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  • Introduction By catalyzing post translational acetylation of

    2019-04-22

    Introduction By catalyzing post-translational acetylation of lysine residues, the histone acetyl transferases (HAT) CREBBP and EP300 moderate the activities of both histone and non-histone proteins. This regulates vital cellular processes such as cell cycling and apoptosis by modulating chromatin structure and by altering the activity of transcription factors. The roles of CREBBP and EP300 in the pathogenesis of leukemia and malignant lymphomas have recently been highlighted.CREBBP and EP300 belong to one of three HAT families based on their catalytic domains. They are capable of acetylating the lysine residues of all 4 core histones as well as over 70 other proteins. It has been shown that acetylation of the tumor suppressor protein p53 leads to its activation, while acetylation of onco-protein BCL6 leads to its functional inactivation. In germinal center B-cells BCL6 inhibits transcription of p53 and p53 mediated apoptosis, which allows the retinoic acid receptor to tolerate DNA strand breaks during immunoglobulin class switch recombination and somatic hypermutation that are necessary for normal B-cell maturation. This implies that loss-of-acetylation may contribute to lymphomagenesis by BCL6 over-expression and p53 inactivation, which will allow for the survival of germinal center cells with aberrant chromosomal breaks. CREBBP mutations have been identified in DLBCL (39%) and follicular lymphoma (FL) (41%), EP300 mutations being found in 10% of the same DLBCLs and in 8.7% of FL samples, respectively. Due to >90% sequence similarity within the two genes, they have been suggested to be functionally homologous; however, co-existing alterations of both CREBBP and EP300 were found in only 6/72 cases. For both genes, a clustering of mutations was observed in the catalytic HAT domains. HAT domain mutant proteins had lost their acetyl transferase activity, while no significant effects were observed when mutations were located outside the HAT domain. While the acetylation activity of the individual mutations was thoroughly described, the consequences for cell survival were not analyzed and is thus the focus of this study. A direct comparison of HDACi induced apoptosis in DLBCL cells with single vs. double mutated vs. wt CREBBP/EP300 genes has so far not been reported. Because of the above mentioned physiologically important function of acetylation in the regulation of apoptosis in germinal center cells and in B-cell lymphomagenesis, we speculated whether different responses to vorinostat could be observed in B-cell tumors with and without CREBBP/EP300 mutations.
    Materials and methods
    Results Screening of the Toledo cell line revealed a single base change of the EP300 gene (Fig. 1a,b). Furthermore, we identified a 3bp deletion of the Toledo CREBBP gene (Fig. 1c). Screening of RL revealed a missense mutation in EP300 (Fig. 1d). The SUDHL-7 harbors two CREBBP mutations both with deleterious effects key to HAT activity as assessed by in vivo acetylation assays in mice (exon 26: Y1450C and exon 30: ΔS1680).
    Discussion Heterogeneous responses have been reported from clinical phase I and II trials of vorinostat in NHL-patients. These data strongly imply that diverse underlying molecular defects in these various tumor types determine the responses to vorinostat. Mutations in the HAT domain proteins are obvious candidates. While experimental data show that HAT mutations did not seem to affect the effectiveness of apoptosis induction by vorinostat in T-ALL cells, our data imply that tumors with concomitant mutations of CREBBP and EP300 may be particularly sensitive to vorinostat induced apoptosis. Given the important role of acetylation in the physiological regulation of BCL6 and p53 activity in germinal center cells, CREBBP/EP300 double mutant cells are likely to have a particularly skewed BCL6/p53-balance, leading to increased cell survival. A plausible theory for the efficacy of HDACi in HAT mutant DLBCL is, that reestablishment of the physiological balance between acetylation and deacetylation in HAT-deficient cells by the use of HDACi, may lead to increased p53 activation and increased apoptosis. Indeed, the double-mutated Toledo cell line exhibited a swift and ultimately more profound degree of apoptosis in comparison with both wild type and single CREBBP/EP300 mutated cell lines.