RESUMO
OBJECTIVE: The prognostic significance of IKZF1plus in adult Philadelphia-positive acute lymphoblastic leukemia (Ph+ ALL) patients had remained to be clarified. METHODS: We conducted a prospective, multicenter study, the ALL/MRD2008 trial, and investigated the clinical significance of IKZF1plus . RESULTS: From December 2008 to November 2013, 38 untreated Ph+ ALL patients were enrolled. At the end of the induction, 97.4% of patients (37/38) achieved complete hematological remission, with MRD-negativity of 48.6% (18/37). There were 19 patients with IKZF1plus , 13 with IKZF1 deletion alone (ΔIKZF1) and 4 with no IKZF1 deletions (no ΔIKZF1). The probability of 3-year DFS and OS in these Ph+ ALL patients were 50% (95% confidence interval [CI], 33-65) and 55% (95% CI, 38-69), respectively. There was no significant difference between IKZF1plus , ΔIKZF1, and no ΔIKZF1 in DFS (47%, 54%, 75% [p = .63]) or OS (47%, 62%, NA [p = .39]). CONCLUSIONS: We revealed no relationship between IKZF1plus status and survival outcomes in Ph+ ALL patients treated with imatinib/dasatinib combination chemotherapy. Further investigations are warranted to clarify the prognostic significance of IKZF1plus in adult Ph+ ALL patients.
Assuntos
Fator de Transcrição Ikaros , Leucemia-Linfoma Linfoblástico de Células Precursoras , Adulto , Humanos , Estudos Prospectivos , Fator de Transcrição Ikaros/genética , Leucemia-Linfoma Linfoblástico de Células Precursoras/diagnóstico , Leucemia-Linfoma Linfoblástico de Células Precursoras/tratamento farmacológico , Leucemia-Linfoma Linfoblástico de Células Precursoras/genética , Mesilato de Imatinib/uso terapêutico , Dasatinibe/uso terapêutico , PrognósticoRESUMO
BACKGROUND: Factor V (FV) deficiency is a monogenic inherited coagulation disorder considered to be an ideal indication for gene therapy. To investigate the possibility of therapeutic application of genome editing, we generated induced pluripotent stem cells (iPSCs) from a FV-deficient patient and repaired the mutation of factor V gene (F5) using a clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated 9 (Cas9). METHODS: The patient's peripheral blood mononuclear cells were reprogrammed for iPSCs. The targeting vector was designed with homology arms against F5 containing the corrected sequence. Cas9 ribonucleoprotein (RNP) complex and targeting vector were electroporated into iPSCs. Gene-edited iPSCs were differentiated into hepatocyte-like cells (HLCs). RESULTS: The mutation of F5 in patient-derived iPSCs was repaired by CRISPR/Cas9. In concentrated culture supernatants of patient-derived iPS-HLCs, neither FV antigen nor activity was detected, while in those of gene-corrected iPS-HLCs, FV antigen and specific activity were 67.0 ± 13.1 ng/mL and 173.2 ± 41.1 U/mg, respectively. CONCLUSIONS: We successfully repaired the mutation of F5 using the CRISPR/Cas9 and confirmed the recovery of FV activity with gene-corrected iPS-HLCs. Gene-edited iPSCs are promising for elucidating the pathophysiology as well as for a modality of gene therapy.
Assuntos
Deficiência do Fator V/genética , Edição de Genes/métodos , Células-Tronco Pluripotentes Induzidas/metabolismo , Diferenciação Celular , Células Cultivadas , Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas , Feminino , Humanos , Pessoa de Meia-IdadeRESUMO
The single-nucleotide polymorphism (SNP) rs12459419 is located at the intron/exon junction of CD33 exon2. When exon2 is skipped by this CD33 SNP, the full-length CD33 (CD33FL) is converted to a short CD33 isoform (CD33D2). Since gemtuzumab ozogamicin (GO) only recognizes CD33FL, the CD33 SNP may affect the clinical efficacy of GO. To elucidate the significance of CD33 SNP on GO reactivity, we leveraged the CRISPR/Cas9 genome-editing system to create OCI-AML3 cell lines with specifically modified CD33 SNPs. Levels of CD33 D2 mRNA were significantly higher in the T/T clone (p < 0.001), but CD33D2 protein was not detectable in any clones. There was no significant difference in CD33FL mRNA expression across edited clones, and CD33FL protein expression was lowest in T/T clones, followed by T/C and C/C. Cytotoxicity assays revealed that the IC50 of GO was significantly lower in T/C and C/C clones than in the T/T clone (p < 0.001). Our study demonstrated a difference in GO-induced cytotoxicity in CD33 SNP-edited clones, clearly indicating that at least one CD33 SNP allele, rs12459419 C, is important for sensitivity to GO.
Assuntos
Sistemas CRISPR-Cas , Gemtuzumab , Edição de Genes , Polimorfismo de Nucleotídeo Único , Lectina 3 Semelhante a Ig de Ligação ao Ácido Siálico , Humanos , Lectina 3 Semelhante a Ig de Ligação ao Ácido Siálico/genética , Edição de Genes/métodos , Linhagem Celular Tumoral , Leucemia Mieloide Aguda/genética , Leucemia Mieloide Aguda/tratamento farmacológicoRESUMO
Previous prospective randomized trials have investigated the efficacy of gemtuzumab ozogamicin in the frontline treatment of acute myeloid leukemia (AML). We evaluated the efficacy of high-dose cytarabine with GO as consolidation therapy in 20 patients with favorable- or intermediate-risk AML in first complete remission. They included six patients with wild-type nucleophosmin (NPM1) core binding factor (CBF), ten with NPM1-mutated non-CBF, and four with wild-type NPM1 non-CBF. The median follow-up for the entire cohort was 62.0 months. The three-year overall survival (OS) and relapse-free survival (RFS) rates were 72.2% and 77.8%, respectively. OS and RFS were significantly higher for NPM1-mutated non-CBF AML than for wild-type NPM1 non-CBF AML (p = 0.001). We also examined the CD33 single-nucleotide polymorphism (SNP) rs12459419, which has been reported to influence the therapeutic efficacy of GO and CD33 expression. The CD33 expression ratio was higher in CD33 SNP C/C than in C/T (83.1% vs. 49.8%, p = 0.035), but 3-year OS and RFS did not differ significantly. These results suggest that consolidation therapy with high-dose cytarabine plus GO is highly effective in transplant-ineligible elderly patients and may be a reasonable treatment, especially for NPM1-mutated AML.
RESUMO
Silent inactivation of L-asparaginase (L-Asp) represents rapid clearance of L-Asp by anti-L-Asp IgG antibodies without clinical symptoms. Measurement of L-Asp activity is the gold standard for diagnosis of silent inactivation, but this test is not commercially available in Japan as of 2023. We evaluated ex vivo and in vivo ammonia production in relation to L-Asp activity. Blood samples from ten adult patients treated with L-Asp were collected to measure ammonia levels and L-Asp activity before the first dose and 24 h after the last dose of L-Asp, during each cycle of treatment. Plasma ammonia levels were analyzed immediately and 1 h after incubation at room temperature, and ex vivo ammonia production was defined as the increase in ammonia concentration. Ex vivo ammonia production correlated with L-Asp activity (R2 = 0.741), and ammonia levels measured immediately after blood collection were moderately correlated with L-Asp activity (R2 = 0.709). One patient with extranodal NK/T-cell lymphoma showed an increase in ammonia levels during the first cycle, but no increase in ammonia levels or L-Asp activity after L-Asp administration during the second cycle. Both ex vivo and in vivo ammonia production and surrogate markers are used for L-Asp biological activity.
Assuntos
Asparaginase , Leucemia-Linfoma Linfoblástico de Células Precursoras , Adulto , Humanos , Asparaginase/efeitos adversos , Amônia/uso terapêutico , Leucemia-Linfoma Linfoblástico de Células Precursoras/tratamento farmacológico , Anticorpos , BiomarcadoresRESUMO
Dopamine increases/decreases synaptic vesicle recycling and in schizophrenia the proteins/mRNA is decreased. We isolated cDNA clone, similar to amphiphysin 1 (vesicle protein) mRNA from the neocortex of rats injected repeatedly with methamphetamine using polymerase chain reaction (PCR) differential display. This clone is highly homologous to the 3' region of the human amphiphysin gene. PCR extension study using a primer specific for the rat amphiphysin 1 gene and a primer located within the clone revealed that it is the 3' UTR region of the rat amphiphysin 1 gene. Furthermore, in situ hybridization revealed that amphiphysin 1 mRNA is expressed in the cerebrum, medial thalamus, hippocampus and cerebellum. In the cerebellum, amphiphysin mRNA expression was confined to upper granule cell layer. Repeated methamphetamine administration increased amphiphysin I mRNA expression in both anterior part of the cerebrum, and the cerebellum. However, the repeated administration did not alter mRNA expression of the other vesicle proteins, synaptotagmin I, synapsin I, synaptojanin and dynamin I, we conclude that the repeated administration selectively increased amphiphysin 1 mRNA expression. Thus, amphiphysin 1 does not work as synaptic recycling, but it is suggested, as a part of pathogenesis of brain tissue injury (under Ca²âº and Mg²âº devoid environment) in repeated methamphetamine-injected states, the gene regulate actin-asssembly, learning, cell stress signaling and cell polarity.
Assuntos
Estimulantes do Sistema Nervoso Central/administração & dosagem , Cerebelo/efeitos dos fármacos , Cérebro/efeitos dos fármacos , Metanfetamina/administração & dosagem , Proteínas do Tecido Nervoso/genética , Monoéster Fosfórico Hidrolases/genética , RNA Mensageiro/metabolismo , Regulação para Cima/efeitos dos fármacos , Análise de Variância , Animais , Cerebelo/metabolismo , Cérebro/metabolismo , Esquema de Medicação , Dinamina I/genética , Dinamina I/metabolismo , Masculino , Proteínas do Tecido Nervoso/metabolismo , Monoéster Fosfórico Hidrolases/metabolismo , Ratos , Ratos Sprague-DawleyRESUMO
BCR-ABL1-like acute lymphoblastic leukemia (ALL) is a neoplasm of lymphoblasts committed to the B-cell lineage that lack the BCR-ABL1 translocation but show a pattern of gene expression very similar to that seen in ALL with BCR-ABL1 with poor prognosis. A 22-year-old female was diagnosed with common-B-cell-ALL positive for CD10, CD19, CD22, CD79a, CD34, HLA-DR, and TdT in January 2017, and achieved complete remission (CR) with induction therapy, followed by consolidation therapy and maintenance therapy. In March 2020, 6 months after the completion of maintenance therapy, she relapsed. Inotuzumab ozogamicin (IO) was administered, and on day 28, bone marrow evaluation showed a morphologic CR. She had an HLA-identical sibling, and transplantation in her 2nd CR was planned. Because her ALL had been identified as BCR-ABL1-like ALL with CCDC88C-PDGFRB fusion, she was treated with imatinib for 2 months accompanied by 2 intrathecal methotrexate therapies, and 1 course of L-asparaginase, vincristine, and prednisolone in an outpatient setting. MRD analysis revealed potent efficacy of 2 months imatinib therapy; IgH MRD decreased from 1 × 10-2 to 1 × 10-3, and CCDC88C-PDGFRB/104ABL from 37.3 to 0. It is earnestly desired that well-designed clinical trials of TKI in ABL class-mutant BCR-ABL1-like ALL be conducted in Japan.
Assuntos
Peptídeos e Proteínas de Sinalização Intracelular/genética , Proteínas dos Microfilamentos/genética , Proteínas de Fusão Oncogênica/genética , Leucemia-Linfoma Linfoblástico de Células Precursoras/tratamento farmacológico , Leucemia-Linfoma Linfoblástico de Células Precursoras/genética , Inibidores de Proteínas Quinases/uso terapêutico , Receptor beta de Fator de Crescimento Derivado de Plaquetas/genética , Biomarcadores , Terapia Combinada , Feminino , Proteínas de Fusão bcr-abl/genética , Testes Genéticos , Humanos , Terapia de Alvo Molecular/efeitos adversos , Terapia de Alvo Molecular/métodos , Neoplasia Residual/diagnóstico , Leucemia-Linfoma Linfoblástico de Células Precursoras/diagnóstico , Inibidores de Proteínas Quinases/administração & dosagem , Inibidores de Proteínas Quinases/efeitos adversos , Recidiva , Retratamento , Resultado do Tratamento , Adulto JovemRESUMO
In the cerebellum, there are numerous cholecystokinin (CCK-8)-containing fibers. Since systemic CCK-8 injection-induced anxiety (psychological stress) activates the locus coeruleus cells that send mossy fiber inputs to the cerebellum, we examined whether systemic CCK-8 injections activate the rat and mouse cerebellum. First, injections of CCK-8 were found to induce c-fos mRNA expression in a vague patchy pattern that is different from single methamphetamine-induced Zebrin band-like c-fos mRNA expression, suggesting that the CCK-8 activating mossy fibers induce gene expression differently from the dopamine-containing mossy fibers in the ventral tegmental area. Second, since CCK-8 facilitates neural activity of dopamine in the midbrain, we examined whether repeated methamphetamine administration that induced behavioral sensitization had similar effects on the cerebellar CCK system. Repeated administration of methamphetamine suppressed the CCK-8-induced c-fos mRNA expression in the rat cerebellum. Third, capsaicin injections (physical stress) into a hind limb of the rat increased junD mRNA expression with no effect on c-fos mRNA expression, and repeated methamphetamine injections had no effect on the capsaicin-induced expression of junD mRNA. Fourth, either single injection of methamphetamine or CCK-8 to mice increased c-fos mRNA expression in the locus coeruleus, and so noradrenalin, but not dopamine, might interact with CCK-8-activating system. However, we considered the possibility unlikely. Thus, we conclude that repeated methamphetamine administration though dopamine selectively inhibits the c-fos mRNA expression after CCK-8 injection in the cerebellum.
Assuntos
Capsaicina/farmacologia , Cerebelo/efeitos dos fármacos , Metanfetamina/administração & dosagem , Proteínas Proto-Oncogênicas c-fos/metabolismo , Sincalida/farmacologia , Fatores de Transcrição/metabolismo , Análise de Variância , Animais , Northern Blotting , Cerebelo/metabolismo , Hibridização In Situ , Camundongos , Fibras Nervosas/efeitos dos fármacos , Fibras Nervosas/metabolismo , Neurônios/efeitos dos fármacos , Neurônios/metabolismo , Proteínas Proto-Oncogênicas c-fos/genética , Proteínas Proto-Oncogênicas c-jun , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Ratos , Ratos Wistar , Estatísticas não Paramétricas , Fatores de Transcrição/genéticaRESUMO
The clustered regulatory interspaced short palindromic repeats (CRISPR)/CRISPR-associated (Cas) system is an efficient genome-editing tool that holds potential for gene therapy. Here, we report an application of this system for gene repair in hemophilia B (HB) using induced pluripotent stem cells (iPSCs). We prepared targeting plasmids with homology arms containing corrected sequences to repair an in-frame deletion in exon 2 of the factor IX (F9) gene and transfected patient-derived iPSCs with the Cas9 nuclease and a guide RNA expression vector. To validate the expression of corrected F9, we attempted to induce the differentiation of iPSCs toward hepatocyte-like cells (HLCs) in vitro. We successfully repaired a disease-causing mutation in HB in patient-derived iPSCs. The transcription product of corrected F9 was confirmed in HLCs differentiated from gene-corrected iPSCs. Although further research should be undertaken to obtain completely functional hepatocytes with secretion of coagulation factor IX, our study provides a proof-of-principle for HB gene therapy using the CRISPR/Cas9 system.
Assuntos
Sistemas CRISPR-Cas/genética , Edição de Genes , Terapia Genética/métodos , Hemofilia B/genética , Hemofilia B/terapia , Células-Tronco Pluripotentes Induzidas , HumanosRESUMO
In hematopoiesis, the expression of critical genes is regulated in a stage-specific manner to maintain normal hematopoiesis. Notch1 is an essential gene involved in the commitment and development of the T-cell lineage. However, the regulation of Notch1 in hematopoiesis is controversial, particularly at the level of hematopoietic stem cell (HSC). Here, we found that the expression of Notch1 is controlled at the post-transcriptional level in HSCs. HSCs express a considerable level of Notch1 mRNA, but its protein level is very low, suggesting a post-transcriptional suppression for Notch1. Using a retroviral sensor vector expressing a fusion mRNA of GFP and 3' untranslated region (3'UTR) of a target gene, we demonstrated that the Notch1-3'UTR had a post-translational suppressive effect only at the HSC but not in the downstream progenitor stages. The sequence motif AUnA was required for this post-transcriptional regulation by the Notch1-3'UTR. Interestingly, this Notch1-3'UTR-mediated suppressive effect was relieved when HSCs were placed in the thymus, but not in the bone marrow. Thus, the expression of Notch1 in HSCs is regulated by microenvironment at the post-transcriptional level, which may control T lymphoid lineage commitment from HSCs.
Assuntos
Regiões 3' não Traduzidas/fisiologia , Expressão Gênica/genética , Hematopoese/genética , Células-Tronco Hematopoéticas/citologia , Células-Tronco Hematopoéticas/metabolismo , Receptor Notch1/genética , Receptor Notch1/metabolismo , Transcrição Gênica/genética , Animais , Divisão Celular/genética , Células Cultivadas , Camundongos Congênicos , Camundongos Endogâmicos C57BL , RNA Mensageiro/metabolismo , Receptor Notch1/fisiologia , Nicho de Células-Tronco/genéticaAssuntos
Leucemia Mieloide Aguda , Proteínas Nucleares , Humanos , Proteínas Nucleares/genética , Compostos Bicíclicos Heterocíclicos com Pontes/uso terapêutico , Leucemia Mieloide Aguda/tratamento farmacológico , Leucemia Mieloide Aguda/genética , Azacitidina/uso terapêutico , Mutação , Tirosina Quinase 3 Semelhante a fms/genética , PrognósticoRESUMO
Peripheral T-cell lymphoma (PTCL) is a biologically diverse lymphoid malignancy. The clinical aggressiveness associated with hemophagocytic syndrome (HS) is a characteristic of PTCL, being more distinctive in CD8(+) PTCL. However, the underlying mechanism of PTCL-associated HS has not yet been fully investigated. We newly established a novel IL-2-dependent CD8(+) PTCL lymphoma cell line (T8ML-1) from a patient with CD8(+) PTCL who suffered recurrent HS accompanying disease flare-up. Focusing on the lymphoma cell T-cell receptor (TCR), we examined the lymphoma cell functions responsible for such clinical manifestations. First, T8ML-1.1 in which endogenous TCR-α/ß chains were silenced by siRNAs, and T8ML-1.2 in which endogenous TCR-α/ß chains were replaced with HLA-A*24:02-restricted and WT1(235-243)-specific TCR-α/ß, were established. T8ML-1 exerted phytohemagglutinin (PHA)-dependent cytotoxicity via granular exocytosis. Additionally, soluble factors produced by PHA-stimulated T8ML-1, which included INF-γ and TNF-α, but not by simple-cultured T8ML-1, caused human monocytes to exhibit erythrophagocytosis and thrombophagocytosis in vitro. PHA binding induced phosphorylation of CD3ζ chain. Furthermore, both cytotoxicity and hemophagocytosis were completely inhibited by T8ML-1.1, but eventually restored by T8ML-1.2. These data suggest that exogenous activation of TCR signaling in PTCL cells might play an important role in the formation of PTCL-associated HS.
Assuntos
Células Sanguíneas/patologia , Citofagocitose , Linfoma de Células T Periférico/fisiopatologia , Receptores de Antígenos de Linfócitos T/metabolismo , Transdução de Sinais , Linhagem Celular Tumoral , Humanos , Linfoma de Células T Periférico/patologiaRESUMO
Human acute myeloid leukemia (AML) originates from rare leukemia stem cells (LSCs). Because these chemotherapy-resistant LSCs are thought to underlie disease relapse, effective therapeutic strategies specifically targeting these cells may be beneficial. Here, we report identification of a primary human LSC gene signature and functional characterization of human LSC-specific molecules in vivo in a mouse xenotransplantation model. In 32 of 61 (53%) patients with AML, either CD32 or CD25 or both were highly expressed in LSCs. CD32- or CD25-positive LSCs could initiate AML and were cell cycle-quiescent and chemotherapy-resistant in vivo. Normal human hematopoietic stem cells depleted of CD32- and CD25-positive cells maintained long-term multilineage hematopoietic reconstitution capacity in vivo, indicating the potential safety of treatments targeting these molecules. In addition to CD32 and CD25, quiescent LSCs within the bone marrow niche also expressed the transcription factor WT1 and the kinase HCK. These molecules are also promising targets for LSC-specific therapy.
Assuntos
Antineoplásicos/farmacologia , Resistencia a Medicamentos Antineoplásicos , Leucemia/terapia , Células-Tronco Neoplásicas/metabolismo , Animais , Transplante de Medula Óssea/métodos , Ciclo Celular , Linhagem da Célula , Separação Celular , Desenho de Fármacos , Citometria de Fluxo , Regulação Leucêmica da Expressão Gênica , Humanos , Subunidade alfa de Receptor de Interleucina-2/biossíntese , Camundongos , Camundongos SCID , Receptores de IgG/biossíntese , Transplante HeterólogoRESUMO
The mechanism of lineage specification in multipotent stem cells has not been fully understood. We recently isolated progenitors with the eosinophil, basophil, or mast cell lineage potential, all of which originate from granulocyte/monocyte progenitors (GMPs). By using these prospectively purified progenitors, we show here that the expression timing of GATA-2 and CCAAT enhancer-binding protein alpha (C/EBPalpha) can differentially control their lineage commitment. The expression of GATA-2 instructed C/EBPalpha-expressing GMPs to commit exclusively into the eosinophil lineage, while it induced basophil and/or mast cell lineage commitment if C/EBPalpha was suppressed at the GMP stage. Furthermore, simply by switching the order of C/EBPalpha and GATA-2 transduction, even lymphoid-committed progenitors recaptured these developmental processes to be reprogrammed into each of these lineages. We propose that the order of expression of key transcription factors is critical for their interplay to selectively drive lineage specification programs, by which stem cells could generate multiple lineage cells in a hierarchical manner.
Assuntos
Proteína alfa Estimuladora de Ligação a CCAAT/metabolismo , Fator de Transcrição GATA2/metabolismo , Regulação da Expressão Gênica no Desenvolvimento , Hematopoese/genética , Células-Tronco Hematopoéticas/metabolismo , Animais , Proteína alfa Estimuladora de Ligação a CCAAT/genética , Diferenciação Celular/genética , Linhagem da Célula/genética , Fator de Transcrição GATA2/genética , Granulócitos/citologia , Granulócitos/metabolismo , Células-Tronco Hematopoéticas/citologia , Mastócitos/citologia , Mastócitos/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Dados de Sequência Molecular , Células Progenitoras Mieloides/citologia , Células Progenitoras Mieloides/metabolismoRESUMO
Basophils and mast cells, which are selectively endowed with the high-affinity IgE receptor and mediate a range of adaptive and innate immune responses, have an unknown developmental relationship. Here, by evaluating the expression of the beta7 integrin, a molecule that is required for selective homing of mast cell progenitors (MCPs) to the periphery, we identified bipotent progenitors that are capable of differentiating into either cell type in the mouse spleen. These basophil/mast cell progenitors (BMCPs) gave rise to basophils and mast cells at the single-cell level and reconstituted both mucosal and connective tissue mast cells. We also identified the basophil progenitor (BaP) and the MCP in the bone marrow and the gastrointestinal mucosa, respectively. We further show that the granulocyte-related transcription factor CCAAT/enhancer-binding protein alpha (C/EBPalpha) plays a primary role in the fate decision of BMCPs, being expressed in BaPs but not in MCPs. Thus, circulating basophils and tissue mast cells share a common developmental stage at which their fate decision might be controlled principally by C/EBPalpha.