复发性急性淋巴细胞白血病（ALL）患者的预后较差，需要新型治疗方法来改善预后。针对WEE1的小分子抑制剂adavosertib（AZD1775）已成为一种治疗策略，可使癌细胞对损伤DNA的化疗药物产生敏感性，尤其是对于TP53突变肿瘤。然而，WEE1抑制作为一种潜在的治疗策略，尚未明确评估其在高危和复发ALL患者中的疗效，包括那些携带TP53突变的患者。本研究使用复发性TP53等基因突变的细胞模型系统、原发患者样本以及由患者提供的ALL样本（n=27）在骨髓来源间充质干细胞的外体共培养模型系统中，研究了adavosertib的抗白血病作用。通过过量布利斯分析，定量评估了其与当前用于复发性ALL治疗的药物的协同效应。细胞周期和凋亡的变化以及相关蛋白的变化通过流式细胞术和Western blot进行了检测。我们的研究结果表明，临床上先进的WEE1抑制剂adavosertib在大多数高危和复发ALL标本（n=18/27）中都表现出强效的抗白血病活性，而且其浓度低于临床可达到的浓度，与TP53突变状态无关。我们发现，即使在无DNA损伤剂的情况下，adavosertib处理也会导致S期失调，并且过早进入有丝分裂并不是其抗白血病作用的先决条件。进一步研究表明，WEE1抑制剂能够增强多种传统化疗药物在复发ALL治疗中的抗白血病效应，具有协同作用。特别是，我们展示了adavosertib与核苷类类似物cytarabine的高度协同和细胞毒性组合，并深入揭示了这种联合活性的作用机制，显示了细胞凋亡死亡优势于细胞周期停滞。我们的发现强烈支持在复发和高危ALL的异种移植模型中对adavosertib与cytarabine的体内研究。综上，我们的数据强调了WEE1在复发ALL细胞中的功能重要性，并将WEE1视为一个有前途的p53非依赖性治疗靶点，用于改善高危和复发ALL的治疗。© 2023. BioMed Central Ltd., part of Springer Nature.

Outcomes for patients with relapsed acute lymphoblastic leukemia (ALL) are poor and there is a need for novel therapies to improve outcomes. Targeted inhibition of WEE1 with small-molecule inhibitor adavosertib (AZD1775) has emerged as a therapeutic strategy to sensitize cancer cells to DNA-damaging chemotherapeutics, particularly in the context of TP53-mutated tumors. However, WEE1 inhibition as a potential therapeutic strategy for patients with high-risk and relapsed ALL, including those with TP53 mutations, has not been definitively evaluated.Anti-leukemic effects of adavosertib were investigated using a relapsed TP53 isogenic cell model system, primary patient, and patient-derived ALL samples (n = 27) in an ex vivo co-culture model system with bone marrow-derived mesenchymal stem cells. Combination effects with drugs currently used for relapsed ALL were quantified by Excess over Bliss analyses. Investigations for alterations of cell cycle and apoptosis as well as related proteins were examined by flow cytometry and Western blot, respectively.Our study demonstrates the potent anti-leukemic activity of the clinically advanced WEE1 inhibitor adavosertib in a large majority (n = 18/27) of high-risk and relapsed ALL specimens at lower than clinically attainable concentrations, independent of TP53 mutation status. We show that treatment with adavosertib results in S-phase disruption even in the absence of DNA-damaging agents and that premature mitotic entry is not a prerequisite for its anti-leukemic effects. We further demonstrate that WEE1 inhibition additively and synergistically enhances the anti-leukemic effects of multiple conventional chemotherapeutics used in the relapsed ALL treatment setting. Particularly, we demonstrate the highly synergistic and cytotoxic combination of adavosertib with the nucleoside analog cytarabine and provide mechanistic insights into the combinational activity, showing preferential engagement of apoptotic cell death over cell cycle arrest. Our findings strongly support in vivo interrogation of adavosertib with cytarabine in xenograft models of relapsed and high-risk ALL.Together, our data emphasize the functional importance of WEE1 in relapsed ALL cells and show WEE1 as a promising p53-independent therapeutic target for the improved treatment of high-risk and relapsed ALL.© 2023. BioMed Central Ltd., part of Springer Nature.