Thakur Varsha, Thakur Vijay S, Wang Dazhi, de Freitas Juliano Tiburcio, Bianchi Anna, Nivelo Luis Alberto, Umland Oliver, Welford Scott M, Bedogni Barbara
Dr. Phillip Frost Department of Dermatology and Cutaneous Surgery, University of Miami Miller School of Medicine, Sylvester Comprehensive Cancer Center, Miami, FL, 33136, USA.
Department of Radiation Oncology, University of Miami Miller School of Medicine, Sylvester Comprehensive Cancer Center, Miami, FL, 33136, USA.
J Exp Clin Cancer Res. 2025 May 28;44(1):163. doi: 10.1186/s13046-025-03411-w.
Melanoma brain metastases (MBM) are a leading cause of death in patients with advanced disease. MBM treatment relay on targeted and immunotherapy and on stereotactic radiosurgery as gold standard. Life expectancy has improved significantly with these therapies however, targeted therapy is short lived and only about half of the patients respond to immunotherapy, while radiation is limited by melanoma cells intrinsic resistance to DNA damage. New therapeutic approaches are therefore needed to treat MBM. Here we investigate a new role of Notch1 in genomic instability and demonstrate that blockade of both Notch1 and the DNA repair factor ChK1 causes extensive DNA damage and tumor cell death increasing survival in MBM bearing mice.
Anti-Notch1 (anti-N1) was previously described. Prexaserib, a ChK1 inhibitor, is currently in clinical trials. K457 and A375 melanoma cells were used. RNA sequencing was performed in K457 cells treated with anti-N1 and Gene Set Enrichment Analysis performed. DNA damage was evaluated by a DNA fiber assay to assess replication fork speed; and γH2AX foci count and neutral comet assay to quantify double strand breaks. Cell survival was evaluated by trypan blue and a colony formation assay. Luciferase expressing A375 cells were orthotopically inoculated in the right cerebral cortex of athymic nude mice, for in vivo evaluation of a therapy with anti-N1 and prexasertib. Survival was assessed by Kaplan-Meyer survival curves and significance assessed by a Log-rank test.
Notch1 blockade caused genomic instability by reducing histone availability, leading to DNA replication stress and DNA damage. This in turn, resulted in the activation of the DNA Damage Response pathway ATR/ChK1 to counter the damage. Co-inhibition of Notch1, via anti-N1, and ChK1, via prexasertib (prex), exacerbated DNA damage increasing melanoma cell death. Importantly, combination anti-N1/prex significantly improved survival of mice bearing MBMs.
A therapy with anti-N1/prexasertib could represent a novel treatment strategy, alone or in combination with current treatment regimens, for melanoma brain metastases.
黑色素瘤脑转移（MBM）是晚期疾病患者死亡的主要原因。MBM治疗依赖于靶向治疗、免疫治疗以及作为金标准的立体定向放射外科手术。这些疗法显著提高了患者的预期寿命，然而，靶向治疗持续时间短，只有约一半的患者对免疫治疗有反应，而放疗则受到黑色素瘤细胞对DNA损伤的固有抗性的限制。因此，需要新的治疗方法来治疗MBM。在此，我们研究了Notch1在基因组不稳定性中的新作用，并证明Notch1和DNA修复因子ChK1的双重阻断会导致广泛的DNA损伤和肿瘤细胞死亡，从而提高荷MBM小鼠的存活率。
抗Notch1（抗N1）已在之前有所描述。Prexaserib是一种ChK1抑制剂，目前正在进行临床试验。使用了K457和A375黑色素瘤细胞。对用抗N1处理的K457细胞进行RNA测序，并进行基因集富集分析。通过DNA纤维测定评估DNA损伤以评估复制叉速度；通过γH2AX焦点计数和中性彗星试验量化双链断裂。通过台盼蓝和集落形成试验评估细胞存活率。将表达荧光素酶的A375细胞原位接种到无胸腺裸鼠的右大脑皮层，用于体内评估抗N1和prexasertib的治疗效果。通过Kaplan - Meyer生存曲线评估存活率，并通过对数秩检验评估显著性。
Notch1阻断通过减少组蛋白可用性导致基因组不稳定，从而导致DNA复制应激和DNA损伤。这反过来又导致DNA损伤反应途径ATR/ChK1的激活以对抗损伤。通过抗N1对Notch1和通过prexasertib（prex）对ChK1的联合抑制加剧了DNA损伤，增加了黑色素瘤细胞死亡。重要的是，抗N1/prex联合治疗显著提高了荷MBM小鼠的存活率。
抗N1/prexasertib治疗可能代表一种新的治疗策略，单独或与当前治疗方案联合用于黑色素瘤脑转移。
