Epstein-Barr virus reactivation induces MYC-IGH spatial proximity and t(8;14) in B cells.

Burkitt lymphoma (BL) is a B cell malignancy associated with the Epstein-Barr virus (EBV). Most BL cases are characterized by a t(8;14) chromosomal translocation involving the MYC oncogene and the immunoglobulin heavy chain gene (IGH). The role of EBV in promoting this translocation remains largely unknown. Here we provide the experimental evidence that EBV reactivation from latency leads to an increase in the proximity between the MYC and IGH loci, otherwise located far away in the nuclear space both in B-lymphoblastoid cell lines and in patients' B-cells. Specific DNA damage within the MYC locus, followed by the MRE11-dependent DNA repair plays a role in this process. Using a CRISPR/Cas9-based B cell model to induce specific DNA double strand breaks in MYC and IGH loci, we have shown that the MYC-IGH proximity induced by EBV reactivation leads to an increased t(8;14) translocation frequency.

Mechanisms of Secondary Leukemia Development Caused by Treatment with DNA Topoisomerase Inhibitors.

Leukemia is a blood cancer originating in the blood and bone marrow. Therapy-related leukemia is associated with prior chemotherapy. Although cancer therapy with DNA topoisomerase II inhibitors is one of the most effective cancer treatments, its side effects include development of secondary leukemia characterized by the chromosomal rearrangements affecting AML1 or MLL genes. Recurrent chromosomal translocations in the therapy-related leukemia differ from chromosomal rearrangements associated with other neoplasias. Here, we reviewed the factors that drive chromosomal translocations induced by cancer treatment with DNA topoisomerase II inhibitors, such as mobility of ends of double-strand DNA breaks formed before the translocation and gain of function of fusion proteins generated as a result of translocation.

Recurrent Translocations in Topoisomerase Inhibitor-Related Leukemia Are Determined by the Features of DNA Breaks Rather Than by the Proximity of the Translocating Genes.

Topoisomerase inhibitors are widely used in cancer chemotherapy. However, one of the potential long-term adverse effects of such therapy is acute leukemia. A key feature of such therapy-induced acute myeloid leukemia (t-AML) is recurrent chromosomal translocations involving AML1 (RUNX1) or MLL (KMT2A) genes. The formation of chromosomal translocation depends on the spatial proximity of translocation partners and the mobility of the DNA ends. It is unclear which of these two factors might be decisive for recurrent t-AML translocations. Here, we used fluorescence in situ hybridization (FISH) and chromosome conformation capture followed by sequencing (4C-seq) to investigate double-strand DNA break formation and the mobility of broken ends upon etoposide treatment, as well as contacts between translocation partner genes. We detected the separation of the parts of the broken AML1 gene, as well as the increased mobility of these separated parts. 4C-seq analysis showed no evident contacts of AML1 and MLL with loci, implicated in recurrent t-AML translocations, either before or after etoposide treatment. We suggest that separation of the break ends and their increased non-targeted mobility-but not spatial predisposition of the rearrangement partners-plays a major role in the formation of these translocations.

Cytogenetic and molecular genetic methods for chromosomal translocations detection with reference to the KMT2A/MLL gene.

Acute leukemias (ALs) are often associated with chromosomal translocations, in particular, KMT2A/MLL gene rearrangements. Identification or confirmation of these translocations is carried out by a number of genetic and molecular methods, some of which are routinely used in clinical practice, while others are primarily used for research purposes. In the clinic, these methods serve to clarify diagnoses and monitor the course of disease and therapy. On the other hand, the identification of new translocations and the confirmation of known translocations are of key importance in the study of disease mechanisms and further molecular classification. There are multiple methods for the detection of rearrangements that differ in their principle of operation, the type of problem being solved, and the cost-result ratio. This review is intended to help researchers and clinicians studying AL and related chromosomal translocations to navigate this variety of methods. All methods considered in the review are grouped by their principle of action and include karyotyping, fluorescence in situ hybridization (FISH) with probes for whole chromosomes or individual loci, PCR and reverse transcription-based methods, and high-throughput sequencing. Another characteristic of the described methods is the type of problem being solved. This can be the discovery of new rearrangements, the determination of unknown partner genes participating in the rearrangement, or the confirmation of the proposed rearrangement between the two genes. We consider the specifics of the application, the basic principle of each method, and its pros and cons. To illustrate the application, examples of studying the rearrangements of the KMT2A/MLL gene, one of the genes that are often rearranged in AL, are mentioned.

Specificity of cancer-related chromosomal translocations is linked to proximity after the DNA double-strand break and subsequent selection.

Most cancer-related chromosomal translocations appear to be cell type specific. It is currently unknown why different chromosomal translocations occur in different cells. This can be due to either the occurrence of particular translocations in specific cell types or adaptive survival advantage conferred by translocations only in specific cells. We experimentally addressed this question by double-strand break (DSB) induction at MYC, IGH, AML and ETO loci in the same cell to generate chromosomal translocations in different cell lineages. Our results show that any translocation can potentially arise in any cell type. We have analyzed different factors that could affect the frequency of the translocations, and only the spatial proximity between gene loci after the DSB induction correlated with the resulting translocation frequency, supporting the 'breakage-first' model. Furthermore, upon long-term culture of cells with the generated chromosomal translocations, only oncogenic MYC-IGH and AML-ETO translocations persisted over a 60-day period. Overall, the results suggest that chromosomal translocation can be generated after DSB induction in any type of cell, but whether the cell with the translocation would persist in a cell population depends on the cell type-specific selective survival advantage that the chromosomal translocation confers to the cell.

Visualizing the Genome: Experimental Approaches for Live-Cell Chromatin Imaging.

Over the years, our vision of the genome has changed from a linear molecule to that of a complex 3D structure that follows specific patterns and possesses a hierarchical organization. Currently, genomics is becoming "four-dimensional": our attention is increasingly focused on the study of chromatin dynamics over time, in the fourth dimension. Recent methods for visualizing the movements of chromatin loci in living cells by targeting fluorescent proteins can be divided into two groups. The first group requires the insertion of a special sequence into the locus of interest, to which proteins that recognize the sequence are recruited (e.g., FROS and ParB-INT methods). In the methods of the second approach, "programmed" proteins are targeted to the locus of interest (i.e., systems based on CRISPR/Cas, TALE, and zinc finger proteins). In the present review, we discuss these approaches, examine their strengths and weaknesses, and identify the key scientific problems that can be studied using these methods.

Direct ENIT: An easy and reliable tool for gRNA efficacy verification by tracking induced chromosomal translocation.

CRISPR/Cas systems (Clustered regularly interspaced palindromic repeats / CRISPR-associated) are rapidly becoming a commonplace and popular tool for gene editing in research and clinical contexts. However, the quality of CRISPR/Cas experiments depends heavily on the guide RNA (gRNA) design; therefore, a reliable, easy, and rapid method for verifying gRNA cleavage efficacy is necessary. Engineered nuclease-induced translocations (ENIT) are an easy and cost-efficient method for the verification of gRNA efficacy, which involves tracking induced chromosomal mutations, using polymerase chain reaction (PCR). We have customized this method using both direct PCR and nested PCR approaches and have been able to reduce the sample preparation time. We present a simple and reliable gRNA testing approach that requires no specific enzymes or equipment.•The approach requires only routinely used enzymes and equipment.•Cost- and time-efficient, requiring approximately 30 min for PCR sample preparation, without requiring DNA purification.•High sensitivity, with induced translocation detected in 100 of 10,000 cells in the general population.