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1405 Suppression of Super-Enhancer-Driven TAL1 Expression By KLF4 in T-Cell Acute Lymphoblastic Leukemia

Program: Oral and Poster Abstracts
Session: 603. Lymphoid Oncogenesis: Basic: Poster I
Hematology Disease Topics & Pathways:
Poster-only abstracts
Saturday, December 9, 2023, 5:30 PM-7:30 PM

Mina Noura, PhD1*, Takahiko Yasuda, MD, PhD2*, Shinobu Tsuzuki, MD, PhD3*, Hidemasa Matsuo, PhD4*, Hitoshi Kiyoi, MD, PhD5 and Fumihiko Hayakawa, MD, PhD6

1Division of Cellular and Genetic Sciences, Department of Integrated Health Sciences, Graduate School of Medicine, Nagoya University, Nagoya, Japan
2Clinical Research Center, National Hospital Organization Nagoya Medical Center, Nagoya, Japan
3Aichi Medical University, Nagakute, JPN
4Department of Human Health Sciences, Graduate School of Medicine, Kyoto University, Kyoto, Japan
5Nagoya University Graduate School of Medicine, Nagoya, Japan
6Department of Pathophysiological Laboratory Sciences, Nagoya University Graduate School of Medicine, Nagoya, JPN

T-cell acute lymphoblastic leukemia (T-ALL) is an aggressive malignancy characterized by differentiation arrest and clonal proliferation of immature thymocytes. Many oncogenes in T-ALL are genes encoding transcription factors. TAL bHLH transcription factor 1 (TAL1) is one of the most frequently dysregulated transcription factors in T-ALL and is ectopically overexpressed in approximately 50% of T-ALL cases, owing to chromosome translocations, SIL (SCL-interrupting locus)-TAL1 fusion, and 5’ super-enhancer (SE)-generating mutations. TAL1 overexpression caused by 5’ TAL1 SE is found in 5% of patients with T-ALL and is associated with unfavorable clinical outcomes. However, no clinically available drugs specifically inhibit TAL1 or TAL1 SE components.

Kruppel-like factor 4 (KLF4) is a member of the KLF family of transcription factors that exhibits both oncogenic and tumor-suppressive functions in a context-dependent manner. In T-cell malignancies, KLF4 is regarded as a tumor suppressor because KLF4 expression is silenced by promoter methylation, and the induction of KLF4 promotes apoptosis through the BCL-2/BCL-XL pathway. However, whether KLF4 affects the expression of T-ALL-related transcription factors remains unclear.

We first analyzed three independent previously reported microarray datasets to explore the essential downstream targets of KLF4 in T-ALL cells. The following groups of genes were extracted: genes upregulated in patients with T-ALL compared to normal thymocytes (GSE46170), genes upregulated in Klf4-deficient T-ALL mice (GSE75663), and genes downregulated in KLF4-overexpressing human T-ALL cells (Li W et al. Mol. Cancer. 2015). Overlapping genes in these groups were identified as potential downstream targets of KLF4. Among these, we focused on TAL1, a well-known oncogenic transcription factor gene in T-ALL. We performed a functional analysis of KLF4 using TAL1-positive T-ALL cell lines (Jurkat, MOLT-3, CCRF-CEM, and RPMI-8402). Genetic induction of KLF4 showed stronger anti-leukemic effects in Jurkat and MOLT-3 cells, 5’TAL1 SE-positive T-ALL cell lines, compared with CCRF-CEM and RPMI-8402 cells, SIL-TAL1-positive T-ALL cell lines. KLF4 overexpression significantly reduced mRNA expression levels of TAL1 in Jurkat and MOLT-3 cells but not in CCRF-CEM and RPMI-8402 cells. Thus, we hypothesized that KLF4 specifically suppressed SE-driven TAL1 expression by impairing mutated TAL1 enhancer activity. The luciferase reporter assay confirmed that the activity of the mutated TAL1 enhancer was significantly reduced by the exogenous expression of KLF4, whereas that of the wild-type reporter was not affected. According to previous studies, mutations in TAL1 enhancers in Jurkat and MOLT-3 cells create MYB primary motifs, and MYB binding at this location is essential for the formation of 5’ TAL1 SE. Therefore, we examined whether KLF4 represses MYB expression. The chromatin immunoprecipitation assay and qPCR analysis revealed that KLF4 bound to the proximal gene promoter of MYB and directly downregulated its expression. The luciferase reporter assay using the MYB promoter showed a significant reduction of reporter activity upon KLF4 overexpression.

Finally, we investigated whether pharmacological induction of KLF4 using APTO-253, a small-molecule inducer of KLF4, could control leukemia. APTO-253 inhibited the growth of all TAL1-positive T-ALL cell lines at nanomolar concentrations. The GI50 values of APTO-253 for Jurkat and MOLT-3 cells were lower than those for CCRF-CEM and RPMI-8402 cells, which was consistent with the KLF4-overexpression-induced growth inhibition of T-ALL cell lines. APTO-253 induced KLF4 expression and decreased TAL1 and MYB expression in Jurkat and MOLT-3 cells. Furthermore, exogenous MYB expression rescued growth inhibition by APTO-253. Collectively, these results indicated that APTO-253 suppressed the growth of T-ALL cells by targeting SE-driven TAL1 expression via the induction of KLF4.

In summary, we revealed that KLF4 suppressed SE-driven TAL1 expression via the direct inhibition of MYB. Moreover, pharmacological induction of KLF4 demonstrated anti-leukemic activity in T-ALL cells. These findings propose a promising strategy for patients with T-ALL and 5’ TAL1 SE.

Disclosures: Kiyoi: Perseus Proteomics: Research Funding; SymBio: Honoraria; Asahi Kasei: Research Funding; Ono Pharmaceuticals: Honoraria; Eisai: Honoraria, Research Funding; AstraZeneca: Honoraria; Daiichi Sankyo: Honoraria, Research Funding; Meiji Seika: Honoraria; Amgen: Honoraria; Sumitomo Dainippon Pharma: Research Funding; Nippon Kayaku: Honoraria; Zenyaku Kogyo: Research Funding; Bristol-Myers Squibb: Honoraria; Novartis: Honoraria; Chugai: Honoraria, Research Funding; Astellas Pharma: Honoraria, Research Funding; CURED: Research Funding; AbbVie: Honoraria, Research Funding; Otsuka Pharmaceutical: Research Funding; Kyowa Hakko Kirin: Research Funding; Pfizer: Honoraria; Towa: Honoraria; MSD: Honoraria; Pharma Essentia Japan: Honoraria.

*signifies non-member of ASH