Journal Article

CTCF Is Essential for the Development and Maintenance of CALM‐AF10‐Induced Leukemia

Kuroki, Yoko, Shima, Yutaka, Yamagata, Kazutsune, Kitabayashi, Issay, Shioneri, Hisafumi, Aikawa, Yukiko

2025

Overview

CALM (Clathrin Assembly Lymphoid Myeloid Leukemia)‐AF10, a fusion gene commonly associated with acute myeloid leukemia (AML), arises from the t(10;11) translocation and is linked to poor prognosis. In this study, we demonstrate that the CCCTC‐binding factor (CTCF) plays a critical role in both the initiation and maintenance of CALM‐AF10‐induced AML (CALM‐AF10 AML). In vivo, CTCF deficiency significantly extended the survival of CALM‐AF10 AML mice. In vitro, CTCF knockout (KO) reduced the colony‐forming capacity of CALM‐AF10 AML cells and induced their differentiation into macrophage‐like cells. RNA sequencing (RNA‐seq) of CTCF KO cells revealed that Transglutaminase 2 (TGM2) was the most significantly downregulated gene. Chromatin immunoprecipitation sequencing (ChIP‐seq) analysis indicated increased repressive histone mark, H3K27 trimethylation (H3K27me3), accompanied by reduced active histone marks, including H3K4 trimethylation (H3K4me3) and H3K27 acetylation (H3K27ac), at the transcription start site (TSS) of Tgm2 following CTCF KO. Knockdown of Tgm2 using short hairpin RNA (shRNA) in CALM‐AF10 AML cells and in the human leukemic cell line U937 harboring the CALM‐AF10 fusion gene resulted in reduced colony formation and proliferation. This also promoted differentiation into macrophage‐like cells, recapitulating the effects observed with CTCF KO. Similarly, comparable outcomes were observed with GK921, a TGM2 inhibitor, highlighting the impact of TGM2 blockade. These findings suggest that CTCF regulates TGM2 expression by modulating post translational modifications of histones at its TSS, thereby preserving the undifferentiated state of CALM‐AF10 AML cells. The demonstrated efficacy of TGM2 inhibitors further underscores the importance of TGM2 in this subtype of leukemia. CTCF regulates TGM2 gene expression by modulating key histone modifications, including increased acetylation of H3K27 (H3K27ac) and trimethylation of H3K4 (H3K4me3), along with decreased trimethylation of H3K27 (H3K27me3). These epigenetic changes are critical for maintaining the stem‐like properties of CALM‐AF10‐induced AML. Pharmacological inhibition of TGM2 activity using GK921 effectively suppresses the growth of CALM‐AF10‐induced AML cells, highlighting TGM2 as a promising therapeutic target for this leukemia subtype.

• Publisher: John Wiley & Sons, Inc
• Subject: Acetylation / Acute myeloid leukemia / Animals / Bone cancer / CALM‐AF10 / CCCTC-Binding Factor - genetics / CCCTC-Binding Factor - metabolism / CCCTC‐binding factor / Cell differentiation / Cell Differentiation - genetics / Cell Line, Tumor / Cell proliferation / Chromatin / Clathrin / epigenetic regulation / Fusion protein / Gene expression / Gene Expression Regulation, Leukemic / Gene fusion / Genomes / Glucose / GTP-Binding Proteins - genetics / GTP-Binding Proteins - metabolism / Histones / Histones - metabolism / Humans / Immunoprecipitation / Laboratory animals / Leukemia / Leukemia, Myeloid, Acute - genetics / Leukemia, Myeloid, Acute - metabolism / Leukemia, Myeloid, Acute - pathology / Macrophages / Medical prognosis / Methylation / Mice / Mice, Knockout / Oncogene Proteins, Fusion - genetics / Oncogene Proteins, Fusion - metabolism / Penicillin / Protein Glutamine gamma Glutamyltransferase 2 / Ribonucleic acid / RNA / Transcription initiation / Transglutaminase 2 / Transglutaminases - genetics / Transglutaminases - metabolism / Transplants & implants