mTOR作为一种丝氨酸/苏氨酸激酶，是广泛追求的抗癌靶点。当前正在进行多项mTOR激酶抑制剂的临床试验，但它们的特异性和安全性仍然不足。在本研究中，我们采用了一种诱导性激酶不活化的D2338A mTOR基因敲入小鼠模型（mTOR-/KI），结合mTOR条件性敲除模型（mTOR-/-），评估了mTOR在造血过程中的激酶依赖/非依赖功能以及mTOR激酶抑制剂AZD2014的靶内/靶外效应。尽管mTOR-/KI小鼠在造血过程中表现出许多与mTOR-/-小鼠类似的表型，但mTOR-/KI小鼠存活时间更长，并且与mTOR-/-小鼠相比，在造血干细胞中显示出差异，表明mTOR在造血过程中具有激酶非依赖功能。血液干细胞（HSCs）中的基因表达特征进一步揭示了mTOR的激酶依赖和非依赖效应。mTOR激酶抑制剂AZD2014主要通过抑制mTOR激酶活性发挥作用，模拟了mTOR-/KI HSCs的转录组分析结果，但在mTOR-/KI HSCs中也诱导了一小部分靶外反应。在小鼠和人类髓系白血病中，除了具有激酶抑制靶内效应外，AZD2014还显示出类似的靶外抑制和细胞停滞生长效应。这些研究为mTOR在造血过程中的激酶依赖/非依赖效应提供了新的洞察，并提出了一种基因手段来精确评估mTOR激酶抑制剂的特异性。© 2023. 作者，Springer Nature Limited独家许可。

mTOR, as a serine/threonine kinase, is a widely pursued anticancer target. Multiple clinical trials of mTOR kinase inhibitors are ongoing, but their specificity and safety features remain lacking. Here, we have employed an inducible kinase-inactive D2338A mTOR knock-in mouse model (mTOR-/KI) together with a mTOR conditional knockout model (mTOR-/-) to assess the kinase-dependent/-independent function of mTOR in hematopoiesis and the on-/off-target effects of mTOR kinase inhibitor AZD2014. Despite exhibiting many similar phenotypes to mTOR-/- mice in hematopoiesis, the mTOR-/KI mice survived longer and showed differences in hematopoietic progenitor cells compared to mTOR-/- mice, suggesting a kinase-independent function of mTOR in hematopoiesis. Gene expression signatures in hematopoietic stem cells (HSCs) further revealed both kinase-dependent and independent effects of mTOR. AZD2014, a lead mTOR kinase inhibitor, appeared to work mostly on-target in suppressing mTOR kinase activity, mimicking that of mTOR-/KI HSCs in transcriptome analysis, but it also induced a small set of off-target responses in mTOR-/KI HSCs. In murine and human myeloid leukemia, besides kinase-inhibitory on-target effects, AZD2014 displayed similar off-target and growth-inhibitory cytostatic effects. These studies provide new insights into kinase-dependent/-independent effects of mTOR in hematopoiesis and present a genetic means for precisely assessing the specificity of mTOR kinase inhibitors.© 2023. The Author(s), under exclusive licence to Springer Nature Limited.