BACKGROUND Activation of cyclin-dependent kinases (CDKs) contributes to the uncontrolled proliferation of tumour cells. Genomic alterations that lead to the constitutive activation of CDKs are a feature of many tumours including glioblastoma (GBM), the most common and aggressive primary brain tumour. Patient resistance to the current standard of care, temozolomide and radiotherapy, is common and highlights the need to discover more effective treatment strategies. Additionally, glioma stem cells (GSCs), tumour initiating cells contribute to treatment resistance in GBM. Targeting GBM tumour cells and GSCs using transcriptional CDK inhibitors, CYC065 and THZ1 is a potential novel treatment to prevent relapse of the tumour. MATERIAL AND METHODS The therapeutic efficacy of two CDK inhibitors (CKIs) was tested in a panel of ten low-passage GBM patient-derived gliomasphere cultures and semi-in vivo chick embryo xenograft models. Specifically, transcriptional inhibitors targeting CDK9/2 (CYC065) and CDK7 (THZ1) were used. Mechanism of cell death was examined following CKI treatment. Additionally, dependence on anti-apoptotic proteins was studied using genetic depletion and BH3 profiling. Fluorescence activated cell sorting (FACS) and light-sheet fluorescence microscopy (LSFM) were employed to study stem cell populations in recurrent GBM and evaluation of CKI efficacy against GSCs. RESULTS We here demonstrate that CYC065 and THZ1 treatments cause loss of cell viability and induce caspase-dependent apoptosis in primary and recurrent patient-derived gliomaspheres while sparing primary cortical neurons. Importantly, apoptosis responses manifested across a range of time points that significantly correlated with the cell doubling time. Mechanistically, CYC065 and THZ1 downregulate the anti-apoptotic protein Mcl-1, which suffices to sensitise gliomasphere cultures to treatment-induced apoptosis in a Bim-dependent manner. Additionally, high expression levels of chemo- and radio-resistant GSCs were found in recurrent gliomaspheres. CKIs induced apoptosis in isolated CD133 and CD44 biomarker-positive cells while TMZ was ineffective, highlighting the potential of these drugs to overcome resistance to conventional chemotherapy. Additionally, using LSFM we shown that CD133, CD44 GSC biomarker-negative cells convert into GSC biomarker-positive cells and contribute to the enrichment in GSCs in recurrent GBM which could potentially explain the TMZ ineffectiveness. Finally, CKIs reduced proliferation and promoted apoptosis in chick embryo xenograft models of primary and recurrent GBM. CONCLUSION Collectively, these data demonstrate that CYC065 and THZ1 display high anti-cancer activity in primary and recurrent GBM and provide scientific rationale for the further development of CDK inhibitors to potentiate their clinical utilization in the future.

中文翻译：

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P13.11 转录 CDK 抑制剂、CYC065 和 THZ1 在原发性和复发性 GBM 肿瘤细胞和胶质瘤干细胞的临床前模型中促进细胞凋亡

背景技术细胞周期蛋白依赖性激酶(CDK)的激活导致肿瘤细胞不受控制的增殖。导致 CDK 组成型激活的基因组改变是许多肿瘤的特征，包括胶质母细胞瘤 (GBM)，这是最常见和侵袭性的原发性脑肿瘤。患者对目前的护理标准、替莫唑胺和放射治疗的抗药性很常见，这突出了发现更有效治疗策略的必要性。此外，胶质瘤干细胞 (GSC)、肿瘤起始细胞有助于 GBM 的治疗耐药性。使用转录 CDK 抑制剂、CYC065 和 THZ1 靶向 GBM 肿瘤细胞和 GSCs 是预防肿瘤复发的潜在新疗法。材料和方法 两种 CDK 抑制剂 (CKI) 的治疗效果在一组 10 个低通道 GBM 患者来源的胶质瘤培养物和半体内鸡胚胎异种移植模型中进行了测试。具体而言，使用了靶向 CDK9/2 (CYC065) 和 CDK7 (THZ1) 的转录抑制剂。在 CKI 治疗后检查细胞死亡的机制。此外，使用遗传耗竭和 BH3 分析研究了对抗凋亡蛋白的依赖性。荧光激活细胞分选 (FACS) 和光片荧光显微镜 (LSFM) 用于研究复发性 GBM 中的干细胞群并评估 CKI 对 GSCs 的功效。结果 我们在此证明，CYC065 和 THZ1 治疗会导致原发性和复发性患者来源的胶质瘤球细胞活力丧失并诱导半胱天冬酶依赖性细胞凋亡，同时保留原发性皮层神经元。重要的是，细胞凋亡反应表现在与细胞倍增时间显着相关的一系列时间点。从机制上讲，CYC065 和 THZ1 下调抗凋亡蛋白 Mcl-1，这足以使胶质瘤培养物以 Bim 依赖性方式对治疗诱导的细胞凋亡敏感。此外，在复发性胶质瘤球中发现了高表达水平的化学和放射抗性 GSC。CKI 在孤立的 CD133 和 CD44 生物标志物阳性细胞中诱导细胞凋亡，而 TMZ 无效，突出了这些药物克服常规化疗耐药性的潜力。此外，使用 LSFM，我们发现 CD133、CD44 GSC 生物标志物阴性细胞转化为 GSC 生物标志物阳性细胞，并有助于 GSCs 在复发性 GBM 中的富集，这可能解释 TMZ 无效。最后，在原发性和复发性 GBM 的鸡胚异种移植模型中，CKIs 减少了增殖并促进了细胞凋亡。结论 总的来说，这些数据表明 CYC065 和 THZ1 在原发性和复发性 GBM 中显示出高抗癌活性，并为进一步开发 CDK 抑制剂以增强其在未来的临床应用提供科学依据。最后，在原发性和复发性 GBM 的鸡胚异种移植模型中，CKIs 减少了增殖并促进了细胞凋亡。结论 总的来说，这些数据表明 CYC065 和 THZ1 在原发性和复发性 GBM 中显示出高抗癌活性，并为进一步开发 CDK 抑制剂以增强其在未来的临床应用提供科学依据。最后，在原发性和复发性 GBM 的鸡胚异种移植模型中，CKIs 减少了增殖并促进了细胞凋亡。结论 总的来说，这些数据表明 CYC065 和 THZ1 在原发性和复发性 GBM 中显示出高抗癌活性，并为进一步开发 CDK 抑制剂以增强其在未来的临床应用提供科学依据。