Stage-specific Chromosomal Deletions Activate the IRX3 Oncogene in T-cell Acute Lymphoblastic Leukemia

In the field of cancer research, understanding regulatory mechanisms of the noncoding genome remains a key focus, especially concerning how oncogenes can be aberrantly activated through non-canonical regulatory pathways. This paper, authored by Sunniyat Rahman et al. and published in the journal Blood in 2024, provides a comprehensive account of a novel mechanism for oncogene activation through focal deletions in the noncoding genome. Specifically, it investigates how the IRX3 oncogene is aberrantly activated in T-cell acute lymphoblastic leukemia (T-ALL). The study is the result of collaboration among researchers from several leading international institutes, including University College London, The University of Melbourne, Ghent University, and Dana-Farber/Harvard Cancer Center. The findings introduce a previously unappreciated “promoter tethering” phenomenon and propose new tumor suppression mechanisms, expanding our understanding of how the noncoding genome impacts cancer development.

Background and Purpose

The noncoding genome in cancer contains critical cis-regulatory elements such as enhancers, promoters, and insulators which ensure precise control of gene expression. However, somatic mutations in the noncoding genome in cancer can disrupt regulatory processes, leading to oncogene activation through mechanisms such as enhancer hijacking or de novo enhancer formation.

In the context of T-ALL, the mechanisms underlying activation of well-characterized oncogenes like TAL1, LMO2, TLX1, and TLX3 have been explored and shown to involve structural rearrangements like boundary deletions or enhancer amplification. However, the mechanisms behind the activation of other potential oncogenes, such as IRX3, remain elusive. This study aimed to elucidate the regulatory mechanism authorizing IRX3 activation in T-ALL and to investigate regulatory principles in the noncoding genome.

Research Workflow and Methodology

The study employed multiple experimental phases, including genomic data exploration, cellular model validation, and elucidation of underlying functional mechanisms.

1. Data Mining and Abnormal Gene Identification

The researchers conducted RNA sequencing analyses on 264 pediatric T-ALL cases, screening for genes that are silenced in normal developmental T cells but aberrantly overexpressed in leukemia. IRX3 emerged as a top candidate, exhibiting significant overexpression, particularly within TAL1 and HOXA subgroups.

Further investigation into genomic variation associated with IRX3 overexpression identified recurrent focal deletions in intron 8 of the FTO (fat mass and obesity-associated) gene (referred to as ftont8del). These deletions consistently intersected with a CTCF-binding site and were strongly associated with IRX3 dysregulation.

2. Investigating the Functional Impact of Focal Deletions

Through CRISPR/Cas9 gene-editing in human T-ALL cells, the authors mimicked the effects of ftont8del to validate its contribution to IRX3 activation. Key findings include: - Deletion of the CTCF-binding site in FTO intron 8 led to a significant upregulation of IRX3 transcription, comparable to levels observed in patient leukemia samples. - Deletion of a co-occurring MYB-binding site alone was insufficient to drive IRX3 activation, highlighting the critical role of the CTCF-binding site in this context.

These experiments demonstrated that regulatory disruptions localized to the CTCF-binding site are the primary mechanisms underpinning ftont8del-mediated IRX3 overexpression.

3. High-Dimensional Genomic Analysis

Using three-dimensional genomic tools such as UMI-4C and HiChIP, the authors showed that, in normal conditions, the CTCF site in FTO intron 8 tethers the IRX3 promoter to a genomically inert region, preventing its interaction with high-activity enhancers. Upon deletion of the CTCF site in ftont8del, the IRX3 promoter was untethered, creating aberrant interactions with a powerful upstream CRNDE super-enhancer.

Interestingly, the CRNDE locus exhibits stage-specific expression during normal T-cell differentiation, providing additional evidence of its role as a developmental enhancer.

Major Findings

1. Aberrant Expression of IRX3
   IRX3 was aberrantly expressed in 49% of adult T-ALL and 42% of pediatric T-ALL cases. This dysregulation was strongly linked to focal deletions in FTO intron 8 affecting a CTCF-binding site.

2. Inverse Regulatory Role of “Promoter Tethering”
   The researchers identified a tumor-suppressive mechanism termed “promoter tethering” whereby the IRX3 oncogene was sequestered to a genomically inert region through interactions with the CTCF-binding site in FTO intron 8.

3. Mechanism of Enhancer Hijacking by CRNDE
   Deletion of the CTCF tether caused the IRX3 promoter to interact with the CRNDE super-enhancer, resulting in transcriptional activation of IRX3. This represents a novel enhancer hijack mechanism.

4. Effect of CTCF Mutations
   The study also discovered that mutations in CTCF itself induced similar IRX3 activation, highlighting the significance of the CTCF-IRX3 regulatory axis.

Significance and Implications

This study is the first to propose a tumor-suppressive “promoter tethering” mechanism, whereby oncogene promoters are anchored to nonproductive genomic regions, isolating them from active enhancer elements. Specifically, CTCF at FTO intron 8 prevents IRX3 from being driven by proximal super-enhancers like CRNDE.

This research introduces a non-canonical model of oncogene regulation that could apply to other cancers where large-scale focal deletions in noncoding regions remain unexplained. The findings also underscore the potential complexity in regulatory relationships between FTO and IRX3, which have previously been implicated in obesity-related genetic studies.

Clinical Relevance

While the study does not directly address therapeutic strategies, its identification of regulatory regions essential for IRX3 activation suggests potential targets for gene therapy or small-molecule interventions. Moreover, FTO deletions could serve as biomarkers to monitor minimal residual disease in T-ALL patients.

Key Highlights

1. Discovery of “promoter tethering” as a tumor suppressor regulatory mechanism in the noncoding genome.
   
2. Elucidation of a unique enhancer hijack pathway driving IRX3 oncogene activation in T-ALL.
   
3. Identification of CRNDE as a developmentally active super-enhancer involved in aberrant IRX3 transcription.

Future Prospects

Integrative approaches combining three-dimensional genomic technologies and functional assays, as exemplified in this study, could uncover additional atypical mechanisms of oncogene activation, offering novel insights for cancer diagnostics, therapeutics, and personalized medicine strategies.