RESUMEN
Recirculation of chronic lymphocytic leukemia (CLL) cells between the peripheral blood and lymphoid niches plays a critical role in disease pathophysiology, and inhibiting this process is one of the major mechanisms of action for B-cell receptor (BCR) inhibitors such as ibrutinib and idelalisib. Migration is a complex process guided by chemokine receptors and integrins. However, it remains largely unknown how CLL cells integrate multiple migratory signals while balancing survival in the peripheral blood and the decision to return to immune niches. Our study provided evidence that CXCR4/CD5 intraclonal subpopulations can be used to study the regulation of migration of CLL cells. We performed RNA profiling of CXCR4dimCD5bright vs CXCR4brightCD5dim CLL cells and identified differential expression of dozens of molecules with a putative function in cell migration. GRB2-associated binding protein 1 (GAB1) positively regulated CLL cell homing capacity of CXCR4brightCD5dim cells. Gradual GAB1 accumulation in CLL cells outside immune niches was mediated by FoxO1-induced transcriptional GAB1 activation. Upregulation of GAB1 also played an important role in maintaining basal phosphatidylinositol 3-kinase (PI3K) activity and the "tonic" AKT phosphorylation required to sustain the survival of resting CLL B cells. This finding is important during ibrutinib therapy, because CLL cells induce the FoxO1-GAB1-pAKT axis, which represents an adaptation mechanism to the inability to home to immune niches. We have demonstrated that GAB1 can be targeted therapeutically by novel GAB1 inhibitors, alone or in combination with BTK inhibition. GAB1 inhibitors induce CLL cell apoptosis, impair cell migration, inhibit tonic or BCR-induced AKT phosphorylation, and block compensatory AKT activity during ibrutinib therapy.
Asunto(s)
Proteínas Adaptadoras Transductoras de Señales/biosíntesis , Movimiento Celular , Proteína Forkhead Box O1/metabolismo , Regulación Leucémica de la Expresión Génica , Leucemia Linfocítica Crónica de Células B/metabolismo , Proteínas Proto-Oncogénicas c-akt/metabolismo , Transducción de Señal , Regulación hacia Arriba , Adenina/análogos & derivados , Adenina/farmacología , Línea Celular Tumoral , Humanos , Leucemia Linfocítica Crónica de Células B/tratamiento farmacológico , Leucemia Linfocítica Crónica de Células B/patología , Piperidinas/farmacologíaRESUMEN
B-cell receptor (BCR) signaling and T-cell interactions play a pivotal role in chronic lymphocytic leukemia (CLL) pathogenesis and disease aggressiveness. CLL cells can use microRNAs (miRNAs) and their targets to modulate microenvironmental interactions in the lymph node niches. To identify miRNA expression changes in the CLL microenvironment, we performed complex profiling of short noncoding RNAs in this context by comparing CXCR4/CD5 intraclonal cell subpopulations (CXCR4dimCD5bright vs CXCR4brightCD5dim cells). This identified dozens of differentially expressed miRNAs, including several that have previously been shown to modulate BCR signaling (miR-155, miR-150, and miR-22) but also other candidates for a role in microenvironmental interactions. Notably, all 3 miR-29 family members (miR-29a, miR-29b, miR-29c) were consistently down-modulated in the immune niches, and lower miR-29(a/b/c) levels associated with an increased relative responsiveness of CLL cells to BCR ligation and significantly shorter overall survival of CLL patients. We identified tumor necrosis factor receptor-associated factor 4 (TRAF4) as a novel direct target of miR-29s and revealed that higher TRAF4 levels increase CLL responsiveness to CD40 activation and downstream nuclear factor-κB (NF-κB) signaling. In CLL, BCR represses miR-29 expression via MYC, allowing for concurrent TRAF4 upregulation and stronger CD40-NF-κB signaling. This regulatory loop is disrupted by BCR inhibitors (bruton tyrosine kinase [BTK] inhibitor ibrutinib or phosphatidylinositol 3-kinase [PI3K] inhibitor idelalisib). In summary, we showed for the first time that a miRNA-dependent mechanism acts to activate CD40 signaling/T-cell interactions in a CLL microenvironment and described a novel miR-29-TRAF4-CD40 signaling axis modulated by BCR activity.
Asunto(s)
Adenina/análogos & derivados , Antígenos CD40/metabolismo , Regulación Neoplásica de la Expresión Génica , Leucemia Linfocítica Crónica de Células B/patología , MicroARNs/genética , Piperidinas/farmacología , Proteínas Proto-Oncogénicas c-bcr/antagonistas & inhibidores , Factor 4 Asociado a Receptor de TNF/metabolismo , Adenina/farmacología , Adulto , Anciano , Biomarcadores de Tumor/genética , Biomarcadores de Tumor/metabolismo , Antígenos CD40/genética , Femenino , Estudios de Seguimiento , Humanos , Leucemia Linfocítica Crónica de Células B/tratamiento farmacológico , Leucemia Linfocítica Crónica de Células B/genética , Leucemia Linfocítica Crónica de Células B/metabolismo , Masculino , Persona de Mediana Edad , Pronóstico , Proteínas Proto-Oncogénicas c-myc/genética , Proteínas Proto-Oncogénicas c-myc/metabolismo , Tasa de Supervivencia , Factor 4 Asociado a Receptor de TNF/genética , Células Tumorales CultivadasRESUMEN
Introduction of small-molecule inhibitors of B-cell receptor signaling and BCL2 protein significantly improves therapeutic options in chronic lymphocytic leukemia. However, some patients suffer from adverse effects mandating treatment discontinuation, and cases with TP53 defects more frequently experience early progression of the disease. Development of alternative therapeutic approaches is, therefore, of critical importance. Here we report details of the anti-chronic lymphocytic leukemia single-agent activity of MU380, our recently identified potent, selective, and metabolically robust inhibitor of checkpoint kinase 1. We also describe a newly developed enantioselective synthesis of MU380, which allows preparation of gram quantities of the substance. Checkpoint kinase 1 is a master regulator of replication operating primarily in intra-S and G2/M cell cycle checkpoints. Initially tested in leukemia and lymphoma cell lines, MU380 significantly potentiated efficacy of gemcitabine, a clinically used inducer of replication stress. Moreover, MU380 manifested substantial single-agent activity in both TP53-wild type and TP53-mutated leukemia and lymphoma cell lines. In chronic lymphocytic leukemia-derived cell lines MEC-1, MEC-2 (both TP53-mut), and OSU-CLL (TP53-wt) the inhibitor impaired cell cycle progression and induced apoptosis. In primary clinical samples, MU380 used as a single-agent noticeably reduced the viability of unstimulated chronic lymphocytic leukemia cells as well as those induced to proliferate by anti-CD40/IL-4 stimuli. In both cases, effects were comparable in samples harboring p53 pathway dysfunction (TP53 mutations or ATM mutations) and TP53-wt/ATM-wt cells. Lastly, MU380 also exhibited significant in vivo activity in a xenotransplant mouse model (immunodeficient strain NOD-scid IL2Rγnull ) where it efficiently suppressed growth of subcutaneous tumors generated from MEC-1 cells.
Asunto(s)
Quinasa 1 Reguladora del Ciclo Celular (Checkpoint 1)/antagonistas & inhibidores , Sinergismo Farmacológico , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Leucemia Linfocítica Crónica de Células B/tratamiento farmacológico , Mutación , Piperidinas/farmacología , Pirazoles/farmacología , Pirimidinas/farmacología , Proteína p53 Supresora de Tumor/genética , Animales , Antimetabolitos Antineoplásicos/farmacología , Apoptosis , Biomarcadores de Tumor/genética , Ciclo Celular , Proliferación Celular , Desoxicitidina/análogos & derivados , Desoxicitidina/farmacología , Resistencia a Antineoplásicos/efectos de los fármacos , Femenino , Humanos , Leucemia Linfocítica Crónica de Células B/genética , Leucemia Linfocítica Crónica de Células B/patología , Ratones , Ratones Endogámicos NOD , Ratones SCID , Inhibidores de Proteínas Quinasas/farmacología , Células Tumorales Cultivadas , Ensayos Antitumor por Modelo de Xenoinjerto , GemcitabinaRESUMEN
Agents targeting B-cell receptor (BCR) signaling-associated kinases such as Bruton tyrosine kinase (BTK) or phosphatidylinositol 3-kinase can induce mobilization of neoplastic B cells from the lymphoid tissues into the blood, which makes them potentially ideal to combine with anti-CD20 monoclonal antibodies (such as rituximab, obinutuzumab, or ofatumumab) for treatment of B-cell lymphomas and chronic lymphocytic leukemia (CLL). Here we show that interactions between leukemia cells and stromal cells (HS-5) upregulate CD20 on CLL cells and that administering ibrutinib downmodulates CD20 (MS4A1) expression in vivo. We observed that CLL cells that have recently exited the lymph node microenvironment and moved into the peripheral blood (CXCR4(dim)CD5(bright) subpopulation) have higher cell surface levels of CD20 than the cells circulating in the bloodstream for a longer time (CXCR4(bright)CD5(dim) cells). We found that CD20 is directly upregulated by CXCR4 ligand stromal cell-derived factor 1 (SDF-1α, CXCL12) produced by stromal cells, and BTK-inhibitor ibrutinib and CXCR4-inhibitor plerixafor block SDF-1α-mediated CD20 upregulation. Ibrutinib also downmodulated Mcl1 levels in CLL cells in vivo and in coculture with stromal cells. Overall, our study provides a first detailed mechanistic explanation of CD20 expression regulation in the context of chemokine signaling and microenvironmental interactions, which may have important implications for microenvironment-targeting therapies.
Asunto(s)
Antígenos CD20/química , Quimiocina CXCL12/metabolismo , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Leucemia Linfocítica Crónica de Células B/tratamiento farmacológico , Pirazoles/farmacología , Pirimidinas/farmacología , Receptores CXCR4/metabolismo , Adenina/análogos & derivados , Antígenos CD20/genética , Antígenos CD20/metabolismo , Quimiocina CXCL12/genética , Humanos , Leucemia Linfocítica Crónica de Células B/metabolismo , Leucemia Linfocítica Crónica de Células B/patología , Piperidinas , Receptores CXCR4/genética , Transducción de Señal , Células Tumorales Cultivadas , Regulación hacia ArribaRESUMEN
The canonical Wnt pathway, dependent on ß-catenin-controlled transcription, is the most explored Wnt pathway, known to drive the malignant transformation of multiple cell types. Several reports have suggested that this pathway also participates in chronic lymphocytic leukaemia (CLL) pathogenesis. To get a better insight into the role of the Wnt/ß-catenin pathway in CLL we analysed in detail the expression of the most overexpressed Wnt ligand, encoded by the WNT3 gene, in a well-defined cohort of 137 CLL patients. Our analysis demonstrated that (i) untreated patients with more aggressive disease (with a notable exception of patients with 11q deletion) express less WNT3, (ii) WNT3 declines with disease progression in a significant proportion of patients and (iii) low WNT3 was identified as a strong independent marker indicating shorter treatment-free survival in CLL patients with IGHV mutation. Interestingly, CLL-related lymphoid cell lines, but not stromal cells, failed to respond to the ligand-induced activation of the Wnt/ß-catenin pathway. This opens the possibility that CLL cells use Wnt-3 to communicate with the cells in the microenvironment. We thus propose that the Wnt/ß-catenin pathway plays a more complex role in CLL pathogenesis than previously anticipated.
Asunto(s)
Regulación Neoplásica de la Expresión Génica , Leucemia Linfocítica Crónica de Células B/diagnóstico , Proteína Wnt3/genética , Comunicación Celular , Línea Celular , Estudios de Cohortes , Progresión de la Enfermedad , Femenino , Genes de las Cadenas Pesadas de las Inmunoglobulinas/genética , Humanos , Leucemia Linfocítica Crónica de Células B/genética , Leucemia Linfocítica Crónica de Células B/metabolismo , Masculino , Mutación , Pronóstico , Vía de Señalización WntRESUMEN
MicroRNAs (miRNAs) play a key role in chronic lymphocytic leukemia as well as in normal B cells. Notably, miRNA gene encoding miR-650 and its homologs overlap with several variable (V) subgenes coding for lambda immunoglobulin (IgLλ). Recent studies describe the role of miR-650 in solid tumors, but its role in chronic lymphocytic leukemia (CLL) has not yet been studied. Our experiments demonstrate that miR-650 expression is regulated by coupled expression with its host gene for IgLλ. This coupling provides a unique yet unobserved mechanism for microRNA gene regulation. We determine that higher expression of miR-650 is associated with a favorable CLL prognosis and influences the proliferation capacity of B cells. We also establish that in B cells, miR-650 targets proteins important in cell proliferation and survival: cyclin dependent kinase 1 (CDK1), inhibitor of growth 4 (ING4), and early B-cell factor 3 (EBF3). This study underscores the importance of miR-650 in CLL biology and normal B-cell physiology.
Asunto(s)
Regulación de la Expresión Génica , Reordenamiento Génico , Genes de Inmunoglobulinas , Leucemia Linfocítica Crónica de Células B/genética , MicroARNs/genética , Adulto , Anciano , Anciano de 80 o más Años , Ciclo Celular/genética , Línea Celular Tumoral , Femenino , Genes de las Cadenas Ligeras de las Inmunoglobulinas , Humanos , Leucemia Linfocítica Crónica de Células B/mortalidad , Masculino , Persona de Mediana Edad , Estadificación de Neoplasias , PronósticoRESUMEN
BACKGROUND: Isoelectric focusing (IEF) is a method with an exquisite resolution, and coupled with affinity immunoblotting (AIB), it can provide superior sensitivity to detect monoclonal free light chains (FLC). METHODS: We tested the hypothesis that IEF/AIB is more sensitive and specific for monoclonal FLC detection in serum and urine samples than conventional methods, that is, electrophoresis (ELP), immunofixation (IF) and serum FLC ratio assessment. Investigation included 107 samples of 68 patients, among which 21 multiple myeloma patients were recently tested for minimal residual disease and 18 patients with AL amyloidosis. RESULTS: Monoclonal FLC were detected by IEF/AIB in 37% of serum samples negative for monoclonal FLC on ELP/IF. As for urine samples, significant advantage of the IEF/AIB over ELP/IF was not demonstrated. Considering both serum and urine results, IEF/AIB definitely revealed monoclonal FLC in 20/83 (24%) of ELP/IF-negative samples. FLC ratio was abnormally high (>1.65) in all 11 patients definitely positive for monoclonal FLC kappa by IEF/AIB but also in 16/47 (34%) IEF/AIB-negative samples. Abnormally low values (<0.26) were found only in 10/28 samples (36%) positive for monoclonal FLC lambda. Appropriate use of renal FLC ratio reference range reduced the number of presumably false positives (6/47, i.e. 13%) but not false negatives (17/28, i.e. 61%). CONCLUSIONS: The IEF/AIB method is more sensitive than IF and might be used in patients with negative IF results before deciding whether to proceed to minimal residual disease testing.
Asunto(s)
Leucemia Linfocítica Crónica de Células B/diagnóstico , Leucemia Linfocítica Crónica de Células B/genética , Receptor Notch1/genética , Proteína p53 Supresora de Tumor/genética , Alelos , Evolución Clonal/genética , Análisis Mutacional de ADN/métodos , Genotipo , Humanos , Leucemia Linfocítica Crónica de Células B/tratamiento farmacológico , Análisis de la Célula Individual/métodosRESUMEN
Genome methylation profiles define naïve-like (n-CLL), memory-like (m-CLL), and intermediate (i-CLL) subsets of chronic lymphocytic leukaemia (CLL). The profiles can be easily determined by the analysis of the five-CpG signature. m-CLL, i-CLL, and n-CLL with the good, intermediate, and poor prognoses, respectively, differ by the somatic hypermutation status of the immunoglobulin heavy chain variable gene (IGHV), a widely used prognostic predictor in CLL. We have previously shown that the expression of WNT5A, encoding a ROR1 ligand, distinguishes patients with the worse outcome within the prognostically favourable IGHV-mutated subgroup. To analyse the mechanisms controlling WNT5A expression, we investigated the methylation status of 54 CpG sites within the WNT5A promoter and its relation to the WNT5A gene expression. In a cohort of 59 CLL patients balanced for combinations of IGHV and WNT5A statuses, we identified three promoter CpG sites whose methylation level correlated with the WNT5A expression within the IGHV-mutated subgroup. Further, we complemented our data with the methylation status of the five-CpG signature. IGHV-mutated/WNT5A-negative and IGHV-mutated/WNT5A-positive cases overlapped with mCLL and iCLL methylation subgroups, respectively, while most IGHVunmutated samples were assigned to n-CLL. Median methylation levels of all the three CpG sites in the WNT5A promoter were lowest in i-CLL. Finally, a detailed analysis of m-CLL and i-CLL showed that undetectable WNT5A expression predicts longer treatment-free survival with higher statistical significance than the classification according to the five-CpG signature. To conclude, a favourable m-CLL subgroup is associated with mutated IGHV and undetectable WNT5A expression due to its promoter methylation.
Asunto(s)
Leucemia Linfocítica Crónica de Células B , Humanos , Leucemia Linfocítica Crónica de Células B/genética , Cadenas Pesadas de Inmunoglobulina/genética , Cadenas Pesadas de Inmunoglobulina/metabolismo , Ligandos , Metilación de ADN , Regiones Promotoras Genéticas , Pronóstico , Mutación , Proteína Wnt-5a/genética , Proteína Wnt-5a/metabolismoRESUMEN
The in vivo rituximab effects in B cell malignancies are only partially understood. Here we analyzed in a large chronic lymphocytic leukemia (CLL) cohort (n = 80) the inter-patient variability in CLL cell count reduction within the first 24 h of rituximab administration in vivo, and a phenomenon of blood repopulation by malignant cells after anti-CD20 antibody therapy. Larger CLL cell elimination after rituximab infusion was associated with lower pre-therapy CLL cell counts, higher CD20 levels, and the non-exhausted capacity of complement-dependent cytotoxicity (CDC). The absolute amount of cell-surface CD20 molecules (CD20 density x CLL lymphocytosis) was a predictor for complement exhaustion during therapy. We also describe that a highly variable decrease in CLL cell counts at 5 h (88 %-2%) following rituximab infusion is accompanied in most patients by peripheral blood repopulation with CLL cells at 24 h, and in â¼20 % of patients, this resulted in CLL counts higher than before therapy. We provide evidence that CLL cells recrudescence is linked with i) CDC exhaustion, which leads to the formation of an insufficient amount of membrane attack complexes, likely resulting in temporary retention of surviving rituximab-opsonized cells by the mononuclear-phagocyte system (followed by their release back to blood), and ii) CLL cells regression from immune niches (CXCR4dimCD5bright intraclonal subpopulation). Patients with major peripheral blood CLL cell repopulation exhibited a longer time-to-progression after chemoimmunotherapy compared to patients with lower or no repopulation, suggesting chemotherapy vulnerability of CLL cells that repopulate the blood.
Asunto(s)
Antineoplásicos Inmunológicos/uso terapéutico , Proteínas del Sistema Complemento/inmunología , Citotoxicidad Inmunológica/inmunología , Leucemia Linfocítica Crónica de Células B/inmunología , Rituximab/uso terapéutico , Estudios de Seguimiento , Humanos , Leucemia Linfocítica Crónica de Células B/sangre , Leucemia Linfocítica Crónica de Células B/tratamiento farmacológico , Leucemia Linfocítica Crónica de Células B/patologíaRESUMEN
OBJECTIVES: Abnormalities of the TP53 or ATM, cooperating tumor-suppressor genes, significantly worsen the treatment options for chronic lymphocytic leukemia (CLL) patients. Although the aberrations seem to be mutually exclusive in this leukemia, inactivation of the former gene leads to worse prognosis. We tested the in vitro sensitivity of the CLL samples with heterozygous ATM deletion to fludarabine and combination of fludarabine and rituximab; the responses were compared with the TP53-abnormal and wild-type (wt) cells to delimitate relative significance of ATM deletion. METHODS: In vitro analysis was performed on fifty-nine characterized CLL samples using viability assay WST-1. Western blot and real-time RT-PCR were used to monitor the activation of the ATM/p53 pathway. RESULTS AND CONCLUSIONS: At the clinically relevant concentration of fludarabine, TP53-abnormal samples exhibited markedly higher resistance to fludarabine than the remaining CLL samples (P = 0.012); cohort with ATM deletion was not more resistant than wt cells. A similar induction of the p53 protein and its downstream target genes PUMA and BAX in ATM-deleted and wt cells confirmed that the former subgroup has preserved a critical pro-apoptotic response. Proportions of the samples, which had been sensitized to fludarabine by rituximab pretreatment, were insignificantly lower (P = 0.22) in the TP53-abnormal and ATM-deleted subgroups compared to the wt cases (30%; 29%; 50%, respectively). The presence of ATM (11q22-23) deletion in the CLL cells should not be considered an indication of resistance to fludarabine or its combination with rituximab.
Asunto(s)
Antineoplásicos/farmacología , Proteínas de Ciclo Celular/genética , Proteínas de Unión al ADN/genética , Eliminación de Gen , Leucemia Linfocítica Crónica de Células B/patología , Proteínas Serina-Treonina Quinasas/genética , Proteínas Supresoras de Tumor/genética , Vidarabina/análogos & derivados , Proteínas de la Ataxia Telangiectasia Mutada , Western Blotting , Daño del ADN , Humanos , Leucemia Linfocítica Crónica de Células B/genética , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Proteína p53 Supresora de Tumor/metabolismo , Vidarabina/farmacologíaRESUMEN
PURPOSE: ROR1, a receptor in the noncanonical Wnt/planar cell polarity (PCP) pathway, is upregulated in malignant B cells of chronic lymphocytic leukemia (CLL) patients. It has been shown that the Wnt/PCP pathway drives pathogenesis of CLL, but which factors activate the ROR1 and PCP pathway in CLL cells remains unclear. EXPERIMENTAL DESIGN: B lymphocytes from the peripheral blood of CLL patients were negatively separated using RosetteSep (StemCell) and gradient density centrifugation. Relative expression of WNT5A, WNT5B, and ROR1 was assessed by quantitative real-time PCR. Protein levels, protein interaction, and downstream signaling were analyzed by immunoprecipitation and Western blotting. Migration capacity of primary CLL cells was analyzed by the Transwell migration assay. RESULTS: By analyzing the expression in 137 previously untreated CLL patients, we demonstrate that WNT5A and WNT5B genes show dramatically (five orders of magnitude) varying expression in CLL cells. High WNT5A and WNT5B expression strongly associates with unmutated IGHV and shortened time to first treatment. In addition, WNT5A levels associate, independent of IGHV status, with the clinically worst CLL subgroups characterized by dysfunctional p53 and mutated SF3B1. We provide functional evidence that WNT5A-positive primary CLL cells have increased motility and attenuated chemotaxis toward CXCL12 and CCL19 that can be overcome by inhibitors of Wnt/PCP signaling. CONCLUSIONS: These observations identify Wnt-5a as the crucial regulator of ROR1 activity in CLL and suggest that the autocrine Wnt-5a signaling pathway allows CLL cells to overcome natural microenvironmental regulation.
Asunto(s)
Comunicación Autocrina/fisiología , Quimiotaxis/fisiología , Leucemia Linfocítica Crónica de Células B/metabolismo , Proteínas Proto-Oncogénicas/metabolismo , Proteínas Wnt/metabolismo , Vía de Señalización Wnt/fisiología , Linfocitos B/metabolismo , Movimiento Celular/fisiología , Regulación Neoplásica de la Expresión Génica/fisiología , Células HEK293 , Humanos , Receptores Huérfanos Similares al Receptor Tirosina Quinasa/metabolismo , Regulación hacia Arriba/fisiología , Proteína Wnt-5aRESUMEN
Little is known about the function and phenotype of leukemic stem cells (LSCs) in chronic myeloid leukemia (CML) or about specific markers that discriminate LSCs from normal hematopoietic stem cells (HSCs). CD26 has recently been described as a specific marker of CML LSCs. In the current study, we investigated this marker in a cohort of 31 unselected CML patients. BCR/ABL1 positivity was analyzed in highly enriched stem cell fractions using fluorescence in situ hybridization (FISH) and reverse transcription PCR (RT-PCR). The proportion of CD26+ LSCs and CD26- HSCs varied considerably among the patients analyzed, and the percentage of CD26+ cells correlated with leukocyte count. The CD26 expression robustly discriminated LSCs from HSCs. This required a strict gating of the stem cell compartment. Thus, in patients with very low LSC or HSC numbers, only the highly sensitive RT-PCR method discriminated between clonal and non-clonal cells, while a robust FISH analysis required larger numbers of cells in both compartments. Finally, our data show that the numbers of CD26+ CML LSCs correlate with responses to treatment with BCR-ABL1 inhibitors.
Asunto(s)
Dipeptidil Peptidasa 4/biosíntesis , Leucemia Mielógena Crónica BCR-ABL Positiva/patología , Células Madre Neoplásicas/inmunología , Dipeptidil Peptidasa 4/inmunología , Humanos , Inmunofenotipificación , Leucemia Mielógena Crónica BCR-ABL Positiva/inmunología , Células Madre Neoplásicas/patología , PronósticoRESUMEN
Treatment options for TP53-mutated lymphoid tumors are very limited. In experimental models, TP53-mutated lymphomas were sensitive to direct inhibition of checkpoint kinase 1 (Chk1), a pivotal regulator of replication. We initially tested the potential of the highly specific Chk1 inhibitor SCH900776 to synergize with nucleoside analogs (NAs) fludarabine, cytarabine and gemcitabine in cell lines derived from B-cell malignancies. In p53-proficient NALM-6 cells, SCH900776 added to NAs enhanced signaling towards Chk1 (pSer317/pSer345), effectively blocked Chk1 activation (Ser296 autophosphorylation), increased replication stress (p53 and γ-H2AX accumulation) and temporarily potentiated apoptosis. In p53-defective MEC-1 cell line representing adverse chronic lymphocytic leukemia (CLL), Chk1 inhibition together with NAs led to enhanced and sustained replication stress and significantly potentiated apoptosis. Altogether, among 17 tested cell lines SCH900776 sensitized four of them to all three NAs. Focusing further on MEC-1 and co-treatment of SCH900776 with fludarabine, we disclosed chromosome pulverization in cells undergoing aberrant mitoses. SCH900776 also increased the effect of fludarabine in a proportion of primary CLL samples treated with pro-proliferative stimuli, including those with TP53 disruption. Finally, we observed a fludarabine potentiation by SCH900776 in a T-cell leukemia 1 (TCL1)-driven mouse model of CLL. Collectively, we have substantiated the significant potential of Chk1 inhibition in B-lymphoid cells.
Asunto(s)
Linfocitos B/citología , Quinasa 1 Reguladora del Ciclo Celular (Checkpoint 1)/antagonistas & inhibidores , Nucleósidos/genética , Proteína p53 Supresora de Tumor/genética , Animales , Apoptosis , Ciclo Celular , Línea Celular Tumoral , Proliferación Celular , Supervivencia Celular , Citarabina/administración & dosificación , Desoxicitidina/administración & dosificación , Desoxicitidina/análogos & derivados , Ensayos de Selección de Medicamentos Antitumorales , Humanos , Leucemia Linfocítica Crónica de Células B/tratamiento farmacológico , Leucemia Linfocítica Crónica de Células B/genética , Leucemia Linfocítica Crónica de Células B/metabolismo , Ratones , Ratones Transgénicos , Mitosis , Mutación , Pirazoles/farmacología , Pirimidinas/farmacología , Transducción de Señal , Vidarabina/administración & dosificación , Vidarabina/análogos & derivados , GemcitabinaRESUMEN
BACKGROUND: Telomerase, a ribonucleoprotein complex catalysing synthesis of telomeric DNA, is an essential cellular immortalizing factor whose activation is a critical step in the progression to malignancy. An important agent maintaining the balance between proliferation, differentiation and apoptosis of intestinal epithelial cells in crypts is butyrate, which is formed in the gastrointestinal tract by anaerobic bacterial fermentation. It inhibits cell growth, induces differentiation and triggers apoptosis in neoplastic colonocytes. MATERIALS AND METHODS: In this study, the responses of adenocarcinoma (HT-29) and fetal (FHC) human colon cells to 5 mM sodium butyrate (NaBt) have been compared. RESULTS: Despite the similar general response of both cell lines to NaBt, i.e., G0/G1 arrest, decrease of growth rate and increase of differentiation (as indicated by alkaline phosphatase activity), they differ in the level and dynamics of the measured parameters. Telomerase activity and the level of mRNA for its catalytic subunit (hTERT) decline significantly after 48 hours, reaching a complete inhibition after 144 hours. While both cell lines show similar kinetics of hTERT transcriptional silencing, the down-regulation of telomerase activity is faster in FHC cells. Correspondingly, we show that a candidate posttranscriptional regulation step, differential splicing of hTERT mRNA, may be involved in the faster loss of telomerase activity in FHC cells. CONCLUSION: Differences in hTERT mRNA splicing may represent a useful marker of telomere metabolism in normal and malignant colon cells and that these changes may be connected with different cytokinetic patterns of these cells.
Asunto(s)
Adenocarcinoma/metabolismo , Colon/metabolismo , Neoplasias del Colon/patología , Regulación Neoplásica de la Expresión Génica , Proteínas de Neoplasias/metabolismo , Empalme del ARN , Telomerasa/metabolismo , Adenocarcinoma/genética , Apoptosis/efectos de los fármacos , Butiratos/farmacología , Ciclo Celular/efectos de los fármacos , Diferenciación Celular/efectos de los fármacos , División Celular/efectos de los fármacos , Células Cultivadas/citología , Células Cultivadas/efectos de los fármacos , Células Cultivadas/metabolismo , Colon/embriología , Proteínas de Unión al ADN , Inducción Enzimática/efectos de los fármacos , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Humanos , Proteínas de Neoplasias/genética , ARN Mensajero/metabolismo , ARN Neoplásico/metabolismo , Telomerasa/genética , Telómero/metabolismo , Células Tumorales Cultivadas/citología , Células Tumorales Cultivadas/efectos de los fármacos , Células Tumorales Cultivadas/metabolismoRESUMEN
Abnormalities in ATM and TP53 genes represent important predictive factors in chronic lymphocytic leukemia (CLL); however, the efficacy of CD20 targeting immunotherapy is only poorly defined in the affected patients. Therefore, we tested the in vitro response to ofatumumab (OFA) and rituximab (RTX) in 75 CLL samples with clearly defined p53 or ATM inactivation. Using standard conditions allowing complement-dependent cytotoxicity, i.e., 10 µg/mL of antibodies and 20% active human serum, we observed clear differences among the tested genetic categories: ATM-mutated samples (n = 17) represented the most sensitive, wild-type samples (n = 31) intermediate, and TP53-mutated samples (n = 27) the most resistant group (ATM-mut vs. TP53-mut: P = 0.0005 for OFA and P = 0.01 for RTX). The response correlated with distinct levels of CD20 and critical complement inhibitors CD55 and CD59; CD20 level median was the highest in ATM-mutated and the lowest in TP53-mutated samples (difference between the groups P < 0.01), while the total level of complement inhibitors (CD55 plus CD59) was distributed in the opposite manner (P < 0.01). Negligible response to both OFA and RTX was noted in all cultures (n = 10) tested in the absence of active serum, which strongly indicated that complement-dependent cytotoxicity was a principal cell death mechanism. Our study shows that (1) common genetic defects in CLL cells significantly impact a primary response to anti-CD20 monoclonal antibodies and (2) ATM-mutated patients with currently poor prognosis may potentially benefit from immunotherapy targeting CD20.
Asunto(s)
Anticuerpos Monoclonales de Origen Murino/farmacología , Anticuerpos Monoclonales/farmacología , Antígenos CD20/inmunología , Antígenos de Neoplasias/inmunología , Proteínas de la Ataxia Telangiectasia Mutada/genética , Genes p53 , Leucemia Linfocítica Crónica de Células B/patología , Proteínas de Neoplasias/genética , Adulto , Anciano , Anciano de 80 o más Años , Anticuerpos Monoclonales Humanizados , Antígenos CD20/efectos de los fármacos , Antígenos de Neoplasias/efectos de los fármacos , Proteínas de la Ataxia Telangiectasia Mutada/deficiencia , Proteínas de la Ataxia Telangiectasia Mutada/fisiología , Antígenos CD55/inmunología , Antígenos CD59/inmunología , Proteínas del Sistema Complemento/inmunología , Medios de Cultivo , Reparación del ADN/genética , Resistencia a Antineoplásicos , Ensayos de Selección de Medicamentos Antitumorales , Femenino , Humanos , Masculino , Persona de Mediana Edad , Rituximab , Suero , Células Tumorales Cultivadas , Proteína p53 Supresora de Tumor/deficiencia , Proteína p53 Supresora de Tumor/fisiologíaRESUMEN
The nucleophosmin 1 (NPM1) gene is one of the most commonly mutated genes in acute myeloid leukemia (AML), occurring in approximately 60% of adult cytogenetically normal AML (CN-AML). To date, these mutations have only been detected in cells of the myeloid lineage, whereas the potential clonal involvement of the lymphoid lineage is controversial. In our study, NPM1 mutations were analyzed using the highly sensitive real-time quantitative polymerase chain reaction (RQ-PCR) method on fluorescence-activated cell-sorted (FACS) purified different circulating mature cell populations in patients with NPM1-mutated CN-AML. As expected, NPM1 mutations were found in myeloid blood cells, including CD14(+) monocytes and CD66b(+) granulocytes. However, we were also able to detect NPM1 mutations in CD19(+) B cells and CD3(-)14(-)16(+)56(+) natural killer (NK) cells, albeit at lower levels. Surprisingly, mutations were also detected in CD3(+) T cells from all analyzed patients. Our data demonstrate that NPM1-mutated CN-AML originates in an early stem cell with both lymphoid and myeloid differentiation potential.
Asunto(s)
Leucemia Mieloide Aguda/genética , Mutación , Proteínas Nucleares/genética , Anciano , Anciano de 80 o más Años , Dosificación de Gen , Humanos , Inmunofenotipificación , Cariotipificación , Leucemia Mieloide Aguda/diagnóstico , Leucemia Mieloide Aguda/tratamiento farmacológico , Leucocitos/metabolismo , Persona de Mediana Edad , Nucleofosmina , FenotipoRESUMEN
The technology of array comparative genomic hybridization (array-CGH/aCGH) enabled the identification of novel genomic aberrations in chronic lymphocytic leukemia (CLL) including the monoallelic and biallelic deletions affecting 22q11 locus. In contrast to previous publications, we hypothesized that the described 22q11 deletions are a consequence of the rearrangement of immunoglobulin lambda light chain locus (IGL) segments surrounding several protein-coding genes located in this region. Indeed, using array-CGH and PCR analysis we show that all deletions (n=7) affecting the 22q11 locus in our cohort (n=40) are based on the physiological mechanism of IGL rearrangement. This demonstrates that this loss of genetic material is likely not pathogenic and in fact is merely a marker of IGL rearrangement.