Deciphering translocation-based genome topology effects and their role in lymphoma formation

Project Information

LymphoTOP

Grant agreement ID: 101039265

DOI

10.3030/101039265

EC signature date 15 June 2022

Start date 1 October 2022

End date 30 September 2027

Funded under

• European Research Council (ERC)

Total cost

€ 1 499 993,00

EU contribution

€ 1 499 993,00

Coordinated by

FUNDACIO CENTRE DE REGULACIO GENOMICA
Spain

Periodic Reporting for period 1 - LymphoTOP (Deciphering translocation-based genome topology effects and their role in lymphoma formation)

Reporting period: 2022-10-01 to 2025-03-31

Summary of the context and overall objectives of the project

Cancer is often associated with chromosomal translocations, a phenomenon whereby a chromosome breaks, and its fragments reattach to another chromosome. This may interrupt tumour-suppressor genes or more often lead to expression of fusion genes or proto-oncogenes located at the translocation breakpoint, resulting in tumor formation. The working hypothesis of the EU-funded LymphoTOP project is that chromosomal translocations disturb genome architecture and may cause further deregulation. We will focus on non-Hodgkin lymphoma (NHL) and determine how translocations in immunoglobulin loci (IGH), which are frequently found in NHL patients, affect the genomic and epigenetic landscape and lead to disease development. Results will provide fundamental knowledge on the function of the nucleus in the cell, in which the DNA is tightly packed in a highly organised manner, and carcinogenesis, whereby the organisation of the nucleus is disturbed.

Work performed from the beginning of the project to the end of the period covered by the report and main results achieved so far

To assess the effect of translocations in the context of NHL, we employed a variety of techniques allowing us to perform genome-editing and thus make translocations in our laboratory, to read out genome architecture and to map the positioning of DNA loci in the nucleus. We combined these with methods to study the gene expression landscape in manipulated cell systems and patient samples, to not only map altered genome organisation in the nucleus but also understand its effect on downstream gene expression, which will provided us clues regarding tumor formation.

We optimised genome editing strategies using CRISPR/Cas9 to generate translocations in healthy mature B cells that we culture in the lab and to delete the regulatory elements at these loci to more specifically understand their effect. Because our methods cannot target 100% of the cells in our system, we end up with mixed populations in which some cells carry the genetic alterations that we aimed to induce. To study the effect on gene expression in these correctly edited cells we turned to single-cell methods, allowing us to study for each cell in our system the gene expression profile.

We also implemented systems to study B-cell differentiation in the lab, using hematopoeitic stem cells from cord blood as the initial cell state. These systems are important, because in NHL patient samples, IGH translocations occur in very early B cells. Besides setting up these systems in the lab, we managed to make translocations in this system with the overall aim to study their impact in early B cells.
Third, we optimised microscopy-based technologies to visualise and measure the positioning of translocated and normal chromosomes in cell lines representing NHL. The optimisation included defining the right microscopy settings and the proper measurement read outs allowing us to study this biological phenomenon.

Progress beyond the state of the art and expected potential impact (including the socio-economic impact and the wider societal implications of the project so far)

The only reported regulatory effects of NHL-associated translocations so far are those affecting single genes close to the breakpoint. However, we show that these translocations can upregulate a large set of genes affected entire chromosome arms. Our finding has large implications for understanding the global functioning of the nucleus, both in the context of healthy cells as well as in the context of lymphoma formation. Especially because overexpressed genes in MCL patients are enriched in the exact same chromosomal regions as those genes affected when we generate translocations in the laboratory. Overall, our results thus allow us thus to provide a clear set of genes to be explored further for their tumorigenic effects, their use to build better murine MCL models, as well as their therapeutic potential in the context of NHL.

A second important result is that we show that the activation and organization of the DNA prior to translocation formation influence the effect of translocations on gene expression. In other words, pre-existing genomic characteristics determine the potential of translocations to generate effects that can induce tumor formation. This finding is especially important now that single-cell approaches enable the identification of more and more cell subtypes. Based on our findings, we will be able to predict translocation-induced effect in these cell subtypes, allowing to better pinpoint the cell type of origin of NHLs.

While our study focuses on understanding the very early effects of lymphoma formation, we believe that our findings have a far more general implication as explained next. Overall, we show that the effects of translocations are far broader than initally thought. As many tumors harbour translocations or other large genomic aberrations, known as structural variants, that usually occur very early during disease development, our results can have a wide impact for the understanding of tumor formation.