Therapeutic Exploitation of Mutant BRAF for Astrocytoma

描述(由申请人提供)：儿童低级别星形细胞瘤(LGA)是一个重大的临床挑战。虽然化疗加放疗的5年无进展生存率约为68%，但肿瘤残留和当前治疗方法(包括神经内分泌认知障碍、视力障碍、血管病变和继发性肿瘤)的存在与显著的发病率有关。此外，转移潜能和向高级别星形细胞瘤的恶变进一步导致预后不良。最近的数据显示，BRAF在~23%的LGA和60%的黄色星形细胞瘤中发生突变。我们已经建立了两个仅有的儿童LGA模型作为小鼠的直接患者-异种移植。这些肿瘤保留了原始患者肿瘤特有的表达特征和基因组变化，因此提供了独特的模式来开发替代的、较少虚弱的治疗方法。BT-40异种移植瘤为突变BRAF(V600E)杂合子，而BT-35异种移植瘤为野生型BRAF。BT-40肿瘤对MEK抑制剂AZD6244非常敏感，而BT-35肿瘤没有反应，这是其他带有野生型BRAF的儿童肿瘤移植的典型。AZD6244耐药的特征是MEK依赖的基因信号增加，IL-6转录和分泌增强，STAT3信号激活。对AZD6244的耐药性是不稳定的，因为在未经治疗的小鼠中传代的肿瘤恢复到药物敏感性，并与IL-6和STAT3活性降低相关。此外，两个对AZD6244具有内在抗性的BRAF(V600E)突变星形细胞系在MEK被抑制时诱导STAT3激活。目标1将研究STAT3激活在耐药中的作用。对于BT-40肿瘤，对AZD6244的敏感性以完全抑制TORC1信号为特征，提示突变的BRAF控制PI3K/TORC1信号轴。在目标2中，将研究Akt和STAT3信号在MEK调节TORC1中的作用，以及同时抑制MEK和STAT3以防止对AZD6244的耐药性的形成或逆转。我们计划利用突变的BRAF对TORC1信号的控制进行治疗。在BT-40移植瘤中，抑制MEK导致TORC1下游的DNA修复蛋白FANCD2迅速和完全丧失，从而潜在地选择性地使肿瘤细胞对顺铂、依托泊苷和电离辐射敏感。此外，我们将测试顺铂o依托泊苷与AZD6244联合使用将防止AZD6244耐药细胞出现的概念，因为突变的BRAF需要维持与顺铂或依托泊苷暴露的细胞存活相关的Akt信号。我们将建立更多的突变BRAF LGA模型，并确定BT-40异种移植的结果是否普遍适用。这些数据将形成快速转化为新的非遗传毒性和更传统的治疗方法的基础，与目前的治疗方法相比，这些方法既有效又不那么虚弱，适用于复发或无法手术切除的LGA儿童。

DESCRIPTION (provided by applicant): Low-grade astrocytomas (LGA) of childhood present a significant clinical challenge. While 5-year progression-free survival rate for chemotherapy plus radiotherapy is ~ 68%, significant morbidity is associated with the presence of residual tumor and the current therapy that includes neuroendocrine- cognitive deficits, visual deficits, vasculopathy and secondary tumors. Moreover, the metastatic potential and malignant transformation to a high-grade astrocytoma further contributes to the poor prognosis. Recent data show that BRAF is mutated in ~23% of LGA's, and 60% of xanthoastrocytomas. We have established two of the only childhood LGA models as direct patient- xenografts in mice. These tumors retain expression profiles and genomic alterations characteristic of the original patient's tumor, thus present unique models to develop alternative, less debilitating, curative therapy. BT-40 xenografts are heterozygous for mutated BRAF (V600E) whereas BT-35 xenografts have wild type BRAF. BT-40 tumors are exquisitely sensitive to the MEK inhibitor AZD6244, whereas BT-35 tumors are unresponsive, typical of other childhood tumor xenografts with wild type BRAF. Resistance to AZD6244 is characterized by an increase in the MEK-dependent gene signature, enhanced IL-6 transcription and secretion, and activation of STAT3 signaling. Resistance to AZD6244 is unstable, as tumors passaged in untreated mice revert to drug sensitivity, and correlates with decreased IL-6 and STAT3 activation. Further, two BRAF (V600E) mutant astrocytic cell lines intrinsically resistant to AZD6244 induce STAT3 activation as MEK is inhibited. The role of STAT3 activation in resistance will be studied in Aim 1. For BT-40 tumors sensitivity to AZD6244 is characterized by complete inhibition of TORC1 signaling, suggesting that mutant BRAF controls the PI3K/TORC1 signaling axis. The role of Akt and STAT3 signaling in MEK regulation of TORC1, and the simultaneous inhibition of MEK and STAT3 to prevent development of resistance, or reverse resistance to AZD6244, will be studied in Aim 2. We plan to exploit the control of mutant BRAF over TORC1 signaling therapeutically. In BT-40 xenografts, MEK inhibition leads to rapid and complete loss of the DNA repair protein FANCD2, downstream of TORC1, thus potentially selectively sensitizing tumor cells to cisplatin, etoposide and ionizing radiation. Further, we will test the concept that combination of cisplatin o etoposide with AZD6244 will prevent the emergence of AZD6244 resistant cells, as mutant BRAF is required to maintain Akt signaling associated with survival in cells exposed to cisplatin or etoposide. We will establish additional models of mutant BRAF LGA's, and determine whether results from BT-40 xenografts are generally applicable. These data will form the basis for rapid translation of novel non-genotoxic and also more conventional therapies that are both effective and less debilitating compared to current therapeutic approaches, for children with recurrent or surgically non-resectable LGA.