Formation Of Chromosomal Translocations Research Articles

Abstract C076: Increased AID results in mutations at the CRLF2 locus implicated in Latin American ALL health disparities

Abstract Activation-induced cytidine deaminase (AID) is a B cell-specific mutator required for antibody diversification. However, it is also implicated in the etiology of several B cell malignancies. Evaluating the AID-induced mutation load in patients at-risk for certain blood cancers is critical in assessing disease severity and treatment options. We have developed a digital PCR (dPCR) assay that allows us to quantify mutations resulting from AID modification or DNA double-strand break (DSB) formation and repair at sites known to be prone to DSBs. Implementation of this new assay showed that increased AID levels in immature B cells increased genome instability at loci linked to chromosomal translocation formation. This included the CRLF2 locus that is often involved in translocations associated with a subtype of acute lymphoblastic leukemia (ALL) that disproportionately affects Hispanics, particularly those with Latin American ancestry. Using dPCR, we characterized the CRLF2 locus in B cell-derived genomic DNA from both Hispanic ALL patients and healthy Hispanic donors and found increased mutations in both, suggesting that vulnerability to DNA damage at CRLF2 may be driving this health disparity. Our ability to detect and quantify these mutations will potentiate future risk identification, early detection of cancers, and reduction of associated cancer health disparities. Citation Format: Valeria Rangel, Fabiola Quintero-Rivera, Sora Tanjasiri, Selma Masri, Angela Fleischman, Nicholas R. Pannunzio. Increased AID results in mutations at the CRLF2 locus implicated in Latin American ALL health disparities [abstract]. In: Proceedings of the 17th AACR Conference on the Science of Cancer Health Disparities in Racial/Ethnic Minorities and the Medically Underserved; 2024 Sep 21-24; Los Angeles, CA. Philadelphia (PA): AACR; Cancer Epidemiol Biomarkers Prev 2024;33(9 Suppl):Abstract nr C076.

Increased AID results in mutations at the CRLF2 locus implicated in Latin American ALL health disparities

Activation-induced cytidine deaminase (AID) is a B cell-specific mutator required for antibody diversification. However, it is also implicated in the etiology of several B cell malignancies. Evaluating the AID-induced mutation load in patients at-risk for certain blood cancers is critical in assessing disease severity and treatment options. We have developed a digital PCR (dPCR) assay that allows us to quantify mutations resulting from AID modification or DNA double-strand break (DSB) formation and repair at sites known to be prone to DSBs. Implementation of this assay shows that increased AID levels in immature B cells increase genome instability at loci linked to chromosomal translocation formation. This includes the CRLF2 locus that is often involved in translocations associated with a subtype of acute lymphoblastic leukemia (ALL) that disproportionately affects Hispanics, particularly those with Latin American ancestry. Using dPCR, we characterize the CRLF2 locus in B cell-derived genomic DNA from both Hispanic ALL patients and healthy Hispanic donors and found increased mutations in both, suggesting that vulnerability to DNA damage at CRLF2 may be driving this health disparity. Our ability to detect and quantify these mutations will potentiate future risk identification, early detection of cancers, and reduction of associated cancer health disparities.

Abstract 403: APOBEC3 enzymes induce chromosomal translocations in solid cancer

Abstract Introduction: Chromosomal translocations are common oncogenic drivers in human cancers, and an increasing number of translocations are being considered as crucial diagnostic and prognostic markers in clinics. In B-cell lymphoma, a number of chromosomal translocations are directed by the off-target activity of the activation-induced cytidine deaminase (AID; a B-cell specific apolipoprotein B mRNA-editing enzyme, catalytic polypeptode-like (APOBEC) family enzyme). However, in solid tumors, no such enzymes capable of directing chromosomal translocations have been identified. Given recent findings on APOBEC-mediated mutagenesis in solid cancers, APOBECs may be involved in the formation of chromosomal translocations in solid tumors, like AID does in B cells. In human, the APOBEC3 (A3) family consists of seven members (A3A-A3H) and is associated with C <t or="" c<g="" substitutions.="" recent="" evidence="" shows="" that="" a3a="" might="" be="" involved="" in="" chromosomal="" instability="" leading="" to="" drug="" resistance="" lung="" cancer.="" however,="" it="" has="" not="" been="" explored="" whether="" a3s="" generate="" dna="" dsbs,="" eventually="" translocations.="" this="" study,="" we="" investigated="" the="" role="" of="" and="" their="" mechanisms="" translocations="" solid="" cancer.Methods: We applied high-throughput genome-wide translocation sequencing (HTGTS) approach to map genome-wide chromosomal translocations induced by human A3s in mouse fibroblast and human epithelial cells. We correlated patterns of translocations induced by A3s with several other cellular features including transcription, DNA accessibility, DNA replication timing, and mutational signatures. We validated the contribution of A3 to the generation of oncogenic translocations in lung cancer. Results: Ectopic expression of A3s significantly increased chromosomal translocations in mouse fibroblasts and human epithelial cells. A3s and AID preferentially induced translocations in different parts of the genome, but some of the translocation hotspots were shared among them. Hierarchical clustering of hotspots showed that A3-mediated translocations were distinguished from AID-mediated translocations. Notably, the junction distribution of breakpoint clusters showed three distinct translocation patterns based on the locations of breakpoint clusters: (1) AID-preferred; (2) A3-preferred; and (3) AID/A3-mixed. AID-mediated translocations were heavily enriched near the transcription start site, whereas A3-mediated translocations were dispersed across the gene body and transcription end site. Furthermore, DNA replication timing and genomic architectures were linked to the formation of A3-mediated chromosomal translocations in solid cancer. Finally, A3 contributed to the formation of oncogenic rearrangements in lung cancer. Conclusions: A3 enzymes induced chromosomal translocations with distinct patterns compared to AID and facilitated chromosomal translocation formation in solid cancers. Citation Format: Taek-Chin Cheong, Qi Wang, Ahram Jang, Elif Karaca-Atabay, Roberto Chiarle. APOBEC3 enzymes induce chromosomal translocations in solid cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 403.

TDP1 suppresses chromosomal translocations and cell death induced by abortive TOP1 activity during gene transcription

DNA topoisomerase I (TOP1) removes torsional stress by transiently cutting one DNA strand. Such cuts are rejoined by TOP1 but can occasionally become abortive generating permanent protein-linked single strand breaks (SSBs). The repair of these breaks is initiated by tyrosyl-DNA phosphodiesterase 1 (TDP1), a conserved enzyme that unlinks the TOP1 peptide from the DNA break. Additionally, some of these SSBs can result in double strand breaks (DSBs) either during replication or by a poorly understood transcription-associated process. In this study, we identify these DSBs as a source of genome rearrangements, which are suppressed by TDP1. Intriguingly, we also provide a mechanistic explanation for the formation of chromosomal translocations unveiling an error-prone pathway that relies on the MRN complex and canonical non-homologous end-joining. Collectively, these data highlight the threat posed by TOP1-induced DSBs during transcription and demonstrate the importance of TDP1-dependent end-joining in protecting both gene transcription and genome stability.

Factors That Affect the Formation of Chromosomal Translocations in Cells.

Simple SummaryChromosomal translocations are products of the erroneous repair of DNA double-strand breaks that result in the illegitimate joining of the two broken chromosomal ends from non-homologous chromosomes. Chromosomal translocations have been linked to aneuploidy, infertility, mental retardation, cancer and other diseases. Here we discuss how chromosomal translocations are formed and explore how different cellular factors contribute to their formation.Chromosomal translocations are products of the illegitimate repair of DNA double-strand breaks (DSBs). Their formation can bring about significant structural and molecular changes in the cell that can be physiologically and pathologically relevant. The induced changes may lead to serious and life-threatening diseases such as cancer. As a growing body of evidence suggests, the formation of chromosomal translocation is not only affected by the mere close spatial proximity of gene loci as potential translocation partners. Several factors may affect formation of chromosomal translocations, including chromatin motion to the potential sources of DSBs in the cell. While these can be apparently random events, certain chromosomal translocations appear to be cell-type-specific. In this review, we discuss how chromosomal translocations are formed and explore how different cellular factors contribute to their formation.

Context-dependent regulation of immunoglobulin mutagenesis by p53

p53 plays a major role in genome maintenance. In addition to multiple p53 functions in the control of DNA repair, a regulation of DNA damage bypass via translesion synthesis has been implied in vitro. Somatic hypermutation of immunoglobulin genes for affinity maturation of antibody responses is based on aberrant translesion polymerase action and must be subject to stringent control to prevent genetic alterations and lymphomagenesis. When studying the role of p53 in somatic hypermutation in vivo, we found altered translesion polymerase-mediated A:T mutagenesis in mice lacking p53 in all organs, but notably not in mice with B cell-specific p53 inactivation, implying that p53 functions in non-B cells may alter mutagenesis in B cells. During class switch recombination, when p53 prevents formation of chromosomal translocations, we in addition detected a B cell-intrinsic role for p53 in altering G:C and A:T mutagenesis. Thus, p53 regulates translesion polymerase activity and shows differential activity during somatic hypermutation versus class switch recombination in vivo. Finally, p53 inhibition leads to increased somatic hypermutation in human B lymphoma cells. We conclude that loss of p53 function may promote genetic instability via multiple routes during antibody diversification in vivo.

Abstract 2393: APOBEC3 enzymes induce chromosomal translocations in solid cancers

Abstract Introduction: Apolipoprotein B mRNA-editing enzyme, catalytic polypeptide-like (APOBEC) family of cytidine deaminases provides a defense for the host genome against exogenous viruses and endogenous retro-elements, but their enzymatic activity may cause host genomic instability via a conversion of cytosine to uracil during RNA and DNA editing. While it is well established that activation-induced cytidine deaminase (AID; a B-cell specific APOBEC family enzyme) induces genomic instability by initiating mutations and oncogenic chromosomal translocations involved in the development and progression of lymphoma, it is unclear whether APOBEC3 enzymes could play a similar role in non-hematopoietic cells. Recently, APOBEC-signature mutation (C-to-T transition) has been identified in almost half of all human cancers, including breast and lung cancers. However, enzymes that trigger chromosomal translocation in solid cancers are largely underdetermined. In this study, we investigated the role of APOBEC3 proteins in chromosomal translocation formation and validated APOBEC3-induced chromosomal translocations in solid cancers. Methods: We generated genome-wide chromosomal translocation maps of human APOBEC3 enzymes in both mouse and human cells by adapting the high-throughput, genome-wide translocation sequencing (HTGTS) technique. We exploited immortalized mouse fibroblasts carrying a 25x I-SceI meganuclease cassette targeted in the c-myc locus and also human MCF10A (breast), Beas-2B (lung), and PC-9 (non-small cell lung cancer) cells by generating DNA DSBs in either the c-myc locus or the EML4 locus by the CRISPR/Cas9 system. Results: All human APOBEC3 enzymes have cytidine deaminase activity on their preferred target motifs with different potency. By overexpressing APOBEC3 proteins in AID−/− B cell, we found that APOBEC3 enzymes, in contrast to AID, did not induce IgG1 class switch recombination (CSR) indicating a different mechanism of action between AID and other APOBEC3 enzymes. By HTGTS, we demonstrated that APOBEC3s and AID induced significantly higher number of translocations in novel hotspots compared to control fibroblasts. Strikingly, bioinformatics analysis of the junctions revealed that APOBEC3 proteins induce chromosomal translocations with similar frequency, but only partially overlap with AID. In addition, by analyzing individual genes or translocation targets, we observed similar but distinct effects on translocation frequency for each member of the APOBEC3 family. Interestingly, AID preferentially targets genomic regions associated with high transcriptional activity, but APOBEC3Cs do not. The analysis of HTGTS from human breast and lung cell lines revealed the similar pattern of translocations found in mouse fibroblasts. Remarkably, APOBEC3 off-target hotspots are distinct and unique in each cell type. Conclusions: We showed that human APOBEC3 enzymes enhance chromosomal translocations in non-hematopoietic cells with similar potency to AID, suggesting that APOBEC3 enzymes could be initiating genomic instability in solid tumors where AID is not expressed. Thus, APOBEC3 expression has to be tightly controlled in non-hematopoietic cells to limit its genotoxic effects. Citation Format: Taek-Chin Cheong, Qi Wang, Roberto Chiarle. APOBEC3 enzymes induce chromosomal translocations in solid cancers [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 2393.

Abstract A09: Characterization of both topoisomerase II-dependent and -independent induction of DNA double-strand breaks, damage signaling pathways, and chromosomal translocations by subgroups of bioflavonoids

Abstract Background: Etoposide is a well-characterized poison of topoisomerase II that promotes DNA double-strand breaks (DSBs) and is associated with therapy-related chromosomal translocation. Bioflavonoids have a similar chemical structure to etoposide, and some evidence from our lab and others suggests that they also induce DSBs and promote genome rearrangements. Since bioflavonoids are found in natural foods as well as in dietary supplements and energy drinks, quantification of their potency has important physiologic impact. The purpose of this study is to determine if subgroups of bioflavonoids induce DSBs by direct or indirect inhibition of topoisomerase II (topo II) and if the downstream damage signaling pathways and illegitimate repair are distinct. Methods: The experiments exposed a mouse embryonic stem cell Reporter cell line to physiologically relevant low and increasing doses of a panel of bioflavonoids. Following exposure, the kinetics of appearance of DNA damage was measured by immunocytochemistry and confocal microscopy to quantify γ-H2AX foci over time. Induction of DNA damage pathways was measured by Western blotting. Finally, appearance of chromosomal translocations was measured by appearance of GFP+ cells. To determine if bioflavonoid-induced DNA damage is directly through inhibition of topo II or an alternative mechanism, all experiments were performed in parallel in the presence of dexrazoxane, a topo II inhibitor. Results: Exposure to subgroups of bioflavonoids generated distinct amount of γ-H2AX foci. In addition, the kinetics of the appearance and repair of DNA damage differed between subgroups. Most bioflavonoid treatments showed persistent damage 8 hours post-exposure, though no treatment group caused as extensive damage as etoposide. Comparison of γ-H2AX foci immediately after bioflavonoid exposure with and without pretreatment with dexrazoxane demonstrated that bioflavonoids differ in their action in a topo II-dependent or -independent manner. Exposure to luteolin and myricetin after pretreatment with dexrazoxane produced a reduction in the presence of γ-H2AX foci. By contrast, exposure to genistein after pretreatment with dexrazoxane produced no change, and exposure to kaempferol or quercetin after pretreatment produced increased presence of γ-H2AX foci. Downstream induction of signaling pathways and formation of chromosomal translocations following exposure to bioflavonoids has been characterized. Current approaches are using pretreatment with dexrazoxane to also determine if signaling and repair patterns are distinct between the bioflavonoid subgroups. Conclusions: Bioflavonoids are known to induce pleiotropic effects on cells. These data suggest that the subgroup of bioflavonoid has a distinct mechanism of action to induce DNA damage in either a topo II-dependent or -independent manner, which may have implications for the potential of individual bioflavonoids to promote genome instability and chromosomal translocations. Citation Format: Donna A. Goodenow, Anindita Ghosh, Kiran Lalwani, Christine Richardson. Characterization of both topoisomerase II-dependent and -independent induction of DNA double-strand breaks, damage signaling pathways, and chromosomal translocations by subgroups of bioflavonoids [abstract]. In: Proceedings of the AACR Special Conference on Environmental Carcinogenesis: Potential Pathway to Cancer Prevention; 2019 Jun 22-24; Charlotte, NC. Philadelphia (PA): AACR; Can Prev Res 2020;13(7 Suppl): Abstract nr A09.

DNA Double Strand Breaks and Chromosomal Translocations Induced by DNA Topoisomerase II.

DNA double strand breaks (DSBs) are the most cytotoxic lesions of those occurring in the DNA and can lead to cell death or result in genome mutagenesis and chromosomal translocations. Although most of these rearrangements have detrimental effects for cellular survival, single events can provide clonal advantage and result in abnormal cellular proliferation and cancer. The origin and the environment of the DNA break or the repair pathway are key factors that influence the frequency at which these events appear. However, the molecular mechanisms that underlie the formation of chromosomal translocations remain unclear. DNA topoisomerases are essential enzymes present in all cellular organisms with critical roles in DNA metabolism and that have been linked to the formation of deleterious DSBs for a long time. DSBs induced by the abortive activity of DNA topoisomerase II (TOP2) are “trending topic” because of their possible role in genome instability and oncogenesis. Furthermore, transcription associated TOP2 activity appears to be one of the most determining causes behind the formation of chromosomal translocations. In this review, the origin of recombinogenic TOP2 breaks and the determinants behind their tendency to translocate will be summarized.

Inhibition of Parp1 by BMN673 Effectively Sensitizes Cells to Radiotherapy by Upsetting the Balance of Repair Pathways Processing DNA Double-Strand Breaks.

Parp inhibitors (Parpi) are commonly used as single agents for the management of tumors with homologous recombination repair (HRR) deficiencies, but combination with radiotherapy (RT) is not widely considered due to the modest radiosensitization typically observed. BMN673 is one of the most recently developed Parpi and has been shown to mediate strong cell sensitization to methylating agents. Here, we explore the mechanisms of BMN673 radiosensitization to killing, aiming to combine it with RT. We demonstrate markedly stronger radiosensitization by BMN673 at concentrations substantially lower (50 nmol/L) than olaparib (3 μmol/L) or AG14361 (0.4 μmol/L) and dramatically lower as compared with second-generation inhibitors such as PJ34 (5 μmol/L). Notably, BMN673 radiosensitization peaks after surprisingly short contact times (∼1 hour) and at pharmacologically achievable concentrations in vivo BMN673 exerts a complex set of effects on DNA double-strand break (DSB) processing, including inhibition of classic nonhomologous end-joining (cNHEJ) and alternative end-joining (altEJ) pathway at high doses of ionizing radiation (IR). BMN673 enhances resection at DSB and favors HRR and altEJ at low clinically relevant IR doses. The combined outcome of these effects is an abrogation in the inherent balance of DSB processing culminating in the formation of chromosomal translocations that underpin radiosensitization. Our observations pave the way to clinical trials exploring inherent benefits in combining BMN673 with RT for the treatment of various forms of cancer. Mol Cancer Ther; 17(10); 2206-16. ©2018 AACR.

Dynamics of Double-Strand Breaks: Implications for the Formation of Chromosome Translocations.

Illegitimate joining of chromosome breaks can lead to the formation of chromosome translocations, a catastrophic type of genome rearrangements that often plays key roles in tumorigenesis. Emerging evidence suggests that the mobility of broken DNA loci can be an important determinant in partner search and clustering of individual breaks, events that can influence translocation frequency. We summarize here the recent literature on the mechanisms that regulate chromatin movement, focusing on studies exploring the motion properties of double-strand breaks in the context of chromatin, the functional consequences for DNA repair, and the formation of chromosome fusions.

The Association between PARP1 and LIG3 Expression Levels and Chromosomal Translocations in Acute Myeloid Leukemia Patients.

Objective Chromosomal translocations are among the most common mutational events in cancer development, especially in hematologic malignancies. However, the precise molecular mechanism of these events is still not clear. It has been recently shown that alternative non-homologous end-joining (alt-NHEJ), a newly described pathway for double-stranded DNA break repair, mediates the formation of chromosomal translocations. Here, we examined the expression levels of the main components of alt-NHEJ (PARP1 and LIG3) in acute myeloid leukemia (AML) patients and assessed their potential correlation with the formation of chromosomal translocations. Materials and Methods This experimental study used reverse transcription-quantitative polymerase chain reaction (RT- qPCR) to quantify the expression levels of PARP1 and LIG3 at the transcript level in AML patients (n=78) and healthy individuals (n=19). Results PARP1 was the only gene overexpressed in the AML group when compared with healthy individuals (P=0.0004), especially in the poor prognosis sub-group. Both genes were, however, found to be up-regulated in AML patients with chromosomal translocations (P=0.04 and 0.0004 respectively). Moreover, patients with one isolated translocation showed an over-expression of only LIG3 (P=0.005), whereas those with two or more translocations over-expressed both LIG3 (P=0.002) and PARP1 (P=0.02). ConclusionThe significant correlations observed between PARP1 and LIG3 expression and the rate of chromosomal translocations in AML patients provides a molecular context for further studies to investigate the causality of this association.

Anaplastic large cell lymphoma in paediatric and young adult patients.

Anaplastic large cell lymphoma (ALCL) is a heterogeneous disease of debateable origin that, in children, is largely anaplastic lymphoma kinase (ALK) positive with aberrant ALK activity induced following the formation of chromosomal translocations. Whilst the survival rates for this disease are relatively high, a significant proportion (20-40%) of patients suffer disease relapse, in some cases on multiple occasions and therefore suffer the toxic side-effects of combination chemotherapy. Traditionally, patients are treated with a combination of agents although recent data from relapse patients have suggested that low risk patients might benefit from single agent vinblastine and, going forward, the addition of ALK inhibitors to the therapeutic regimen may have beneficial consequences. There are also a plethora of other drugs that might be advantageous to patients with ALCL and many of these have been identified through laboratory research although the decision as to which drugs to implement in trials will not be trivial.

PI3Kdelta Inhibitors Increase Genomic Instability By Upregulating Aid Expression

▪Activation-induced cytidine deaminase (AID) is a B cell-specific enzyme that initiates class switch recombination (CSR) and somatic hypermutation (SHM) of immunoglobulin (Ig) genes, essential mechanisms to generate different classes of antibody and antibody diversity for the antigens. At lower frequency, AID also promiscuously introduces DNA structural lesions at non-Ig loci and it is involved in the pathogenesis of B cell lymphoma. Thus, its expression is tightly controlled in B cells to limit its genotoxic effects. Phosphatidylinositol 3-kinase (PI3K) p110δ isoform acts downstream of the B-Cell Receptor (BCR) to suppress AID expression, whereas blockade of PI3K signaling enhances the expression of AID. Potent oral PI3K inhibitors such as the p110δ inhibitor idelalisib (GS-1101, CAL-101) have been recently approved for the treatment of chronic lymphocytic leukemia (CLL), mantle cell lymphoma (MCL) and Waldenstrom macroglobulinemia (WM), whereas dual PI3K p110δ and γ inhibitors such as duvelisib (IPI-145) showed promising results for the treatment of CLL and other hematologic malignancies.Here we investigated whether the increased of AID expression caused by idelalisib or duvelisib induced genomic instability in normal and malignant B cells.We observed that in splenic purified mouse B cells activated with IL4 and αCD40 to induce AID expression and CSR, treatment with idelalisib and duvelisib significantly increased both AID protein and mRNA levels, compared to controls. As a result, both drugs strongly promoted CSR. The regulation of AID expression was strictly dependent on PI3K p110δ activity because a selective p110γ inhibitor (AS-604850) did not have any effect. In contrast, B cells expressing a constitutively active PI3Kδ showed suppression of CSR and reduced AID expression compared to inactive PI3Kδ.To gain insights into the degree of genomic instability induced by increased AID expression upon PI3Kδ inhibition, we applied a genome-wide translocation technique we previously developed (High-Throughput Genomic Translocation Sequencing approach, HTGTS) to identify translocation partners from DNA double strand breaks (DSBs) introduced into the c-myc locus (Chiarle et al, Cell 2011). By HTGTS, we isolated thousands of translocations from activated primary mouse B cells distributed widely across the genome. Remarkably, in B cells treated with idelalisib or duvelisib, we identified a significantly higher number of translocations in known AID off-target genes as well as novel hotspots of translocations (48 for idelalisib, 50 for duvelisib). Unbiased genome-wide analysis of translocation formation revealed a consistency in the translocation patterns, with AID target hotspots localized in the TSS region and predominantly grouped within super-enhancers and regulatory clusters. HTGTS analysis performed on activated AID knock-out (AID KO) B cells showed that the vast majority of these translocations were dependent on AID. Hence, our data demonstrate that in normal B cells PI3Kδ inhibitors increase genomic instability by an AID-dependent mechanism.Finally, we observed that both idelalisib and duvelisib increased AID expression in human EBV-immortalized and lymphoma cell lines (MCL and CLL). We adapted the HTGTS technique to human cells by introducing DSBs in the human myc locus by CRISPR/Cas9 technology and sequencing genome-wide the formation of chromosomal translocations. By this approach, we demonstrated that also in human neoplastic B cells the treatment with idelalisib or duvelisib increased the formation of translocations to known AID off targets.In conclusion, we showed that idelalisib or duvelisib increase genomic instability in normal and neoplastic B cells by enhancing AID expression. Since several B cell malignancies imply treatment with these drugs for years, these effects of PI3Kδ inhibitors on the genomic stability of B cells should be carefully taken into account for therapeutic outcomes and protocol design. DisclosuresNo relevant conflicts of interest to declare.

Alternative end-joining repair pathways are the ultimate backup for abrogated classical non-homologous end-joining and homologous recombination repair: Implications for the formation of chromosome translocations

DNA double strand breaks (DSB) are the most deleterious lesions for the integrity of the genome, as their misrepair can lead to the formation of chromosome translocations. Cells have evolved two main repair pathways to suppress the formation of these genotoxic lesions: homology-dependent, error-free homologous recombination repair (HRR), and potentially error-prone, classical, DNA-PK-dependent non-homologous end-joining (c-NHEJ). The most salient feature of c-NHEJ, speed, will largely suppress chromosome translocation formation, while sequence alterations at the junction remain possible. It is now widely accepted that when c-NHEJ is inactivated, globally or locally, an alternative form of end-joining (alt-EJ) removes DSBs. Alt-EJ operates with speed and fidelity markedly lower than c-NHEJ, causing thus with higher probability chromosome translocations, and generating more extensive sequence alterations at the junction. Our working hypothesis is that alt-EJ operates as a backup to c-NHEJ. Recent results show that alt-EJ can also backup abrogated HRR in G2 phase cells, again at the cost of elevated formation of chromosome translocations. These observations raise alt-EJ to a global rescuing mechanism operating on ends that have lost their chromatin context in ways that compromise processing by HRR or c-NHEJ. While responsible for eliminating from the genome highly cytotoxic DNA ends, alt-EJ provides this function at the price of increased translocation formation. Here, we analyze recent literature on the mechanisms of chromosome translocation formation and propose a functional hierarchy among DSB processing pathways that makes alt-EJ the global backup pathway. We discuss possible ramifications of this model in cellular DSB management and pathway choice, and analyze its implications in radiation carcinogenesis and the design of novel therapeutic approaches.

Histone modifications predispose genome regions to breakage and translocation.

Chromosome translocations are well-established hallmarks of cancer cells and often occur at nonrandom sites in the genome. The molecular features that define recurrent chromosome breakpoints are largely unknown. Using a combination of bioinformatics, biochemical analysis, and cell-based assays, we identify here specific histone modifications as facilitators of chromosome breakage and translocations. We show enrichment of several histone modifications over clinically relevant translocation-prone genome regions. Experimental modulation of histone marks sensitizes genome regions to breakage by endonuclease challenge or irradiation and promotes formation of chromosome translocations of endogenous gene loci. Our results demonstrate that histone modifications predispose genome regions to chromosome breakage and translocations.

A role for epigenetics in the formation of chromosome translocations in acute leukemia

In general, the field of cancer genetics seems to have shifted its focus from cancer-associated genes to cancer-associated epigenetic activity. An abundance of evidence suggests that epigenetic malfunction, such as aberrant histone modification, and altered DNA methylation, is at the root of much, if not most aberrant gene expression associated with cancer. However, a role for epigenetics in physical DNA changes, such as chromosome rearrangements, is less obvious, and certainly less well understood. A growing body of evidence suggests that epigenetics may play a role in many of the steps of aberrant chromosome recombination, especially chromosome translocations, associated with cancers such as acute leukemias.

Nuclear position dictates DNA repair pathway choice.

Faithful DNA repair is essential to avoid chromosomal rearrangements and promote genome integrity. Nuclear organization has emerged as a key parameter in the formation of chromosomal translocations, yet little is known as to whether DNA repair can efficiently occur throughout the nucleus and whether it is affected by the location of the lesion. Here, we induce DNA double-strand breaks (DSBs) at different nuclear compartments and follow their fate. We demonstrate that DSBs induced at the nuclear membrane (but not at nuclear pores or nuclear interior) fail to rapidly activate the DNA damage response (DDR) and repair by homologous recombination (HR). Real-time and superresolution imaging reveal that DNA DSBs within lamina-associated domains do not migrate to more permissive environments for HR, like the nuclear pores or the nuclear interior, but instead are repaired in situ by alternative end-joining. Our results are consistent with a model in which nuclear position dictates the choice of DNA repair pathway, thus revealing a new level of regulation in DSB repair controlled by spatial organization of DNA within the nucleus.

Mechanism of suppression of chromosomal instability by DNA polymerase POLQ.

Although a defect in the DNA polymerase POLQ leads to ionizing radiation sensitivity in mammalian cells, the relevant enzymatic pathway has not been identified. Here we define the specific mechanism by which POLQ restricts harmful DNA instability. Our experiments show that Polq-null murine cells are selectively hypersensitive to DNA strand breaking agents, and that damage resistance requires the DNA polymerase activity of POLQ. Using a DNA break end joining assay in cells, we monitored repair of DNA ends with long 3′ single-stranded overhangs. End joining events retaining much of the overhang were dependent on POLQ, and independent of Ku70. To analyze the repair function in more detail, we examined immunoglobulin class switch joining between DNA segments in antibody genes. POLQ participates in end joining of a DNA break during immunoglobulin class-switching, producing insertions of base pairs at the joins with homology to IgH switch-region sequences. Biochemical experiments with purified human POLQ protein revealed the mechanism generating the insertions during DNA end joining, relying on the unique ability of POLQ to extend DNA from minimally paired primers. DNA breaks at the IgH locus can sometimes join with breaks in Myc, creating a chromosome translocation. We found a marked increase in Myc/IgH translocations in Polq-defective mice, showing that POLQ suppresses genomic instability and genome rearrangements originating at DNA double-strand breaks. This work clearly defines a role and mechanism for mammalian POLQ in an alternative end joining pathway that suppresses the formation of chromosomal translocations. Our findings depart from the prevailing view that alternative end joining processes are generically translocation-prone.

Generation of cell-based systems to visualize chromosome damage and translocations in living cells.

Traditional methods for the generation of DNA damage are not well suited for the observation of spatiotemporal aspects of damaged chromosomal loci. We describe a protocol for the derivation of a cellular system to induce and to visualize chromosome damage at specific sites of the mammalian genome in living cells. The system is based on the stable integration of endonuclease I-SceI recognition sites flanked by bacterial LacO/TetO operator arrays, coupled with retroviral-mediated integration of their fluorescent repressors (LacR/TetR) to visualize the LacO/TetO sites. Expression of the I-SceI endonuclease induces double-strand breaks (DSBs) specifically at the sites of integration, and it permits the dynamics of damaged chromatin to be followed by time-lapse microscopy. Sequential LacO-I-SceI/TetO-I-SceI integrations in multiple chromosomes permit the generation of a system to visualize the formation of chromosome translocations in living cells. This protocol requires intermediate cell culture and molecular biology skills, and it is adaptable to the efficient derivation of any integrated clonal reporter system of interest in ≈ 3-5 months.