The extensively activated Notch signaling pathway in pancreatic cancer cells is important in carcinogenesis, chemoresistance, and recurrence. Targeting this pathway is a promising therapeutic strategy for pancreatic cancer; however, few successful approaches have been reported, and currently used molecular inhibitors of this pathway exhibit limited clinical benefits. In this study, we identified a previously uncharacterized microprotein, Notch1 degradation-associated regulatory polypeptide (N1DARP), encoded by LINC00261. N1DARP knockout accelerated tumor progression and enhanced stem cell properties in pancreatic cancer organoids and LSL-Kras, LSL-Trp53, and Pdx1-Cre (KPC) mice. Mechanistically, N1DARP suppressed canonical and non-canonical Notch1 pathways by competitively disrupting the interaction between N1ICD and ubiquitin-specific peptidase 10 (USP10), thereby promoting K11- and K48-linked polyubiquitination of N1ICD. To evaluate the therapeutic potential of N1DARP, we designed a cell-penetrating stapled peptide, SAH-mAH2-5, with a helical structure similar to that of N1DARP that confers remarkable physicochemical stability. SAH-mAH2-5 interacted with and promoted the proteasome-mediated degradation of N1ICD. SAH-mAH2-5 injection provided substantial therapeutic benefits with limited off-target and systemic adverse effects in Notch1-activated pancreatic cancer models. Taken together, these findings confirm that N1DARP acts as a tumor suppressor and chemosensitizer by regulating USP10-Notch1 oncogenic signaling, and suggest a promising therapeutic strategy targeting the N1DARP–N1ICD interaction in Notch1-activated pancreatic cancer.

胰腺癌细胞中广泛激活的 Notch 信号通路在癌发生、化疗耐药和复发中发挥着重要作用。针对这一途径是一种有前景的胰腺癌治疗策略。然而，很少有成功的方法被报道，并且目前使用的该途径的分子抑制剂表现出有限的临床益处。在这项研究中，我们鉴定了一种以前未表征的微生物蛋白，Notch1 降解相关调节多肽 (N1DARP)，由LINC00261编码。N1DARP 敲除加速了胰腺癌类器官和 LSL-Kras、LSL-Trp53 和 Pdx1-Cre (KPC) 小鼠的肿瘤进展并增强了干细胞特性。从机制上讲，N1DARP 通过竞争性破坏 N1ICD 和泛素特异性肽酶 10 (USP10) 之间的相互作用来抑制经典和非经典 Notch1 通路，从而促进 N1ICD 的 K11 和 K48 连接的多泛素化。为了评估 N1DARP 的治疗潜力，我们设计了一种细胞穿透性钉合肽 SAH-mAH2-5，其螺旋结构与 N1DARP 相似，具有显着的理化稳定性。SAH-mAH2-5 与 N1ICD 相互作用并促进蛋白酶体介导的降解。SAH-mAH2-5 注射液在 Notch1 激活的胰腺癌模型中提供了显着的治疗益处，且脱靶和全身性副作用有限。总而言之，这些发现证实 N1DARP 通过调节 USP10-Notch1 致癌信号传导发挥肿瘤抑制因子和化学增敏剂的作用，并提出了一种针对 Notch1 激活的胰腺癌中 N1DARP-N1ICD 相互作用的有前途的治疗策略。