RÉSUMÉ
Even though SYK and ZAP70 kinases share high sequence homology and serve analogous functions, their expression in B and T cells is strictly segregated throughout evolution. Here, we identified aberrant ZAP70 expression as a common feature in a broad range of B cell malignancies. We validated SYK as the kinase that sets the thresholds for negative selection of autoreactive and premalignant clones. When aberrantly expressed in B cells, ZAP70 competes with SYK at the BCR signalosome and redirects SYK from negative selection to tonic PI3K signaling, thereby promoting B cell survival. In genetic mouse models for B-ALL and B-CLL, conditional expression of Zap70 accelerated disease onset, while genetic deletion impaired malignant transformation. Inducible activation of Zap70 during B cell development compromised negative selection of autoreactive B cells, resulting in pervasive autoantibody production. Strict segregation of the two kinases is critical for normal B cell selection and represents a central safeguard against the development of autoimmune disease and B cell malignancies.
Sujet(s)
Auto-immunité , Tumeurs/enzymologie , Tumeurs/prévention et contrôle , Syk kinase/métabolisme , ZAP-70 Protein-tyrosine kinase/métabolisme , Animaux , Antigènes CD19/métabolisme , Lymphocytes B , Calcium/métabolisme , Différenciation cellulaire , Transformation cellulaire néoplasique , Activation enzymatique , Humains , Tolérance immunitaire , Lymphome B/enzymologie , Lymphome B/anatomopathologie , Souris , Modèles génétiques , Facteurs de transcription NFATC/métabolisme , Protéines tumorales , Phosphatidylinositol 3-kinases/métabolisme , Liaison aux protéines , Récepteurs pour l'antigène des lymphocytes B/métabolisme , Transduction du signalRÉSUMÉ
Interferon-induced transmembrane protein 3 (IFITM3) has previously been identified as an endosomal protein that blocks viral infection1-3. Here we studied clinical cohorts of patients with B cell leukaemia and lymphoma, and identified IFITM3 as a strong predictor of poor outcome. In normal resting B cells, IFITM3 was minimally expressed and mainly localized in endosomes. However, engagement of the B cell receptor (BCR) induced both expression of IFITM3 and phosphorylation of this protein at Tyr20, which resulted in the accumulation of IFITM3 at the cell surface. In B cell leukaemia, oncogenic kinases phosphorylate IFITM3 at Tyr20, which causes constitutive localization of this protein at the plasma membrane. In a mouse model, Ifitm3-/- naive B cells developed in normal numbers; however, the formation of germinal centres and the production of antigen-specific antibodies were compromised. Oncogenes that induce the development of leukaemia and lymphoma did not transform Ifitm3-/- B cells. Conversely, the phosphomimetic IFITM3(Y20E) mutant induced oncogenic PI3K signalling and initiated the transformation of premalignant B cells. Mechanistic experiments revealed that IFITM3 functions as a PIP3 scaffold and central amplifier of PI3K signalling. The amplification of PI3K signals depends on IFITM3 using two lysine residues (Lys83 and Lys104) in its conserved intracellular loop as a scaffold for the accumulation of PIP3. In Ifitm3-/- B cells, lipid rafts were depleted of PIP3, which resulted in the defective expression of over 60 lipid-raft-associated surface receptors, and impaired BCR signalling and cellular adhesion. We conclude that the phosphorylation of IFITM3 that occurs after B cells encounter antigen induces a dynamic switch from antiviral effector functions in endosomes to a PI3K amplification loop at the cell surface. IFITM3-dependent amplification of PI3K signalling, which in part acts downstream of the BCR, is critical for the rapid expansion of B cells with high affinity to antigen. In addition, multiple oncogenes depend on IFITM3 to assemble PIP3-dependent signalling complexes and amplify PI3K signalling for malignant transformation.
Sujet(s)
Lymphocytes B/métabolisme , Protéines membranaires/métabolisme , Phosphatidylinositol 3-kinases/métabolisme , Phosphates phosphatidylinositol/métabolisme , Protéines de liaison à l'ARN/métabolisme , Transduction du signal , Animaux , Antigènes CD19/métabolisme , Lymphocytes B/enzymologie , Lymphocytes B/immunologie , Lymphocytes B/anatomopathologie , Transformation cellulaire néoplasique , Femelle , Centre germinatif/cytologie , Centre germinatif/immunologie , Centre germinatif/anatomopathologie , Humains , Intégrines/métabolisme , Microdomaines membranaires/métabolisme , Souris , Souris de lignée C57BL , Souris de lignée NOD , Modèles moléculaires , Phosphorylation , Récepteurs pour l'antigène des lymphocytes B/métabolismeRÉSUMÉ
Resistance to multimodal chemotherapy continues to limit the prognosis of acute lymphoblastic leukemia (ALL). This occurs in part through a process called adhesion-mediated drug resistance, which depends on ALL cell adhesion to the stroma through adhesion molecules, including integrins. Integrin α6 has been implicated in minimal residual disease in ALL and in the migration of ALL cells to the central nervous system. However, it has not been evaluated in the context of chemotherapeutic resistance. Here, we show that the anti-human α6-blocking Ab P5G10 induces apoptosis in primary ALL cells in vitro and sensitizes primary ALL cells to chemotherapy or tyrosine kinase inhibition in vitro and in vivo. We further analyzed the underlying mechanism of α6-associated apoptosis using a conditional knockout model of α6 in murine BCR-ABL1+ B-cell ALL cells and showed that α6-deficient ALL cells underwent apoptosis. In vivo deletion of α6 in combination with tyrosine kinase inhibitor (TKI) treatment was more effective in eradicating ALL than treatment with a TKI (nilotinib) alone. Proteomic analysis revealed that α6 deletion in murine ALL was associated with changes in Src signaling, including the upregulation of phosphorylated Lyn (pTyr507) and Fyn (pTyr530). Thus, our data support α6 as a novel therapeutic target for ALL.
Sujet(s)
Résistance aux médicaments antinéoplasiques , Délétion de gène , Intégrine alpha6 , Protéines tumorales , Leucémie-lymphome lymphoblastique à précurseurs B , Pyrimidines/pharmacologie , Animaux , Anticorps antitumoraux/pharmacologie , Anticorps neutralisants/pharmacologie , Apoptose/effets des médicaments et des substances chimiques , Apoptose/génétique , Résistance aux médicaments antinéoplasiques/effets des médicaments et des substances chimiques , Résistance aux médicaments antinéoplasiques/génétique , Femelle , Humains , Intégrine alpha6/génétique , Intégrine alpha6/métabolisme , Mâle , Souris , Souris knockout , Protéines tumorales/antagonistes et inhibiteurs , Protéines tumorales/génétique , Protéines tumorales/métabolisme , Leucémie-lymphome lymphoblastique à précurseurs B/génétique , Leucémie-lymphome lymphoblastique à précurseurs B/métabolisme , Leucémie-lymphome lymphoblastique à précurseurs B/thérapieRÉSUMÉ
DNA and histone modifications have notable effects on gene expression1. Being the most prevalent internal modification in mRNA, the N6-methyladenosine (m6A) mRNA modification is as an important post-transcriptional mechanism of gene regulation2-4 and has crucial roles in various normal and pathological processes5-12. However, it is unclear how m6A is specifically and dynamically deposited in the transcriptome. Here we report that histone H3 trimethylation at Lys36 (H3K36me3), a marker for transcription elongation, guides m6A deposition globally. We show that m6A modifications are enriched in the vicinity of H3K36me3 peaks, and are reduced globally when cellular H3K36me3 is depleted. Mechanistically, H3K36me3 is recognized and bound directly by METTL14, a crucial component of the m6A methyltransferase complex (MTC), which in turn facilitates the binding of the m6A MTC to adjacent RNA polymerase II, thereby delivering the m6A MTC to actively transcribed nascent RNAs to deposit m6A co-transcriptionally. In mouse embryonic stem cells, phenocopying METTL14 knockdown, H3K36me3 depletion also markedly reduces m6A abundance transcriptome-wide and in pluripotency transcripts, resulting in increased cell stemness. Collectively, our studies reveal the important roles of H3K36me3 and METTL14 in determining specific and dynamic deposition of m6A in mRNA, and uncover another layer of gene expression regulation that involves crosstalk between histone modification and RNA methylation.
Sujet(s)
Adénosine/analogues et dérivés , Histone/composition chimique , Histone/métabolisme , Lysine/métabolisme , ARN messager/composition chimique , ARN messager/métabolisme , Transcription génétique , Adénosine/métabolisme , Animaux , Différenciation cellulaire , Lignée cellulaire , Cellules souches embryonnaires/métabolisme , Humains , Lysine/composition chimique , Méthylation , Methyltransferases/déficit , Methyltransferases/génétique , Methyltransferases/métabolisme , Souris , RNA polymerase II/métabolisme , Élongation de la transcription , Transcriptome/génétiqueRÉSUMÉ
B cell activation during normal immune responses and oncogenic transformation impose increased metabolic demands on B cells and their ability to retain redox homeostasis. While the serine/threonine-protein phosphatase 2A (PP2A) was identified as a tumor suppressor in multiple types of cancer, our genetic studies revealed an essential role of PP2A in B cell tumors. Thereby, PP2A redirects glucose carbon utilization from glycolysis to the pentose phosphate pathway (PPP) to salvage oxidative stress. This unique vulnerability reflects constitutively low PPP activity in B cells and transcriptional repression of G6PD and other key PPP enzymes by the B cell transcription factors PAX5 and IKZF1. Reflecting B-cell-specific transcriptional PPP-repression, glucose carbon utilization in B cells is heavily skewed in favor of glycolysis resulting in lack of PPP-dependent antioxidant protection. These findings reveal a gatekeeper function of the PPP in a broad range of B cell malignancies that can be efficiently targeted by small molecule inhibition of PP2A and G6PD.
Sujet(s)
Carbone/métabolisme , Glucose/métabolisme , Leucémie-lymphome lymphoblastique à précurseurs B et T/anatomopathologie , Animaux , Lymphocytes B/cytologie , Lymphocytes B/métabolisme , Lignée cellulaire tumorale , Survie cellulaire , Glucose 6-phosphate dehydrogenase/génétique , Glucose 6-phosphate dehydrogenase/métabolisme , Glycolyse , Humains , Facteur de transcription Ikaros/génétique , Facteur de transcription Ikaros/métabolisme , Souris , Souris de lignée C57BL , Souris de lignée NOD , Stress oxydatif , Protéine activatrice spécifique des lymphocytes B/génétique , Protéine activatrice spécifique des lymphocytes B/métabolisme , Voie des pentoses phosphates , Leucémie-lymphome lymphoblastique à précurseurs B et T/métabolisme , Protein Phosphatase 2/déficit , Protein Phosphatase 2/génétique , Protein Phosphatase 2/métabolisme , Protéines proto-oncogènes c-bcl-2/métabolisme , Transcription génétiqueRÉSUMÉ
Studying mechanisms of malignant transformation of human pre-B cells, we found that acute activation of oncogenes induced immediate cell death in the vast majority of cells. Few surviving pre-B cell clones had acquired permissiveness to oncogenic signaling by strong activation of negative feedback regulation of Erk signaling. Studying negative feedback regulation of Erk in genetic experiments at three different levels, we found that Spry2, Dusp6, and Etv5 were essential for oncogenic transformation in mouse models for pre-B acute lymphoblastic leukemia (ALL). Interestingly, a small molecule inhibitor of DUSP6 selectively induced cell death in patient-derived pre-B ALL cells and overcame conventional mechanisms of drug-resistance.
Sujet(s)
Transformation cellulaire néoplasique/génétique , Système de signalisation des MAP kinases , Leucémie-lymphome lymphoblastique à précurseurs B et T/anatomopathologie , Animaux , Antinéoplasiques/pharmacologie , Transformation cellulaire néoplasique/métabolisme , Transformation cellulaire néoplasique/anatomopathologie , Protéines de liaison à l'ADN/génétique , Protéines de liaison à l'ADN/métabolisme , Dual Specificity Phosphatase 6/génétique , Dual Specificity Phosphatase 6/métabolisme , Facteur de prolifération cellulaire HCF , Humains , Protéines et peptides de signalisation intracellulaire/antagonistes et inhibiteurs , Protéines et peptides de signalisation intracellulaire/génétique , Protéines et peptides de signalisation intracellulaire/métabolisme , Système de signalisation des MAP kinases/effets des médicaments et des substances chimiques , Protéines membranaires/antagonistes et inhibiteurs , Protéines membranaires/génétique , Protéines membranaires/métabolisme , Souris , Souris transgéniques , Données de séquences moléculaires , Leucémie-lymphome lymphoblastique à précurseurs B et T/génétique , Leucémie-lymphome lymphoblastique à précurseurs B et T/métabolisme , Pronostic , Protein-Serine-Threonine Kinases , Bibliothèques de petites molécules/pharmacologie , Facteurs de transcription/génétique , Facteurs de transcription/métabolismeRÉSUMÉ
Childhood acute lymphoblastic leukemia (ALL) can often be traced to a pre-leukemic clone carrying a prenatal genetic lesion. Postnatally acquired mutations then drive clonal evolution toward overt leukemia. The enzymes RAG1-RAG2 and AID, which diversify immunoglobulin-encoding genes, are strictly segregated in developing cells during B lymphopoiesis and peripheral mature B cells, respectively. Here we identified small pre-BII cells as a natural subset with increased genetic vulnerability owing to concurrent activation of these enzymes. Consistent with epidemiological findings on childhood ALL etiology, susceptibility to genetic lesions during B lymphopoiesis at the transition from the large pre-BII cell stage to the small pre-BII cell stage was exacerbated by abnormal cytokine signaling and repetitive inflammatory stimuli. We demonstrated that AID and RAG1-RAG2 drove leukemic clonal evolution with repeated exposure to inflammatory stimuli, paralleling chronic infections in childhood.
Sujet(s)
Lymphocytes B/immunologie , Évolution clonale/immunologie , Leucémie-lymphome lymphoblastique à précurseurs B et T/immunologie , Précurseurs lymphoïdes B/immunologie , Adolescent , Animaux , Diversité des anticorps/génétique , Diversité des anticorps/immunologie , Lymphocytes B/métabolisme , Enfant , Enfant d'âge préscolaire , Évolution clonale/génétique , Cytidine deaminase/génétique , Cytidine deaminase/immunologie , Cytidine deaminase/métabolisme , Protéines de liaison à l'ADN/génétique , Protéines de liaison à l'ADN/immunologie , Protéines de liaison à l'ADN/métabolisme , Femelle , Cytométrie en flux , Protéines à homéodomaine/génétique , Protéines à homéodomaine/immunologie , Protéines à homéodomaine/métabolisme , Humains , Immunotransfert , Nourrisson , Mâle , Souris de lignée NOD , Souris knockout , Souris SCID , Souris transgéniques , Microscopie de fluorescence , Leucémie-lymphome lymphoblastique à précurseurs B et T/génétique , Leucémie-lymphome lymphoblastique à précurseurs B et T/métabolisme , Précurseurs lymphoïdes B/métabolisme , RT-PCR , Cellules cancéreuses en cultureRÉSUMÉ
Despite recent advances in the cure rate of acute lymphoblastic leukaemia (ALL), the prognosis for patients with relapsed ALL remains poor. Here we identify FOXM1 as a candidate responsible for an aggressive clinical course. We show that FOXM1 levels peak at the pre-B-cell receptor checkpoint but are dispensable for normal B-cell development. Compared with normal B-cell populations, FOXM1 levels are 2- to 60-fold higher in ALL cells and are predictive of poor outcome in ALL patients. FOXM1 is negatively regulated by FOXO3A, supports cell survival, drug resistance, colony formation and proliferation in vitro, and promotes leukemogenesis in vivo. Two complementary approaches of pharmacological FOXM1 inhibition-(i) FOXM1 transcriptional inactivation using the thiazole antibiotic thiostrepton and (ii) an FOXM1 inhibiting ARF-derived peptide-recapitulate the findings of genetic FOXM1 deletion. Taken together, our data identify FOXM1 as a novel therapeutic target, and demonstrate feasibility of FOXM1 inhibition in ALL.
Sujet(s)
Antinéoplasiques/pharmacologie , Facteurs de transcription Forkhead/antagonistes et inhibiteurs , Régulation de l'expression des gènes dans la leucémie , Peptides/synthèse chimique , Leucémie-lymphome lymphoblastique à précurseurs B et T/traitement médicamenteux , Thiostrepton/pharmacologie , Adulte , Animaux , Lymphocytes B/effets des médicaments et des substances chimiques , Lymphocytes B/métabolisme , Lymphocytes B/anatomopathologie , Prolifération cellulaire/effets des médicaments et des substances chimiques , Survie cellulaire/effets des médicaments et des substances chimiques , Enfant , Essais cliniques comme sujet , Inhibiteur p16 de kinase cycline-dépendante/composition chimique , Résistance aux médicaments antinéoplasiques/génétique , Protéine M1 à motif en tête de fourche , Protéine O3 à motif en tête de fourche , Facteurs de transcription Forkhead/génétique , Facteurs de transcription Forkhead/métabolisme , Humains , Souris , Peptides/pharmacologie , Leucémie-lymphome lymphoblastique à précurseurs B et T/génétique , Leucémie-lymphome lymphoblastique à précurseurs B et T/métabolisme , Leucémie-lymphome lymphoblastique à précurseurs B et T/mortalité , Transduction du signal , Analyse de survie , Tests d'activité antitumorale sur modèle de xénogreffeRÉSUMÉ
B cells are selected for an intermediate level of B-cell antigen receptor (BCR) signalling strength: attenuation below minimum (for example, non-functional BCR) or hyperactivation above maximum (for example, self-reactive BCR) thresholds of signalling strength causes negative selection. In â¼25% of cases, acute lymphoblastic leukaemia (ALL) cells carry the oncogenic BCR-ABL1 tyrosine kinase (Philadelphia chromosome positive), which mimics constitutively active pre-BCR signalling. Current therapeutic approaches are largely focused on the development of more potent tyrosine kinase inhibitors to suppress oncogenic signalling below a minimum threshold for survival. We tested the hypothesis that targeted hyperactivation--above a maximum threshold--will engage a deletional checkpoint for removal of self-reactive B cells and selectively kill ALL cells. Here we find, by testing various components of proximal pre-BCR signalling in mouse BCR-ABL1 cells, that an incremental increase of Syk tyrosine kinase activity was required and sufficient to induce cell death. Hyperactive Syk was functionally equivalent to acute activation of a self-reactive BCR on ALL cells. Despite oncogenic transformation, this basic mechanism of negative selection was still functional in ALL cells. Unlike normal pre-B cells, patient-derived ALL cells express the inhibitory receptors PECAM1, CD300A and LAIR1 at high levels. Genetic studies revealed that Pecam1, Cd300a and Lair1 are critical to calibrate oncogenic signalling strength through recruitment of the inhibitory phosphatases Ptpn6 (ref. 7) and Inpp5d (ref. 8). Using a novel small-molecule inhibitor of INPP5D (also known as SHIP1), we demonstrated that pharmacological hyperactivation of SYK and engagement of negative B-cell selection represents a promising new strategy to overcome drug resistance in human ALL.
Sujet(s)
Lymphocytes B/métabolisme , Lymphocytes B/anatomopathologie , Leucémie-lymphome lymphoblastique à précurseurs B et T/métabolisme , Leucémie-lymphome lymphoblastique à précurseurs B et T/anatomopathologie , Transduction du signal , Motifs d'acides aminés/génétique , Animaux , Antigènes CD/métabolisme , Lymphocytes B/effets des médicaments et des substances chimiques , Mort cellulaire/effets des médicaments et des substances chimiques , Lignée cellulaire tumorale , Transformation cellulaire néoplasique , Modèles animaux de maladie humaine , Résistance aux médicaments antinéoplasiques/effets des médicaments et des substances chimiques , Activation enzymatique/effets des médicaments et des substances chimiques , Femelle , Protéines de fusion bcr-abl/génétique , Délétion de gène , Humains , Inositol polyphosphate 5-phosphatases , Protéines et peptides de signalisation intracellulaire/agonistes , Protéines et peptides de signalisation intracellulaire/métabolisme , Souris , Souris de lignée NOD , Souris SCID , Phosphatidylinositol-3,4,5-trisphosphate 5-phosphatases , Phosphoric monoester hydrolases/antagonistes et inhibiteurs , Phosphoric monoester hydrolases/métabolisme , Antigènes CD31/métabolisme , Leucémie-lymphome lymphoblastique à précurseurs B et T/traitement médicamenteux , Leucémie-lymphome lymphoblastique à précurseurs B et T/génétique , Précurseurs lymphoïdes B/effets des médicaments et des substances chimiques , Précurseurs lymphoïdes B/métabolisme , Précurseurs lymphoïdes B/anatomopathologie , Protein Tyrosine Phosphatase, Non-Receptor Type 6/déficit , Protein Tyrosine Phosphatase, Non-Receptor Type 6/génétique , Protein Tyrosine Phosphatase, Non-Receptor Type 6/métabolisme , Protein-tyrosine kinases/métabolisme , Récepteurs pour l'antigène des lymphocytes B/déficit , Récepteurs pour l'antigène des lymphocytes B/génétique , Récepteurs pour l'antigène des lymphocytes B/métabolisme , Récepteurs immunologiques/génétique , Récepteurs immunologiques/métabolisme , Transduction du signal/effets des médicaments et des substances chimiques , Syk kinase , Tyrosine/métabolisme , Tests d'activité antitumorale sur modèle de xénogreffeRÉSUMÉ
Activation-induced cytidine deaminase (AID) was originally identified as an inducer of somatic hypermutation (SHM) and class switch recombination (CSR) in immunoglobulin genes. However, AID can also cause mutations in host genes and contribute to cancer progression and drug resistance. In this study, molecular docking showed the interaction of free 5-aza-CdR and Zebularine (Zeb) with AID. However, only 5-aza-CdR-incorporated ssDNA bound to the active site of AID and inhibited AID expression through proteasomal degradation. 5-aza-CdR demonstrated cytotoxicity against AID-positive and -negative hematopoietic cancer cells. In contrast, Zeb exhibited a cytotoxic effect only in AID-negative cells due to its inability to inhibit AID expression. This differential effect might be due to the DNMT1 stabilization induced by AID, thus restricting the ability of Zeb to deplete DNMT1 and induce tumor suppressor genes (TSGs), such as p21, in AID-positive cells. Moreover, the in vivo anticancer effect of 5-aza-CdR but not Zeb in AID-positive hematopoietic cancer cells was demonstrated. The study not only displays the association of AID and DNMT1 and identifies a novel biological function of AID, but also provides novel information regarding the use of DNMT inhibitors to treat AID-positive hematopoietic cancers.
Sujet(s)
Azacitidine/analogues et dérivés , Azacitidine/pharmacologie , Cytidine deaminase/métabolisme , DNA (cytosine-5-)-methyltransferase/antagonistes et inhibiteurs , Antienzymes/pharmacologie , Animaux , DNA (Cytosine-5-)-methyltransferase 1 , Décitabine , Régulation négative/effets des médicaments et des substances chimiques , Hétérogreffes , Humains , Souris , Souris de lignée NOD , Souris SCID , Simulation de docking moléculaireRÉSUMÉ
Bone marrow (BM) provides chemoprotection for acute lymphoblastic leukemia (ALL) cells, contributing to lack of efficacy of current therapies. Integrin alpha4 (alpha4) mediates stromal adhesion of normal and malignant B-cell precursors, and according to gene expression analyses from 207 children with minimal residual disease, is highly associated with poorest outcome. We tested whether interference with alpha4-mediated stromal adhesion might be a new ALL treatment. Two models of leukemia were used, one genetic (conditional alpha4 ablation of BCR-ABL1 [p210(+)] leukemia) and one pharmacological (anti-functional alpha4 antibody treatment of primary ALL). Conditional deletion of alpha4 sensitized leukemia cell to nilotinib. Adhesion of primary pre-B ALL cells was alpha4-dependent; alpha4 blockade sensitized primary ALL cells toward chemotherapy. Chemotherapy combined with Natalizumab prolonged survival of NOD/SCID recipients of primary ALL, suggesting adjuvant alpha4 inhibition as a novel strategy for pre-B ALL.
Sujet(s)
Anticorps monoclonaux humanisés/pharmacologie , Résistance aux médicaments antinéoplasiques , Protéines de fusion bcr-abl/physiologie , Intégrine alpha4/composition chimique , Maladie résiduelle/traitement médicamenteux , Leucémie-lymphome lymphoblastique à précurseurs B/traitement médicamenteux , Animaux , Moelle osseuse/effets des médicaments et des substances chimiques , Moelle osseuse/métabolisme , Moelle osseuse/anatomopathologie , Adhérence cellulaire , Enfant , Cytométrie en flux , Humains , Integrases/métabolisme , Intégrine alpha4/génétique , Intégrine alpha4/métabolisme , Souris , Souris de lignée NOD , Souris knockout , Souris SCID , Natalizumab , Maladie résiduelle/métabolisme , Maladie résiduelle/mortalité , Leucémie-lymphome lymphoblastique à précurseurs B/métabolisme , Leucémie-lymphome lymphoblastique à précurseurs B/mortalité , ARN messager/génétique , Réaction de polymérisation en chaine en temps réel , RT-PCR , Cellules stromales/effets des médicaments et des substances chimiques , Cellules stromales/métabolisme , Cellules stromales/anatomopathologieRÉSUMÉ
The t(14;18) chromosomal translocation typically involves breakage at the bcl-2 major breakpoint region (MBR) to cause human follicular lymphoma. A theory to explain the striking propensity of the MBR breaks at three CpG clusters within the 175-bp MBR region invoked activation-induced deaminase (AID). In a test of that theory, we used here minichromosomal substrates in human pre-B cell lines. Consistent with the essential elements of the theory, we found that the MBR breakage process is indeed highly dependent on DNA methylation at the CpG sites and highly dependent on the AID enzyme to create lesions at peak locations within the MBR. Interestingly, breakage of the phosphodiester bonds at the AID-initiated MBR lesions is RAG dependent, but, unexpectedly, most are also dependent on Artemis. We found that Artemis is capable of nicking small heteroduplex structures and is even able to nick single-base mismatches. This raises the possibility that activated Artemis, derived from the unjoined D to J(H) DNA ends at the IgH locus on chromosome 14, nicks AID-generated TG mismatches at methyl CpG sites, and this would explain why the breaks at the chromosome 18 MBR occur within the same time window as those on chromosome 14.
Sujet(s)
Points de cassure de chromosome , Ilots CpG , Cytidine deaminase/métabolisme , Méthylation de l'ADN , Protéines proto-oncogènes c-bcl-2/génétique , Translocation génétique , Lymphocytes B/métabolisme , Lignée cellulaire , Chromosomes humains de la paire 14/génétique , Chromosomes humains de la paire 14/métabolisme , Chromosomes humains de la paire 18/génétique , Chromosomes humains de la paire 18/métabolisme , ADN/génétique , ADN/métabolisme , Protéines de liaison à l'ADN , Endonucleases , Techniques de knock-out de gènes , Gènes bcl-2 , Protéines à homéodomaine/génétique , Protéines à homéodomaine/métabolisme , Humains , Protéines nucléaires/génétique , Protéines nucléaires/métabolismeRÉSUMÉ
Vitamin D deficiency has been linked with increased cancer risk, and vitamin D has been shown to be cytotoxic to some cancer cells in vitro. In the present study we evaluated whether vitamin D would have antiproliferative or cytotoxic effects on human pre-B acute lymphoblastic leukemia cells. Contrary to our hypotheses, calcitriol, the active form of vitamin D, had no effect on leukemia cell proliferation. Calcitriol actually had a modest effect to impair dexamethasone cytotoxicity and induction of apoptosis. Further studies are needed to evaluate the effects of vitamin D on leukemia cells in vivo.
Sujet(s)
Calcitriol/pharmacologie , Dexaméthasone/pharmacologie , Leucémie-lymphome lymphoblastique à précurseurs B/traitement médicamenteux , Apoptose/effets des médicaments et des substances chimiques , Lignée cellulaire tumorale , Prolifération cellulaire/effets des médicaments et des substances chimiques , Humains , Leucémie-lymphome lymphoblastique à précurseurs B/anatomopathologieRÉSUMÉ
Relapse of drug-resistant acute lymphoblastic leukemia (ALL) has been associated with increased expression of survivin/BIRC5, an inhibitor of apoptosis protein, suggesting a survival advantage for ALL cells. In the present study, we report that inhibition of survivin in patient-derived ALL can eradicate leukemia. Targeting survivin with shRNA in combination with chemotherapy resulted in no detectable minimal residual disease in a xenograft model of primary ALL. Similarly, pharmacologic knock-down of survivin using EZN-3042, a novel locked nucleic acid antisense oligonucleotide, in combination with chemotherapy eliminated drug-resistant ALL cells. These findings show the importance of survivin expression in drug resistance and demonstrate that survivin inhibition may represent a powerful approach to overcoming drug resistance and preventing relapse in patients with ALL.
Sujet(s)
Protéines IAP/antagonistes et inhibiteurs , Protéines IAP/génétique , Leucémie-lymphome lymphoblastique à précurseurs B et T/traitement médicamenteux , Leucémie-lymphome lymphoblastique à précurseurs B et T/thérapie , Animaux , Association thérapeutique , Résistance aux médicaments antinéoplasiques/génétique , Expression des gènes , Ciblage de gène , Humains , Protéines IAP/déficit , Souris , Souris de lignée NOD , Souris knockout , Maladie résiduelle , Oligonucléotides/génétique , Oligonucléotides/usage thérapeutique , Leucémie-lymphome lymphoblastique à précurseurs B et T/génétique , Petit ARN interférent/génétique , Protéines de répression/déficit , Protéines de répression/génétique , Survivine , Test clonogénique de cellules souches tumorales , Tests d'activité antitumorale sur modèle de xénogreffeRÉSUMÉ
Tyrosine kinase inhibitors (TKIs) are widely used to treat patients with leukaemia driven by BCR-ABL1 (ref. 1) and other oncogenic tyrosine kinases. Recent efforts have focused on developing more potent TKIs that also inhibit mutant tyrosine kinases. However, even effective TKIs typically fail to eradicate leukaemia-initiating cells (LICs), which often cause recurrence of leukaemia after initially successful treatment. Here we report the discovery of a novel mechanism of drug resistance, which is based on protective feedback signalling of leukaemia cells in response to treatment with TKI. We identify BCL6 as a central component of this drug-resistance pathway and demonstrate that targeted inhibition of BCL6 leads to eradication of drug-resistant and leukaemia-initiating subclones.
Sujet(s)
Protéines de liaison à l'ADN/métabolisme , Résistance aux médicaments antinéoplasiques , Protéines de fusion bcr-abl/antagonistes et inhibiteurs , Leucémie-lymphome lymphoblastique à précurseurs B et T/traitement médicamenteux , Leucémie-lymphome lymphoblastique à précurseurs B et T/anatomopathologie , Inhibiteurs de protéines kinases/pharmacologie , Facteur-1 d'ADP-ribosylation/métabolisme , Animaux , Survie cellulaire/effets des médicaments et des substances chimiques , Protéines de liaison à l'ADN/biosynthèse , Protéines de liaison à l'ADN/déficit , Protéines de liaison à l'ADN/génétique , Régulation de l'expression des gènes tumoraux/effets des médicaments et des substances chimiques , Humains , Souris , Souris de lignée NOD , Souris SCID , Leucémie-lymphome lymphoblastique à précurseurs B et T/génétique , Leucémie-lymphome lymphoblastique à précurseurs B et T/métabolisme , Inhibiteurs de protéines kinases/usage thérapeutique , Protéines proto-oncogènes c-bcl-6 , Transcription génétique , Protéine p53 suppresseur de tumeur/métabolismeRÉSUMÉ
Obesity is associated with an increased incidence of many cancers, including leukemia, although it is unknown whether leukemia incidence is increased directly by obesity or rather by associated genetic, lifestyle, health, or socioeconomic factors. We developed animal models of obesity and leukemia to test whether obesity could directly accelerate acute lymphoblastic leukemia (ALL) using BCR/ABL transgenic and AKR/J mice weaned onto a high-fat diet. Mice were observed until development of progressive ALL. Although obese and control BCR/ABL mice had similar median survival, older obese mice had accelerated ALL onset, implying a time-dependent effect of obesity on ALL. Obese AKR mice developed ALL significantly earlier than controls. The effect of obesity was not explained by WBC count, thymus/spleen weight, or ALL phenotype. However, obese AKR mice had higher leptin, insulin, and interleukin-6 levels than controls, and these obesity-related hormones all have potential roles in leukemia pathogenesis. In conclusion, obesity directly accelerates presentation of ALL, likely by increasing the risk of an early event in leukemogenesis. This is the first study to show that obesity can directly accelerate the progression of ALL. Thus, the observed associations between obesity and leukemia incidence are likely to be directly related to biological effects of obesity.
Sujet(s)
Obésité/complications , Leucémie-lymphome lymphoblastique à précurseurs B et T/complications , Adiponectine/sang , Âge de début , Animaux , Régime alimentaire , Modèles animaux de maladie humaine , Évolution de la maladie , Gènes abl/génétique , Humains , Insuline/sang , Interleukine-6/sang , Leptine/sang , Souris , Souris de lignée AKR , Souris transgéniques , Obésité/sang , Leucémie-lymphome lymphoblastique à précurseurs B et T/génétiqueRÉSUMÉ
BCL6 protects germinal center (GC) B cells against DNA damage-induced apoptosis during somatic hypermutation and class-switch recombination. Although expression of BCL6 was not found in early IL-7-dependent B cell precursors, we report that IL-7Ralpha-Stat5 signaling negatively regulates BCL6. Upon productive VH-DJH gene rearrangement and expression of a mu heavy chain, however, activation of pre-B cell receptor signaling strongly induces BCL6 expression, whereas IL-7Ralpha-Stat5 signaling is attenuated. At the transition from IL-7-dependent to -independent stages of B cell development, BCL6 is activated, reaches expression levels resembling those in GC B cells, and protects pre-B cells from DNA damage-induced apoptosis during immunoglobulin (Ig) light chain gene recombination. In the absence of BCL6, DNA breaks during Ig light chain gene rearrangement lead to excessive up-regulation of Arf and p53. As a consequence, the pool of new bone marrow immature B cells is markedly reduced in size and clonal diversity. We conclude that negative regulation of Arf by BCL6 is required for pre-B cell self-renewal and the formation of a diverse polyclonal B cell repertoire.
Sujet(s)
Lymphocytes B/cytologie , Lymphocytes B/immunologie , Protéines de liaison à l'ADN/immunologie , Facteurs d'ADP-ribosylation/métabolisme , Animaux , Apoptose , Séquence nucléotidique , Prolifération cellulaire , Survie cellulaire , Cellules cultivées , Cytoprotection , Altération de l'ADN/génétique , Régulation négative/génétique , Réarrangement des gènes des chaines légères des lymphocytes B/génétique , Humains , Interleukine-7/métabolisme , Lymphopoïèse , Souris , Données de séquences moléculaires , Récepteurs des lymphocytes pré-B/métabolisme , Précurseurs lymphoïdes B/cytologie , Précurseurs lymphoïdes B/métabolisme , Protéines proto-oncogènes c-bcl-6 , Protéines proto-oncogènes c-myc/métabolisme , Recombinaison génétique/génétique , Transduction du signal , Transcription génétique , Régulation positive/génétiqueRÉSUMÉ
Chronic myeloid leukemia (CML) is induced by BCR-ABL1 and can be effectively treated for many years with Imatinib until leukemia cells acquire drug resistance through BCR-ABL1 mutations and progress into fatal B lymphoid blast crisis (LBC). Despite its clinical significance, the mechanism of progression into LBC is unknown. Here, we show that LBC but not CML cells express the B cell-specific mutator enzyme AID. We demonstrate that AID expression in CML cells promotes overall genetic instability by hypermutation of tumor suppressor and DNA repair genes. Importantly, our data uncover a causative role of AID activity in the acquisition of BCR-ABL1 mutations leading to Imatinib resistance, thus providing a rationale for the rapid development of drug resistance and blast crisis progression.