RESUMO
Somatic hypermutation specifically modifies rearranged immunoglobulin (Ig) genes in germinal center (GC) B cells. However, the bcl-6 gene can also acquire somatic mutations during the GC reaction, indicating that certain non-Ig genes can be targeted by the somatic hypermutation machinery. The CD95 gene, implicated in negative selection of B lymphocytes in GCs, is specifically expressed by GC B cells and was recently identified as a tumor suppressor gene being frequently mutated in (post) GC B cell lymphomas. In this study, the 5' region (5'R) and/or the last exon coding for the death domain (DD) of the CD95 gene were investigated in naive, GC, and memory B cells from seven healthy donors. About 15% of GC and memory, but not naive, B cells carried mutations within the 5'R (mutation frequency 2.5 x 10(-4) per basepair). Mutations within the DD were very rare but could be efficiently selected by inducing CD95-mediated apoptosis: in 22 apoptosis-resistant cells, 12 DD mutations were found. These results indicate that human B cells can acquire somatic mutations of the CD95 gene during the GC reaction, which potentially confers apoptosis resistance and may counteract negative selection through the CD95 pathway.
Assuntos
Linfócitos B/imunologia , Linfócitos B/metabolismo , Centro Germinativo/imunologia , Mutagênese/genética , Receptor fas/genética , Apoptose , Clonagem Molecular , Análise Mutacional de DNA , Éxons/genética , Citometria de Fluxo , Genes Supressores de Tumor/genética , Humanos , Memória Imunológica/genética , Memória Imunológica/imunologia , Reação em Cadeia da Polimerase , Estrutura Terciária de Proteína , Análise de Sequência , Transdução de Sinais , Receptor fas/química , Receptor fas/imunologiaRESUMO
Recent work identified Hodgkin and Reed-Sternberg (H/RS) cells in classical Hodgkin's disease (cHD) as clonal progeny of mature B cells. Therefore, it is generally assumed that cHD homogenously represents a B cell lymphoma. In a subset of cHD, however, H/RS cells expressing T cell-associated proteins may be candidates for alternative lineage derivation. Single H/RS cells with cytotoxic T cell phenotype were micromanipulated from three cases of cHD and analyzed by single cell polymerase chain reaction for immunoglobulin heavy (IgH) and light chain (IgL) gene rearrangements, T cell receptor (TCR)-beta gene rearrangements, and germline configuration of the IgH and TCR-beta loci. H/RS cells from two cases of cHD harbored clonal, somatically mutated Ig gene rearrangements, whereas TCR-beta loci were in germline configuration. In contrast, H/RS cells from an additional case harbored clonal TCR-beta variable/diversity/joining (VDJ) and DJ gene rearrangements, whereas the IgH locus was in germline configuration on both alleles. Thus, in two cases of cHD with H/RS cells expressing cytotoxic T cell molecules, the tumor cells are derived from mature B cells that aberrantly express T cell markers. In a third case, however, H/RS cells were derived from a T cell, demonstrating that cHD can also occur as a T cell lymphoma.
Assuntos
Doença de Hodgkin/imunologia , Linfoma de Células T/imunologia , Células de Reed-Sternberg/imunologia , Adulto , Sequência de Bases , Humanos , Masculino , Pessoa de Meia-Idade , Dados de Sequência MolecularRESUMO
Although substantial progress has been made in the treatment of B-cell acute lymphoblastic leukemia (B-ALL), the prognosis of patients with either refractory or relapsed B-ALL remains dismal. Novel therapeutic strategies are needed to improve the outcome of these patients. KPT-9274 is a novel dual inhibitor of p21-activated kinase 4 (PAK4) and nicotinamide phosphoribosyltransferase (NAMPT). PAK4 is a serine/threonine kinase that regulates a variety of fundamental cellular processes. NAMPT is a rate-limiting enzyme in the salvage biosynthesis pathway of nicotinamide adenine dinucleotide (NAD) that plays a vital role in energy metabolism. Here, we show that KPT-9274 strongly inhibits B-ALL cell growth regardless of cytogenetic abnormalities. We also demonstrate the potent in vivo efficacy and tolerability of KPT-9274 in a patient-derived xenograft murine model of B-ALL. Interestingly, although KPT-9274 is a dual PAK4/NAMPT inhibitor, B-ALL cell growth inhibition by KPT-9274 was largely abolished with nicotinic acid supplementation, indicating that the inhibitory effects on B-ALL cells are mainly exerted by NAD+ depletion through NAMPT inhibition. Moreover, we have found that the extreme susceptibility of B-ALL cells to NAMPT inhibition is related to the reduced cellular NAD+ reserve. NAD+ depletion may be a promising alternative approach to treating patients with B-ALL.
Assuntos
NAD/metabolismo , Leucemia-Linfoma Linfoblástico de Células Precursoras B/metabolismo , Acrilamidas/química , Acrilamidas/farmacologia , Aminopiridinas/química , Aminopiridinas/farmacologia , Animais , Antineoplásicos/química , Antineoplásicos/farmacologia , Apoptose/efeitos dos fármacos , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Citocinas/antagonistas & inibidores , Modelos Animais de Doenças , Feminino , Humanos , Masculino , Camundongos , Nicotinamida Fosforribosiltransferase/antagonistas & inibidores , Leucemia-Linfoma Linfoblástico de Células Precursoras B/tratamento farmacológico , Leucemia-Linfoma Linfoblástico de Células Precursoras B/patologia , Transdução de Sinais/efeitos dos fármacos , Ensaios Antitumorais Modelo de Xenoenxerto , Quinases Ativadas por p21/antagonistas & inibidoresRESUMO
Perpetual V(D)J recombinase activity involving multiple DNA double-strand break events in B-cell lineage leukemia and lymphoma cells may introduce secondary genetic aberrations leading towards malignant progression. Here, we investigated defective negative feedback signaling through the (pre-) B-cell receptor as a possible reason for deregulated V(D)J recombinase activity in B-cell malignancy. On studying 28 cases of pre-B-lymphoblastic leukemia and 27 B-cell lymphomas, expression of the (pre-) B-cell receptor-related linker molecule SLP65 (SH2 domain-containing lymphocyte protein of 65 kDa) was found to be defective in seven and five cases, respectively. SLP65 deficiency correlates with RAG1/2 expression and unremitting V(H) gene rearrangement activity. Reconstitution of SLP65 expression in SLP65-deficient leukemia and lymphoma cells results in downregulation of RAG1/2 expression and prevents both de novo V(H)-DJ(H) rearrangements and secondary V(H) replacement. We conclude that iterative V(H) gene rearrangement represents a frequent feature in B-lymphoid malignancy, which can be attributed to SLP65 deficiency in many cases.
Assuntos
Linfoma de Burkitt/genética , Proteínas de Transporte/genética , Linfoma de Células B/genética , Fosfoproteínas/deficiência , Fosfoproteínas/genética , VDJ Recombinases/metabolismo , Proteínas Adaptadoras de Transdução de Sinal , Sequência de Bases , Linfoma de Burkitt/enzimologia , Linhagem Celular Tumoral , Dano ao DNA , Rearranjo Gênico , Genes de Imunoglobulinas , Humanos , Dados de Sequência Molecular , Deleção de Sequência , VDJ Recombinases/genéticaRESUMO
SLP65 represents a critical component in (pre-) B cell receptor signal transduction but is compromised in a subset of pre-B cell-derived acute lymphoblastic leukemia. Based on these findings, we investigated (i.) whether SLP65-deficiency also occurs in mature B cell-derived lymphoma and (ii.) whether SLP65-deficient B cell lymphoma cells use an alternative B cell receptor signaling pathway in the absence of SLP65. Indeed, expression of SLP65 protein was also missing in a fraction of B cell lymphoma cases. While SLP65 is essential for B cell receptor-induced Ca2+ mobilization in normal B cells, B cell receptor engagement in SLP65-deficient as compared to SLP65-reconstituted B cell lymphoma cells resulted in an accelerated yet shortlived Ca2+-signal. B cell receptor engagement of SLP65-deficient lymphoma cells involves SRC kinase activation, which is critical for B cell receptor-dependent Ca2+-mobilisation in the absence but not in the presence of SLP65. As shown by RNA interference, the SRC kinase LYN is required for B cell receptor-induced Ca2+ release in SLP65-deficient B cell lymphoma cells but dispensable after SLP65-reconstitution. B cell receptor engagement in SLP65-deficient B cell lymphoma cells also resulted in tyrosine-phosphorylation of the proliferation- and survival-related MAPK1 and STAT5 molecules, which was sensitive to silencing of the SRC kinase LYN. Inhibition of SRC kinase activity resulted in growth arrest and cell death specifically in SLP65-deficient lymphoma cells. These findings indicate that LYN can short-circuit conventional B cell receptor signaling in SLP65-deficient B cell lymphoma cells and thereby promote activation of survival and proliferation-related molecules.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal/genética , Linfoma de Células B/enzimologia , Transdução de Sinais , Quinases da Família src/metabolismo , Humanos , Linfoma de Células B/patologia , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Fosforilação , Fator de Transcrição STAT5/metabolismoRESUMO
Pre-B lymphoblastic leukemia cells carrying a BCR-ABL1 gene rearrangement exhibit an undifferentiated phenotype. Comparing the genome-wide gene expression profiles of normal B-cell subsets and BCR-ABL1+ pre-B lymphoblastic leukemia cells by SAGE, the leukemia cells show loss of B lymphoid identity and aberrant expression of myeloid lineage-specific molecules. Consistent with this, BCR-ABL1+ pre-B lymphoblastic leukemia cells exhibit defective expression of IKAROS, a transcription factor needed for early lymphoid lineage commitment. As shown by inducible expression of BCR-ABL1 in human and murine B-cell precursor cell lines, BCR-ABL1 induces the expression of a dominant-negative IKAROS splice variant, termed IK6. Comparing matched leukemia sample pairs from patients before and during therapy with the BCR-ABL1 kinase inhibitor STI571 (Imatinib), inhibition of BCR-ABL1 partially corrected aberrant expression of IK6 and lineage infidelity of the leukemia cells. To elucidate the contribution of IK6 to lineage infidelity in BCR-ABL1+ cell lines, IK6 expression was silenced by RNA interference. Upon inhibition of IK6, BCR-ABL1+ leukemia cells partially restored B lymphoid lineage commitment. Therefore, we propose that BCR-ABL1 induces aberrant splicing of IKAROS, which interferes with lineage identity and differentiation of pre-B lymphoblastic leukemia cells.
Assuntos
Processamento Alternativo , Fator de Transcrição Ikaros/genética , Leucemia-Linfoma Linfoblástico de Células Precursoras B/enzimologia , Leucemia-Linfoma Linfoblástico de Células Precursoras B/genética , Proteínas Tirosina Quinases/metabolismo , Animais , Antineoplásicos/farmacologia , Antineoplásicos/uso terapêutico , Benzamidas , Linhagem Celular Tumoral , Linhagem da Célula/genética , Núcleo Celular/química , Proteínas de Fusão bcr-abl , Perfilação da Expressão Gênica , Inativação Gênica , Humanos , Fator de Transcrição Ikaros/análise , Fator de Transcrição Ikaros/metabolismo , Mesilato de Imatinib , Camundongos , Piperazinas/farmacologia , Leucemia-Linfoma Linfoblástico de Células Precursoras B/dietoterapia , Inibidores de Proteínas Quinases/farmacologia , Inibidores de Proteínas Quinases/uso terapêutico , Proteínas Tirosina Quinases/análise , Pirimidinas/farmacologiaRESUMO
Donor-specific (DST) or nonspecific blood transfusions administered before transplantation can enhance survival of vascularized allografts both in humans and animals but the immunological mechanisms of this effect remain unclear. We have analyzed the expression and the role of endogenous TGF-beta1 in a model of heart allograft tolerance, induced by pregraft DST in adult rats. We reported previously that this tolerance occurs despite a strong infiltration of leukocytes into the graft that are unable to produce both Th1- and Th2-related cytokines in vivo. Allografts from DST-treated rats express high levels of TGF-beta1 mRNA and active protein. This phenomenon is correlated with the rapid infiltration of leukocytes producing high amounts of TGF-beta1. TGF-beta1-producing cells are virtually absent among early infiltrating cells in rejected grafts but are found at a later time point. The induction of allograft tolerance in vivo is abrogated by administration of neutralizing anti-TGF-beta mAb. Moreover, overexpression of active TGF- beta1 in heart allografts using a recombinant adenovirus leads to prolonged graft survival in unmodified recipients. Taken together, our results identify TGF-beta as a critical cytokine involved in the suppression of allograft rejection induced by DST and suggest that TGF-beta-producing regulatory cells are also involved in allograft tolerance.
Assuntos
Transfusão de Sangue , Facilitação Imunológica de Enxerto , Sobrevivência de Enxerto/fisiologia , Transplante de Coração , Fator de Crescimento Transformador beta/fisiologia , Animais , Anticorpos Monoclonais/farmacologia , Rejeição de Enxerto/imunologia , Leucócitos/metabolismo , Teste de Cultura Mista de Linfócitos , Masculino , RNA Mensageiro/biossíntese , Ratos , Ratos Endogâmicos BUF , Ratos Endogâmicos Lew , Baço/imunologia , Doadores de Tecidos , Fator de Crescimento Transformador beta/antagonistas & inibidores , Fator de Crescimento Transformador beta/biossíntese , Fator de Crescimento Transformador beta/imunologia , Transplante HomólogoRESUMO
The frequency of poor outcomes in relapsed leukemia patients underscores the need for novel therapeutic approaches. The Food and Drug Administration-approved immunosuppressant FTY720 limits leukemia progression by activating protein phosphatase 2A and restricting nutrient access. Unfortunately, FTY720 cannot be re-purposed for use in cancer patients due to on-target toxicity associated with S1P receptor activation at the elevated, anti-neoplastic dose. Here we show that the constrained azacyclic FTY720 analog SH-RF-177 lacks S1P receptor activity but maintains anti-leukemic activity in vitro and in vivo. SH-RF-177 was not only more potent than FTY720, but killed via a distinct mechanism. Phosphorylation is dispensable for FTY720's anti-leukemic actions. However, chemical biology and genetic approaches demonstrated that the sphingosine kinase 2 (SPHK2)-mediated phosphorylation of SH-RF-177 led to engagement of a pro-apoptotic target and increased potency. The cytotoxicity of membrane-permeant FTY720 phosphonate esters suggests that the enhanced potency of SH-RF-177 stems from its more efficient phosphorylation. The tight inverse correlation between SH-RF-177 IC50 and SPHK2 mRNA expression suggests a useful biomarker for SH-RF-177 sensitivity. In summary, these studies indicate that FTY720 analogs that are efficiently phosphorylated but fail to activate S1P receptors may be superior anti-leukemic agents compared to compounds that avoid cardiotoxicity by eliminating phosphorylation.
Assuntos
Antineoplásicos/farmacologia , Cloridrato de Fingolimode/farmacologia , Receptores de Lisoesfingolipídeo/metabolismo , Animais , Apoptose/efeitos dos fármacos , Linhagem Celular Tumoral , Modelos Animais de Doenças , Resistencia a Medicamentos Antineoplásicos/genética , Feminino , Humanos , Leucemia/tratamento farmacológico , Leucemia/genética , Leucemia/metabolismo , Leucemia/patologia , Camundongos , Fosforilação , Fosfotransferases (Aceptor do Grupo Álcool)/genética , Receptores de Lisoesfingolipídeo/agonistas , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Hodgkin and Reed Sternberg (H-RS) cells represent the malignant cells in classical Hodgkin's disease. Although derived from germinal center B cells, they do not express surface immunoglobulin. This has been explained by the presence of crippling mutations within the immunoglobulin genes in numerous cases of Hodgkin's disease. As immunoglobulin gene expression in B cells requires an interaction between octamer sites and the transactivating factors Oct-2 and Bob-1, this study addresses the expression of the transcription factors Oct-2 and Bob-1 in H-RS cells. In Hodgkin's disease-derived cell lines, low levels of Oct-2 transcripts but no Oct-2 protein were detected. Transcripts of Bob-1, a B-cell-specific co-factor of Oct-2, could not be observed in these cell lines. Absence of Oct-2 and Bob-1 protein expression in primary H-RS cells was demonstrated by performing immunohistochemistry in 20 cases of classical Hodgkin's disease. H-RS cells stained negative for both proteins in all of the cases analyzed. In conclusion, absence of functional Oct-2 and Bob-1 cells represents a novel mechanism for immunoglobulin gene deregulation in H-RS cells. Lack of Oct-2 and Bob-1 points to a defect in transcription machinery in H-RS cells and is associated with lack of immunoglobulin gene expression in these cells.
Assuntos
Proteínas de Ligação a DNA/deficiência , Doença de Hodgkin/metabolismo , Células de Reed-Sternberg/metabolismo , Transativadores/deficiência , Fatores de Transcrição/deficiência , Células 3T3 , Adolescente , Adulto , Idoso , Animais , Proteínas de Ligação a DNA/biossíntese , Proteínas de Ligação a DNA/genética , Genes de Imunoglobulinas/genética , Humanos , Camundongos , Pessoa de Meia-Idade , Fator 2 de Transcrição de Octâmero , RNA Mensageiro/biossíntese , RNA Mensageiro/genética , Transativadores/biossíntese , Transativadores/genética , Fatores de Transcrição/biossíntese , Fatores de Transcrição/genética , Transcrição Gênica , Células Tumorais CultivadasRESUMO
Hodgkin and Reed-Sternberg (H/RS) cells in classical Hodgkin's disease (cHD) are thought to be derived from preapoptotic germinal center B cells. However, little is known about the transforming events rescuing the precursor of the H/RS cells from apoptosis. Given the importance of CD95 (Apo-1/Fas)-mediated apoptosis for negative selection within the germinal center, single micromanipulated H/RS cells from 10 cases of cHD were analyzed for somatic mutations within the CD95 gene. Three clonal mutations within the 5' regions were amplified from single H/RS cells in one case. From H/RS cells of another case, two mutations within the last exon coding for the death domain were detected. About half of these H/RS cells carried a monoallelic stop-codon; the remaining tumor cells harbored a monoallelic replacement mutation. Both mutations likely impair CD95 function. Because all these H/RS cells also bear clonal mutations inactivating the IkappaB alpha gene, the IkappaB alpha mutations occurred earlier than those of the CD95 gene in the sequence of transforming events leading to cHD. In conclusion, somatic mutations of the CD95 gene occur in a fraction of cHD cases and may favor the escape of the precursor of the H/RS clone from apoptosis.
Assuntos
Doença de Hodgkin/genética , Mutação , Células de Reed-Sternberg/fisiologia , Receptor fas/genética , Adulto , Idoso , Apoptose/genética , Criança , Feminino , Amplificação de Genes , Doença de Hodgkin/patologia , Humanos , Masculino , Pessoa de Meia-Idade , Reação em Cadeia da PolimeraseRESUMO
BCOR is a component of a variant Polycomb group repressive complex 1 (PRC1). Recently, we and others reported recurrent somatic BCOR loss-of-function mutations in myelodysplastic syndrome and acute myelogenous leukemia (AML). However, the role of BCOR in normal hematopoiesis is largely unknown. Here, we explored the function of BCOR in myeloid cells using myeloid murine models with Bcor conditional loss-of-function or overexpression alleles. Bcor mutant bone marrow cells showed significantly higher proliferation and differentiation rates with upregulated expression of Hox genes. Mutation of Bcor reduced protein levels of RING1B, an H2A ubiquitin ligase subunit of PRC1 family complexes and reduced H2AK119ub upstream of upregulated HoxA genes. Global RNA expression profiling in murine cells and AML patient samples with BCOR loss-of-function mutation suggested that loss of BCOR expression is associated with enhanced cell proliferation and myeloid differentiation. Our results strongly suggest that BCOR plays an indispensable role in hematopoiesis by inhibiting myeloid cell proliferation and differentiation and offer a mechanistic explanation for how BCOR regulates gene expression such as Hox genes.
Assuntos
Diferenciação Celular , Proliferação de Células , Células Progenitoras Mieloides/citologia , Proteínas Repressoras/fisiologia , Animais , Regulação da Expressão Gênica , Genes Homeobox/genética , Hematopoese , Humanos , Leucemia Mieloide Aguda/patologia , Camundongos , Mutagênese Sítio-Dirigida , Complexo Repressor Polycomb 1/fisiologia , Proteínas Repressoras/genéticaRESUMO
Precursor-B-cell receptor (pre-BCR) signaling and spleen tyrosine kinase (SYK) recently were introduced as therapeutic targets for patients with B-cell acute lymphoblastic leukemia (B-ALL), but the importance of this pathway in B-ALL subsets and mechanism of downstream signaling have not fully been elucidated. Here, we provide new detailed insight into the mechanism of pre-BCR signaling in B-ALL. We compared the effects of pharmacological and genetic disruption of pre-BCR signaling in vitro and in mouse models for B-ALL, demonstrating exquisite dependency of pre-BCR(+) B-ALL, but not other B-ALL subsets, on this signaling pathway. We demonstrate that SYK, PI3K/AKT, FOXO1 and MYC are important downstream mediators of pre-BCR signaling in B-ALL. Furthermore, we define a characteristic immune phenotype and gene expression signature of pre-BCR(+) ALL to distinguish them from other B-ALL subsets. These data provide comprehensive new insight into pre-BCR signaling in B-ALL and corroborate pre-BCR signaling and SYK as promising new therapeutic targets in pre-BCR(+) B-ALL.
Assuntos
Leucemia-Linfoma Linfoblástico de Células Precursoras B/metabolismo , Células Precursoras de Linfócitos B/química , Receptores de Antígenos de Linfócitos B/metabolismo , Transdução de Sinais , Animais , Linhagem Celular , Proteína Forkhead Box O1/metabolismo , Xenoenxertos , Humanos , Camundongos , Fosfatidilinositol 3-Quinases/metabolismo , Leucemia-Linfoma Linfoblástico de Células Precursoras B/patologia , Proteínas Proto-Oncogênicas c-myc/metabolismo , Quinase Syk/metabolismoRESUMO
The CD95 (Apo-1/Fas) receptor-ligand system is one of the key regulators of apoptosis and is particularly important for the maintenance of lymphocyte homeostasis. There is now broad evidence that susceptibility of tumor cells towards CD95-mediated apoptosis is largely reduced. In the human, germline and somatic mutations of the CD95 gene are associated with a high risk of both lymphoid and solid tumors. Based on these observations a new concept defining CD95 as a tumor suppressor gene is discussed. In addition to CD95, its natural ligand (CD95L) is also implicated in malignant progression. Compared to their nonmalignant counterparts, malignant cells frequently exhibit aberrant de novo expression of CD95L and are able to induce CD95L-mediated apoptosis in bystander cells. The role for neoplastic CD95L expression in local tissue destruction, invasion, and metastatic spread has been established for many tumor types. CD95L expression by malignant cells may counteract the host's antitumor immunity and favors immune escape of the tumor. On this basis, the significance of loss of CD95 and gain of CD95L expression for tumor progression is discussed.
Assuntos
Expressão Gênica , Genes Supressores de Tumor/genética , Glicoproteínas de Membrana/genética , Glicoproteínas de Membrana/metabolismo , Neoplasias/genética , Receptor fas/genética , Receptor fas/metabolismo , Apoptose , Transformação Celular Neoplásica , Proteína Ligante Fas , Humanos , Glicoproteínas de Membrana/imunologia , Mutação , Metástase Neoplásica , Neoplasias/imunologia , Neoplasias/patologia , Evasão Tumoral , Receptor fas/imunologiaRESUMO
BACKGROUND: Cytotoxic T cells can induce target cell lysis and apoptosis by different pathways. The interactions of CD95 antigen (Fas) with its ligand (CD95L) and of tumor necrosis factor (TNF)-alpha with its receptor (TNF-R1) lead to apoptotic cell death. Recently, conflicting studies have been published concerning the expression and the role of CD95L in allograft rejection and tolerance. METHODS: In this study, the intragraft expression of CD95/CD95L and TNF-alpha and the frequency and distribution of apoptotic cells were compared in a model of heterotopic cardiac allograft in the rat in which recipients were either not treated (acute rejection) or pretreated with donor-specific blood transfusion (tolerant). RESULTS: In the acutely rejected allografts, a peak in the expression of CD95L and TNF-alpha and in the number of apoptotic cells was observed during the first week after transplantation; apoptotic cells were confined to graft-infiltrating cells. In the tolerated allografts, however, levels of graft-infiltrating cell apoptosis and CD95L and TNF-alpha expression during the same period of time were dramatically lower. The expression of Fas was constitutive and was not modulated during acute rejection or tolerance. CONCLUSION: This down-regulation of CD95L and TNF-alpha in allografts rendered tolerant by donor-specific transfusion suggests a role for apoptosis-inducing pathways in acute allograft rejection.
Assuntos
Apoptose/fisiologia , Rejeição de Enxerto/metabolismo , Rejeição de Enxerto/fisiopatologia , Transplante de Coração , Tolerância Imunológica/fisiologia , Glicoproteínas de Membrana/metabolismo , Fator de Necrose Tumoral alfa/metabolismo , Doença Aguda , Animais , Transfusão de Sangue , Proteína Ligante Fas , Rejeição de Enxerto/imunologia , Rejeição de Enxerto/patologia , Masculino , Miocárdio/imunologia , Miocárdio/patologia , Ratos , Ratos Endogâmicos Lew , Doadores de Tecidos , Transplante Heterotópico , Transplante Homólogo , Receptor fas/análiseRESUMO
Drug resistance in acute lymphoblastic leukemia (ALL) remains a major problem warranting new treatment strategies. Wnt/catenin signaling is critical for the self-renewal of normal hematopoietic progenitor cells. Deregulated Wnt signaling is evident in chronic and acute myeloid leukemia; however, little is known about ALL. Differential interaction of catenin with either the Kat3 coactivator CREBBP (CREB-binding protein (CBP)) or the highly homologous EP300 (p300) is critical to determine divergent cellular responses and provides a rationale for the regulation of both proliferation and differentiation by the Wnt signaling pathway. Usage of the coactivator CBP by catenin leads to transcriptional activation of cassettes of genes that are involved in maintenance of progenitor cell self-renewal. However, the use of the coactivator p300 leads to activation of genes involved in the initiation of differentiation. ICG-001 is a novel small-molecule modulator of Wnt/catenin signaling, which specifically binds to the N-terminus of CBP and not p300, within amino acids 1-110, thereby disrupting the interaction between CBP and catenin. Here, we report that selective disruption of the CBP/ß- and γ-catenin interactions using ICG-001 leads to differentiation of pre-B ALL cells and loss of self-renewal capacity. Survivin, an inhibitor-of-apoptosis protein, was also downregulated in primary ALL after treatment with ICG-001. Using chromatin immunoprecipitation assay, we demonstrate occupancy of the survivin promoter by CBP that is decreased by ICG-001 in primary ALL. CBP mutations have been recently identified in a significant percentage of ALL patients, however, almost all of the identified mutations reported occur C-terminal to the binding site for ICG-001. Importantly, ICG-001, regardless of CBP mutational status and chromosomal aberration, leads to eradication of drug-resistant primary leukemia in combination with conventional therapy in vitro and significantly prolongs the survival of NOD/SCID mice engrafted with primary ALL. Therefore, specifically inhibiting CBP/catenin transcription represents a novel approach to overcome relapse in ALL.
Assuntos
Antineoplásicos/farmacologia , Compostos Bicíclicos Heterocíclicos com Pontes/farmacologia , Fragmentos de Peptídeos/metabolismo , Leucemia-Linfoma Linfoblástico de Células Precursoras/tratamento farmacológico , Pirimidinonas/farmacologia , Sialoglicoproteínas/metabolismo , beta Catenina/metabolismo , Animais , Asparaginase/farmacologia , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Dexametasona/farmacologia , Regulação para Baixo/efeitos dos fármacos , Resistencia a Medicamentos Antineoplásicos , Sinergismo Farmacológico , Humanos , Proteínas Inibidoras de Apoptose/genética , Proteínas Inibidoras de Apoptose/metabolismo , Camundongos , Camundongos Endogâmicos NOD , Camundongos SCID , Mutação , Fragmentos de Peptídeos/antagonistas & inibidores , Fragmentos de Peptídeos/genética , Sialoglicoproteínas/antagonistas & inibidores , Sialoglicoproteínas/genética , Survivina , Vincristina/farmacologia , Via de Sinalização Wnt , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
ABL gene translocations create constitutively active tyrosine kinases that are causative in chronic myeloid leukemia, acute lymphocytic leukemia and other hematopoietic malignancies. Consistent retention of ABL SH3/SH2 autoinhibitory domains, however, suggests that these leukemogenic tyrosine kinase fusion proteins remain subject to regulation. We resolve this paradox, demonstrating that BCR-ABL1 kinase activity is regulated by RIN1, an ABL SH3/SH2 binding protein. BCR-ABL1 activity was increased by RIN1 overexpression and decreased by RIN1 silencing. Moreover, Rin1(-/-) bone marrow cells were not transformed by BCR-ABL1, ETV6-ABL1 or BCR-ABL1(T315I), a patient-derived drug-resistant mutant, as judged by growth factor independence. Rescue by ectopic RIN1 verified a cell autonomous mechanism of collaboration with BCR-ABL1 during transformation. Sensitivity to the ABL kinase inhibitor imatinib was increased by RIN1 silencing, consistent with RIN1 stabilization of an activated BCR-ABL1 conformation having reduced drug affinity. The dependence on activation by RIN1 to unleash full catalytic and cell transformation potential reveals a previously unknown vulnerability that could be exploited for treatment of leukemic cases driven by ABL translocations. The findings suggest that RIN1 targeting could be efficacious for imatinib-resistant disease and might complement ABL kinase inhibitors in first-line therapy.
Assuntos
Transformação Celular Neoplásica , Proteínas de Fusão bcr-abl , Genes abl , Peptídeos e Proteínas de Sinalização Intracelular/fisiologia , Inibidores de Proteínas Quinases/farmacologia , Animais , Benzamidas , Humanos , Mesilato de Imatinib , Células K562 , Camundongos , Piperazinas/farmacologia , Pirimidinas/farmacologia , Translocação Genética , Domínios de Homologia de srcRESUMO
Dasatinib is a potent dual Abl/Src inhibitor approved for treatment of Philadelphia chromosome-positive (Ph-positive) leukemias. At a once-daily dose and a relatively short half-life of 3-5 h, tyrosine kinase inhibition is not sustained. However, transient inhibition of K562 leukemia cells with a high-dose pulse of dasatinib or long-term treatment with a lower dose was reported to irreversibly induce apoptosis. Here, the effect of dasatinib on treatment of Bcr/Abl-positive acute lymphoblastic leukemia (ALL) cells was evaluated in the presence of stromal support. Dasatinib eradicated Bcr/Abl ALL cells, caused significant apoptosis and eliminated tyrosine phosphorylation on Bcr/Abl, Src, Crkl and Stat-5. However, treatment of mouse ALL cells with lower doses of dasatinib over an extended period of time allowed the emergence of viable drug-resistant cells. Interestingly, dasatinib treatment increased cell-surface expression of CXCR4, which is important for survival of B-lineage cells, but this did not promote survival. Combined treatment of cells with dasatinib and a CXCR4 inhibitor resulted in enhanced cell death. These results do not support the concept that long-term treatment with low-dose dasatinib monotherapy will be effective in causing irreversible apoptosis in Ph-positive ALL, but suggest that combined treatment with dasatinib and drugs such as AMD3100 may be effective.
Assuntos
Resistencia a Medicamentos Antineoplásicos , Proteínas de Fusão bcr-abl/metabolismo , Leucemia Experimental/tratamento farmacológico , Leucemia-Linfoma Linfoblástico de Células Precursoras/tratamento farmacológico , Inibidores de Proteínas Quinases/farmacologia , Proteínas Tirosina Quinases/metabolismo , Pirimidinas/farmacologia , Tiazóis/farmacologia , Animais , Apoptose/efeitos dos fármacos , Western Blotting , Células Cultivadas , Dasatinibe , Embrião de Mamíferos/citologia , Embrião de Mamíferos/efeitos dos fármacos , Embrião de Mamíferos/metabolismo , Fibroblastos/efeitos dos fármacos , Fibroblastos/metabolismo , Proteínas de Fusão bcr-abl/antagonistas & inibidores , Humanos , Leucemia Experimental/metabolismo , Leucemia Experimental/patologia , Camundongos , Camundongos Endogâmicos NOD , Camundongos Knockout , Camundongos SCID , Fosforilação , Leucemia-Linfoma Linfoblástico de Células Precursoras/metabolismo , Leucemia-Linfoma Linfoblástico de Células Precursoras/patologia , Proteínas Tirosina Quinases/antagonistas & inibidores , Receptores CXCR4/metabolismo , Células Estromais/efeitos dos fármacos , Células Estromais/metabolismo , Células Estromais/efeitos da radiação , Quinases da Família src/antagonistas & inibidores , Quinases da Família src/metabolismoRESUMO
Mantle cell lymphoma (MCL) is an IgM-expressing B cell lymphoma that originates from naive B cells and responds poorly to chemotherapy. We show here that several MCLs harbour isotype-switched subclones. Similar to the situation in normal B cells, in vitro stimulation of MCL cell lines with CD40 ligand (CD40L) and interleukin-4 induced expression of activation-induced cytidine deaminase (AID) and germline transcription at the immunoglobulin heavy chain gene locus. Additionally, the occurrence of switch-circle transcripts and mature IgG transcripts after stimulation indicated ongoing class-switch recombination in mantle cell lymphoma cell lines. Furthermore, stimulation of primary MCL cells in vitro induced expression of class-switched IgG mRNA in the tumour cells. Our data indicate that mantle cell lymphomas have retained the ability to undergo class-switch recombination if appropriate stimuli, such as the CD40 ligand, are provided.