RESUMO
BACKGROUND: Adhesion of cancer cells to extracellular matrix laminin through the integrin superfamily reportedly induces drug resistance. Heterodimers of integrin α6 (CD49f) with integrin ß1 (CD29) or ß4 (CD104) are major functional receptors for laminin. Higher CD49f expression is reportedly associated with a poorer response to induction therapy in childhood B-cell precursor acute lymphoblastic leukemia (BCP-ALL). Moreover, a xenograft mouse model transplanted with primary BCP-ALL cells revealed that neutralized antibody against CD49f improved survival after chemotherapy. AIMS: Considering the poor outcomes in Philadelphia chromosome (Ph)-positive ALL treated with conventional chemotherapy without tyrosine kinase inhibitors, we sought to investigate an involvement of the laminin adhesion. METHODS AND RESULTS: Ph-positive ALL cell lines expressed the highest levels of CD49f among the BCP-ALL cell lines with representative translocations, while CD29 and CD104 were ubiquitously expressed in BCP-ALL cell lines. The association of Ph-positive ALL with high levels of CD49f gene expression was also confirmed in two databases of childhood ALL cohorts. Ph-positive ALL cell lines attached to laminin and their laminin-binding properties were disrupted by blocking antibodies against CD49f and CD29 but not CD104. The cell surface expression of CD49f, but not CD29 and CD104, was downregulated by imatinib treatment in Ph-positive ALL cell lines, but not in their T315I-acquired sublines. Consistently, the laminin-binding properties were disrupted by the imatinib pre-treatment in the Ph-positive ALL cell line, but not in its T315I-acquired subline. CONCLUSION: BCR::ABL1 plays an essential role in the laminin adhesion of Ph-positive ALL cells through upregulation of CD49f.
Assuntos
Integrina alfa6 , Laminina , Leucemia-Linfoma Linfoblástico de Células Precursoras , Regulação para Cima , Animais , Humanos , Camundongos , Mesilato de Imatinib/farmacologia , Mesilato de Imatinib/uso terapêutico , Integrina alfa6/genética , Laminina/metabolismo , Leucemia-Linfoma Linfoblástico de Células Precursoras/tratamento farmacológico , Leucemia-Linfoma Linfoblástico de Células Precursoras/genéticaRESUMO
CD19-specific chimeric antigen receptor T (CAR T) immunotherapy is used to treat B-cell malignancies. However, antigen-escape mediated relapse following CAR T therapy has emerged as a major concern. In some relapsed cases, especially KMT2A rearrangement-positive B-acute lymphoblastic leukemia (KMT2A-r B-ALL), most of the B-cell antigens are lost via lineage conversion to the myeloid phenotype, rendering multi-B-cell-antigen-targeted CAR T cell therapy ineffective. Fms-related tyrosine kinase-3 (FLT3) is highly expressed in KMT2A-r B-ALL; therefore, in this study, we aimed to evaluate the antitumor efficacy of CAR T cells targeting both CD19 and FLT3 in KMT2A-r B-ALL cells. We developed piggyBac transposon-mediated CAR T cells targeting CD19, FLT3, or both (dual) and generated CD19-negative KMT2A-r B-ALL models through CRISPR-induced CD19 gene-knockout (KO). FLT3 CAR T cells showed antitumor efficacy against CD19-KO KMT2A-r B-ALL cells both in vitro and in vivo; dual-targeted CAR T cells showed cytotoxicity against wild-type (WT) and CD19-KO KMT2A-r B-ALL cells, whereas CD19 CAR T cells demonstrated cytotoxicity only against WT KMT2A-r B-ALL cells in vitro. Therefore, targeting FLT3-specific CAR T cells would be a promising strategy for KMT2A-r B-ALL cells even with CD19-negative relapsed cases.
Assuntos
Leucemia-Linfoma Linfoblástico de Células Precursoras , Receptores de Antígenos Quiméricos , Humanos , Antígenos CD19/genética , Tirosina Quinase 3 Semelhante a fms/genética , Imunoterapia , Imunoterapia Adotiva , Leucemia-Linfoma Linfoblástico de Células Precursoras/genética , Leucemia-Linfoma Linfoblástico de Células Precursoras/terapia , Receptores de Antígenos Quiméricos/genética , Linfócitos T , Proteína de Leucina Linfoide-Mieloide/metabolismo , Histona-Lisina N-Metiltransferase/metabolismoRESUMO
RATIONALE: The gastrointestinal (GI) tract is a common target organ of graft-vs-host disease (GVHD) in hematopoietic stem cell transplantation (HSCT) patients, and GI tract GVHD is often resistant to standard treatments such as corticosteroids. Moreover, longterm use of systemic corticosteroids sometimes induces adverse events such as infection. Beclomethasone dipropionate (BDP) is a potent, topically active corticosteroid, which is metabolized to an active derivative in the intestinal mucosa. Oral BDP therapy is reportedly effective against GI tract GVHD in adult HSCT patients, but its efficacy and safety in pediatric patients remain undefined. Here, we report three pediatric and young adult cases who were treated with oral BDP. PATIENT CONCERNS: Three (6-, 7-, and 18-year-old) patients developed stage 2 to 4 lower GI tract GVHD, which was resistant to standard immunosuppressive therapies. DIAGNOSIS: Lower GI tract GVHD in these patients was histopathologically proven by endoscopic biopsy. INTERVENTIONS: Oral administration of enteric-coated capsules of BDP (3-8 mg/day) was started for the treatment of lower GI tract GVHD. OUTCOMES: With the introduction of oral BDP therapy, their GI tract symptoms promptly resolved (abdominal pain, within 3-7 days; diarrhea, within 2-3 weeks). Subsequently, systemic immunosuppressive agents such as corticosteroids and mycophenolate mofetil were successfully tapered off. During oral BDP therapy, although cytomegalovirus antigenemia and Acinetobacter Iwoffii sepsis developed in 2 cases, both were curable with conventional treatments. In a young adult case, concomitant BK virus-associated hemorrhagic cystitis resolved after oral BDP was introduced and systemic immunosuppressive agents were reduced. Transient growth restriction was observed in a pediatric case who was treated with oral BDP for approximately 300days. LESSONS: Our experiences suggest that oral BDP therapy is an effective approach for GI tract GVHD that is resistant to standard immunosuppressive therapies. Of clinical importance, our case suggests the possibility that oral BDP therapy may improve the immunosuppressive condition in GI tract GVHD patients by contributing to the reduction of systemic immunosuppressive medications as a result of prompt improvement of GI tract GVHD symptoms.
Assuntos
Gastroenteropatias , Doença Enxerto-Hospedeiro , Transplante de Células-Tronco Hematopoéticas , Beclometasona/efeitos adversos , Beclometasona/uso terapêutico , Criança , Gastroenteropatias/induzido quimicamente , Gastroenteropatias/etiologia , Doença Enxerto-Hospedeiro/tratamento farmacológico , Doença Enxerto-Hospedeiro/etiologia , Transplante de Células-Tronco Hematopoéticas/efeitos adversos , Humanos , Adulto JovemRESUMO
Glucocorticoid (GC) is a key drug in the treatment of B-cell precursor acute lymphoblastic leukemia (BCP-ALL), and the initial GC response is an important prognostic factor. GC receptors play an essential role in GC sensitivity, and somatic mutations of the GC receptor gene, NR3C1, are reportedly identified in some BCP-ALL cases, particularly at relapse. Moreover, associations of somatic mutations of the CREB-binding protein (CREBBP) and Wolf-Hirschhorn syndrome candidate 1 (WHSC1) genes with the GC-resistance of ALL have been suggested. However, the significance of these mutations in the GC sensitivity of BCP-ALL remains to be clarified in the intrinsic genes. In the present study, we sequenced NR3C1, WHSC1, and CREBBP genes in 99 BCP-ALL and 22 T-ALL cell lines (32 and 67 cell lines were known to be established at diagnosis and at relapse, respectively), and detected their mutations in 19 (2 cell lines at diagnosis and 15 cell lines at relapse), 26 (6 and 15), and 38 (11 and 15) cell lines, respectively. Of note, 14 BCP-ALL cell lines with the NR3C1 mutations were significantly more resistant to GC than those without mutations. In contrast, WHSC1 and CREBBP mutations were not associated with GC resistance. However, among the NR3C1 unmutated BCP-ALL cell lines, WHSC1 mutations tended to be associated with GC resistance and lower NR3C1 gene expression. Finally, we successfully established GC-resistant sublines of the GC-sensitive BCP-ALL cell line (697) by disrupting ligand binding and DNA binding domains of the NR3C1 gene using the CRISPR/Cas9 system. These observations demonstrated that somatic mutations of the NR3C1 gene, and possibly the WHSC1 gene, confer GC resistance in BCP-ALL.
Assuntos
Leucemia-Linfoma Linfoblástico de Células Precursoras , Receptores de Glucocorticoides , Glucocorticoides/farmacologia , Glucocorticoides/uso terapêutico , Humanos , Erros Inatos do Metabolismo , Mutação , Leucemia-Linfoma Linfoblástico de Células Precursoras/genética , Receptores de Glucocorticoides/deficiência , Receptores de Glucocorticoides/genética , Receptores de Glucocorticoides/metabolismo , RecidivaRESUMO
Asparaginase therapy is a key component of chemotherapy for patients with T-cell acute lymphoblastic leukemia (T-ALL). Asparaginase depletes serum asparagine by deamination into aspartic acid. Normal hematopoietic cells can survive due to asparagine synthetase (ASNS) activity, whereas leukemia cells are supposed to undergo apoptosis due to silencing of the ASNS gene. Because the ASNS gene has a typical CpG island in its promoter, its methylation status in T-ALL cells may be associated with asparaginase sensitivity. Thus, we investigated the significance of ASNS methylation status in asparaginase sensitivity of T-ALL cell lines and prognosis of childhood T-ALL. Sequencing of bisulfite polymerase chain reaction products using next-generation sequencing technology in 22 T-ALL cell lines revealed a stepwise allele-specific methylation of the ASNS gene, in association with an aberrant methylation of a 7q21 imprinted gene cluster. T-ALL cell lines with ASNS hypermethylation status showed significantly higher in vitro l-asparaginase sensitivity in association with insufficient asparaginase-induced upregulation of ASNS gene expression and lower basal ASNS protein expression. A comprehensive analysis of diagnostic samples from pediatric patients with T-ALL in Japanese cohorts (N = 77) revealed that methylation of the ASNS gene was associated with an aberrant methylation of the 7q21 imprinted gene cluster. In pediatric T-ALL patients in Japanese cohorts (n = 75), ASNS hypomethylation status was significantly associated with poor therapeutic outcome, and all cases with poor prognostic SPI1 fusion exclusively exhibited ASNS hypomethylation status. These observations show that ASNS hypomethylation status is associated with asparaginase resistance and is a poor prognostic biomarker in childhood T-ALL.
Assuntos
Asparaginase , Aspartato-Amônia Ligase , Carbono-Nitrogênio Ligases com Glutamina como Doadora de N-Amida , Leucemia-Linfoma Linfoblástico de Células T Precursoras , Alelos , Asparaginase/uso terapêutico , Asparagina/genética , Asparagina/metabolismo , Aspartato-Amônia Ligase/genética , Aspartato-Amônia Ligase/metabolismo , Carbono-Nitrogênio Ligases com Glutamina como Doadora de N-Amida/genética , Carbono-Nitrogênio Ligases com Glutamina como Doadora de N-Amida/metabolismo , Linhagem Celular Tumoral , Criança , Metilação de DNA , Humanos , Leucemia-Linfoma Linfoblástico de Células T Precursoras/tratamento farmacológico , Leucemia-Linfoma Linfoblástico de Células T Precursoras/genética , PrognósticoRESUMO
In chemotherapy for childhood acute lymphoblastic leukaemia (ALL), maintenance therapy consisting of oral daily mercaptopurine and weekly methotrexate is important. NUDT15 variant genotype is reportedly highly associated with severe myelosuppression during maintenance therapy, particularly in Asian and Hispanic populations. It has also been demonstrated that acquired somatic mutations of the NT5C2 and PRPS1 genes, which are involved in thiopurine metabolism, are detectable in a portion of relapsed childhood ALL. To directly confirm the significance of the NUDT15 variant genotype and NT5C2 and PRPS1 mutations in thiopurine sensitivity of leukaemia cells in the intrinsic genes, we investigated 84 B-cell precursor-ALL (BCP-ALL) cell lines. Three and 14 cell lines had homozygous and heterozygous variant diplotypes of the NUDT15 gene, respectively, while 4 and 2 cell lines that were exclusively established from the samples at relapse had the NT5C2 and PRPS1 mutations, respectively. Both NUDT15 variant genotype and NT5C2 and PRPS1 mutations were significantly associated with DNA-incorporated thioguanine levels after exposure to thioguanine at therapeutic concentration. Considering the continuous exposure during the maintenance therapy, we evaluated in vitro mercaptopurine sensitivity after 7-day exposure. Mercaptopurine concentrations lethal to 50% of the leukaemia cells were comparable to therapeutic serum concentration of mercaptopurine. Both NUDT15 variant genotype and NT5C2 and PRPS1 mutations were significantly associated with mercaptopurine sensitivity in 83 BCP-ALL and 23 T-ALL cell lines. The present study provides direct evidence to support the general principle showing that both inherited genotype and somatically acquired mutation are crucially implicated in the drug sensitivity of leukaemia cells.
Assuntos
5'-Nucleotidase/genética , Resistencia a Medicamentos Antineoplásicos/genética , Mercaptopurina/farmacologia , Mutação , Polimorfismo Genético , Pirofosfatases/genética , Ribose-Fosfato Pirofosfoquinase/genética , Alelos , Antimetabólitos Antineoplásicos/farmacologia , Apoptose/genética , Linhagem Celular Tumoral , Sobrevivência Celular/genética , Relação Dose-Resposta a Droga , Genótipo , HumanosRESUMO
The long-term prognosis of Philadelphia chromosome-positive acute lymphoblastic leukemia (Ph + ALL) is still unsatisfactory even after the emergence of tyrosine kinase inhibitors (TKIs) against chimeric BCR-ABL, and this is associated with the high incidence of genetic alterations of Ikaros family zinc finger 1 (IKZF1), most frequently the hemi-allelic loss of exons 4-7 expressing a dominant-negative isoform Ik6. We found that lenalidomide (LEN), a representative of immunomodulatory drugs (IMiDs), which have been long used for the treatment of multiple myeloma, specifically induced accumulation of Ik6 with the disappearance of functional isoforms within 24 h (i.e., abrupt and complete shut-down of the IKZF1 activity) in Ik6-positive Ph+ALL cells in a neddylation-dependent manner. The functional IKZF3 isoforms expression was also abruptly and markedly downregulated. The LEN treatment specifically suppressed proliferation of Ik6-positive-Ph+ALL cells by inducing cell cycle arrest via downregulation of cyclins D3 and E and CDK2, and of importance, markedly upregulated their apoptosis in synergy with the TKI imatinib (IM). Apoptosis of IM-resistant Ph+ALL cells with T315I mutation of BCR-ABL was also upregulated by LEN in the presence of the newly developed TKI ponatinib. Analyses of flow cytometry, western blot, and oligonucleotide array revealed that apoptosis was caspase-/p53-dependent and associated with upregulation of pro-apoptotic Bax/Bim, enhanced dephosphorylation of BCR-ABL/Akt, and downregulation of oncogenic helicase genes HILLS, CDC6, and MCMs4 and 8. Further, the synergism of LEN with IM was clearly documented as a significant prolongation of survival in the xenograft mice model. Because this synergism was further potentiated in vitro by dexamethasone, a key drug for ALL treatment, the strategy of repositioning IMiDs for the treatment of Ik6-positive Ph+ALL patients certainly shed new light on an outpatient-based treatment option for achieving their long-term durable remission and higher QOL, particularly for those who are not tolerable to intensified therapeutic approaches.
RESUMO
Extensive activation of glial cells during a latent period has been well documented in various animal models of epilepsy. However, it remains unclear whether activated glial cells contribute to epileptogenesis, i.e., the chronically persistent process leading to epilepsy. Particularly, it is not clear whether interglial communication between different types of glial cells contributes to epileptogenesis, because past literature has mainly focused on one type of glial cell. Here, we show that temporally distinct activation profiles of microglia and astrocytes collaboratively contributed to epileptogenesis in a drug-induced status epilepticus model. We found that reactive microglia appeared first, followed by reactive astrocytes and increased susceptibility to seizures. Reactive astrocytes exhibited larger Ca2+ signals mediated by IP3R2, whereas deletion of this type of Ca2+ signaling reduced seizure susceptibility after status epilepticus. Immediate, but not late, pharmacological inhibition of microglial activation prevented subsequent reactive astrocytes, aberrant astrocyte Ca2+ signaling, and the enhanced seizure susceptibility. These findings indicate that the sequential activation of glial cells constituted a cause of epileptogenesis after status epilepticus. Thus, our findings suggest that the therapeutic target to prevent epilepsy after status epilepticus should be shifted from microglia (early phase) to astrocytes (late phase).
Assuntos
Astrócitos/metabolismo , Epilepsia/metabolismo , Receptores de Inositol 1,4,5-Trifosfato/metabolismo , Microglia/metabolismo , Estado Epiléptico/metabolismo , Animais , Astrócitos/efeitos dos fármacos , Astrócitos/patologia , Sinalização do Cálcio , Modelos Animais de Doenças , Progressão da Doença , Suscetibilidade a Doenças , Epilepsia/induzido quimicamente , Epilepsia/patologia , Epilepsia/fisiopatologia , Gliose/metabolismo , Interleucina-1beta/metabolismo , Camundongos , Microglia/efeitos dos fármacos , Microglia/patologia , Agonistas Muscarínicos/toxicidade , Compostos Orgânicos/farmacologia , Pilocarpina/toxicidade , Receptores de Fator Estimulador das Colônias de Granulócitos e Macrófagos/antagonistas & inibidores , Bloqueadores dos Canais de Sódio/toxicidade , Estado Epiléptico/induzido quimicamente , Estado Epiléptico/patologia , Estado Epiléptico/fisiopatologia , Tetrodotoxina/toxicidade , Fatores de Tempo , Fator de Necrose Tumoral alfa/metabolismoRESUMO
Identification of genetic variants associated with glucocorticoids (GC) sensitivity of leukaemia cells may provide insight into potential drug targets and tailored therapy. In the present study, within 72 leukaemic cell lines derived from Japanese patients with B-cell precursor acute lymphoblastic leukaemia (ALL), we conducted genome-wide genotyping of single nucleotide polymorphisms (SNP) and attempted to identify genetic variants associated with GC sensitivity and NR3C1 (GC receptor) gene expression. IC50 measures for prednisolone (Pred) and dexamethasone (Dex) were available using an alamarBlue cell viability assay. IC50 values of Pred showed the strongest association with rs904419 (P = 4.34 × 10-8 ), located between the FRMD4B and MITF genes. The median IC50 values of prednisolone for cell lines with rs904419 AA (n = 13), AG (n = 31) and GG (n = 28) genotypes were 0.089, 0.139 and 297 µmol/L, respectively. For dexamethasone sensitivity, suggestive association was observed for SNP rs2306888 (P = 1.43 × 10-6 ), a synonymous SNP of the TGFBR3 gene. For NR3C1 gene expression, suggestive association was observed for SNP rs11982167 (P = 6.44 × 10-8 ), located in the PLEKHA8 gene. These genetic variants may affect GC sensitivity of ALL cells and may give rise to opportunities in personalized medicine for effective and safe chemotherapy in ALL patients.
Assuntos
Regulação Leucêmica da Expressão Gênica , Variação Genética , Glucocorticoides/farmacologia , Leucemia-Linfoma Linfoblástico de Células Precursoras/tratamento farmacológico , Leucemia-Linfoma Linfoblástico de Células Precursoras/genética , Linhagem Celular Tumoral , Dexametasona/farmacologia , Ensaios de Seleção de Medicamentos Antitumorais , Perfilação da Expressão Gênica , Genótipo , Humanos , Concentração Inibidora 50 , Japão , Farmacogenética , Polimorfismo de Nucleotídeo Único , Prednisolona/farmacologia , Receptores de Glucocorticoides/genéticaRESUMO
Psychological stress activates the hypothalamus, augments the sympathetic nervous output, and elevates blood pressure via excitation of the ventral medullary cardiovascular regions. However, anatomical and functional connectivity from the hypothalamus to the ventral medullary cardiovascular regions has not been fully elucidated. We investigated this issue by tract-tracing and functional imaging in rats. Retrograde tracing revealed the rostral ventrolateral medulla was innervated by neurons in the ipsilateral dorsomedial hypothalamus (DMH). Anterograde tracing showed DMH neurons projected to the ventral medullary cardiovascular regions with axon terminals in contiguity with tyrosine hydroxylase-immunoreactive neurons. By voltage-sensitive dye imaging, dynamics of ventral medullary activation evoked by electrical stimulation of the DMH were analyzed in the diencephalon-lower brainstem-spinal cord preparation of rats. Although the activation of the ventral medulla induced by single pulse stimulation of the DMH was brief, tetanic stimulation caused activation of the DMH sustained into the post-stimulus phase, resulting in delayed recovery. We suggest that prolonged excitation of the DMH, which is triggered by tetanic electrical stimulation and could also be triggered by psychological stress in a real life, induces further prolonged excitation of the medullary cardiovascular networks, and could contribute to the pathological elevation of blood pressure. The connectivity from the DMH to the medullary cardiovascular networks serves as a chronological amplifier of stress-induced sympathetic excitation. This notion will be the anatomical and pathophysiological basis to understand the mechanisms of stress-induced sustained augmentation of sympathetic activity.
Assuntos
Vias Autônomas/fisiologia , Núcleo Hipotalâmico Dorsomedial/fisiologia , Bulbo/fisiologia , Neurônios/metabolismo , Sistema Nervoso Simpático/fisiologia , Animais , Vias Autônomas/anatomia & histologia , Núcleo Hipotalâmico Dorsomedial/anatomia & histologia , Masculino , Bulbo/anatomia & histologia , Neurônios/citologia , Ratos , Ratos Wistar , Sistema Nervoso Simpático/anatomia & histologiaRESUMO
Karyotype is an important prognostic factor in childhood B-cell precursor acute lymphoblastic leukemia (BCP-ALL), but the underlying pharmacogenomics remain unknown. Asparaginase is an integral component in current chemotherapy for childhood BCP-ALL. Asparaginase therapy depletes serum asparagine. Normal hematopoietic cells can produce asparagine by asparagine synthetase (ASNS) activity, but ALL cells are unable to synthesize adequate amounts of asparagine. The ASNS gene has a typical CpG island in its promoter. Thus, methylation of the ASNS CpG island could be one of the epigenetic mechanisms for ASNS gene silencing in BCP-ALL. To gain deep insights into the pharmacogenomics of asparaginase therapy, we investigated the association of ASNS methylation status with asparaginase sensitivity. The ASNS CpG island is largely unmethylated in normal hematopoietic cells, but it is allele-specifically methylated in BCP-ALL cells. The ASNS gene is located at 7q21, an evolutionally conserved imprinted gene cluster. ASNS methylation in childhood BCP-ALL is associated with an aberrant methylation of the imprinted gene cluster at 7q21. Aberrant methylation of mouse Asns and a syntenic imprinted gene cluster is also confirmed in leukemic spleen samples from ETV6-RUNX1 knockin mice. In 3 childhood BCP-ALL cohorts, ASNS is highly methylated in BCP-ALL patients with favorable karyotypes but is mostly unmethylated in BCP-ALL patients with poor prognostic karyotypes. Higher ASNS methylation is associated with higher L-asparaginase sensitivity in BCP-ALL through lower ASNS gene and protein expression levels. These observations demonstrate that silencing of the ASNS gene as a result of aberrant imprinting is a pharmacogenetic mechanism for the leukemia-specific activity of asparaginase therapy in BCP-ALL.
Assuntos
Asparaginase/uso terapêutico , Aspartato-Amônia Ligase/genética , Variantes Farmacogenômicos/genética , Leucemia-Linfoma Linfoblástico de Células Precursoras B/tratamento farmacológico , Leucemia-Linfoma Linfoblástico de Células Precursoras B/genética , Animais , Criança , Aberrações Cromossômicas , Metilação de DNA/genética , Impressão Genômica/genética , Humanos , CamundongosAssuntos
Proteínas de Fusão Oncogênica , Leucemia-Linfoma Linfoblástico de Células Precursoras B , Linhagem Celular Tumoral , Humanos , Fatores de Transcrição MEF2/genética , Fatores de Transcrição MEF2/metabolismo , Proteínas de Fusão Oncogênica/genética , Proteínas de Fusão Oncogênica/metabolismo , Leucemia-Linfoma Linfoblástico de Células Precursoras B/genética , Leucemia-Linfoma Linfoblástico de Células Precursoras B/metabolismo , Leucemia-Linfoma Linfoblástico de Células Precursoras B/patologiaRESUMO
BACKGROUND: The genetic variants of the ARID5B gene have recently been reported to be associated with disease susceptibility and treatment outcome in childhood acute lymphoblastic leukemia (ALL). However, few studies have explored the association of ARID5B with sensitivities to chemotherapeutic agents. METHODS: We genotyped susceptibility-linked rs7923074 and rs10821936 as well as relapse-linked rs4948488, rs2893881, and rs6479778 of ARDI5B by direct sequencing of polymerase chain reaction (PCR) products in 72 B-cell precursor-ALL (BCP-ALL) cell lines established from Japanese patients. We also quantified their ARID5B expression levels by real-time reverse transcription PCR, and determined their 50% inhibitory concentration (IC50) values by alamarBlue assays in nine representative chemotherapeutic agents used for ALL treatment. RESULTS: No significant associations were observed in genotypes of the susceptibility-linked single nucleotide polymorphisms (SNPs) and the relapsed-linked SNPs with ARID5B gene expression levels. Of note, IC50 values of vincristine (VCR) (median IC50: 39.6 ng/ml) in 12 cell lines with homozygous genotype of risk allele (C) in the relapse-linked rs4948488 were significantly higher (p = 0.031 in Mann-Whitney U test) than those (1.04 ng/ml) in 60 cell lines with heterozygous or homozygous genotypes of the non-risk allele (T). Furthermore, the IC50 values of mafosfamide [Maf; active metabolite of cyclophosphamide (CY)] and cytarabine (AraC) tended to be associated with the genotype of rs4948488. Similar associations were observed in genotypes of the relapse-linked rs2893881 and rs6479778, but not in those of the susceptibility-linked rs7923074 and rs10821936. In addition, the IC50 values of methotrexate (MTX) were significantly higher (p = 0.023) in 36 cell lines with lower ARID5B gene expression (median IC50: 37.1 ng/ml) than those in the other 36 cell lines with higher expression (16.9 ng/ml). CONCLUSION: These observations in 72 BCP-ALL cell lines suggested that the risk allele of the relapse-linked SNPs of ARID5B may be involved in a higher relapse rate because of resistance to chemotherapeutic agents such as VCR, CY, and AraC. In addition, lower ARID5B gene expression may be associated with MTX resistance.
Assuntos
Endocardite Bacteriana/microbiologia , Infecções por Haemophilus/microbiologia , Haemophilus influenzae/patogenicidade , Ceftriaxona/administração & dosagem , Pré-Escolar , Endocardite Bacteriana/complicações , Endocardite Bacteriana/tratamento farmacológico , Infecções por Haemophilus/complicações , Infecções por Haemophilus/tratamento farmacológico , Haemophilus influenzae/classificação , Humanos , Masculino , Resultado do TratamentoAssuntos
Arabinonucleosídeos/farmacologia , Biomarcadores Tumorais/biossíntese , Desoxicitidina Quinase/biossíntese , Transportador Equilibrativo 1 de Nucleosídeo/biossíntese , Regulação Leucêmica da Expressão Gênica/efeitos dos fármacos , Proteínas de Neoplasias/biossíntese , Leucemia-Linfoma Linfoblástico de Células T Precursoras/tratamento farmacológico , Linhagem Celular Tumoral , Humanos , Leucemia-Linfoma Linfoblástico de Células T Precursoras/metabolismo , Leucemia-Linfoma Linfoblástico de Células T Precursoras/patologia , Valor Preditivo dos TestesRESUMO
t(17;19)(q21-q22;p13), responsible for TCF3-HLF fusion, is a rare translocation in childhood B-cell precursor acute lymphoblastic leukemia(BCP-ALL). t(1;19)(q23;p13), producing TCF3-PBX1 fusion, is a common translocation in childhood BCP-ALL. Prognosis of t(17;19)-ALL is extremely poor, while that of t(1;19)-ALL has recently improved dramatically in intensified chemotherapy. In this study, TCF3-HLF mRNA was detectable at a high level during induction therapy in a newly diagnosed t(17;19)-ALL case, while TCF3-PBX1 mRNA was undetectable at the end of induction therapy in most newly diagnosed t(1;19)-ALL cases. Using 4 t(17;19)-ALL and 16 t(1;19)-ALL cell lines, drug response profiling was analyzed. t(17;19)-ALL cell lines were found to be significantly more resistant to vincristine (VCR), daunorubicin (DNR), and prednisolone (Pred) than t(1;19)-ALL cell lines. Sensitivities to three (Pred, VCR, and l-asparaginase [l-Asp]), four (Pred, VCR, l-Asp, and DNR) and five (Pred, VCR, l-Asp, DNR, and cyclophosphamide) agents, widely used in induction therapy, were significantly poorer for t(17;19)-ALL cell lines than for t(1;19)-ALL cell lines. Consistent with poor responses to VCR and DNR, gene and protein expression levels of P-glycoprotein (P-gp) were higher in t(17;19)-ALL cell lines than in t(1;19)-ALL cell lines. Inhibitors for P-gp sensitized P-gp-positive t(17;19)-ALL cell lines to VCR and DNR. Knockout of P-gp by CRISPRCas9 overcame resistance to VCR and DNR in the P-gp-positive t(17;19)-ALL cell line. A combination of cyclosporine A with DNR prolonged survival of NSG mice inoculated with P-gp-positive t(17;19)-ALL cell line. These findings indicate involvement of P-gp in resistance to VCR and DNR in Pgp positive t(17;19)-ALL cell lines. In all four t(17;19)-ALL cell lines, RAS pathway mutation was detected. Furthermore, among 16 t(1;19)-ALL cell lines, multiagent resistance was usually observed in the cell lines with RAS pathway mutation in comparison to those without it, suggesting at least a partial involvement of RAS pathway mutation in multiagent resistance of t(17;19)-ALL.
Assuntos
Cromossomos Humanos Par 17 , Cromossomos Humanos Par 19 , Resistencia a Medicamentos Antineoplásicos/genética , Leucemia-Linfoma Linfoblástico de Células Precursoras/genética , Translocação Genética , Membro 1 da Subfamília B de Cassetes de Ligação de ATP/genética , Membro 1 da Subfamília B de Cassetes de Ligação de ATP/metabolismo , Alelos , Animais , Antineoplásicos/farmacologia , Antineoplásicos/uso terapêutico , Linhagem Celular , Modelos Animais de Doenças , Relação Dose-Resposta a Droga , Feminino , Frequência do Gene , Genótipo , Humanos , Imunofenotipagem , Camundongos , Neoplasia Residual/diagnóstico , Neoplasia Residual/genética , Leucemia-Linfoma Linfoblástico de Células Precursoras/diagnóstico , Leucemia-Linfoma Linfoblástico de Células Precursoras/tratamento farmacológicoRESUMO
Individual busulfan (BU) dosing based on pharmacokinetic (PK) data is preferable for hematopoietic stem cell transplantation (HSCT) conditioning, but information on BU PK in infants is scarce. We report BU PK data on HSCT conditioning for infants with KMT2A-gene-rearrangement-positive acute lymphoblastic leukemia (MLL-r ALL). Infants showed wide variation in BU PK indices, such as clearance (CL) and volume of distribution (Vd) value, which are distributed more widely among those who received oral, rather than intravenous (IV), BU. Because the steady state concentration (Css) fluctuates readily in infants, dose re-adjustment based on PK at the initial administration was important even if the initial dose was determined by a PK test. HSCT can be performed safely within the Css range of 600-900 ng/mL per dose, although it was difficult to fit within the therapeutic index of BU. The clinical outcome of engraftment, graft-versus-host disease, adverse events, including sinusoidal obstruction syndrome, and survival did not correlate with the BU PK data, which paradoxically suggests that remaining within this Css range helped minimize transplant-related toxicities, while securing engraftment in infants with MLL-r ALL.
Assuntos
Bussulfano , Transplante de Células-Tronco Hematopoéticas , Leucemia-Linfoma Linfoblástico de Células Precursoras , Condicionamento Pré-Transplante , Aloenxertos , Bussulfano/administração & dosagem , Bussulfano/farmacocinética , Intervalo Livre de Doença , Feminino , Humanos , Lactente , Masculino , Leucemia-Linfoma Linfoblástico de Células Precursoras/sangue , Leucemia-Linfoma Linfoblástico de Células Precursoras/mortalidade , Leucemia-Linfoma Linfoblástico de Células Precursoras/terapia , Taxa de SobrevidaRESUMO
JMML is an aggressive hematopoietic malignancy of early childhood, and allogeneic HSCT is the only curative treatment for this disease. Umbilical cord blood is one of donor sources for HSCT in JMML patients who do not have an HLA-compatible relative, but engraftment failure remains a major problem. Here, we report two cases of JMML who were successfully rescued by HSCT from an HLA-mismatched parent after development of primary engraftment failure following unrelated CBT. Both patients had severe splenomegaly and underwent unrelated CBT from an HLA-mismatched donor. Immediately after diagnosis of engraftment failure, both patients underwent HSCT from their parent. For the second HSCT, we used RIC regimens consisting of FLU, CY, and a low dose of rabbit ATG with or without TBI and additionally administered ETP considering their persistent severe splenomegaly. Both patients achieved engraftment without severe treatment-related adverse effects. After engraftment of second HSCT, their splenomegaly was rapidly regressed, and both patients showed no sign of relapse for over 4 years. These observations demonstrate that HSCT from an HLA-mismatched parent could be a feasible salvage treatment for primary engraftment failure in JMML patients.
Assuntos
Transplante de Células-Tronco de Sangue do Cordão Umbilical/métodos , Antígenos HLA/imunologia , Transplante de Células-Tronco Hematopoéticas/métodos , Células-Tronco Hematopoéticas/citologia , Leucemia Mielomonocítica Juvenil/terapia , Pré-Escolar , Feminino , Sangue Fetal , Hepatomegalia/cirurgia , Humanos , Imunossupressores/uso terapêutico , Lactente , Masculino , Mutação , Recidiva , Esplenomegalia/cirurgia , Condicionamento Pré-Transplante , Resultado do TratamentoRESUMO
In Kawasaki disease (KD), the effect of plasma exchange (PE) on immune cells has not been fully elucidated. Therefore, we examined the changes in the number of CD14+ CD16+ activated monocytes, regulatory T (Treg ), and T-helper type 17 (Th17) cells in KD patients treated with PE. The percentage of total monocytes and subclasses of lymphocytes, including CD4+ and CD8+ T cells, and CD19+ B cells, showed no significant difference before and after PE. However, the percentage of CD14+ CD16+ monocytes in total leukocytes decreased significantly after PE (1.1% ± 1.5% vs. 2.1% ± 2.3%, P < 0.05). Furthermore, while the percentage of Th17 cells in CD4+ T cells did not change, the percentage of Treg cells in CD4+ T cells increased significantly after PE (11.1% ± 5.1% vs. 8.0% ± 4.4%, P < 0.05). Therefore, PE downregulates activated monocytes and upregulates Treg cells toward normal levels and thus attenuates inflammation in KD.