Therapeutic Exploitation of Mutant BRAF for Astrocytoma

描述（由申请人提供）：儿童低度星形细胞瘤（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%，但显著的发病率与残留肿瘤的存在有关，目前的治疗包括神经内分泌-认知缺陷、视力缺陷、血管病变和继发性肿瘤。此外，转移潜力和恶性转化为高级星形细胞瘤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。我们建立了两种儿童LGA模型作为直接患者异种移植小鼠。这些肿瘤保留了原始患者肿瘤的表达谱和基因组改变特征，因此提供了独特的模型来开发替代的，更少的衰弱性治疗。BT-40异种移植物是突变BRAF （V600E）的杂合，而BT-35异种移植物是野生型BRAF。BT-40肿瘤对MEK抑制剂AZD6244非常敏感，而BT-35肿瘤无反应，这是其他野生型BRAF儿童肿瘤异种移植物的典型特征。AZD6244耐药的特点是mek依赖性基因特征增加，IL-6转录和分泌增强，STAT3信号通路激活。对AZD6244的耐药是不稳定的，因为未经治疗的小鼠的肿瘤传代恢复到药物敏感性，并且与IL-6和STAT3激活降低相关。此外，两种BRAF （V600E）突变型星形细胞细胞系对AZD6244具有内在抗性，在MEK被抑制的情况下诱导STAT3激活。STAT3激活在耐药性中的作用将在Aim 1中进行研究。对于BT-40肿瘤，AZD6244的敏感性以完全抑制TORC1信号传导为特征，表明突变BRAF控制PI3K/TORC1信号传导轴。Akt和STAT3信号在MEK调控TORC1中的作用，以及同时抑制MEK和STAT3以防止AZD6244的耐药或反向耐药的发展，将在Aim 2中进行研究。我们计划在治疗上利用突变BRAF对TORC1信号的控制。在BT-40异种移植物中，MEK抑制导致TORC1下游DNA修复蛋白FANCD2的快速和完全丧失，从而可能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，我们将验证顺铂或依托泊苷联合AZD6244将阻止AZD6244耐药细胞的出现，因为在暴露于顺铂或依托泊苷的细胞中，突变BRAF是维持与存活相关的Akt信号通路所必需的。我们将建立突变BRAF LGA的其他模型，并确定BT-40异种移植的结果是否普遍适用。这些数据将为快速转化新的无基因毒性和更传统的治疗方法奠定基础，这些治疗方法与目前的治疗方法相比既有效又不那么衰弱，适用于复发性或手术不可切除的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.