Cancers overcome replicative immortality by activating either telomerase or an alternative lengthening of telomeres (ALT) mechanism. ALT occurs in ~25% of high-risk neuroblastomas, and progression in patients with ALT neuroblastoma during or after front-line therapy is frequent and often fatal. Temozolomide + irinotecan is commonly used as salvage therapy for neuroblastoma. Patient-derived cell lines and xenografts established from patients with relapsed ALT neuroblastoma demonstrated de novo resistance to temozolomide + irinotecan [SN-38 in vitro, P < 0.05; in vivo mouse event-free survival (EFS), P < 0.0001] vs. telomerase-positive neuroblastomas. We observed that ALT neuroblastoma cells manifested constitutive ataxia-telangiectasia mutated (ATM) activation due to spontaneous telomere dysfunction which was not observed in telomerase-positive neuroblastoma cells. We demonstrated that induction of telomere dysfunction resulted in ATM activation that, in turn, conferred resistance to temozolomide + SN-38 (4.2-fold change in IC₅₀, P < 0.001). ATM knockdown (shRNA) or inhibition using a clinical-stage small-molecule inhibitor (AZD0156) reversed resistance to temozolomide + irinotecan in ALT neuroblastoma cell lines in vitro (P < 0.001) and in four ALT xenografts in vivo (EFS, P < 0.0001). AZD0156 showed modest to no enhancement of temozolomide + irinotecan activity in telomerase-positive neuroblastoma cell lines and xenografts. Ataxia telangiectasia and Rad3 related (ATR) inhibition using AZD6738 did not enhance temozolomide + SN-38 activity in ALT neuroblastoma cells. Thus, ALT neuroblastoma chemotherapy resistance occurs via ATM activation and is reversible with ATM inhibitor AZD0156. Combining AZD0156 with temozolomide + irinotecan warrants clinical testing for neuroblastoma.

癌症通过激活端粒酶或替代端粒延长 (ALT) 机制来克服复制性永生。ALT 发生在约 25% 的高危神经母细胞瘤中，而 ALT 神经母细胞瘤患者在一线治疗期间或之后的进展频繁且通常是致命的。替莫唑胺+伊立替康通常用作神经母细胞瘤的抢救疗法。从复发性 ALT 神经母细胞瘤患者中建立的患者来源细胞系和异种移植物显示出对替莫唑胺 + 伊立替康的从头耐药 [SN-38 体外，P < 0.05；体内 小鼠无事件生存 (EFS), P< 0.0001] 与端粒酶阳性神经母细胞瘤相比。我们观察到 ALT 神经母细胞瘤细胞由于自发性端粒功能障碍而表现出组成性共济失调 - 毛细血管扩张症突变 (ATM) 激活，这在端粒酶阳性神经母细胞瘤细胞中未观察到。我们证明了端粒功能障碍的诱导会导致 ATM 激活，进而导致对替莫唑胺 + SN-38 的耐药性（IC ₅₀变化 4.2 倍，P < 0.001）。ATM 敲低 (shRNA) 或使用临床阶段小分子抑制剂 (AZD0156) 的抑制逆转了体外 ALT 神经母细胞瘤细胞系 ( P < 0.001) 和四种 ALT 异种移植物 (EFS, P< 0.0001)。AZD0156 在端粒酶阳性神经母细胞瘤细胞系和异种移植物中显示出适度或无增强的替莫唑胺 + 伊立替康活性。使用 AZD6738 抑制共济失调毛细血管扩张症和 Rad3 相关 (ATR) 不会增强 ALT 神经母细胞瘤细胞中的替莫唑胺 + SN-38 活性。因此，ALT 神经母细胞瘤化疗耐药通过 ATM 激活发生，并且可通过 ATM 抑制剂 AZD0156 逆转。将 AZD0156 与替莫唑胺 + 伊立替康联合使用需要对神经母细胞瘤进行临床试验。