Relapsed and refractory acute myeloid leukemia (AML) carries a dismal prognosis. CAR T cells have shown limited efficacy in AML, partially due to dysfunctional autologous T cells and the extended time for generation of patient specific CAR T cells. Allogeneic NK cell therapy is a promising alternative, but strategies to enhance efficacy and persistence may be necessary. Proteasome inhibitors (PI) induce changes in the surface proteome which may render malignant cells more vulnerable to NK mediated cytotoxicity. Here, we investigated the potential benefit of combining PIs with CAR-expressing allogeneic NK cells against AML. We established the IC50 concentrations for Bortezomib and Carfilzomib against several AML cell lines. Surface expression of class-I HLA molecules and stress-associated proteins upon treatment with proteasome inhibitors was determined by multiparameter flow cytometry. Using functional in vitro assays, we explored the therapeutic synergy between pre-treatment with PIs and the anti-leukemic efficacy of NK cells with or without expression of AML-specific CAR constructs against AML cell lines and primary patient samples. Also, we investigated the tolerability and efficacy of a single PI application strategy followed by (CAR-) NK cell infusion in two different murine xenograft models of AML. AML cell lines and primary AML patient samples were susceptible to Bortezomib and Carfilzomib mediated cytotoxicity. Conditioned resistance to Azacitidine/Venetoclax did not confer primary resistance to PIs. Treating AML cells with PIs reduced the surface expression of class-I HLA molecules on AML cells in a time-and-dose dependent manner. Stress-associated proteins were upregulated on the transcriptional level and on the cell surface. NK cell mediated killing of AML cells was enhanced in a synergistic manner. PI pre-treatment increased effector-target cell conjugate formation and Interferon-γ secretion, resulting in enhanced NK cell activity against AML cell lines and primary samples in vitro. Expression of CD33- and CD70-specific CARs further improved the antileukemic efficacy. In vivo, Bortezomib pre-treatment followed by CAR-NK cell infusion reduced AML growth, leading to prolonged overall survival. PIs enhance the anti-leukemic efficacy of CAR-expressing allogeneic NK cells against AML in vitro and in vivo, warranting further exploration of this combinatorial treatment within early phase clinical trials.

中文翻译：

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蛋白酶体抑制增强表达嵌合抗原受体（CAR）的 NK 细胞对抗急性髓系白血病的抗白血病功效


复发性难治性急性髓系白血病（AML）的预后很差。 CAR T 细胞在 AML 中的疗效有限，部分原因是自体 T 细胞功能失调以及患者特异性 CAR T 细胞生成时间延长。同种异体 NK 细胞疗法是一种有前途的替代方案，但可能需要提高疗效和持久性的策略。蛋白酶体抑制剂 (PI) 会诱导表面蛋白质组发生变化，这可能会使恶性细胞更容易受到 NK 介导的细胞毒性的影响。在这里，我们研究了将 PI 与表达 CAR 的同种异体 NK 细胞结合起来对抗 AML 的潜在益处。我们确定了硼替佐米和卡非佐米针对几种 AML 细胞系的 IC50 浓度。通过多参数流式细胞术测定用蛋白酶体抑制剂处理后 I 类 HLA 分子和应激相关蛋白的表面表达。通过功能性体外测定，我们探索了 PI 预处理与 NK 细胞（无论是否表达 AML 特异性 CAR 构建体）针对 AML 细胞系和原发性患者样本的抗白血病功效之间的治疗协同作用。此外，我们还研究了两种不同的 AML 小鼠异种移植模型中单一 PI 应用策略随后输注 (CAR-) NK 细胞的耐受性和有效性。 AML 细胞系和原发性 AML 患者样本对硼替佐米和卡非佐米介导的细胞毒性敏感。对阿扎胞苷/维奈托克的条件性耐药不会赋予对 PI 的原发耐药性。用 PI 处理 AML 细胞，以时间和剂量依赖性方式减少 AML 细胞上 I 类 HLA 分子的表面表达。应激相关蛋白在转录水平和细胞表面上表达上调。 NK 细胞介导的 AML 细胞杀伤作用以协同方式得到增强。 PI 预处理增加了效应器-靶细胞缀合物的形成和干扰素-γ 的分泌，从而增强了 NK 细胞针对 AML 细胞系和体外初级样品的活性。 CD33和CD70特异性CAR的表达进一步提高了抗白血病功效。在体内，硼替佐米预处理后进行 CAR-NK 细胞输注可减少 AML 的生长，从而延长总体生存期。 PI 增强了表达 CAR 的同种异体 NK 细胞在体外和体内对抗 AML 的抗白血病功效，值得在早期临床试验中进一步探索这种组合治疗。