Functional Analysis of BTG1 and BTG2 Variants in Non-Hodgkin Lymphoma

Abstract

Non-Hodgkin's lymphoma (NHL) represents a highly heterogeneous group of lympho-proliferative disorders and accounts for more than half of the cases of haematological malignancies. These disorders are characterised by multiple steps of accumulating genetic mutations resulting in the selection of a malignant clone. Recently, mutations in the anti-proliferative genes B-cell translocation 1 gene (BTG1) and B-cell translocation 2 gene (BTG2) have been identified in approximately 10-15% of NHL cases, which suggests a direct involvement of BTG1 and BTG2 in malignant transformation.BTG1 and BTG2 are characterised by the conserved amino-terminal BTG domain, which mediates interactions with the Caf1 catalytic subunit of the Ccr4-Not deadenylase complex. In addition, the BTG domain binds to the poly(A)-binding protein PABPC1. This complex plays a critical role in mRNA deadenylation and degradation as well as translational repression.It is currently unclear how, or indeed whether, mutations in BTG1 and BTG2 affect the function of the gene products. Therefore, we used a combination of sequence analysis and molecular modelling to predict the functional consequences of mutations previously identified in NHL. Sorting intolerant from tolerant (SIFT) prediction tool was used to predict amino acidic residues potentially interfering with the protein function. In total we assessed 45 mutations in BTG1 and BTG2 derived from NHL samples, including diffuse large B-cell lymphoma, germinal centre B-cell-like, activated B-cell-like lymphoma, follicular lymphoma and Burkitt'slymphoma. Of the mutations analysed, 25 were predicted to interfere with the function of BTG1 and BTG2. We therefore analysed the ability of these protein variants to interact with known partners and functionally assessed the role of the mutated proteins in cell cycle progression, translational repression and mRNA degradation. Using a yeast two-hybrid system, fourteen mutations were shown to affect the interaction of BTG1 or BTG2 with the Caf1catalytic subunit of the Ccr4-Not deadenylase complex. In addition, when we transfected into mammalian cells, these BTG1 and BTG2 variants, unlike the wild-type proteins, were not able to inhibit cell cycle progression. Interestingly, our findings show that several mutations (M11I, F25C, P58L, I115V) on BTG1 do not require interaction with the Caf1 deadenylase enzyme to reduce reporter activity as established using 3' UTR tethering assays. This suggests that BTG1 and BTG2 may also have a role in regulating cell cycle progression and RNA degradation via Ccr4-Not-independent mechanisms.The data show that mutations in BTG1 and BTG2, commonly found in NHL, are functionally significant and are likely to contribute to malignant transformation and tumour cell grow. DisclosuresAlmasmoum:Umm Al-Qura University: Employment, Research Funding.