DNA crosslinking and recombination‐activating genes 1/2 (RAG1/2) are required for oncogenic splicing in acute lymphoblastic leukemia

Abstract

Background Abnormal alternative splicing is frequently associated with carcinogenesis. In B‐cell acute lymphoblastic leukemia (B‐ALL), double homeobox 4 fused with immunoglobulin heavy chain (DUX4/IGH) can lead to the aberrant production of E‐26 transformation‐specific family related gene abnormal transcript (ERGalt) and other splicing variants. However, the molecular mechanism underpinning this process remains elusive. Here, we aimed to know how DUX4/IGH triggers abnormal splicing in leukemia. Methods The differential intron retention analysis was conducted to identify novel DUX4/IGH‐driven splicing in B‐ALL patients. X‐ray crystallography, small angle X‐ray scattering (SAXS), and analytical ultracentrifugation were used to investigate how DUX4/IGH recognize double DUX4 responsive element (DRE)‐DRE sites. The ERGalt biogenesis and B‐cell differentiation assays were performed to characterize the DUX4/IGH crosslinking activity. To check whether recombination‐activating gene 1/2 (RAG1/2) was required for DUX4/IGH‐driven splicing, the proximity ligation assay, co‐immunoprecipitation, mammalian two hybrid characterizations, in vitro RAG1/2 cleavage, and shRNA knock‐down assays were performed. Results We reported previously unrecognized intron retention events in C‐type lectin domain family 12, member A abnormal transcript (CLEC12Aalt) and chromosome 6 open reading frame 89 abnormal transcript (C6orf89alt), where also harbored repetitive DRE‐DRE sites. Supportively, X‐ray crystallography and SAXS characterization revealed that DUX4 homeobox domain (HD)1‐HD2 might dimerize into a dumbbell‐shape trans configuration to crosslink two adjacent DRE sites. Impaired DUX4/IGH‐mediated crosslinking abolishes ERGalt, CLEC12Aalt, and C6orf89alt biogenesis, resulting in marked alleviation of its inhibitory effect on B‐cell differentiation. Furthermore, we also observed a rare RAG1/2‐mediated recombination signal sequence‐like DNA edition in DUX4/IGH target genes. Supportively, shRNA knock‐down of RAG1/2 in leukemic Reh cells consistently impaired the biogenesis of ERGalt, CLEC12Aalt, and C6orf89alt. Conclusions All these results suggest that DUX4/IGH‐driven DNA crosslinking is required for RAG1/2 recruitment onto the double tandem DRE‐DRE sites, catalyzing V(D)J‐like recombination and oncogenic splicing in acute lymphoblastic leukemia.

Genomic and Clinical Analysis of Children with Acute Lymphoblastic Leukemia

“…Second-generation sequencing technology has been rapidly developed in the field of precision medicine due to its advantages of high throughput, high sensitivity, ease of operation, and relative quantification [ 6 , 7 ]. Its development provides a large amount of data for medical research, which can be transformed into clinically relevant information to allow researchers to determine how genes affect phenotypes, following which genome editing technology can be used to directly manipulate almost any gene in various cell types and organisms to develop therapeutic strategies for diseases at the genetic level [ 8 , 9 ]. Mutations have been shown to play a vital role in various diseases, including cancer.…”

Dithiocarbazate ligands and their Ni(II) complexes with potential biological activity: Structural, antitumor and molecular docking study

“…The DNA-binding double homeobox 4 fused with immunoglobulin domain 1 and 2 (DUX4 1-150 -DNA ERG HD1-HD2) protein complexed with the expression recombinant gene (ERG ALT ) has its catalytic activity related to the interaction between Frontiers in Molecular Biosciences frontiersin.org the residues of the Linker A and Linker B of the DUX4 1-150 protein (Zhang et al, 2021). The mechanism of oncogenic biogenesis of ERG alt is activated when the atom OE2 of the residue Glu93 of Linker A establishes a strong hydrogen bond (around 2.20 Å) with the atom ND2 of the residue Asn41 of Linker B.…”

A typical bedside-to-bench investigation of leukemogenic driver MEF2D fusion reveals new targeted therapy in B-cell acute lymphoblastic leukemia

“…More importantly, the unexpected DUX4/IHG-mediated genomic instability is often associated with recombination-activating genes 1/2 (RAG1/2) recruitment onto the double tandem DRE-DRE sites, catalyzing V(D)J-like recombination and oncogenic splicings such as ERG alt , CLEC12A alt , and C6orf89 alt in leukemia. 4 Moreover, compared with the good prognosis for DUX4/ IGH, the recurrent MEF2D fusions are with a poor clinical outcome. The lack of effective therapy might be related to our poor understanding of the disease mechanism.…”

“… 3 In addition, we also demonstrated that impaired DUX4-DRE interaction disrupted ERG alternative splicing (ERG alt ), also known as an important secondary hit in DUX4/IGH-type leukemia. In 2021, Zhang and co-workers 4 further showed that DUX4 homeobox domain HD1-HD2 might dimerize into a dumbbell-shaped trans configuration to crosslink two adjacent DRE sites. More importantly, the unexpected DUX4/IHG-mediated genomic instability is often associated with recombination-activating genes 1/2 (RAG1/2) recruitment onto the double tandem DRE-DRE sites, catalyzing V(D)J-like recombination and oncogenic splicings such as ERG alt , CLEC12A alt , and C6orf89 alt in leukemia.…”

“…More importantly, the unexpected DUX4/IHG-mediated genomic instability is often associated with recombination-activating genes 1/2 (RAG1/2) recruitment onto the double tandem DRE-DRE sites, catalyzing V(D)J-like recombination and oncogenic splicings such as ERG alt , CLEC12A alt , and C6orf89 alt in leukemia. 4 …”