RESUMO
Although the unfavourable prognostic role of complex karyotype (CK) in chronic lymphocytic leukaemia (CLL) patients treated with chemoimmunotherapy has been clarified, its impact on the outcome of patients being treated with novel targeted agents, and especially with venetoclax-based regimens, remains to be resolved. In fact, only few studies, utilizing data derived from clinical trials (e.g. MURANO, CLL14, GAIA-CLL13), specifically focus on this topic while real-word evidence is missing. In our real-life retrospective study conducted on 61 patients with CLL and treated with venetoclax-based regimens in any therapeutic line, we documented a remarkable lower progression-free survival in patients harbouring both CK and high CK, while overall response rate (including complete remissions and partial remissions) and overall survival are not affected by CK in our population.
Assuntos
Compostos Bicíclicos Heterocíclicos com Pontes , Leucemia Linfocítica Crônica de Células B , Sulfonamidas , Humanos , Leucemia Linfocítica Crônica de Células B/tratamento farmacológico , Leucemia Linfocítica Crônica de Células B/genética , Leucemia Linfocítica Crônica de Células B/mortalidade , Compostos Bicíclicos Heterocíclicos com Pontes/uso terapêutico , Compostos Bicíclicos Heterocíclicos com Pontes/administração & dosagem , Sulfonamidas/uso terapêutico , Sulfonamidas/administração & dosagem , Feminino , Masculino , Idoso , Pessoa de Meia-Idade , Prognóstico , Estudos Retrospectivos , Protocolos de Quimioterapia Combinada Antineoplásica/uso terapêutico , Idoso de 80 Anos ou mais , Cariótipo , AdultoRESUMO
Chronic lymphocytic leukemia (CLL) is the most common leukemia in western societies, recognized by clinical and molecular heterogeneity. Despite the success of targeted therapies, acquired resistance remains a challenge for relapsed and refractory CLL, as a consequence of mutations in the target or the upregulation of other survival pathways leading to the progression of the disease. Research on proteins that can trigger such pathways may define novel therapies for a successful outcome in CLL such as the receptor tyrosine kinase-like orphan receptor 1 (ROR1). ROR1 is a signaling receptor for Wnt5a, with an important role during embryogenesis. The aberrant expression on CLL cells and several types of tumors, is involved in cell proliferation, survival, migration as well as drug resistance. Antibody-based immunotherapies and small-molecule compounds emerged to target ROR1 in preclinical and clinical studies. Efforts have been made to identify new prognostic markers having predictive value to refine and increase the detection and management of CLL. ROR1 can be considered as an attractive target for CLL diagnosis, prognosis, and treatment. It can be clinically effective alone and/or in combination with current approved agents. In this review, we summarize the scientific achievements in targeting ROR1 for CLL diagnosis, prognosis, and treatment.
Assuntos
Leucemia Linfocítica Crônica de Células B , Receptores Órfãos Semelhantes a Receptor Tirosina Quinase , Leucemia Linfocítica Crônica de Células B/metabolismo , Leucemia Linfocítica Crônica de Células B/tratamento farmacológico , Leucemia Linfocítica Crônica de Células B/patologia , Humanos , Receptores Órfãos Semelhantes a Receptor Tirosina Quinase/metabolismo , Prognóstico , Terapia de Alvo Molecular , Animais , Biomarcadores Tumorais/metabolismoRESUMO
Signalling events downstream the B-cell receptor (BCR) are central for the survival and progression of chronic lymphocytic leukaemia (CLL) cells. Focal adhesion kinase (FAK), regulated through calpain, interacts with molecules of BCR signalling, cytoskeletal modelling and disease progression, such as Src/Lyn, cortactin and HS1. Hypothesizing that FAK might play a key role in CLL pathogenesis, we observed a down-modulation of FAK whole form, associated with FAK cleavage due to calpain activity upon BCR stimulation. Patients, whose cells were able to release Ca++ after BCR stimulation, had less amount of full-length FAK, which translated into a higher presence of cleaved/activated form of the protein phosphorylated at Y397, these features being mostly shown by immunoglobulin heavy chain (IGHV)-unmutated poor-prognosis patients. Moreover, we found that cortactin and HS1 proteins were overexpressed in those cells, suggesting a possible interplay with FAK. Treatment with the FAK inhibitor Defactinib was able to induce apoptosis in CLL cells. In conclusion, the malignant phenotype in unfavourable-prognosis patients seems to be encouraged by the overexpression of cortactin and HS1, that, together with FAK, may be involved in a druggable pathogenetic pathway in CLL.
Assuntos
Leucemia Linfocítica Crônica de Células B , Humanos , Proteína-Tirosina Quinases de Adesão Focal/metabolismo , Leucemia Linfocítica Crônica de Células B/genética , Calpaína/metabolismo , Cortactina/metabolismo , Cálcio/metabolismo , Fosforilação , Receptores de Antígenos de Linfócitos B/metabolismoRESUMO
The stromal microenvironment is central to chronic lymphocytic leukemia (CLL) pathogenesis. How leukemic cells condition the stroma to enhance its chemoattractant properties remains elusive. Here, we show that mouse and human CLL cells promote the contact-independent stromal expression of homing chemokines. This function was strongly enhanced in leukemic cells from Eµ-TCL1 mice lacking the pro-oxidant p66Shc adaptor, which develop an aggressive disease with organ infiltration. We identified interleukin-9 (IL-9) as the soluble factor, negatively modulated by p66Shc, that is responsible for the chemokine-elevating activity of leukemic cells on stromal cells. IL-9 blockade in Eµ-TCL1/p66Shc-/- mice resulted in a decrease in the nodal expression of homing chemokines, which correlated with decreased leukemic cell invasiveness. IL-9 levels were found to correlate inversely with residual p66Shc in p66Shc-deficient human CLL cells (n = 52 patients). p66Shc reconstitution in CLL cells normalized IL-9 expression and neutralized their chemokine-elevating activity. Notably, high IL-9 expression in CLL cells directly correlates with lymphadenopathy, liver infiltration, disease severity, and overall survival, emerging as an independent predictor of disease outcome. Our results demonstrate that IL-9 modulates the chemokine landscape in the stroma and that p66Shc, by regulating IL-9 expression, fine tunes the ability of leukemic cells to shape the microenvironment, thereby contributing to CLL pathogenesis.
RESUMO
Complex karyotype (CK) at chronic lymphocytic leukemia (CLL) diagnosis is a negative biomarker of adverse outcome. Since the impact of CK and its subtypes, namely type-2 CK (CK with major structural abnormalities) or high-CK (CK with ≥5 chromosome abnormalities), on the risk of developing Richter syndrome (RS) is unknown, we carried out a multicenter real-life retrospective study to test its prognostic impact. Among 540 CLL patients, 107 harbored a CK at CLL diagnosis, 78 were classified as CK2 and 52 as high-CK. Twenty-eight patients developed RS during a median follow-up of 6.7 years. At the time of CLL diagnosis, CK2 and high-CK were more common and predicted the highest risk of RS transformation, together with advanced Binet stage, unmutated (U)-IGHV, 11q-, and TP53 abnormalities. We integrated these variables into a hierarchical model: high-CK and/or CK2 patients showed a 10-year time to RS (TTRS) of 31%; U-IGHV/11q- /TP53 abnormalities/Binet stage B-C patients had a 10-year TTRS of 12%; mutated (M)-IGHV without CK and TP53 disruption a 10-year TTRS of 3% (P<0.0001). We herein demonstrate that CK landscape at CLL diagnosis allows the risk of RS transformation to be refined and we recapitulated clinico-biological variables into a prognostic model.
Assuntos
Leucemia Linfocítica Crônica de Células B , Linfoma Difuso de Grandes Células B , Humanos , Cariótipo , Leucemia Linfocítica Crônica de Células B/diagnóstico , Leucemia Linfocítica Crônica de Células B/genética , Mutação , Estudos RetrospectivosRESUMO
Cancer cells undergo changes in metabolic and survival pathways that increase their malignancy. Isoform 2 of the glycolytic enzyme hexokinase (HK2) enhances both glucose metabolism and resistance to death stimuli in many neoplastic cell types. Here, we observe that HK2 locates at mitochondria-endoplasmic reticulum (ER) contact sites called MAMs (mitochondria-associated membranes). HK2 displacement from MAMs with a selective peptide triggers mitochondrial Ca2+ overload caused by Ca2+ release from ER via inositol-3-phosphate receptors (IP3Rs) and by Ca2+ entry through plasma membrane. This results in Ca2+ -dependent calpain activation, mitochondrial depolarization and cell death. The HK2-targeting peptide causes massive death of chronic lymphocytic leukemia B cells freshly isolated from patients, and an actionable form of the peptide reduces growth of breast and colon cancer cells allografted in mice without noxious effects on healthy tissues. These results identify a signaling pathway primed by HK2 displacement from MAMs that can be activated as anti-neoplastic strategy.
Assuntos
Hexoquinase , Neoplasias , Animais , Morte Celular , Retículo Endoplasmático/metabolismo , Hexoquinase/genética , Hexoquinase/metabolismo , Humanos , Camundongos , Mitocôndrias , Membranas Mitocondriais/metabolismo , Neoplasias/metabolismoRESUMO
Hairy cell leukemia variant (HCLv) is a provisional disease in the 2016 WHO classification of lymphomas, characterized by unfavorable prognosis and early relapse following conventional purine analog-based regimens. In this study, we report 2 patients with relapsed HCLv treated with ibrutinib. The first patient achieved a partial response following ibrutinib treatment and received the drug for 16 months, without severe adverse events. However, at disease progression venetoclax was not clinically active. The second patient discontinued the drug early due to intolerance. Ibrutinib was active in our patients with HCLv and deserve further investigations.
Assuntos
Adenina/análogos & derivados , Leucemia de Células Pilosas/diagnóstico , Leucemia de Células Pilosas/tratamento farmacológico , Piperidinas/uso terapêutico , Inibidores de Proteínas Quinases/uso terapêutico , Adenina/administração & dosagem , Adenina/efeitos adversos , Adenina/uso terapêutico , Idoso de 80 Anos ou mais , Protocolos de Quimioterapia Combinada Antineoplásica/efeitos adversos , Protocolos de Quimioterapia Combinada Antineoplásica/uso terapêutico , Biomarcadores , Biópsia , Feminino , Humanos , Imuno-Histoquímica , Imunofenotipagem , Leucemia de Células Pilosas/terapia , Pessoa de Meia-Idade , Piperidinas/administração & dosagem , Piperidinas/efeitos adversos , Inibidores de Proteínas Quinases/administração & dosagem , Inibidores de Proteínas Quinases/efeitos adversos , Retratamento , Tomografia Computadorizada por Raios X , Resultado do TratamentoRESUMO
Considering the role played by the heat shock protein of 70 kDa (HSP70) in cancer, we characterized this protein and its major regulator, the heat shock factor 1 (HSF1), in chronic lymphocytic leukemia (CLL). We found both HSP70 and HSF1 overexpressed in CLL patients, correlated to poor prognosis and abnormally localized in the nucleus of leukemic B cells. The two proteins were strictly correlated each other and their levels decreased consensually in those patients responding to in vivo therapeutic regimens. HSP70 and HSF1 inhibition was proved to be effective in inducing a dose-dependent in vitro apoptosis of CLL B cells. Considering that HSF1 is finely regulated by kinases belonging to pathways triggered by rat sarcoma (RAS), we benefited from a previous proteomic study performed in CLL patients aiming to assess the activation/expression of key signaling proteins. We found that patients showing high levels of HSP70 also expressed high Akt-Ser473, thus activating HSF1. Inhibition of PI3K, which activates AKT, reduced the expression of HSF1 and HSP70. By contrast, HSP70-low patients displayed high activation of MEK1/2 and ERK1/2, known to negatively regulate HSF1. These data demonstrate that the HSP70 expression is regulated by the modulation of HSF1 activity through the activation of RAS-regulated pathways and suggest the HSP70/HSF1 interplay as an interesting target for antileukemic therapies. Finally, inhibition of PI3K, that activates AKT, reduced the expression of HSF1 and HSP70.
Assuntos
Núcleo Celular/metabolismo , Proteínas de Choque Térmico HSP70/metabolismo , Fatores de Transcrição de Choque Térmico/metabolismo , Leucemia Linfocítica Crônica de Células B/metabolismo , Transdução de Sinais , Adenina/análogos & derivados , Estudos de Casos e Controles , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Relação Dose-Resposta a Droga , Flavonoides/farmacologia , Flavonóis , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Humanos , Leucemia Linfocítica Crônica de Células B/tratamento farmacológico , Terapia de Alvo Molecular , Fosfatidilinositol 3-Quinases/metabolismo , Piperidinas , Prognóstico , Proteômica/métodos , Proteínas Proto-Oncogênicas c-akt/metabolismo , Pirazóis/farmacologia , Pirimidinas/farmacologia , Transdução de Sinais/efeitos dos fármacos , Regulação para Cima/efeitos dos fármacosRESUMO
BACKGROUND: Complex karyotype (CK) is a heterogeneous category with a negative impact in chronic lymphocytic leukaemia (CLL). Our group has recently reported that CK patients with major structural abnormalities (i.e. CK2) are characterised by a worse prognosis, as compared to other lesions within CK(CK1). METHODS: We performed a multicentre retrospective study to test whether the combination of CK subtypes with IGHV status could be a relevant prognostic and predictive tool. RESULTS: Among 522 patients 13% harboured CK2, 41% CK1 and/or U-IGHV (U-CK1) and 46% M-IGHV without any CK subtypes (M-noCK). After a median follow-up of 5.8 years, CK2 patients had the shortest TTFT (5-year TTFT 31%, 39 and 81%, p < 0.0001) and OS (5-year OS 67%, 85 and 93%, p < 0.0001) as compared to U-CK1 or M-noCK cases, regardless of TP53 abnormalities. CK2 patients also had the worst outcome after chemoimmunotherapy. In fact, the median TTNT after FCR or BR was 1.86 and 4.79 years for CK2 and U-CK1, but not reached for M-noCK patients (p < 0.0005). CONCLUSIONS: We herein suggest that the combined assessment of the IGHV mutational status and CK subtypes refines the prognostication of CLL, allowing to identify M-IGHV patients without any CK subtypes who are characterised by an indolent disease and excellent outcome after chemoimmunotherapy.
Assuntos
Aberrações Cromossômicas , Cadeias Pesadas de Imunoglobulinas/genética , Leucemia Linfocítica Crônica de Células B/genética , Mutação , Idoso , Idoso de 80 Anos ou mais , Bandeamento Cromossômico , Feminino , Humanos , Cariótipo , Leucemia Linfocítica Crônica de Células B/mortalidade , Masculino , Pessoa de Meia-Idade , Prognóstico , Estudos RetrospectivosRESUMO
The Shc family adaptor p66Shc acts as a negative regulator of proliferative and survival signals triggered by the B-cell receptor and, by enhancing the production of reactive oxygen species, promotes oxidative stress-dependent apoptosis. Additionally, p66Shc controls the expression and function of chemokine receptors that regulate lymphocyte traffic. Chronic lymphocytic leukemia cells have a p66Shc expression defect which contributes to their extended survival and correlates with poor prognosis. We analyzed the impact of p66Shc ablation on disease severity and progression in the Eµ-TCL1 mouse model of chronic lymphocytic leukemia. We showed that Eµ-TCL1/p66Shc-/- mice developed an aggressive disease that had an earlier onset, occurred at a higher incidence and led to earlier death compared to that in Eµ-TCL1 mice. Eµ-TCL1/p66Shc-/- mice displayed substantial leukemic cell accumulation in both nodal and extranodal sites. The target organ selectivity correlated with upregulation of chemokine receptors whose ligands are expressed therein. This also applied to chronic lymphocytic leukemia cells, where chemokine receptor expression and extent of organ infiltration were found to correlate inversely with these cells' level of p66Shc expression. p66Shc expression declined with disease progression in Eµ-TCL1 mice and could be restored by treatment with the Bruton tyrosine kinase inhibitor ibrutinib. Our results highlight p66Shc deficiency as an important factor in the progression and severity of chronic lymphocytic leukemia and underscore p66Shc expression as a relevant therapeutic target.
Assuntos
Carcinogênese/metabolismo , Leucemia Linfocítica Crônica de Células B/metabolismo , Proteínas de Neoplasias/metabolismo , Neoplasias Experimentais/metabolismo , Receptores de Quimiocinas/metabolismo , Proteína 1 de Transformação que Contém Domínio 2 de Homologia de Src/deficiência , Animais , Carcinogênese/genética , Carcinogênese/patologia , Leucemia Linfocítica Crônica de Células B/genética , Leucemia Linfocítica Crônica de Células B/patologia , Camundongos , Camundongos Knockout , Proteínas de Neoplasias/genética , Neoplasias Experimentais/genética , Neoplasias Experimentais/patologia , Receptores de Quimiocinas/genética , Proteína 1 de Transformação que Contém Domínio 2 de Homologia de Src/metabolismoRESUMO
Light chain amyloidosis is characterized by the progressive deposition of immunoglobulin light chains into the extracellular tissue, leading to organ dysfunction. Usually, it is associated with an underlying clonal plasma cell dyscrasia and rarely with chronic lymphocytic leukaemia. Herein, we described the first report of a patient with relapsed chronic lymphocytic leukaemia harbouring TP53 abnormalities who developed, histologically proven, systemic light chain amyloidosis who was treated with the PI3K inhibitor, idelalisib, and rituximab. Unfortunately, the patient had sudden death during sleep, likely caused by arrhythmia secondary to amyloid cardiomyopathy. Idelalisib was at least effective in reducing secretory free light chain, chronic lymphocytic leukaemia burden, and to improve the survival of patient.
Assuntos
Antineoplásicos Imunológicos/uso terapêutico , Antineoplásicos/uso terapêutico , Leucemia Linfocítica Crônica de Células B/tratamento farmacológico , Purinas/uso terapêutico , Quinazolinonas/uso terapêutico , Rituximab/uso terapêutico , Antineoplásicos/administração & dosagem , Antineoplásicos/farmacologia , Humanos , Masculino , Pessoa de Meia-Idade , Purinas/administração & dosagem , Purinas/farmacologia , Quinazolinonas/administração & dosagem , Quinazolinonas/farmacologiaRESUMO
Cortactin (CTTN) is a substrate of the Src kinase Lyn that is known to play an actin cytoskeletal regulatory role involved in cell migration and cancer progression following its phosphorylation at Y421. We recently demonstrated that Cortactin is overexpressed in patients with chronic lymphocytic leukaemia (CLL). This work was aimed at defining the functional role of Cortactin in these patients. We found that Cortactin is variably expressed in CLL patients both in the peripheral blood and lymph nodes and that its expression correlates with the release of matrix metalloproteinase 9 (MMP-9) and the motility of neoplastic cells. Cortactin knockdown, by siRNA, induced a reduction in MMP-9 release as well as a decrease of migration capability of leukaemic B cells in vitro, also after chemotactic stimulus. Furthermore, Cortactin phosphorylation was lowered by the Src kinase-inhibitor PP2 with a consequent decrease of MMP-9 release in culture medium. An impaired migration, as compared to control experiments without Cortactin knockdown, was observed following CXCL12 triggering. Reduced Cortactin expression and phosphorylation were also detected both in vivo and in vitro after treatment with Ibrutinib, a Btk inhibitor. Our results highlight the role of Cortactin in CLL as a check-point molecule between the BCR and CXCR4 signalling pathways.
Assuntos
Pontos de Checagem do Ciclo Celular/fisiologia , Cortactina/fisiologia , Leucemia Linfocítica Crônica de Células B/metabolismo , Proteínas Proto-Oncogênicas c-bcr/fisiologia , Receptores CXCR4/fisiologia , Adenina/análogos & derivados , Adulto , Tirosina Quinase da Agamaglobulinemia , Idoso , Idoso de 80 Anos ou mais , Antineoplásicos/farmacologia , Antineoplásicos/uso terapêutico , Movimento Celular/fisiologia , Feminino , Humanos , Leucemia Linfocítica Crônica de Células B/tratamento farmacológico , Leucemia Linfocítica Crônica de Células B/patologia , Masculino , Metaloproteinase 9 da Matriz/metabolismo , Pessoa de Meia-Idade , Proteínas de Neoplasias/fisiologia , Fosforilação/efeitos dos fármacos , Piperidinas , Proteínas Tirosina Quinases/antagonistas & inibidores , Pirazóis/farmacologia , Pirazóis/uso terapêutico , Pirimidinas/farmacologia , Pirimidinas/uso terapêutico , Transdução de Sinais/fisiologia , Células Tumorais Cultivadas , Quinases da Família src/fisiologiaRESUMO
Aberrant protein kinase activities, and the consequent dramatic increase of Ser/Thr and -Tyr phosphorylation, promote the deregulation of the survival pathways in chronic lymphocytic leukemia (CLL), which is crucial to the pathogenesis and progression of the disease. In this study, we show that the tumor suppressor protein phosphatase 2A (PP2A), one of the major Ser/Thr phosphatases, is in an inhibited form because of the synergistic contribution of 2 events, the interaction with its physiologic inhibitor SET and the phosphorylation of Y307 of the catalytic subunit of PP2A. The latter event is mediated by Lyn, a Src family kinase previously found to be overexpressed, delocalized, and constitutively active in CLL cells. This Lyn/PP2A axis accounts for the persistent high level of phosphorylation of the phosphatase's targets and represents a key connection linking phosphotyrosine- and phosphoserine/threonine-mediated oncogenic signals. The data herein presented show that the disruption of the SET/PP2A complex by a novel FTY720-analog (MP07-66) devoid of immunosuppressive effects leads to the reactivation of PP2A, which in turn triggers apoptosis of CLL cells. When used in combination with SFK inhibitors, the action of MP07-66 is synergistically amplified, providing a new option in the therapeutic strategy for CLL patients.
Assuntos
Chaperonas de Histonas/metabolismo , Imunossupressores/farmacologia , Leucemia Linfocítica Crônica de Células B/tratamento farmacológico , Leucemia Linfocítica Crônica de Células B/metabolismo , Propilenoglicóis/farmacologia , Proteína Fosfatase 2/metabolismo , Esfingosina/análogos & derivados , Fatores de Transcrição/metabolismo , Quinases da Família src/metabolismo , Apoptose/efeitos dos fármacos , Proteínas de Ligação a DNA , Cloridrato de Fingolimode , Proteínas de Choque Térmico HSP90/metabolismo , Humanos , Imunossupressores/química , Fosforilação/efeitos dos fármacos , Fosfotirosina/metabolismo , Propilenoglicóis/química , Mapas de Interação de Proteínas/efeitos dos fármacos , Transdução de Sinais/efeitos dos fármacos , Esfingosina/química , Esfingosina/farmacologia , Células Tumorais CultivadasRESUMO
Lyn, a member of the Src family of kinases, is a key factor in the dysregulation of survival and apoptotic pathways of malignant B cells in chronic lymphocytic leukemia. One of the effects of Lyn's action is spatial and functional segregation of the tyrosine phosphatase SHP-1 into two pools, one beneath the plasma membrane in an active state promoting pro-survival signals, the other in the cytosol in an inhibited conformation and unable to counter the elevated level of cytosolic tyrosine phosphorylation. We herein show that SHP-1 activity can be elicited directly by nintedanib, an agent also known as a triple angiokinase inhibitor, circumventing the phospho-S591-dependent inhibition of the phosphatase, leading to the dephosphorylation of pro-apoptotic players such as procaspase-8 and serine/threonine phosphatase 2A, eventually triggering apoptosis. Furthermore, the activation of PP2A by using MP07-66, a novel FTY720 analog, stimulated SHP-1 activity via dephosphorylation of phospho-S591, which unveiled the existence of a positive feedback signaling loop involving the two phosphatases. In addition to providing further insights into the molecular basis of this disease, our findings indicate that the PP2A/SHP-1 axis may emerge as an attractive, novel target for the development of alternative strategies in the treatment of chronic lymphocytic leukemia.
Assuntos
Apoptose/efeitos dos fármacos , Leucemia Linfocítica Crônica de Células B/patologia , Proteína Fosfatase 2/metabolismo , Proteína Tirosina Fosfatase não Receptora Tipo 6/metabolismo , Transdução de Sinais/efeitos dos fármacos , Retroalimentação Fisiológica , Humanos , Indóis/farmacologia , Leucemia Linfocítica Crônica de Células B/tratamento farmacológico , Terapia de Alvo Molecular/métodos , Células Tumorais CultivadasRESUMO
Lyn, a member of the group of tyrosine kinases named the Src family kinases (SFKs), is overexpressed, associated with an aberrant multiprotein complex and constitutively active in B-cell chronic lymphocytic leukemia (B-CLL) cells, resulting in a high level of tyrosine phosphorylation and contributing to their resistance to apoptosis. By using biochemical and bioinformatics tools, we identified procaspase-8 (procasp8), the caspase-8 zymogen, as a cytosolic target for Lyn in B-CLL cells, the phosphorylation of which at Tyr380 promotes the formation of an inactive procasp8 homodimer. This complex remains segregated in the cytosol and appears to be crucial in mediating the antiapoptotic function of Lyn in this disease. The significance of the Lyn-procasp8 axis in impairing apoptosis in B-CLL cells was further confirmed by pharmacological and genetic inhibition of procasp8, which drastically reduced the apoptosis induced by the SFK inhibitors PP2 and dasatinib. Our data highlight that Lyn's dysregulated expression, activity, and localization in B-CLLs support resistance to cell demise by inhibiting an early player of apoptotic signaling, and potentially broaden the perspectives of developing new strategies for the treatment of this disease.
Assuntos
Apoptose , Caspase 8/química , Leucemia Linfocítica Crônica de Células B/patologia , Quinases da Família src/metabolismo , Western Blotting , Caspase 8/metabolismo , Proliferação de Células , Biologia Computacional , Citosol/metabolismo , Eletroforese em Gel Bidimensional , Humanos , Leucemia Linfocítica Crônica de Células B/metabolismo , Fosforilação , Multimerização Proteica , Proteoma/análise , Células Tumorais Cultivadas , Tirosina/metabolismoRESUMO
The JAK/STAT pathway is altered in T-cell large granular lymphocytic leukemia. In all patients, leukemic LGLs display upregulation of phosphorylated STAT3 (P-STAT3) that activates expression of many antiapoptotic genes. To investigate the mechanisms maintaining STAT3 aberrantly phosphorylated using transcriptional protein and functional assays, we analyzed interleukin (IL)-6 and suppressor of cytokine signaling-3 (SOCS3), 2 key factors of the JAK/STAT pathway that induce and inhibit STAT3 activation, respectively. We showed that IL-6 was highly expressed and released by the patients' peripheral blood LGL-depleted population, accounting for a trans-signaling process. By neutralizing IL-6 or its specific receptor with specific antibodies, a significant reduction of P-STAT3 levels and, consequently, LGL survival was demonstrated. In addition, we found that SOCS3 was down-modulated in LGL and unresponsive to IL-6 stimulation. By treating neoplastic LGLs with a demethylating agent, IL-6-mediated SOCS3 expression was restored with consequent P-STAT3 and myeloid cell leukemia-1 down-modulation. Methylation in the SOCS3 promoter was not detectable, suggesting that an epigenetic inhibition mechanism occurs at a different site. Our data indicate that loss of the inhibitor SOCS3 cooperates with IL-6 to maintain JAK/STAT pathway activation, thus contributing to leukemic LGL survival, and suggest a role of demethylating agents in the treatment of this disorder.
Assuntos
Janus Quinases/metabolismo , Leucemia Linfocítica Granular Grande/metabolismo , Fatores de Transcrição STAT/metabolismo , Transdução de Sinais/fisiologia , Idoso , Células Cultivadas , Feminino , Humanos , Janus Quinases/genética , Leucemia Linfocítica Granular Grande/genética , Masculino , Pessoa de Meia-Idade , Mutação/fisiologia , Fosforilação , Fatores de Transcrição STAT/genética , Fator de Transcrição STAT3/genética , Fator de Transcrição STAT3/metabolismo , Transdução de Sinais/genética , Proteína 3 Supressora da Sinalização de Citocinas , Proteínas Supressoras da Sinalização de Citocina/genética , Proteínas Supressoras da Sinalização de Citocina/metabolismoAssuntos
Protocolos de Quimioterapia Combinada Antineoplásica/uso terapêutico , Leucemia Linfocítica Crônica de Células B/diagnóstico , Leucemia Linfocítica Crônica de Células B/tratamento farmacológico , Adenina/análogos & derivados , Protocolos de Quimioterapia Combinada Antineoplásica/efeitos adversos , Humanos , Leucemia Linfocítica Crônica de Células B/mortalidade , Piperidinas , Inibidores de Proteínas Quinases/administração & dosagem , Purinas/administração & dosagem , Pirazóis/administração & dosagem , Pirimidinas/administração & dosagem , Quinazolinonas/administração & dosagem , Resultado do TratamentoRESUMO
Functional abnormalities of chronic lymphocytic leukaemia (CLL) cells may be related to the microtubular network of cell cytoskeleton; specifically tubulin involvement in cells after B-cell receptor engagement. As microtubule inhibitors could represent a therapeutic strategy for CLL, this study investigated the capability of nocodazole, a synthetic depolymerizing agent, to kill CLL leukaemic cells. We demonstrated that nocodazole was highly specific for the in vitro induction of apoptosis in leukaemic cells from 90 CLL patients, without affecting the viability of T-cells and/or mesenchymal stromal cells (MSCs) recovered from the same patients. Nocodazole was observed to overcome the pro-survival signals provided by MSCs. Competing with ATP for the nucleotide-binding site, nocodazole has been observed to turn off the high basal tyrosine phosphorylation of leukaemic cells mediated by the Src-kinase Lyn. Considering that most anti-microtubule drugs have limited clinical use because of their strong toxic effects, the high selectivity of nocodazole for leukaemic cells in CLL and its capability to bypass microenvironmental pro-survival stimuli, suggests the use of this inhibitor for designing new therapeutic strategies in CLL treatment.
Assuntos
Antineoplásicos/farmacologia , Leucemia Linfocítica Crônica de Células B/patologia , Microtúbulos/efeitos dos fármacos , Nocodazol/farmacologia , Moduladores de Tubulina/farmacologia , Quinases da Família src/antagonistas & inibidores , Adulto , Idoso , Idoso de 80 Anos ou mais , Antineoplásicos/metabolismo , Apoptose/efeitos dos fármacos , Apoptose/fisiologia , Linfócitos B/efeitos dos fármacos , Linfócitos B/fisiologia , Comunicação Celular/fisiologia , Técnicas de Cocultura , Ensaios de Seleção de Medicamentos Antitumorais/métodos , Feminino , Humanos , Leucemia Linfocítica Crônica de Células B/metabolismo , Masculino , Células-Tronco Mesenquimais/efeitos dos fármacos , Células-Tronco Mesenquimais/fisiologia , Microscopia Confocal , Pessoa de Meia-Idade , Nocodazol/metabolismo , Prognóstico , Proteínas Proto-Oncogênicas c-bcr/metabolismo , Linfócitos T/efeitos dos fármacos , Linfócitos T/fisiologia , Moduladores de Tubulina/metabolismo , Células Tumorais CultivadasRESUMO
Although intrinsic apoptosis defects are causal to the extended survival of chronic lymphocytic leukemia (CLL) B cells, several lines of evidence support a contribution of the peripheral lymphoid organs and BM microenvironment to the extended lifespan of leukemic B cells. Lymphocyte trafficking is controlled by homing signals provided by stromal cell-derived chemokines and egress signals provided by sphingosine-1-phosphate (S1P). In the present study, we show that expression of S1P1, the S1P receptor responsible for lymphocyte egress, is selectively reduced in CLL B cells with unmutated IGHV. Expression of S1P2, which controls B-cell homeostasis, is also impaired in CLL B cells but independently of the IGHV mutational status. We provide evidence herein that p66Shc, a Shc adaptor family member the deficiency of which is implicated in the apoptosis defects of CLL B cells, controls S1P1 expression through its pro-oxidant activity. p66Shc also controls the expression of the homing receptor CCR7, which opposes S1P1 by promoting lymphocyte retention in peripheral lymphoid organs. The results of the present study provide insights into the regulation of S1P1 expression in B cells and suggest that defective egress caused by impaired S1P1 expression contributes to the extended survival of CLL B cells by prolonging their residency in the prosurvival niche of peripheral lymphoid organs.
Assuntos
Leucemia Linfocítica Crônica de Células B/diagnóstico , Leucemia Linfocítica Crônica de Células B/genética , Receptores de Lisoesfingolipídeo/genética , Proteínas Adaptadoras da Sinalização Shc/fisiologia , Adulto , Animais , Feminino , Regulação da Expressão Gênica/genética , Regulação Leucêmica da Expressão Gênica , Humanos , Leucemia Linfocítica Crônica de Células B/metabolismo , Leucemia Linfocítica Crônica de Células B/mortalidade , Masculino , Camundongos , Camundongos Knockout , Oxidantes/metabolismo , Prognóstico , Receptores de Lisoesfingolipídeo/fisiologia , Proteínas Adaptadoras da Sinalização Shc/genética , Proteínas Adaptadoras da Sinalização Shc/metabolismo , Proteína 1 de Transformação que Contém Domínio 2 de Homologia de Src , Células Tumorais CultivadasRESUMO
Cortactin, an actin binding protein and Lyn substrate, is up-regulated in several cancers and its level is associated with increased cell migration, metastasis and poor prognosis. The identification that the Src kinase Lyn and its substrate HS1 are over-expressed in B-cell chronic lymphocytic leukemia and involved in resistance to chemotherapy and poor prognosis, prompted us to investigate the role of cortactin, an HS1 homolog, in the pathogenesis and progression of this disorder. In this study, we observed that cortactin is over-expressed in leukemic cells of patients (1.10 ± 0.12) with respect to normal B lymphocytes (0.19 ± 0.06; P=0.0065). Fifty-three percent of our patients expressed the WT mRNA and p80/85 protein isoforms, usually lacking in normal B lymphocytes which express the SV1 variant and the p70/75 protein isoforms. Moreover, we found an association of the cortactin overexpression and negative prognostic factors, including ZAP-70 (P<0.01), CD38 (P<0.01) and somatic hypermutations in the immunoglobulin heavy-chain variable region (P<0.01). Our results show that patients with B-cell chronic lymphocytic leukemia express high levels of cortactin with a particular overexpression of the WT isoform that is lacking in normal B cells, and a correlation to poor prognosis, suggesting that this protein could be relevant in the pathogenesis and aggressiveness of the disease.