携带酪氨酸激酶受体FLT3（FLT3-ITD）突变的急性髓系白血病（AML）患者预后差，易复发。FLT3-ITD保留在内质网（ER）并产生内在的蛋白质毒性应激。我们设计了一种基于蛋白质毒性应激的策略，通过低剂量的分化剂维甲酸（R）、蛋白酶体抑制剂硼替佐米（B）和氧化应激诱导剂三氧化二砷（A）的联合使用产生。我们将FLT3-ITD阳性的AML细胞用上述药物的低剂量治疗，单独或联合使用，并研究内质网和氧化应激的诱导情况。然后，我们在 vitro 平台上进行了相同实验，使用包括FLT3-ITD阳性AML细胞和骨髓基质细胞（BMSCs）在内的共培养系统，以评估微环境对AML克隆细胞的保护作用。最后，我们在人类FLT3-ITD阳性AML的同位型小鼠模型中测试了药物联合应用。RBA联合应用对FLT3-ITD阳性的AML细胞系和从患者中分离的原发细胞具有强烈的细胞毒活性，由于ER平衡紊乱和氧化应激的生成。当AML细胞与BMSCs共培养时，对RBA联合应用变得完全耐药。然而，我们可以通过使用高剂量的抗坏血酸（维生素C）作为辅助剂克服此类保护效应。重要的是，RBA加抗坏血酸的联合应用显著延长了人类FLT3-ITD阳性AML小鼠模型的寿命，且无毒副作用。此外，我们首次显示了治疗后AML与BMSCs之间的相互作用，涉及动力转导和YAP信号通路，表现为细胞骨架和细胞帽破坏、细胞核厚度增加以及转录共调控因子YAP在BMSCs的细胞质内重定位。我们的发现加强了我们先前的工作，指出诱导蛋白质毒性应激可能是FLT3-ITD阳性AML治疗的一种可能策略，并为在AML与BMSCs之间识别新的治疗靶点，包括机械转导和YAP信号通路，打开了可能性。 © 2023意大利国家癌症研究所“玛丽娜埃琳娜”。

Acute myeloid leukemia (AML) patients bearing the ITD mutation in the tyrosine kinase receptor FLT3 (FLT3-ITD) present a poor prognosis and a high risk of relapse. FLT3-ITD is retained in the endoplasmic reticulum (ER) and generates intrinsic proteotoxic stress. We devised a strategy based on proteotoxic stress, generated by the combination of low doses of the differentiating agent retinoic acid (R), the proteasome inhibitor bortezomib (B), and the oxidative stress inducer arsenic trioxide (A).We treated FLT3-ITD+ AML cells with low doses of the aforementioned drugs, used alone or in combinations and we investigated the induction of ER and oxidative stress. We then performed the same experiments in an in vitro co-culture system of FLT3-ITD+ AML cells and bone marrow stromal cells (BMSCs) to assess the protective role of the niche on AML blasts. Eventually, we tested the combination of drugs in an orthotopic murine model of human AML.The combination RBA exerts strong cytotoxic activity on FLT3-ITD+ AML cell lines and primary blasts isolated from patients, due to ER homeostasis imbalance and generation of oxidative stress. AML cells become completely resistant to the combination RBA when treated in co-culture with BMSCs. Nonetheless, we could overcome such protective effects by using high doses of ascorbic acid (Vitamin C) as an adjuvant. Importantly, the combination RBA plus ascorbic acid significantly prolongs the life span of a murine model of human FLT3-ITD+ AML without toxic effects. Furthermore, we show for the first time that the cross-talk between AML and BMSCs upon treatment involves disruption of the actin cytoskeleton and the actin cap, increased thickness of the nuclei, and relocalization of the transcriptional co-regulator YAP in the cytosol of the BMSCs.Our findings strengthen our previous work indicating induction of proteotoxic stress as a possible strategy in FLT3-ITD+ AML therapy and open to the possibility of identifying new therapeutic targets in the crosstalk between AML and BMSCs, involving mechanotransduction and YAP signaling.© 2023. Italian National Cancer Institute ‘Regina Elena’.