阻断SHH信号通路药物对髓母细胞瘤及其干细胞的抑制作用研究

Malignant brain tumors are devastating illnesses that even if cured leave patients with long term morbidities as a result of current therapies. Medulloblastoma usually originates in the cerebellum and can metastasize to other CNS sites and bone. Up-regulation of the Hedgehog (Hh) signaling pathway plays a key role in the pathogenesis of medulloblastoma as well as a variety of other devastating malignancies. Upstream members of the Hh pathway include the Hh ligands themselves, Patched, Smoothened, arrestins et al. Dysregulations in the activities of these members of the Hh pathway that contribute to increased downstream Hh transcriptional signaling produce a predisposition to malignant transformation. For example, in mouse models, inactivation of both Ptc alleles results in a dramatic increase of medulloblastoma incidence. Additionally, patients with Gorlin syndrome harbor germline mutations in the Ptc gene and like the mice tend to develop neoplasms, including medulloblastoma due to inappropriate Smo activation. If up regulation of Hh signaling produces susceptibility to cancers, then suppression of Hh activity should have an opposite effect. In the past few years the Smo antagonist cyclopamine was shown to dramatically repress the growth in vivo of murine medulloblastoma allografts. In recognizing the need to develop new therapies for medulloblastom and childhood brain tumors in general, we have taken advantage of our laboratory's discoveries in the Hh field to initiate a program with an aim to identify novel compounds demonstrating better pharmacological properties than cyclopamine that have ability to block Hh signaling. We have created a state-of-the-art drug discovery platform based upon our research to systematically search for compounds with anti-cancer properties. Recently, as part of our ongoing screening project, we have identified by primary and secondary screening 32 potent small molecule inhibitors of Hh signaling that inhibit the Smo receptor and we anticipate more hit compounds will be discovered as we are expanding our screening capacity. We hypothesize based upon what has been discovered about the Hh signaling pathway that pharmacological inhibition of Hh signaling will become an effective therapy for brain tumors including medulloblastoma. We have designed experiments with the following aims: 1. To discover new Hh/Smo inhibitors by screening small molecule libraries at Tiantan Hospital. 2. To determine the tumor inhibitory effect of ZM compound and other Smoothened inhibitor in inhibiting the self-renewal and tumorigenicity of cancer stem cells from meduloblastma patients. 3. Test the effect of combined ZM and Temozolomide treatment on cancer stem cells from meduloblastma patients. These studies will help guide the clinical development of Hh (Smo) inhibitors and enabling novel therapies for the treatment of brain tumors in China.

髓母细胞瘤是最常见的恶性儿童脑肿瘤。Hedgehog信号通路的上调在髓母细胞瘤的发病过程中发挥关键的作用。我们建立了一个高通量的新药筛选平台，并已筛选出了多个具有阻断Hedgehog信号通路的化合物。其中小分子化合物ZM在细胞及动物体内对Hedgehog信号通路都具有明显的抑制作用。我们进一步应用PtcFlox/Flox/GFAP-cre髓母细胞瘤小鼠模型证实，ZM治疗能够明显延长模型动物的存活时间。我们将继续开发Hedgehog信号通路的抑制剂用来在临床上治疗髓母胶质瘤。本申请书的具体目标包括：1,利用高通量筛选方法探寻新的SHH/Smo拮抗剂；2,检测ZM抑制人脑瘤肿瘤干细胞的自我更新能力及抑制肿瘤发生的能力；3,研究ZM和临床常用药替莫唑胺对肿瘤干细胞及肿瘤模型的抑制作用。我们的研究工作将为新的Hedgehog信号通路抑制剂提供有关的临床前数据，并为髓母细胞瘤患者提供新的治疗方法。

髓母细胞瘤是最常见的恶性儿童脑肿瘤。Hedgehog信号通路的上调在髓母细胞瘤的发病过程中发挥关键的作用。 我们建立了一个人髓母细胞瘤干细胞的培养系统，成功的从术中取得的髓母细胞瘤组织进行了体外肿瘤干细胞的培养。另外，我们建立了一个基于SHH 信号通路的高通量新药筛选平台，筛选出了具有阻断Hedgehog信号通路的化合物，ZM336372。 它可以结合到Smothened受体上，抑制Smothened和Gli受体的活性，抑制sonic hedgehog导致的颗粒细胞前体的增值。在U2OS细胞中，它能抑制βarrestin2-GFP的聚集。同时，我们发现ZM336372可以抑制由PDGF导致的髓母细胞瘤的浸润和迁移，降低髓母细胞瘤细胞中PDGF、AKT、ERK的磷酸化水平。在体内Ptchflox/floxed /GFAP-cre髓母细胞瘤小鼠实验中，ZM336372有利于小鼠的长期生存。为髓母细胞瘤患者提供新的治疗方法。