RESUMEN
Bcl-2 inhibits apoptosis by two distinct mechanisms but only one is targeted to treat Bcl-2-positive malignancies. In this mechanism, the BH1-3 domains of Bcl-2 form a hydrophobic pocket, binding and inhibiting pro-apoptotic proteins, including Bim. In the other mechanism, the BH4 domain mediates interaction of Bcl-2 with inositol 1,4, 5-trisphosphate receptors (IP3Rs), inhibiting pro-apoptotic Ca2+ signals. The current anti-Bcl-2 agents, ABT-263 (Navitoclax) and ABT-199 (Venetoclax), induce apoptosis by displacing pro-apoptotic proteins from the hydrophobic pocket, but do not inhibit Bcl-2-IP3R interaction. Therefore, to target this interaction we developed BIRD-2 (Bcl-2 IP3 Receptor Disruptor-2), a decoy peptide that binds to the BH4 domain, blocking Bcl-2-IP3R interaction and thus inducing Ca2+-mediated apoptosis in chronic lymphocytic leukemia, multiple myeloma, and follicular lymphoma cells, including cells resistant to ABT-263, ABT-199, or the Bruton's tyrosine kinase inhibitor Ibrutinib. Moreover, combining BIRD-2 with ABT-263 or ABT-199 enhances apoptosis induction compared to single agent treatment. Overall, these findings provide strong rationale for developing novel therapeutic agents that mimic the action of BIRD-2 in targeting the BH4 domain of Bcl-2 and disrupting Bcl-2-IP3R interaction.
Asunto(s)
Linfoma Folicular/patología , Mieloma Múltiple/patología , Péptidos/química , Proteínas Proto-Oncogénicas c-bcl-2/metabolismo , Compuestos de Anilina/uso terapéutico , Animales , Apoptosis , Compuestos Bicíclicos Heterocíclicos con Puentes/uso terapéutico , Señalización del Calcio , Línea Celular Tumoral , Supervivencia Celular , Resistencia a Antineoplásicos , Células HEK293 , Humanos , Interacciones Hidrofóbicas e Hidrofílicas , Inmunohistoquímica , Receptores de Inositol 1,4,5-Trifosfato/química , Leucemia Linfocítica Crónica de Células B/tratamiento farmacológico , Linfoma Folicular/tratamiento farmacológico , Ratones , Ratones Desnudos , Mieloma Múltiple/tratamiento farmacológico , Células 3T3 NIH , Trasplante de Neoplasias , Estructura Terciaria de Proteína , Sulfonamidas/uso terapéutico , Proteína X Asociada a bcl-2/metabolismoRESUMEN
Acute leukemias caused by translocations of the MLL gene at chromosome 11 band q23 (11q23) are characterized by a unique gene expression profile. More recently, data from several laboratories indicate that the most commonly encountered MLL fusion proteins, MLLT1, MLLT3, and AFF1 are found within a molecular complex that facilitates the elongation phase of mRNA transcription. Mutational analyses suggest that interaction between the MLLT1/3 proteins and AFF family proteins are required for experimental transformation of hematopoietic progenitor cells (HPCs). Here, we define a specific pairing of two amino acids that creates a salt bridge between MLLT1/3 and AFF proteins that is critically important for MLL-mediated transformation of HPCs. Our findings, coupled with the newly defined structure of MLLT3 in complex with AFF1, should facilitate the development of small molecules that block this amino acid interaction and interfere with the activity of the most common MLL oncoproteins.
Asunto(s)
Aminoácidos/genética , Proteínas de Unión al ADN/genética , Leucemia Experimental/genética , Proteínas Nucleares/genética , Factores de Transcripción/genética , Secuencia de Aminoácidos , Animales , Proteínas de Unión al ADN/química , Células HEK293 , Humanos , Ratones , Datos de Secuencia Molecular , Proteínas Nucleares/química , Homología de Secuencia de Aminoácido , Factores de Transcripción/química , Factores de Elongación TranscripcionalRESUMEN
Bcl-2 interacts with the inositol 1,4,5-trisphosphate receptor (InsP3R) and thus prevents InsP3-induced Ca(2+) elevation that induces apoptosis. Here we report that Bcl-2 binds dopamine- and cAMP-regulated phosphoprotein of 32 kDa (DARPP-32), a protein kinase A (PKA)-activated and calcineurin (CaN)-deactivated inhibitor of protein phosphatase 1 (PP1). Bcl-2 docks DARPP-32 and CaN in a complex on the InsP3R, creating a negative feedback loop that prevents exaggerated Ca(2+) release by decreasing PKA-mediated InsP3R phosphorylation. T-cell activation increases PKA activity, phosphorylating both the InsP3R and DARPP-32. Phosphorylated DARPP-32 inhibits PP1, enhancing InsP3R phosphorylation and Ca(2+) release. Elevated Ca(2+) activates CaN, which dephosphorylates DARPP-32 to dampen Ca(2+) release by eliminating PP1 inhibition to enable it to dephosphorylate the InsP3R. Knocking down either Bcl-2 or DARPP-32 abrogates this feedback mechanism, resulting in increased Ca(2+) elevation and apoptosis. This feedback mechanism appears to be exploited by high levels of Bcl-2 in chronic lymphocytic leukemia cells, repressing B-cell receptor-induced Ca(2+) elevation and apoptosis.
Asunto(s)
Fosfoproteína 32 Regulada por Dopamina y AMPc/metabolismo , Regulación de la Expresión Génica , Receptores de Inositol 1,4,5-Trifosfato/metabolismo , Proteínas Proto-Oncogénicas c-bcl-2/metabolismo , Animales , Apoptosis , Encéfalo/metabolismo , Calcineurina/metabolismo , Calcio/metabolismo , Línea Celular Tumoral , Supervivencia Celular , Proteínas Quinasas Dependientes de AMP Cíclico/metabolismo , Humanos , Células Jurkat , Leucemia Linfocítica Crónica de Células B/metabolismo , Ratones , Fosforilación , Unión Proteica , Estructura Terciaria de Proteína , Interferencia de ARN , Transducción de SeñalRESUMEN
The tumor microenvironment is generally an acidic environment, yet the effect of extracellular acidosis on chronic lymphocytic leukemia (CLL) is not well established. Here we are the first to report that the extracellular acid sensing G-protein coupled receptor, GPR65, is expressed in primary CLL cells where its level correlate strongly with anti-apoptotic Bcl-2 family member levels. GPR65 expression is found normally within the lymphoid lineage and has not been previously reported in CLL. We demonstrate a wide range of GPR65 mRNA expression among CLL 87 patient samples. The correlation between GPR65 mRNA levels and Bcl-2 mRNA levels is particularly strong (r=0.8063, p= <0.001). The correlation extends to other anti-apoptotic Bcl-2 family members, Mcl-1 (r=0.4847, p=0.0010) and Bcl-xl (r=0.3411, p=0.0252), although at lower levels of significance. No correlation is detected between GPR65 and levels of the pro-apoptotic proteins BIM, PUMA or NOXA. GPR65 expression also correlates with the favorable prognostic marker of 13q deletion. The present findings suggest the acid sensing receptor GPR65 may be of significance to allow CLL tolerance of extracellular acidosis. The correlation of GPR65 with Bcl-2 suggests a novel cytoprotective mechanism that enables CLL cell adaptation to acidic extracellular conditions. These findings suggest the potential value of targeting GPR65 therapeutically.
RESUMEN
Chimeric oncoproteins resulting from fusion of MLL to a wide variety of partnering proteins cause biologically distinctive and clinically aggressive acute leukemias. However, the mechanism of MLL-mediated leukemic transformation is not fully understood. Dot1, the only known histone H3 lysine 79 (H3K79) methyltransferase, has been shown to interact with multiple MLL fusion partners including AF9, ENL, AF10, and AF17. In this study, we utilize a conditional Dot1l deletion model to investigate the role of Dot1 in hematopoietic progenitor cell immortalization by MLL fusion proteins. Western blot and mass spectrometry show that Dot1-deficient cells are depleted of the global H3K79 methylation mark. We find that loss of Dot1 activity attenuates cell viability and colony formation potential of cells immortalized by MLL oncoproteins but not by the leukemic oncoprotein E2a-Pbx1. Although this effect is most pronounced for MLL-AF9, we find that Dot1 contributes to the viability of cells immortalized by other MLL oncoproteins that are not known to directly recruit Dot1. Cells immortalized by MLL fusions also show increased apoptosis, suggesting the involvement of Dot1 in survival pathways. In summary, our data point to a pivotal requirement for Dot1 in MLL fusion protein-mediated leukemogenesis and implicate Dot1 as a potential therapeutic target.