A Tumor Suppressor Enhancer Of Pten In T-Cell Development And Transformation

Recent studies have dissected the mutational landscape of T-cell acute lymphoblastic leukemia (T-ALL), identifying several oncogenes and tumor suppressors implicated in T-cell transformation. However, most genetic lesions in cancer are located in intergenic regions, whose role remains poorly understood. In this context, 20% of T-ALL patients show loss of expression of the tumor suppressor PTEN, but not all can be explained by mutations/deletions of its coding region, defective splicing or promoter hypermethylation. Thus, we hypothesized that alterations of yet unidentified enhancers might play a critical role in the regulation of PTEN expression in T-ALL. To test this, we first performed 4C-seq of the PTEN promoter in human T-ALL cells. Integration of our results with epigenetic profiling revealed a distal non-coding region (named PE for PTEN enhancer) that shows concomitant interaction with the PTEN promoter and bona fide enhancer marks. Moreover, reciprocal 4C-seq assays using PE as viewpoint confirmed its interaction with the PTEN promoter. Next, multispecies DNA sequence alignment revealed notable conservation of PE in mammals and detailed analysis of H3K27ac across 64 cell lines and tissues revealed that PE is specifically active in T-cells and other hematopoietic cells, but is largely repressed in non-hematopoietic cells. Moreover, luciferase assays showed strong, orientation-independent activation of reporter constructs containing either the mouse or human PE sequence in Jurkat cells, and CRISPR/Cas9-induced deletion of PE in human T-ALL cells led to reduced PTEN levels, increased phospho-AKT and enhanced cellular proliferation. To formally test the functional relevance of PE in T-cell development and transformation, we generated PE knockout and conditional knockout mice. PE-null animals are viable and show a marked increase in thymus size and cellularity as their only developmental alteration. Mechanistically, this phenotype is associated with a marked reduction in Pten expression. Next, we analyzed the role of PE in the induction of NOTCH1-driven T-ALLs by transplanting mice with PE wild-type or knockout hematopoietic progenitors infected with retroviruses expressing a mutant constitutively active form of NOTCH1. In this context, mice transplanted with NOTCH-infected PE-null cells showed 100% penetrance and developed T-ALL ~50 days after transplant. However, mice transplanted with NOTCH-infected PE wild-type cells showed delayed T-ALL kinetics and reduced penetrance. In addition, secondary loss of PE in already established NOTCH1-induced T-ALLs from PE conditional knockout mice led to accelerated T-ALL progression and a gene expression signature driven by Pten loss. Finally, analyses of human T-ALL patient samples uncovered recurrent deletions encompassing PE without concomitant deletion of the PTEN coding sequence. Altogether, our results identify PE as the first long-range tumor suppressor enhancer directly implicated in the pathogenesis of human leukemia. Citation Format: Luca Tottone, Olga Lancho, Maria Victoria da Silva-Diz, Shirley Luo, Shreeta Chakraborty, Pedro P. Rocha, Daniel Herranz. A tumor suppressor enhancer of PTEN in T-cell development and transformation [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 3434.