DNA MODIFICATIONS AND LYMPHOMAGENESIS

GCs are sites of oligoclonal B cell growth. DNA modifications that affect Ig genes of B cells during GC phase include SHM, which alters the V region of Ig genes during the proliferation phase of centroblasts, and CSR that occurs in the light zone of centrocytes.

All these mechanisms require DNA strand breaks process and the activity of activation-induced cytidine deaminase (AID), which is an enzyme that catalyses the deamination of cytidines directly in the VDJ and switch regions of the Ig genes, that is the starting point for DNA strand breaks [58, 59]. However, AID is not always specific for the Ig locus, and can cause mutations in oncogenes or double strand breaks leading to a genomic instability. These events importantly contribute to lymphomagenesis and I will describe them more in detail.

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2.2.2.1. Somatic hypermutation (SHM)

The GC, dark zone in particular, is the main structure where SHM occur. The physiological role of SHM is to introduce point mutations in the V region of rearranged Ig genes. SHM leads to affinity maturation, which results in the preferential outgrowth of B cells expressing an Ig with high affinity for its cognate antigene [60]. The mutations introduced by SHM are predominantly point mutations, although insertions and deletions are occasionally observed. The base changes are distributed throughout the V region, but not completely randomly: there are ‘hotspots’ of mutations that indicate a preference for characteristic short motifs of four or five nucleotides, and, perhaps, for secondary structural features as well. Some of the mutant Ig molecules bind antigen better than the original BCR, and the B cell clones that express them are preferentially selected to mature into antibody-secreting cells.

The process of SHM is closely linked to transcription [60]. The mutation rate of an Ig gene is proportional to the transcription rate of the locus, and mutations are confined to a 1-2 kilobase (kb) region downstream of the transcription start site [60].

As mention before, AID is required for this process. This enzyme acts directly on DNA, converting cytidine (C) to uridine (U) in Ig V regions. After AID initiates SHM by the deamination of C nucleotides, the resulting U-G mismatch may lead to mutations in several different ways. If the mismatch is not repaired before the onset of DNA replication, DNA polymerases will insert an A nucleotide opposite to U, thus creating a C T and G A transition (interchanges of two ring purines, G A, or one-ring pyrimidines, C T). Conversely, if the U is removed by uracil DNA glycosilase (UNG), an abasic site is created, and its replication may give rise to either transitions (interchanges of two- ring purines, A-G, or one-ring pyrimidines, C-T) or transvertions (interchanges of purine for pyrimidine bases, which therefore involve exchange of one-ring with two-ring structure).

In addition, U-G mismatch recruits the mismatch repair (MMR) machinery, which creates mutations at A-T near the initiating U-G lesion.

The rate of mutation in the Ig V regions during SHM in the GC is estimated to be as high as 103 mutations per base pair per generation [61, 62].

As above mentioned, SHM is not strictly limited to Ig genes, but, in normal B cells, it targets also other genes expressed in the GC, including, for example, BCL6 and CD95/FAS [63, 64]. In some lymphomas deriving from GC B cells, not only DLBCL, SHM may target several proto-oncogenes, thus contributing to neoplastic transformation.

Malfunctioning of SHM in the context of lymphoma is termed aberrant SHM and the proto-oncogenes most frequently affected include BCL6, PIM1, MYC, RHOH (Ras homologue gene-familiy member H), and PAX5 [65-67]. Aberrant SHM can contribute to

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lymphomagenesis by mutating both regulatory and coding sequences in proto-oncogenes [65-67]. For example, mutations in MYC frequently target important functional domains and regulatory sequences, and mutations in the BCL6 regulatory region have been shown to deregulate BCL6 expression [68-70]. Also, SHM may promote chromosomal translocations of the targeted proto-oncogenes in the regions where SHM-derived mutations are located.

2.2.2.2. Class switch recombination (CSR)

During CSR of the membrane-expressed BCR, which is primarily encoded during development and maturation, the C regions are replaced. In particular, this modification replaces the expressed constant region (C ) of the heavy-chain locus with a downstream isotype C , C , or C . The process alters the effectors functions of an antibody. CSR is an irreversible somatic recombination mechanism by which B cells can switch their Ig class expression from IgM and IgD to IgG, IgA and IgE. CSR occurs via DNA recombination involving non-homologous end-joining processes between specific repetitive regions of several hundred base pairs (known as switch regions) that precede the Ig constant region genes. CSR, as well as SHM, is initiated by the activity of AID selectively expressed in GC B cells [71] and it can occur in both T cell dependent and T cell independent manner. It is the combination of specific cytokines and co-stimulatory signals that determine the nature of resulting Ig class. Different cytokines that regulate heavy-chain class switching are made by different subsets of helper T cells that are generated in response to distinct types of microbes. TGF- that is produced by many cell types, in association with T cell derived IL-5 stimulates production of IgA in mucosal lymphoid tissue, resulting in production of local immunity, IL-4 alone is necessary for IgE, while when in association with IL-6, IL2 and INF- induces IgG switch.

For CSR, CD40-CD154 interaction is critical, and IRF4, a downstream target of NF-kB, regulates AID expression through intermediate molecules rather than through a direct mechanism [71].

A hallmark of mature B cell lymphomas is the presence of reciprocal chromosomal translocations that juxtapose loci encoding the Ig genes to a proto-oncogene. In the process of CSR, DNA double-strand break intermediates generated in Ig genes by this reaction are potent substrates for translocations by recombining with double-strand breaks on a nonhomologous chromosome. The formal proof that AID is required for GC-derived lymphomagenesis is based on the observation that AID deficiency prevents the formation of BCL6-dependent GC-derived B cell NHL in lymphoma prone animal models [72]. Regarding DLBCL, some of the most commonly translocated genes are: BCL2, BCL6

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and c-MYC, whose coding domain undergoes under the control of Ig promoter, leading to deregulation of their expression.

Translocations t(14;18)(q32;q21) involving the Ig heavy-chain locus and the BCL2 proto-oncogene are characteristic of follicular lymphoma (FL). As centroblasts do not normally express BCL2, the ectopic expression of BCL2 in these cells promotes lymphomagenesis by overriding the characteristic pro-apoptotic program of the centroblast. In contrast to BCL2, BCL6 is normally expressed in GC B cells, but its expression has to be switched off for the post-GC differentiation of B cells to take place. Chromosomal translocations involving the BCL6 gene locus (3q27) is commonly associated with 30-40% of DLBCL and less frequently with FL and it causes the deregulation of BCL6 expression by preventing its silencing at the conclusion of the GC response. The BCL6 translocation might result in a block in PC differentiation maintaining GC conditions with high proliferation rate, DNA-damage tolerance with the possible consequence that the cells may be subjected to further genetic alterations, eventually contributing to lymphomagenesis.

Translocations of MYC into the Ig heavy- or light-chain loci are associated with 100% of Burkitt lymphoma (BL) cases and up to 10% of DLBCL cases. Normally, GC B cells do not express MYC and this means that expression of MYC in BL and DLBCL is ectopic. MYC deregulation has been associated with abnormal cell growth, as well as genomic instability. Thus, it is conceivable that the ectopic expression of MYC in GC B cells contributes to lymphomagenesis.

I will come back to the chromosomal translocations occurring in DLBCL in the next chapter.

2.2.3. ROLE OF CHEMOKINES IN GC FORMATION AND