The mechanisms of human lymphoid chromosomal translocations and their medical relevance

Abstract

The most common human lymphoid chromosomal translocations involve concurrent failures of the recombination activating gene (RAG) complex and Activation-Induced Deaminase (AID). These are two enzymes that are normally expressed for purposes of the two site-specific DNA recombination processes: V(D)J recombination and class switch recombination (CSR). First, though it is rare, a low level of expression of AID can introduce long-lived T:G mismatch lesions at 20-600 bp fragile zones. Second, the V(D)J recombination process can occasionally fail to rejoin coding ends, and this failure may permit an opportunity for Artemis:DNA-dependent kinase catalytic subunit (DNA-PKcs) to convert the T:G mismatch sites at the fragile zones into double-strand breaks. The 20-600 bp fragile zones must be, at least transiently, in a single-stranded DNA (ssDNA) state for the first step to occur, because AID only acts on ssDNA. Here we discuss the key DNA sequence features that lead to AID action at a fragile zone, which are (a) the proximity and density of strings of cytosine nucleotides (C-strings) that cause a B/A-intermediate DNA conformation; (b) overlapping AID hotspots that contain a methyl CpG (WRCG), which AID converts to a long-lived T:G mismatch; and (c) transcription, which, though not essential, favors increased ssDNA in the fragile zone. We also summarize chromosomal features of the focal fragile zones in lymphoid malignancies and discuss the clinical relevance of understanding the translocation mechanisms. Many of the key principles covered here are also relevant to chromosomal translocations in non-lymphoid somatic cells as well.

Keywords: B/A-intermediate DNA; Chromosomal translocations; activation-induced deaminase (AID); double-strand breaks; lymphoid leukemia; lymphoma; non-B DNA structure; single-stranded DNA.

Figure 1.

Illustration of factors causing double-strand DNA breaks (DSBs) and chromosomal translocation consequences. (A) Causes and repair of DSBs. Physiological and pathological causes of DSBs in mammalian somatic cells are listed at the top. At any time in the cell cycle, DSBs can be repaired by non-homologous DNA end-joining (NHEJ). During S and G2 of the cell cycle, homology-directed repair is common because the two sister chromatids are in close proximity, providing a nearby homology donor. Homology-directed repair includes homologous recombination (HR) and single-strand annealing (SSA). Proteins involved in the repair pathways are also shown. RAG: recombination activating gene; AID: Activation-Induced Deaminase; UDG: uracil-DNA glycosylase; APE1: apurinic-apyrimidinic endonuclease 1; DNA-PKcs: DNA-dependent kinase catalytic subunit; Pol: DNA polymerase; XLF: XRCC4-like factor; NBS1: Nijmegen breakage syndrome 1; MRE11A: meiotic recombination 11 homolog A. (B) Consequences of chromosomal translocations. The reciprocal chromosomal translocation results in two derivative chromosomes with exchanged segments. In the case where each chromosome contains one centromere as shown in the left panel, the two derivative chromosomes are usually stable. However, if one of the two derivative chromosomes contains two centromeres and the other has none as shown in the right panel, they are usually lost or unstable during cell growth.

Figure 2.

Common chromosomal translocations in B cells. Most of the lymphoid translocations in the early B cell (pro-B/pre-B) stage (A,B,D,E) involve a double-strand DNA break (DSB) that is a RAG-induced event (termed RAG-type break) at the immunoglobulin locus and a second DSB that is an AID-induced event (termed AID-type break) on an oncogene. The E2A-PBX1 translocation in early B cells (C) involves an AID-type break on E2A and a second break due to random causes, such as oxidative stress, ionizing radiation, or others on PBX1. Mature B cell translocations (F,G) commonly involve an AID-type event on one chromosome and a second event involving a failed IGH class switch recombination (CSR) event, which is a physiologic AID-type event. MALT1: mucosa-associated lymphoid tissue lymphoma translocation 1; CRLF2: cytokine receptor-like factor 2.

Figure 3.

Breakpoint distributions on oncogenes involved in lymphoid translocations in early B cells. (A) BCL2 breakpoints in BCL2-IGH translocations. The BCL2 breakpoints are scattered within a 30 kb region with three cluster regions (red starbursts). The breakpoints that do not fall into cluster regions are plotted as dark gray vertical lines. The 175 bp BCL2 major break region (MBR), located at the 3′ untranslated region (UTR) of BCL2, contains 50% of all BCL2 patient breakpoints. The breakage frequency within this 175 bp region is 300-fold higher than what one would expect to occur randomly. Patient breaks within the MBR are not uniformly distributed across the entire 175 bp, but rather are focused in three peaks centered around the CG motif. The 105 bp intermediate cluster region (ICR) and 561 bp minor cluster region (MCR) of BCL2 contains 13 and 5% of the BCL2 breakpoints, respectively. The breakpoints within MBR, ICR, and MCR are illustrated below the figure in zoomed-in views. Human lymphomas are clinically indistinguishable regardless of the position of the breakpoint within the 30 kb. (B) BCL1 breakpoints in BCL1-IGH translocations. The major translocation cluster (MTC) of BCL1 (also known as CCND1) is located 109 kb upstream of the CCND1 gene on chromosome 11 and contains 30% of all BCL1 breakpoints. The rest of the BCL1 breakpoints are scattered within a 344 kb intergenic region between the CCND1 gene at the telomeric end and the MYEOV gene on the centromeric side. (C) E2A breakpoints in E2A-PBX1 and E2A-HLF translocations. The E2A breakpoints in the E2A-PBX1 translocation (top panel) and E2A-HLF translocation (bottom panel) are illustrated. Over 75% of the E2A breakpoints are in a 23 bp region within E2A intron 16, making the 23 bp more than 400-fold more fragile compared with other regions of the same E2A intron. (D) MALT1 breakpoints in IGH-MALT1 and API2-MALT1 translocations. The breakpoints of MALT1 are focused on an 86 bp region upstream of the MALT1 gene in IGH-MALT1 translocations which are shown in the zoomed-in view. In contrast, the breakpoints of MALT1 in the API2-MALT1 translocation are scattered in a 29 kb region in several introns and exons of the MALT1 gene. For all figures, each triangle represents a single breakpoint from a patient. Triangles above and below the sequence are sequenced from two derivative chromosomes that resulted from the translocation. The CpG sites are highlighted with a red background. Within each fragile zone, the actual translocations occur at DNA sequence motifs, consisting of either the sequence CG (CpG) or mCG (when methylated) or WGCW where W = A or T. We are trying to determine why these small fragile zones are highly preferred for DNA breakage and translocation.

Figure 3.

Breakpoint distributions on oncogenes involved in lymphoid translocations in early B cells. (A) BCL2 breakpoints in BCL2-IGH translocations. The BCL2 breakpoints are scattered within a 30 kb region with three cluster regions (red starbursts). The breakpoints that do not fall into cluster regions are plotted as dark gray vertical lines. The 175 bp BCL2 major break region (MBR), located at the 3′ untranslated region (UTR) of BCL2, contains 50% of all BCL2 patient breakpoints. The breakage frequency within this 175 bp region is 300-fold higher than what one would expect to occur randomly. Patient breaks within the MBR are not uniformly distributed across the entire 175 bp, but rather are focused in three peaks centered around the CG motif. The 105 bp intermediate cluster region (ICR) and 561 bp minor cluster region (MCR) of BCL2 contains 13 and 5% of the BCL2 breakpoints, respectively. The breakpoints within MBR, ICR, and MCR are illustrated below the figure in zoomed-in views. Human lymphomas are clinically indistinguishable regardless of the position of the breakpoint within the 30 kb. (B) BCL1 breakpoints in BCL1-IGH translocations. The major translocation cluster (MTC) of BCL1 (also known as CCND1) is located 109 kb upstream of the CCND1 gene on chromosome 11 and contains 30% of all BCL1 breakpoints. The rest of the BCL1 breakpoints are scattered within a 344 kb intergenic region between the CCND1 gene at the telomeric end and the MYEOV gene on the centromeric side. (C) E2A breakpoints in E2A-PBX1 and E2A-HLF translocations. The E2A breakpoints in the E2A-PBX1 translocation (top panel) and E2A-HLF translocation (bottom panel) are illustrated. Over 75% of the E2A breakpoints are in a 23 bp region within E2A intron 16, making the 23 bp more than 400-fold more fragile compared with other regions of the same E2A intron. (D) MALT1 breakpoints in IGH-MALT1 and API2-MALT1 translocations. The breakpoints of MALT1 are focused on an 86 bp region upstream of the MALT1 gene in IGH-MALT1 translocations which are shown in the zoomed-in view. In contrast, the breakpoints of MALT1 in the API2-MALT1 translocation are scattered in a 29 kb region in several introns and exons of the MALT1 gene. For all figures, each triangle represents a single breakpoint from a patient. Triangles above and below the sequence are sequenced from two derivative chromosomes that resulted from the translocation. The CpG sites are highlighted with a red background. Within each fragile zone, the actual translocations occur at DNA sequence motifs, consisting of either the sequence CG (CpG) or mCG (when methylated) or WGCW where W = A or T. We are trying to determine why these small fragile zones are highly preferred for DNA breakage and translocation.

Figure 4.

Models for DNA breakage in early B cells. (A) Schematic models for the DNA breakage in early B cells. The fragile zones and their nearby regions usually contain a high density of C-strings that predispose the regions to a B/A intermediate (non-B) DNA structure. The DNA in the non-B structure tends to show increased thermal fluctuation that predisposes the fragile zones to AID activity without transcription. Frequent transcription through these fragile zones can increase their single-stranded character. RNA polymerase tends to move slowly and accumulates in regions adopting a non-B DNA structure, which further increases the duration of ssDNA. The cytosines in regions of ssDNA due to the transcription through these non-B regions are preferred substrates of AID deamination activity when they are in AID hotspot motifs. A long-lived DNA lesion can result if the cytosines are within a WRCG motif and in a methylated state. The persistent DNA lesions can be subject to nuclease attack inside the cells and then DSBs. (B) Steps leading to DNA breakage in fragile zones involved in early B cell translocations. The high density of C-strings around the fragile zones predisposes the fragile regions to a B/A intermediate DNA structure and then increased single-stranded character. Slowed RNA polymerase during transcription in regions adopting a non-B DNA structure and slippage between DNA direct repeats further increase their single-stranded character. The cytosines in regions of ssDNA are preferred sites of AID deamination activity when they are in AID hotspot motifs. As in panel A, a long-lived DNA lesion can result if the cytosines are within a WRCG motif in a methylated state, which is vulnerable to nuclease attack inside the cells.

Figure 5.

Fraction of human hematopoietic malignancies explained by AID-type breaks. Each human hematopoietic malignancy is shown to reflect the fraction of all hematopoietic malignancies. The numbers of events reflect the incidence of all events per year in the United States, including all ages (adults and children) (Teras et al. 2016; Cancer Facts & Figures 2021). The translocations and their percentage in each malignancy are estimated from Swerdlow et al. (2008). The portion of translocations that involve at least one AID-type event, often at the oncogene, are shown within the red. Some are AID-type/AID-type events, such as translocations between BCL6 or MYC and the IGH switch regions. But most are AID-type/RAG-type events, such as BCL1, BCL2, MALT1, or CRLF2, to the IGH locus during failed D_H to J_H joining. The small fraction colored in blue is RAG-type/RAG-type events. Note that many other translocations are not included [for example, BCR–ABL1 translocations also occur in some B cell lineage acute lymphoblastic leukemia (ALL); and mixed-lineage leukemia (MLL)–AF9 occurs in some cases of acute myeloid leukemia (AML)] as this figure is not intended to be comprehensive; readers are referred to the current American Cancer Society website or the current version of the WHO text cited here (Swerdlow et al. 2008) for further details. CCND1: cyclin D1; CDKN1A: cyclin-dependent kinase inhibitor 1 A; ETO: eight twenty one protein; HOX11: homeobox; NPM1: nucleophosmin; PBX: pre B cell leukemia homeobox 1; PML: promyelocytic leukemia; RARA: retinoic acid receptor-α; SIL: SCL interrupting locus; TAF8: TATA-box binding protein associated factor 8; TCR: T cell receptor; WHSC1: Wolf–Hirschhorn syndrome candidate 1; WWOX: WW domain-containing oxidoreductase.