Department of Obstetrics and Gynecology, University of Texas Health San Antonio, San Antonio, Texas, 78229, USA.
Department of Biochemistry and Structural Biology, University of Texas Health San Antonio, San Antonio, Texas, 78229, USA.
Neuro Oncol. 2023 Jul 6;25(7):1249-1261. doi: 10.1093/neuonc/noad018.
Efficient DNA repair in response to standard chemo and radiation therapies often contributes to glioblastoma (GBM) therapy resistance. Understanding the mechanisms of therapy resistance and identifying the drugs that enhance the therapeutic efficacy of standard therapies may extend the survival of GBM patients. In this study, we investigated the role of KDM1A/LSD1 in DNA double-strand break (DSB) repair and a combination of KDM1A inhibitor and temozolomide (TMZ) in vitro and in vivo using patient-derived glioma stem cells (GSCs).
Brain bioavailability of the KDM1A inhibitor (NCD38) was established using LS-MS/MS. The effect of a combination of KDM1A knockdown or inhibition with TMZ was studied using cell viability and self-renewal assays. Mechanistic studies were conducted using CUT&Tag-seq, RNA-seq, RT-qPCR, western blot, homologous recombination (HR) and non-homologous end joining (NHEJ) reporter, immunofluorescence, and comet assays. Orthotopic murine models were used to study efficacy in vivo.
TCGA analysis showed KDM1A is highly expressed in TMZ-treated GBM patients. Knockdown or knockout or inhibition of KDM1A enhanced TMZ efficacy in reducing the viability and self-renewal of GSCs. Pharmacokinetic studies established that NCD38 readily crosses the blood-brain barrier. CUT&Tag-seq studies showed that KDM1A is enriched at the promoters of DNA repair genes and RNA-seq studies confirmed that KDM1A inhibition reduced their expression. Knockdown or inhibition of KDM1A attenuated HR and NHEJ-mediated DNA repair capacity and enhanced TMZ-mediated DNA damage. A combination of KDM1A knockdown or inhibition and TMZ treatment significantly enhanced the survival of tumor-bearing mice.
Our results provide evidence that KDM1A inhibition sensitizes GBM to TMZ via attenuation of DNA DSB repair pathways.
标准化疗和放疗的有效 DNA 修复通常导致胶质母细胞瘤（GBM）治疗耐药。了解耐药机制并确定增强标准治疗疗效的药物可能会延长 GBM 患者的生存时间。在这项研究中，我们使用患者来源的神经胶质瘤干细胞（GSCs）研究了 KDM1A/LSD1 在 DNA 双链断裂（DSB）修复中的作用以及 KDM1A 抑制剂与替莫唑胺（TMZ）联合使用的作用。
使用 LS-MS/MS 确定 KDM1A 抑制剂（NCD38）的脑生物利用度。使用细胞活力和自我更新测定研究 KDM1A 敲低或抑制与 TMZ 联合使用的效果。使用 CUT&Tag-seq、RNA-seq、RT-qPCR、western blot、同源重组（HR）和非同源末端连接（NHEJ）报告、免疫荧光和彗星分析进行机制研究。使用原位小鼠模型研究体内疗效。
TCGA 分析表明，KDM1A 在接受 TMZ 治疗的 GBM 患者中高表达。敲低或敲除或抑制 KDM1A 增强了 TMZ 降低 GSCs 活力和自我更新的功效。药代动力学研究表明，NCD38 容易穿过血脑屏障。CUT&Tag-seq 研究表明，KDM1A 富集在 DNA 修复基因的启动子上，RNA-seq 研究证实 KDM1A 抑制降低了它们的表达。敲低或抑制 KDM1A 减弱了 HR 和 NHEJ 介导的 DNA 修复能力，并增强了 TMZ 介导的 DNA 损伤。KDM1A 敲低或抑制与 TMZ 治疗的联合使用显著提高了荷瘤小鼠的存活率。
我们的研究结果提供了证据，表明 KDM1A 抑制通过减弱 DNA DSB 修复途径使 GBM 对 TMZ 敏感。
