Functional Interrogation of Non-coding DNA Sequences in leukemia development and drug resistance

Project Information

FIND-seq

Grant agreement ID: 947803

DOI

10.3030/947803

EC signature date 9 November 2020

Start date 1 March 2021

End date 28 February 2027

Funded under

• EXCELLENT SCIENCE - European Research Council (ERC)

Total cost

€ 1 784 000,00

EU contribution

€ 1 784 000,00

Coordinated by

ST. ANNA KINDERKREBSFORSCHUNG GMBH
Austria

Periodic Reporting for period 3 - FIND-seq (Functional Interrogation of Non-coding DNA Sequences in leukemia development and drug resistance)

Reporting period: 2024-03-01 to 2025-08-31

Summary of the context and overall objectives of the project

How regulatory elements orchestrate gene expression to give rise to different phenotypes, how the genome is physically organized and how enhancers work to recruit specific combinations of transcription factors are fascinating problems that in-situ targeted perturbations and next-generation sequencing (NGS) are helping to solve. Functional non-coding regions such as enhancers and insulators play an essential role in the regulation of cell type-specific gene expression programs, and mutations at non-coding elements can drive observable phenotypic changes comparable to those driven by mutations at coding sequences. Furthermore, genetic studies suggested that most of the genomic regions associated with disease are non-coding, including instances in cancer. This project specifically investigates the contribution of non-coding element, their sequence variation and 3D genome organization in leukemia. We focus on two aspects: leukemia development and acquisition of drug resistance. Previous work highlighted that non-coding elements and their physical interactions are linked to drug resistance, and are associated with transcriptional changes that remark a new cell identity. This observation opened new opportunites in understanding (and overcoming) drug resistance through the lense of the non-coding genome. For this reason, we investigate non-coding sequences (Objective 1) and chromatin structures (Objective 2) that drive drug resistance in pediatric acute leukemias. We are developing technologies that can be adapted to study drug resistance in other malignancies as well as other biological processes. Several GWAS studies identified non-coding regions whose sequence variation is associated with risk of developing B-cell derived acute lymphoblastic leukemia (B-ALL); genetic associations with B-ALL are solid, and validated across large cohorts of different etnicity. However, very little is known about how variation at these loci predisposes to leukemia development. This project fills this gap and provide mechanistic insights into leukemia development (Objective 3) by dissecting disease-associated enhancers and the network of associated transcription factors.

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 uncover regulatory elements involved in drug resistance, we've developed a method that allows us to make specific changes in the non-coding regions of our DNA. This method is genome-wide and doesn't require specific design. We introduce short DNA tags randomly, then employ CRISPR technology to modify the epigenetic features of nearby regulatory elements. To ensure the effectiveness of our approach, we've determined the minimum number of cells required for comprehensive genome analysis. By examining the CRISPR enzyme's behavior in a specific genomic site, we found that it can influence DNA within a 10 kb range. This insight allows us to divide the genome into manageable 10 kb bins. We've also optimized the number of DNA tag insertions per cell and developed a versatile DNA tool for recruiting CRISPR enzymes and recording integration sites. We've identified efficient delivery methods and are currently working on the sequencing part. Our next step is to conduct screenings and validate the regulatory elements we've identified. Our research has further focused on three specific genomic regions: CD273/CD274, CD19, and IKZF1. In the CD273/CD274 region, we've identified a critical 2.5 kb core area within a super enhancer, which controls the expression of genes related to immunotherapy. We've used CRISPRi to study this area and are preparing for more detailed investigations by employing a set of base editors for high-resolution mapping of critical motifs. By introducing single nucleotide substitution at the CD19 enhancer, we've discovered important motifs that control CD19 expression. Our findings have led to the development of a computational tool to assist in data analysis. Additionally, we're studying DNA regions associated with leukemia predisposition by perfirming tiling screenings and investigating which nucleotides and transcription factors are involved in this condition.

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)

Identification of disease-associated non-coding variants and regions has led to innovative therapies, but technological limitations have hindered the study of non-coding elements. The proposed research, using FIND-seq, aims to overcome these limitations. FIND-seq enables the genome-wide screening of coding and non-coding elements without requiring large sgRNA libraries. It's not affected by sequence variants and can be used in various organisms. FIND-seq can identify unannotated functional elements, particularly in the case of enhancers. The research focuses on understanding drug resistance from a non-coding genome perspective, aiming to identify target genes and their regulating transcription factors through TF-binding motifs. The results may reveal new approaches to combat drug resistance, often linked to distinct epigenetic changes. The FIND-base concept represents a transformative approach in contrast to current CRISPR screens. Enrichment scores in traditional screens link sgRNAs to target genes and indicate a gene's importance in a phenotype, but they are also influenced by sgRNA cleavage efficiencies. FIND-base, however, delves directly into mutations linked to functional changes in cell behavior, particularly in non-coding DNA. This method can provide detailed information, such as discriminating between mutations that disrupt a critical binding motif within an enhancer and those that do not alter transcription factor recruitment capacity. Furthermore, incorporating base editors and advanced nucleases enhances the resolution of mutagenesis screens. FIND-base will be applied to study oncogenic non-coding mutations in B-cell derived acute lymphoblastic leukemia, focusing on non-coding regions linked to SNPs associated with the disease based on multiple GWAS studies.