Abstract 3652: USP7 heterozygous loss-of-function affects T-cell differentiation in pediatric T-ALL

Abstract

Abstract Ubiquitin-specific-processing protease 7 (USP7), a protein deubiquitinase, is one of the most frequently mutated genes (33%) in the TAL1 subtype of pediatric T-lineage acute lymphoblastic leukemia (T-ALL). However, the functional effect of USP7 haploinsufficiency on T-ALL pathogenesis remains elusive. To understand USP7 haploinsufficiency’s impact on T-ALL, we performed gene expression analysis on 42 non-early T-cell precursor T-ALL RNAseq samples downloaded from phs000218. The gene expression analysis suggested that USP7 haploinsufficiency (USP7-mut N = 12; USP7-wt N = 30) down-regulated the expression of T-cell maturation markers (e.g. RAG1, RAG2, and CD1B), which were negatively regulated by TAL1 [1]. RNAseq on a T-ALL cell line with USP7 knocked down by shRNA also found the same TAL1 negatively regulated gene set, further supporting an increase of TAL1 activity in the USP7 mutated T-ALLs. To examine whether the T-cell maturation was affected by USP7 heterozygous knockout, we generated a conditional knockout (cKO) mouse model by cross-breeding the transgenic vav1-cre mice with the USP7fl/fl mice to obtain heterozygous USP7fl/wt-vav1-cre. Thymocytes isolated from cKO mice were co-cultured with the OP9-Δ1 cells in the medium supplied with cytokines. The cell surface differentiation markers, CD4 and CD8 were stained for flow cytometry detection, and we observed an increase of double-negative cells and a decrease of double-positive cells in USP7-het-KO mice versus control (N = 4 in each group; p-value < 0.05), consolidating the disrupted T-cell development ex vivo. To further understand USP7’s mechanism to regulate TAL1, we analyzed proteins that interacted with USP7 by affinity purification-mass spectrometry (AP-MS) and immune-precipitated followed by western-blotting (IP/WB). AP-MS with the anti-USP7 antibody revealed that USP7 directly interacts with TAL1 in Jurkat cells; AP-MS with the anti-TAL1 antibody also reciprocated the USP7-TAL1 interactions. Whole proteome analysis, through tandem-mass-tag and two-dimensional liquid chromatography-tandem mass spectrometry, revealed that USP7 knockdown down-regulated TRIM27, a deubiquitin target of USP7. IP/WB further confirmed the interaction between USP7, TRIM27, and TAL1, suggesting a possible synergistic relationship between USP7 and TRIM27 to regulate TAL1. In conclusion, our finding demonstrates heterozygous loss of function of USP7 dysregulates T-cell maturation by enhancing TAL1 activity. Future work on TRIM27 could further shed light on the mechanism underlying USP7’s ability to regulate TAL1. Reference: [1] Sanda et al. Cancer Cell 22, 209 (2012). Citation Format: Timothy I. Shaw, Li Dong, Anthony High, Yu Liu, Bensheng Ju, Kanisha Kavdia, Vishwajeeth Pagala, Bridget Shaner, John Easton, Chenxi Qian, Jiyang Yu, Janke Janke, John Kim Choi, Junmin Peng, Wei Gu, James R. Downing, Jinghui Zhang. USP7 heterozygous loss-of-function affects T-cell differentiation in pediatric T-ALL [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 3652.