Mechanism of TAL1 Activation by Alterations in the Downstream Enhancer in Childhood T-lineage Acute Lymphoblastic Leukemia

The transcription factor (TF) TAL1 is a master regulator expressed at the early stages of normal thymocyte development. It is one of the major drivers in childhood T-lineage acute lymphoblastic leukemia (T-ALL) and is aberrantly activated in 30-40% of T-ALL. While somatic alterations in upstream and intronic cis-regulatory regions of TAL1 are known mechanisms of activation, the role of downstream enhancer alterations (DEAs) remains less understood. We investigated somatic alterations in an intergenic region 11Kb downstream of TAL1, identifying a complex indel that created a de novo MYB transcription factor binding site (TFBS), and a 2.4Kb internal tandem duplication (ITD) encompassing a MYB TFBS. Alterations in this region were found to be present in 2% of the samples in a recent T-ALL landscape study involving 1,335 cases. Using the patient-derived xenograft (PDX) mouse models of the two T-ALLs harboring the complex indel and ITD DEA, we performed multi-omic profiling, including H3K27ac, H3K4me1, H3K27me3 and MYB chromatin immunoprecipitation sequencing (ChIP-seq); assay for transposase-accessible chromatin using sequencing (ATAC-seq), Hi-C, RNA-seq and full-length transcriptome sequencing (Iso-seq). Enhancer activity by DEA was validated by presence of H3K27ac peaks and cis-activation of TAL1 which exhibits mono-allelic expression in both the primary tumors and PDX models. Involvement of MYB in enhancer activity is validated by MYB ChIP-seq peaks centered around the de novo and existing MYB TFBSs. Interestingly, we found that TAL1 activation via DEA led to preferential initiation of transcription from the promoter located at the exon 4, resulting in predominant expression of the TAL1-short isoform in cases with DEA. Indeed, TAL1-short expression accounted for 78% of the TAL1 expression in T-ALLs harboring DEA in contrast to the 37% frequency in other TAL1-activated T-ALLs (Wilcoxon two-sided test P=0.001). Furthermore, using the full-length transcripts generated by PacBio Iso-seq platform on the PDX samples, only TAL1-short transcripts were detected. By comparing Hi-C data generated from our PDX samples with those from Jurkat (TAL1+, with upstream enhancer region) and DND41 (TAL1-) cell lines, we identified a re-organization of chromatin looping structure showing distinct interaction between DEA region and TAL1 promoter at exon 4. This study provides a detailed view of the regulatory mechanism of TAL1 activation by DEA, indicating that preferential expression of TAL1-short isoform, protein product of which is known to bind more strongly to TAL1 E-protein partners than that of TAL1-long isoform, is active in initiating and maintaining the TAL1 core regulatory circuit in the transformation of pre-T cell progenitors in T-ALL. Nadezhda V. Terekhanova, Xiaolong Chen, Wentao Yang, Ying Shao, Li Dong, Bensheng Ju, Yu Liu, John Easton, A. Thomas Look, Jinghui Zhang. Mechanism of TAL1 activation by alterations in the downstream enhancer in childhood T-lineage acute lymphoblastic leukemia [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 4057.