Pre-Clinical Study of Panobinostat in Xenograft and Genetically Engineered Murine Diffuse Intrinsic Pontine Glioma Models

Background
Diffuse intrinsic pontine glioma (DIPG), or high-grade brainstem glioma (BSG), is one of the major causes of brain tumor-related deaths in children. Its prognosis has remained poor despite numerous efforts to improve survival. Panobinostat, a histone deacetylase inhibitor, is a targeted agent that has recently shown pre-clinical efficacy and entered a phase I clinical trial for the treatment of children with recurrent or progressive DIPG.
Methods
A collaborative pre-clinical study was conducted using both a genetic BSG mouse model driven by PDGF-B signaling, p53 loss, and ectopic H3.3-K27M or H3.3-WT expression and an H3.3-K27M orthotopic DIPG xenograft model to confirm and extend previously published findings regarding the efficacy of panobinostat in vitro and in vivo.
Results
In vitro, panobinostat potently inhibited cell proliferation, viability, and clonogenicity and induced apoptosis of human and murine DIPG cells. In vivo analyses of tissue after short-term systemic administration of panobinostat to genetically engineered tumor-bearing mice indicated that the drug reached brainstem tumor tissue to a greater extent than normal brain tissue, reduced proliferation of tumor cells and increased levels of H3 acetylation, demonstrating target inhibition. Extended consecutive daily treatment of both genetic and orthotopic xenograft models with 10 or 20 mg/kg panobinostat consistently led to significant toxicity. Reduced, well-tolerated doses of panobinostat, however, did not prolong overall survival compared to vehicle-treated mice.
Conclusion
Our collaborative pre-clinical study confirms that panobinostat is an effective targeted agent against DIPG human and murine tumor cells in vitro and in short-term in vivo efficacy studies in mice but does not significantly impact survival of mice bearing H3.3-K27M-mutant tumors. We suggest this may be due to toxicity associated with systemic administration of panobinostat that necessitated dose de-escalation.

背景
弥漫内生型桥脑胶质瘤（Diffuse intrinsic pontine glioma, DIPG），又称高级别脑干胶质瘤（high-grade brainstem glioma, BSG），是儿童脑肿瘤相关死亡的主要病因之一。尽管学界已投入大量研究以改善患者生存预后，但该疾病的预后仍极差。帕比司他（Panobinostat）是一种组蛋白去乙酰化酶抑制剂（histone deacetylase inhibitor），作为靶向治疗药物，近期已展现出临床前疗效，并进入针对复发或进展性DIPG儿童患者的I期临床试验（phase I clinical trial）。

方法
本研究开展了一项合作性临床前研究，采用两类模型体系：一是由PDGF-B信号通路驱动、p53缺失并异位表达H3.3-K27M或H3.3-WT的基因工程BSG小鼠模型，二是H3.3-K27M原位DIPG异种移植模型（orthotopic DIPG xenograft model），旨在验证并拓展此前已发表的关于帕比司他体内外疗效的研究结果。

结果
体外实验中，帕比司他可强效抑制人源及鼠源DIPG细胞的增殖、存活能力与克隆形成能力，并诱导细胞凋亡（apoptosis）。对基因工程荷瘤小鼠进行短期全身给药（systemic administration）后，体内组织分析结果显示：该药物相较于正常脑组织，更易富集于脑干肿瘤组织；可降低肿瘤细胞的增殖水平，并提升H3乙酰化（H3 acetylation）水平，证实了其靶点抑制效应。采用10或20 mg/kg的帕比司他对基因工程模型及原位异种移植模型进行连续每日给药时，持续引发显著的毒性反应。然而，使用剂量下调且耐受良好的帕比司他给药方案，相较于赋形剂处理组小鼠，并未显著延长受试小鼠的总生存期（overall survival）。

结论
本项合作临床前研究证实：帕比司他在体外实验及小鼠短期体内疗效研究中，对人源及鼠源DIPG肿瘤细胞均展现出有效的靶向抑制作用，但无法显著延长携带H3.3-K27M突变肿瘤的小鼠的生存期。我们推测，这一现象可能与帕比司他全身给药引发的毒性反应相关，该毒性反应迫使研究人员采用剂量递减（dose de-escalation）的给药策略。