RESUMEN
The BCL2 inhibitor venetoclax has been approved to treat different hematological malignancies. Because there is no common genetic alteration causing resistance to venetoclax in chronic lymphocytic leukemia (CLL) and B-cell lymphoma, we asked if epigenetic events might be involved in venetoclax resistance. Therefore, we employed whole-exome sequencing, methylated DNA immunoprecipitation sequencing, and genome-wide clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 screening to investigate venetoclax resistance in aggressive lymphoma and high-risk CLL patients. We identified a regulatory CpG island within the PUMA promoter that is methylated upon venetoclax treatment, mediating PUMA downregulation on transcript and protein level. PUMA expression and sensitivity toward venetoclax can be restored by inhibition of methyltransferases. We can demonstrate that loss of PUMA results in metabolic reprogramming with higher oxidative phosphorylation and adenosine triphosphate production, resembling the metabolic phenotype that is seen upon venetoclax resistance. Although PUMA loss is specific for acquired venetoclax resistance but not for acquired MCL1 resistance and is not seen in CLL patients after chemotherapy-resistance, BAX is essential for sensitivity toward both venetoclax and MCL1 inhibition. As we found loss of BAX in Richter's syndrome patients after venetoclax failure, we defined BAX-mediated apoptosis to be critical for drug resistance but not for disease progression of CLL into aggressive diffuse large B-cell lymphoma in vivo. A compound screen revealed TRAIL-mediated apoptosis as a target to overcome BAX deficiency. Furthermore, antibody or CAR T cells eliminated venetoclax resistant lymphoma cells, paving a clinically applicable way to overcome venetoclax resistance.
Asunto(s)
Neoplasias Hematológicas , Leucemia Linfocítica Crónica de Células B , Linfoma de Células B Grandes Difuso , Humanos , Leucemia Linfocítica Crónica de Células B/tratamiento farmacológico , Leucemia Linfocítica Crónica de Células B/genética , Leucemia Linfocítica Crónica de Células B/patología , Proteína 1 de la Secuencia de Leucemia de Células Mieloides/genética , Proteínas Proto-Oncogénicas c-bcl-2/genética , Proteínas Proto-Oncogénicas c-bcl-2/metabolismo , Proteína X Asociada a bcl-2/metabolismo , Resistencia a Antineoplásicos/genética , Proteínas Reguladoras de la Apoptosis/genética , Compuestos Bicíclicos Heterocíclicos con Puentes/farmacología , Compuestos Bicíclicos Heterocíclicos con Puentes/uso terapéutico , Linfoma de Células B Grandes Difuso/patología , Neoplasias Hematológicas/tratamiento farmacológico , Neoplasias Hematológicas/genética , Epigénesis GenéticaRESUMEN
The pathogenesis of chronic lymphocytic leukemia (CLL) has been linked to constitutive NF-κB activation but the underlying mechanisms are poorly understood. Here we show that alternative splicing of the negative regulator of NF-κB and tumor suppressor gene CYLD regulates the pool of CD5+ B cells through sustained canonical NF-κB signaling. Reinforced canonical NF-κB activity leads to the development of B1 cell-associated tumor formation in aging mice by promoting survival and proliferation of CD5+ B cells, highly reminiscent of human B-CLL. We show that a substantial number of CLL patient samples express sCYLD, strongly implicating a role for it in human B-CLL. We propose that our new CLL-like mouse model represents an appropriate tool for studying ubiquitination-driven canonical NF-κB activation in CLL. Thus, inhibition of alternative splicing of this negative regulator is essential for preventing NF-κB-driven clonal CD5+ B-cell expansion and ultimately CLL-like disease.
Asunto(s)
Enzima Desubiquitinante CYLD/genética , Genes Supresores de Tumor/fisiología , Leucemia Linfocítica Crónica de Células B/genética , FN-kappa B/genética , Empalme del ARN/genética , Transducción de Señal/genética , Animales , Linfocitos B/metabolismo , Antígenos CD5/genética , Proliferación Celular/genética , Supervivencia Celular/genética , Humanos , Ratones , Ubiquitinación/genéticaAsunto(s)
Antineoplásicos/farmacología , Linfocitos B/efectos de los fármacos , Compuestos de Bifenilo/farmacología , Compuestos Bicíclicos Heterocíclicos con Puentes/farmacología , Regulación Leucémica de la Expresión Génica , Proteína 1 de la Secuencia de Leucemia de Células Mieloides/antagonistas & inhibidores , Nitrofenoles/farmacología , Sulfonamidas/farmacología , Proteínas Quinasas p38 Activadas por Mitógenos/genética , Linfocitos B/metabolismo , Linfocitos B/patología , Clorhidrato de Bendamustina , Ligando de CD40/genética , Ligando de CD40/metabolismo , Hipoxia de la Célula , Células Cultivadas , Resistencia a Antineoplásicos , Activación Enzimática , Humanos , Leucemia Linfocítica Crónica de Células B/genética , Leucemia Linfocítica Crónica de Células B/metabolismo , Leucemia Linfocítica Crónica de Células B/patología , Proteína 1 de la Secuencia de Leucemia de Células Mieloides/genética , Proteína 1 de la Secuencia de Leucemia de Células Mieloides/metabolismo , Compuestos de Mostaza Nitrogenada/farmacología , Oxígeno/farmacología , Piperazinas/farmacología , Proteínas Proto-Oncogénicas c-bcl-2/genética , Proteínas Proto-Oncogénicas c-bcl-2/metabolismo , Transducción de Señal , Vidarabina/análogos & derivados , Vidarabina/farmacología , Proteína bcl-X/genética , Proteína bcl-X/metabolismo , Proteínas Quinasas p38 Activadas por Mitógenos/metabolismoRESUMEN
The pharmacological induction of apoptosis in neoplastic B cells presents a promising therapeutic avenue for the treatment of chronic lymphocytic leukemia (CLL). We profiled a panel of clinical multi-kinase inhibitors for their ability to induce apoptosis in primary CLL cells. Whereas inhibitors targeting a large number of receptor and intracellular tyrosine kinases including c-KIT, FLT3, BTK and SYK were comparatively inactive, the CDK inhibitors BMS-387032 and flavopiridol showed marked efficacy similar to staurosporine. Using the kinobeads proteomics method, kinase expression profiles and binding profiles of the inhibitors to target protein complexes were quantitatively monitored in CLL cells. The targets most potently affected were CDK9, cyclin T1, AFF3/4 and MLLT1, which may represent four subunits of a deregulated positive transcriptional elongation factor (p-TEFb) complex. Albeit with lower potency, both drugs also bound the basal transcription factor BTF2/TFIIH containing CDK7. Staurosporine and geldanamycin do not affect these targets and thus seem to exhibit a different mechanism of action. The data support a critical role of p-TEFb inhibitors in CLL that supports their future clinical development.
Asunto(s)
Leucemia Linfocítica Crónica de Células B/tratamiento farmacológico , Inhibidores de Proteínas Quinasas/uso terapéutico , Proteómica , Apoptosis/efectos de los fármacos , Flavonoides/uso terapéutico , Humanos , Leucemia Linfocítica Crónica de Células B/patología , Oxazoles/uso terapéutico , Piperidinas/uso terapéutico , Factor B de Elongación Transcripcional Positiva/antagonistas & inhibidores , Tiazoles/uso terapéuticoRESUMEN
Constitutively activated pathways contribute to apoptosis resistance in chronic lymphocytic leukemia (CLL). Little is known about the metabolism of lipids and function of lipases in CLL cells. Performing gene expression profiling including B-cell receptor (BCR) stimulation of CLL cells in comparison to healthy donor CD5+ B cells, we found significant overexpression of lipases and phospholipases in CLL cells. In addition, we observed that the recently defined prognostic factor lipoprotein lipase (LPL) is induced by stimulation of BCR in CLL cells but not in CD5+ normal B cells. CLL cellular lysates exhibited significantly higher lipase activity compared to healthy donor controls. Incubation of primary CLL cells (n=26) with the lipase inhibitor orlistat resulted in induction of apoptosis, with a half-maximal dose (IC(50)) of 2.35 microM. In healthy B cells a significantly higher mean IC(50) of 148.5 microM of orlistat was observed, while no apoptosis was induced in healthy peripheral blood mononuclear cells (PBMCs; P<0.001). Orlistat-mediated cytotoxicity was decreased by BCR stimulation. Finally, the cytotoxic effects of orlistat on primary CLL cells were enhanced by the simultaneous incubation with fludarabine (P=0.003). In summary, alterations of lipid metabolism are involved in CLL pathogenesis and might represent a novel therapeutic target in CLL.
Asunto(s)
Apoptosis/efectos de los fármacos , Lactonas/farmacología , Leucemia Linfocítica Crónica de Células B/patología , Metabolismo de los Lípidos/efectos de los fármacos , Lipoproteína Lipasa/antagonistas & inhibidores , Proteínas de Neoplasias/antagonistas & inhibidores , Linfocitos B/citología , Linfocitos B/efectos de los fármacos , Ensayos de Selección de Medicamentos Antitumorales , Sinergismo Farmacológico , Ácidos Grasos no Esterificados/metabolismo , Perfilación de la Expresión Génica , Humanos , Concentración 50 Inhibidora , Leucocitos Mononucleares/citología , Leucocitos Mononucleares/efectos de los fármacos , Familia de Multigenes/efectos de los fármacos , Proteínas de Neoplasias/biosíntesis , Proteínas de Neoplasias/genética , Orlistat , Fosfolipasas/biosíntesis , Fosfolipasas/genética , Proteínas Proto-Oncogénicas c-bcr/fisiología , Células Tumorales Cultivadas/efectos de los fármacos , Células Tumorales Cultivadas/enzimología , Vidarabina/análogos & derivados , Vidarabina/farmacologíaRESUMEN
Glioma constitutes the most frequent brain tumour in man with glioblastoma as the most prevalent and malignant type. The average survival time of less than 16 months underlines the need for improvements in diagnosis and therapy. Here, we report the identification of a novel antigen termed glioma-expressed antigen 2 (GLEA2) causing a frequent immune response in glioma patients. Screening of 450 000 clones from a glioblastoma lambda zap expression library with autologous patient serum revealed a group of five serum-positive clones sharing a high sequence homology. Further sequence analysis showed a sequence homology to a hepatocellular carcinoma associated antigen 58 (HCA58). We localized the novel HCA homologous gene termed glioma-expressed antigen 2 (GLEA2) on chromosome 20 by somatic cell hybrid panel mapping. Using allogenic sera from 39 glioblastoma patients, we found an immune response against GLEA2 in 17 patients (43%). In addition, screening with allogenic sera from other glioma patients revealed GLEA2 directed antibodies in two out of five pilocytic astrocytomas and in one out of two astrocytomas. Unrelated tumour sera revealed no immune response and sera from healthy persons showed an immune response in two out of 14 cases (14%). Northern blot hybridization and RT-PCR showed ubiquitous GLEA2 gene expression in glioma and normal tissues. The novel HCA homologous gene, GLEA2, appears to induce a frequent immune response in glioma. In the light of the lack of useful glioma markers, it appears reasonable to consider GLEA2 as a potential future diagnostic marker.