https://orcid.org/0000-0002-6691-7191
In this issue of Blood, Hilton et al1 show that the architecture of MYC translocations is strikingly different in B-cell non-Hodgkin lymphomas (B-NHL) with sole MYC translocations in comparison with double-hit lymphomas carrying a BCL2 and a MYC chromosomal translocation, which has important implications for the underlying selective pressures and oncogenic mechanisms.
Many B-cell lymphomas in humans carry chromosomal translocations involving an immunoglobulin locus, mostly the immunoglobulin heavy chain (IGH) locus, and a protooncogene.2 For example, most follicular lymphomas carry BCL2::IGH translocations, and nearly all Burkitt lymphomas harbor MYC::immunoglobulin translocations.2 These translocations mediate their oncogenic effects by bringing the oncogene under the transcriptional control of the strong immunoglobulin enhancers, causing its constitutive and often increased expression.
The generation of immunoglobulin locus-associated translocations is closely linked to the immunoglobulin gene remodeling processes V(D)J recombination, class-switch recombination, and somatic hypermutation (SHM).2 V(D)J recombination in B-cell precursors leads to the assembly of variable, diversity and joining gene segments to form functional V region genes. These encode the highly diverse antigen-binding sites of antibodies. SHM and class-switch recombination occur in antigen-activated B cells, and both are initiated by activation-induced cytidine deaminase (AID). SHM occurs in germinal center B cells and enables the generation of antibodies with improved affinity to the cognate antigen. Class switching is also mostly linked to the germinal center reaction. Through exchange of the originally expressed immunoglobulin M (IgM) and IgD heavy chains by 1 of the 4 IgG, 2 IgA, or the IgE constant region genes, antibodies with distinct effector functions are generated. Aberrant activity of AID outside of the immunoglobulin loci, in conjunction either with the SHM or the class-switching enzymatic machineries,3 contributes to lymphoma pathogenesis by causing mutations in oncogenes or tumor suppressor genes and also promotes translocations between nonimmunoglobulin AID targets and protooncogenes.4
In single translocation events involving immunoglobulin loci, the translocation nearly always occurs on the nonproductively rearranged immunoglobulin allele, so that the B cells can still express a functional B-cell receptor (BCR) (see figure panels A-B).2 This indicates a strong selection for BCR expression when the translocation happened. This poses intriguing questions, namely, what is the structure of translocations when a B-cell lymphoma carries 2 or even 3 oncogene translocations, and which selection forces are active in such situations? The study by Hilton and colleagues is the largest and most detailed analysis of translocation structures in B-NHL. The study encompasses 883 cases of follicular lymphomas, Burkitt lymphomas, diffuse large B-cell lymphomas, and double-hit high-grade lymphomas with either an IGH::BCL2 and MYC translocation (HGBCL-DH-BCL2) or a BCL6 and MYC translocation. The translocations were characterized by fluorescence in situ hybridization (FISH) and either whole genome or targeted sequencing. A key finding is that in lymphomas with IGH::BCL2 translocations and a MYC rearrangement, the functionality of the BCR with few exceptions is also preserved. BCR functionality is preserved via 2 distinct methods. First, over 50% of MYC rearrangements in HGBCL-DH-BCL2 are translocated to nonimmunoglobulin loci, thereby leaving the functional BCR rearrangement and the IGH::BCL2 translocation unaffected (see figure panel C). The partner genes (eg, BCL6, PAX5) are mostly targets of aberrant SHM, meaning that off-target AID activity is also involved in their generation. The second way is that in HGBCL-DH-BCL2 the MYC rearrangement is targeted to the switch region of a downstream-located IGH constant region gene. Thereby, 1 IGH locus is disrupted, but both parts can still drive expression of the affected genes: The Eμ enhancer in the intron between IGHJ genes and Cμ and the a1 3′ regulatory region (RR) enhancer in the middle of the IGH constant locus drive expression of the BCR and the translocated BCL2 gene, whereas the a2 3′ RR enhancer downstream of the last constant gene controls expression of MYC (see figure panel D). Thus, the architecture of the chromosomal breaks in HGBCL-DH-BCL2 provides a further strong argument that BCR expression is essential for the vast majority of B-cell lymphomas.2
Distinct MYC translocation architectures in lymphomas with sole MYC translocations vs double-hit lymphomas with BCL2 and MYC translocations. In Burkitt lymphomas and diffuse large B-cell lymphomas with sole MYC translocations, these translocations are mostly targeted to the nonproductively rearranged IGH locus, either into or near to a nonproductively rearranged IGHV gene as a mistake of SHM (A), or into one of the switch regions (often switch-μ) as a mistake of class switching (B). In double-hit high-grade lymphomas with MYC and IGH::BCL2 translocations, the MYC gene is in most instances either translocated to a nonimmunoglobulin gene, for example BCL6, coming under control of the strong BCL6 super enhancer (C), or to a downstream switch region (often switch-ε) on either the allele with the productive IGHV gene (as shown in D), or the allele with the IGH::BCL2 rearrangement. Importantly, in all these constellations of MYC translocations in HGBCL-DH-BCL2, expression of the BCR and of the translocated BCL2 gene is retained. The Eμ and the 2 IGH locus 3′ RR enhancers (red squares) drive the expression of the genes juxtaposed to them. Only 4 of the 9 IGH constant region genes are shown as yellow squares. Arrows indicate the transcriptional orientation of the genes. chr, chromosome.
Distinct MYC translocation architectures in lymphomas with sole MYC translocations vs double-hit lymphomas with BCL2 and MYC translocations. In Burkitt lymphomas and diffuse large B-cell lymphomas with sole MYC translocations, these translocations are mostly targeted to the nonproductively rearranged IGH locus, either into or near to a nonproductively rearranged IGHV gene as a mistake of SHM (A), or into one of the switch regions (often switch-μ) as a mistake of class switching (B). In double-hit high-grade lymphomas with MYC and IGH::BCL2 translocations, the MYC gene is in most instances either translocated to a nonimmunoglobulin gene, for example BCL6, coming under control of the strong BCL6 super enhancer (C), or to a downstream switch region (often switch-ε) on either the allele with the productive IGHV gene (as shown in D), or the allele with the IGH::BCL2 rearrangement. Importantly, in all these constellations of MYC translocations in HGBCL-DH-BCL2, expression of the BCR and of the translocated BCL2 gene is retained. The Eμ and the 2 IGH locus 3′ RR enhancers (red squares) drive the expression of the genes juxtaposed to them. Only 4 of the 9 IGH constant region genes are shown as yellow squares. Arrows indicate the transcriptional orientation of the genes. chr, chromosome.
Interestingly, somatic mutations in the BCL2 gene were only found in cases with an IGH::BCL2 translocation. Apparently, BCL2 is not itself targeted by aberrant SHM. Only upon translocation can its 5′ region be mutated, even if the translocation breakpoint is more than 200 kilobases away. Finding ≥4 point mutations in BCL2 strongly indicates the presence of an IGH::BCL2 break and even identified IGH::BCL2 translocations, which standard FISH failed to do. Even though BCL2 sequence analysis will likely not replace FISH to detect IGH::BCL2 translocations in B-NHL, this is a straightforward approach to detect such translocations.
The frequent targeting of switch regions for the IGH::MYC translocations in HGBCL-DH-BCL2 appears at first glance puzzling, because most diffuse large B-cell lymphomas express IgM, that is, have not undergone productive class-switch recombination.5 However, class-switch recombination is initiated by transcription through the switch regions, and this early step is already sufficient to attract AID to these regions and induce DNA strand breaks, even if class switching is not completed. There is also indication for illegitimate switch events in the IGH locus of diffuse large B-cell lymphomas.5 These processes, together with the selection forces to keep the productive IGH gene rearrangement and the IGH::BCL2 translocation active, would favor translocations into downstream switch regions. The surprisingly frequent targeting of switch-ε may be due to particular cytokine signals in germinal center B cells that promote class switching to Cε.6
In conclusion, Hilton et al provide strong indication that even in B-cell lymphomas with dual oncogene translocations there is strong selection to retain BCR expression. This demonstrates the importance of a functional BCR for malignant B cells, which is therapeutically exploited for several types of B-cell lymphomas by targeting BCR signaling, for example, with inhibitors of the BCR signaling kinase BTK.7 Furthermore, the study deepens our understanding that not only translocation events targeting rearranged IGV genes or switch regions in the immunoglobulin locus are mediated by AID activity, but also most nonimmunoglobulin-targeted translocations in germinal center B cell–derived lymphomas are caused by off-target AID activity. Thus, misguided AID activity is a major contributor to B-cell lymphomagenesis.
Conflict-of-interest disclosure: R.K. declares no competing financial interests.
