-Author name in bold denotes the presenting author
-Asterisk * with author name denotes a Non-ASH member
Clinically Relevant Abstract denotes an abstract that is clinically relevant.

PhD Trainee denotes that this is a recommended PHD Trainee Session.

Ticketed Session denotes that this is a ticketed session.

501 Elevated Enhancer-Oncogene Contacts and Higher Oncogene Expression Levels By Recurrent CTCF inactivating Mutations in T Cell Acute Lymphoblastic Leukemia

Program: Oral and Poster Abstracts
Type: Oral
Session: 602. Disordered Gene Expression and Epigenetics in Hematologic Malignancies: Basic: New mechanisms of epigenetic dysregulation in hematologic cancers
Hematology Disease Topics & Pathways:
Genomics, Translational Research, Biological Processes, Pathogenesis
Sunday, December 12, 2021: 5:00 PM

Willem K. Smits1*, Carlo Vermeulen, PhD2,3*, Rico Hagelaar1*, Shunsuke Kimura, MD, PhD4*, Eric Vroegindeweij, PhD1*, Jessica G.C.A.M. Buijs-Gladdines1*, Ellen Van De Geer1*, Marjon J.A.M. Verstegen2*, Erik Splinter, PhD5*, Simon V. van Reijmersdal, PhD1*, Arjan Buijs, PhD6*, Niels Galjart, PhD7*, Winfried Van Eyndhoven, PhD8*, Max Jan Van Min, PhD5*, Roland P Kuiper, PhD1,6, Patrick Kemmeren, PhD1*, Charles G. Mullighan, MBBS, MD9, Wouter De Laat, PhD2* and Jules P.P. Meijerink, PhD1*

1Princess Máxima Center for Pediatric Oncology, Utrecht, Netherlands
2Oncode Institute, Hubrecht Institute-KNAW, Utrecht, Netherlands
3Center for Molecular Medicine, University Medical Center Utrecht, Utrecht, Netherlands
4St. Jude Children's Research Hospital, Memphis, TN
5Cergentis BV, Utrecht, Netherlands
6Department of Genetics, University Medical Center Utrecht, Utrecht, Netherlands
7Erasmus University Medical Center Rotterdam, Rotterdam, Netherlands
8Agilent Technologies, Amstelveen, Netherlands
9Department of Pathology, St. Jude Children's Research Hospital, Memphis, TN

Introduction. The CCCTC-binding factor (CTCF) regulates the 3D chromatin architecture by facilitating chromosomal loops and forming the boundaries of structural domains. In addition, CTCF is an important transcription factor and regulator of antigen receptor and T cell receptor recombination events. CTCF inactivating events have been found in various human cancers. Loss-of-heterozygosity (LOH) or inactivating missense mutations in specific zinc- fingers have been identified in many human cancers including sporadic breast cancer, prostate cancer, Wilms-tumors and acute lymphoblastic leukemia (ALL). Heterozygous deletions or point mutations have been identified in over half of the patients with breast cancer or uterine endometrial cancers, deregulating global gene expression by altering methylated genomic states and poor survival. Here, we investigated the functional significance and molecular-cytogenetic associations of CTCF aberrations in T-cell acute lymphoblastic leukemia patients.

Methods. Biopsies from a cohort of 181 pediatric T-ALL patients who enrolled on DCOG or COALL protocols and/or their derivative patient-derived xenograft models were screened for alterations in global DNA copy number, methylation status, topologically associating domain organization and CTCF and cohesion binding patterns and changes in local TLX3 and BCL11B promoter enhancer loops using array-comparative genomic hybridization, single molecule Molecular Inversion Probe sequencing, targeted locus amplification, gene expression and DNA methylation microarrays, Hi-C sequencing, Chromatin Immunoprecipitation and/or real-time quantitative PCR. Ctcff/fl mice1 were crossed on a the Lck-cre transgenic background2 to study the impact of Ctcf loss during early T-cell development.

Results. We here describe that inactivation of CTCF can drive subtle and local genomic effects that elevate oncogene expression levels from driver chromosomal rearrangements. We find that for T cell acute lymphoblastic leukemia (T-ALL), heterozygous CTCF deletions or inactivating mutations are present in nearly 50 percent of t(5;14)(q35;q32.2) rearranged patients that positions the TLX3 oncogene in the vicinity of the BCL11B enhancer. Functional CTCF loss results in diminished expression of the αβ-lineage commitment factor BCL11B from the non-rearranged allele and γδ-lineage development. Unexpectedly, it also drives higher levels of the TLX3 oncogene from the translocated allele. We demonstrate that heterozygous CTCF aberrations specifically occur in TLX3-rearranged patients with distal breakpoints that preserve CTCF bindings sites in the translocation breakpoint areas in between the BCL11B enhancer and the TLX3 oncogene. We show that these intervening CTCF sites insulate TLX3 from the enhancer by forming competitive loops with TLX3. Upon loss of CTCF, or the deletion of the intervening CTCF sites, these competitive loops are weakened and loops with the BCL11B enhancer are stimulated, boosting TLX3 oncogene expression levels and leukemia burden in these T-ALL patients.

Conclusions. CTCF aberrations are especially associated with t(5;14)(q35;q32.2) rearranged T-ALL patients who maintain TLX3-proximal CTCF sites reflects a necessity to neutralize these sites in order to topologically enable the distal BCL11B enhancer to interact with the TLX3 oncogene and to boost its expression. Collectively, this provides direct demonstration of a mechanism in which loss of CTCF result in removal of enhancer insulation that facilitates elevated levels of an oncogene in leukemia.


  1. Heath H, Ribeiro de Almeida C, Sleutels F, et al. CTCF regulates cell cycle progression of alphabeta T cells in the thymus. EMBO J. 2008;27(21):2839-2850.
  2. Lee PP, Fitzpatrick DR, Beard C, et al. A critical role for Dnmt1 and DNA methylation in T cell development, function, and survival. Immunity. 2001;15(5):763-774.

Disclosures: Splinter: Cergentis BV: Current Employment. Van Eyndhoven: Agilent Technologies Netherland: Current Employment. Van Min: Cergentis BV: Current Employment. Mullighan: AbbVie: Research Funding; Illumina: Membership on an entity's Board of Directors or advisory committees; Amgen: Current equity holder in publicly-traded company; Pfizer: Research Funding.

*signifies non-member of ASH