primary human hematopoietic progenitor cells after expression of shRNAs targeting RPS14, the ribosomal protein gene deleted in the 5q-syndrome, or RPS19, the most commonly mutated gene in DBA. Induction of p53 led to lineage-specific accumulation of p21 and consequent cell cycle arrest in erythroid progenitor cells. Pharmacologic inhibition of p53 rescued the erythroid defect, whereas nutlin-3, a compound that activates p53 through inhibition of HDM2, selectively impaired erythropoiesis. In bone marrow biopsies from patients with DBA or del(5q) myelodysplastic syndrome, we found an accumulation of nuclear p53 staining in erythroid progenitor cells that was not present in control samples. Our findings indicate that the erythroid lineage has a low threshold for the induction of p53, providing a basis for the failure of erythropoiesis in the 5q-syndrome, DBA, and perhaps other bone marrow failure syndromes.
Assuntos
Células Precursoras Eritroides/metabolismo , Haploinsuficiência/genética , Proteínas Ribossômicas/genética , Proteína Supressora de Tumor p53/metabolismo , Anemia de Diamond-Blackfan/genética , Anemia de Diamond-Blackfan/patologia , Anemia Macrocítica/genética , Anemia Macrocítica/patologia , Animais , Benzotiazóis/farmacologia , Ciclo Celular/efeitos dos fármacos , Linhagem da Célula/efeitos dos fármacos , Nucléolo Celular/efeitos dos fármacos , Nucléolo Celular/metabolismo , Deleção Cromossômica , Cromossomos Humanos Par 5/genética , Inibidor de Quinase Dependente de Ciclina p21/metabolismo , Células Precursoras Eritroides/efeitos dos fármacos , Células Precursoras Eritroides/patologia , Hematopoese/efeitos dos fármacos , Humanos , Imidazóis/metabolismo , Camundongos , Camundongos Endogâmicos BALB C , Síndromes Mielodisplásicas/genética , Síndromes Mielodisplásicas/patologia , Piperazinas/metabolismo , Ligação Proteica/efeitos dos fármacos , Proteínas Proto-Oncogênicas c-mdm2/metabolismo , RNA Interferente Pequeno/metabolismo , Proteínas Ribossômicas/deficiência , Proteínas Ribossômicas/metabolismo , Tolueno/análogos & derivados , Tolueno/farmacologiaRESUMO
BACKGROUND: Thrombocytopenia is a significant problem in patients with relapsed or refractory multiple myeloma, precipitating a need for supportive platelet transfusions and necessitating decreases in delivered doses of chemotherapy. Eltrombopag is a non-peptide, small molecule thrombopoietin (TPO) receptor agonist that promotes megakaryopoiesis similar to endogenous human TPO and may be an effective agent for thrombocytopenia in this patient population. METHODS: We examined the effects of eltrombopag on megakaryocyte colony-forming capacity in CD34+ cells in patients with multiple myeloma and investigated its impact on proliferation, viability, and apoptosis in primary CD138+ human myeloma cells and myeloma cell lines. RESULTS: Eltrombopag at doses of 0.1 to 100 µM did not enhance proliferation of primary human CD138+ multiple myeloma cells from patients with relapsed disease or myeloma cell lines when used alone or in combination with erythropoietin (EPO) and granulocyte colony-stimulating factor (G-CSF) and did not alter cell viability nor apoptosis of human myeloma cells exposed to bortezomib and lenalidomide. Eltrombopag stimulated megakaryopoiesis in human CD34+ cells from normal individuals and from patients with relapsed multiple myeloma via activation of Akt signaling pathways. CONCLUSIONS: These results provide proof-of-principle supporting the design of future clinical studies examining eltrombopag for the treatment of thrombocytopenia in patients with advanced multiple myeloma.
Assuntos
Benzoatos/farmacologia , Hematopoese/efeitos dos fármacos , Hidrazinas/farmacologia , Megacariócitos/efeitos dos fármacos , Mieloma Múltiplo/complicações , Pirazóis/farmacologia , Trombocitopenia/tratamento farmacológico , Linhagem Celular Tumoral , Humanos , Técnicas In Vitro , Recidiva Local de Neoplasia/complicações , Receptores de Trombopoetina/agonistas , Trombocitopenia/etiologiaRESUMO
CASK is the mammalian ortholog of LIN2, a component of the LIN2/7/10 protein complex that targets epidermal growth factor receptor (EGFR) to basolateral membranes in Caenorhabditis elegans. A member of the MAGUK family of scaffolding proteins, CASK resides at basolateral membranes in polarized epithelia. Its interaction with LIN7 is evolutionarily conserved. In addition, CASK forms a complex with another MAGUK, the DLG1 tumor suppressor. Although complete knockout of CASK is lethal, the gene is X-linked, enabling us to generate heterozygous female adults that are mosaic for its expression. We also generated intestine-specific CASK knockout mice. Immunofluorescence analysis revealed that in intestine, CASK is not required for epithelial polarity or differentiation but is necessary for the basolateral localization of DLG1 and LIN7C. However, the subcellular distributions of DLG1 and LIN7C are independent of CASK in the stomach. Moreover, CASK and LIN7C show normal localization in dlg1(-/-) intestine. Despite the disappearance of basolateral LIN7C in CASK-deficient intestinal crypts, this epithelium retains normal localization of LIN7A/B, EGFR and ErbB-2. Finally, crypt-to-villus migration rates are unchanged in CASK-deficient intestinal epithelium. Thus, CASK expression and the appropriate localization of DLG1 are not essential for either epithelial polarity or intestinal homeostasis in vivo.