Triple-negative breast cancer (TNBC) is a breast cancer subtype that lacks targeted treatment options. The activation of the Notch developmental signaling pathway, which is a feature of TNBC, results in the secretion of proinflammatory cytokines and the recruitment of protumoral macrophages to the tumor microenvironment. While the Notch pathway is an obvious therapeutic target, its activity is ubiquitous, and predictably, anti-Notch therapies are burdened with significant on-target side effects. Previously, we discovered that, under conditions of cellular stress commonly found in the tumor microenvironment, the deubiquitinase USP9x forms a multiprotein complex with the pseudokinase tribbles homolog 3 (TRB3) that together activate the Notch pathway. Herein, we provide preclinical studies that support the potential of therapeutic USP9x inhibition to deactivate Notch. Using a murine TNBC model, we show that USP9x knockdown abrogates Notch activation, reducing the production of the proinflammatory cytokines, C-C motif chemokine ligand 2 (CCL2) and interleukin-1 beta (IL-1β). Concomitant with these molecular changes, a reduction in tumor inflammation, the augmentation of antitumor immune response, and the suppression of tumor growth were observed. The pharmacological inhibition of USP9x using G9, a partially selective, small-molecule USP9x inhibitor, reduced Notch activity, remodeled the tumor immune landscape, and reduced tumor growth without associated toxicity. Proving the role of Notch, the ectopic expression of the activated Notch1 intracellular domain rescued G9-induced effects. This work supports the potential of USP9x inhibition to target Notch in metabolically vulnerable tissues like TNBC, while sparing normal Notch-dependent tissues.

三阴性乳腺癌 (TNBC) 是一种缺乏靶向治疗方案的乳腺癌亚型。作为 TNBC 特征的 Notch 发育信号通路的激活导致促炎细胞因子的分泌和原瘤巨噬细胞向肿瘤微环境的募集。虽然 Notch 通路是一个明显的治疗靶点，但它的活性无处不在，而且可以预见的是，抗 Notch 疗法具有显着的靶向副作用。此前，我们发现，在肿瘤微环境中常见的细胞应激条件下，去泛素酶 USP9x 与假激酶 tribbles 同源物 3 (TRB3) 形成多蛋白复合物，共同激活 Notch 通路。在此处，我们提供临床前研究，支持治疗性 USP9x 抑制使 Notch 失活的潜力。使用鼠 TNBC 模型，我们表明 USP9x 敲低消除了 Notch 激活，减少了促炎细胞因子、CC 基序趋化因子配体 2 (CCL2) 和白细胞介素 1 β (IL-1β) 的产生。伴随这些分子变化，观察到肿瘤炎症减少、抗肿瘤免疫反应增强和肿瘤生长抑制。使用 G9（一种部分选择性的小分子 USP9x 抑制剂）对 USP9x 进行药理抑制，降低了 Notch 活性，重塑了肿瘤免疫景观，并减少了肿瘤生长而没有相关毒性。证明 Notch 的作用，激活的 Notch1 细胞内结构域的异位表达挽救了 G9 诱导的作用。