Genome-wide expression profiling of CUTTL1 cells upon miR-17-92 cluster overexpression with or without gamma-secretase inhibitor treatment. Genome-wide expression profiling of CUTTL1 cells upon miR-17-92 cluster overexpression with or without gamma-secretase inhibitor treatment

T-cell acute lymphoblastic leukemia (T-ALL) is an aggressive T-cell malignancy characterized by genotypically-defined and phenotypically divergent cell populations, governed by adaptive landscapes. Clonal expansions are associated to genetic and epigenetic events, and modulation of external stimuli that affect the hierarchical structure of subclones and support the dynamics of leukemic subsets. Recently, extracellular vesicles (EVs) such exosomes were also shown to play a role in leukemia. Here, we explored the role of EVs in the realm of Notch-driven T-ALLs and found that T-ALL cells secrete Notch-dependent EVs containing microRNAs (EV-miRs), which control oncogenic pathways acting as autocrine stimuli and promote in vitro the expansion/survival of cell subsets of human T-cell leukemias. Of interest, the highlighted Notch-dependent EV-miRs mostly comprised members of miR-17-92a cluster and paralogues, which rescued in vitro the proliferation of T-ALL cells induced by γ-secretase inhibitors (GSI). Additionally, single-cell RNA sequencing analysis of leukemia cells from primary T-ALL patients revealed cell subsets differently responsive to Notch-dependent EV-miRs. All these findings suggest that restricted EV-miRs may sustain the growth/survival of immunophenotypically defined cell populations, altering the cell heterogeneity and the dynamics of T-cell leukemias in response to conventional therapies. Overall design: A total of 4 different experimental conditions were analyzed in this study. We performed biological replicates for a total of 8 samples profiled.

T细胞急性淋巴细胞白血病（T-cell acute lymphoblastic leukemia, T-ALL）是一类侵袭性T细胞恶性肿瘤，以基因分型明确且表型异质性的细胞群体为特征，其发生发展受适应性景观调控。克隆扩增与遗传及表观遗传事件、以及可影响亚克隆层级结构并维持白血病细胞亚群动态平衡的外部刺激调控密切相关。近年来，细胞外囊泡（extracellular vesicles, EVs）如外泌体在白血病中的作用已得到证实。本研究探究了细胞外囊泡在Notch信号驱动型T-ALL中的作用，发现T-ALL细胞可分泌依赖Notch信号的携带微小RNA的细胞外囊泡（EV-miRs），这类囊泡可作为自分泌刺激物调控致癌通路，并在体外促进人T细胞白血病细胞亚群的扩增与存活。值得关注的是，本次研究重点筛选出的Notch依赖型EV-miRs大多包含miR-17-92a基因簇及其旁系同源基因簇成员，它们可恢复γ分泌酶抑制剂（γ-secretase inhibitors, GSI）诱导的T-ALL细胞体外增殖能力。此外，对原发性T-ALL患者白血病细胞的单细胞RNA测序分析显示，不同细胞亚群对Notch依赖型EV-miRs的响应存在显著差异。上述研究结果表明，特定的EV-miRs可维持免疫表型明确的细胞群体的生长与存活，进而在常规治疗响应过程中改变细胞异质性与T细胞白血病的动态平衡。整体实验设计：本研究共设置4种不同实验条件，对共计8份样本开展了生物学重复实验。