STAT5/OLIG2 regulation of GBM therapeutic resistance and recurrence - Resubmit 3.22

项目摘要 胶质母细胞瘤(GBM)是人类最致命的癌症之一，具有广泛的遗传和细胞异质性。 GBM的遗传异质性是通过激活多个受体酪氨酸激酶(RTK)来实现的，包括 EGFR。大约50%-60%的GBM肿瘤存在EGFR基因的扩增，包括 成分活性突变体EGFRvIII和EGFR胞外区内的点突变。临床努力 靶向EGFR本身未能提高存活率，部分原因是下游信号转导中的冗余 通过EGFR或其他致癌RTK触发的通路。我们的数据显示，EGFRvIII和可能的热 点突变通过下游激活致癌的STAT5信号来促进治疗耐药。 因此，了解STAT5如何作为EGFR等致癌RTK的汇聚点将 为GBM患者的治疗干预提供新的机会。STAT5的作用的原因之一 在GBM中信号转导被低估是因为长期培养的GBM细胞经常失去EGFRvIII 表达，并且不显示STAT5激活。然而，保留EGFRvIII表达的原代GBM 显示激活的STAT5信号。我们的数据表明，EGFRvIII信号通过JAK1/2激活STAT5- 独立的机制，并代表了一个吸引人的RTK模型，用于理解STAT5在 GBM。我们还发现，保留EGFRvIII表达的耐药胶质瘤干细胞(GSCs)也 增加了STAT5的激活。在GSCs中，EGFR参与了一个具有关键细胞命运的前馈循环 和促有丝分裂转录因子OLIG2。缺失OLIG2的GSC显示其磷酸化水平降低 Stat5及其下游靶标的表达(如Fn14)。STAT5和OLIG2都具有二聚体的功能 在GSC中，我们发现STAT5与OLIG2相互作用。此外，抑制STAT5可降低OLIG2- 依赖GSC的侵袭。然而，OLIG2依赖的上游分子机制 必须确定信号转导通路5的激活和受OLIG2-STAT5复合体调控的信号通路(S 以有效靶向浸润性和耐药的胶质瘤干细胞。我们假设激活了 由致癌RTK介导的Stat5-OLIG2信号，如EGFRvIII，促进侵袭性增加， GBM的治疗抵抗力和茎特性。目标1将确定STAT5的机械作用 GBM细胞中EGFR异构体背景下的异型信号转导。目标2将确定分子 OLIG2调节GSCs中STAT5激活的机制(S)。目标3将评估 基底膜中STAT5-OLIG2信号轴的功能及临床意义这项提议的成功将 识别、验证STAT5/OLIG2信号通路并将其置于临床有意义的上下文中 治疗靶点为浸润性细胞，这些细胞通常是导致GBM死亡的基础。

Project Summary Glioblastoma (GBM) is among the most lethal human cancers with extensive genetic and cellular heterogeneity. Genetic heterogeneity in GBM is seen through activation of multiple receptor tyrosine kinases (RTKs) including EGFR. Approximately ~50-60% of GBM tumors harbor amplification of the EGFR gene, including the constitutively active variant EGFRvIII, and point mutations within the extracellular domain of EGFR. Clinical effort at targeting EGFR itself has failed to increase survival, in part due to redundancy in the downstream signaling pathways triggered through EGFR or other oncogenic RTKs. Our data shows that EGFRvIII and possibly the hot spot mutations promote therapeutic resistance through downstream activation of oncogenic STAT5 signaling. Thus, understanding how STAT5 can serve as a point of convergence for oncogenic RTKs, such as EGFR will provide new opportunities for therapeutic interventions in GBM patients. One of the reasons why role of STAT5 signaling has been underappreciated in GBM is because long-term cultured GBM cells often lose EGFRvIII expression and do not show STAT5 activation. However, primary GBMs that retain EGFRvIII expression exhibit active STAT5 signaling. Our data indicate that EGFRvIII signaling activates STAT5 through JAK1/2- independent mechanisms and represents an attractive RTK model for understanding the role of STAT5 in GBM. We also find that therapy-resistant glioma stem cells (GSCs) that retain EGFRvIII expression also have increased STAT5 activation. In GSCs, EGFR participates in a feed-forward loop with a key cell fate and pro-mitogenic transcription factor, OLIG2. GSCs depleted of OLIG2 show decreased levels of phospho- STAT5 and expression of its downstream targets, (e.g. Fn14). Both STAT5 and OLIG2 function as dimers and in GSCs we find that STAT5 interacts with OLIG2. Furthermore, STAT5 inhibition decreases OLIG2- dependent GSC invasion. However, the upstream molecular mechanisms involved in OLIG2-dependent activation of STAT5 and signaling pathway(s) regulated by the OLIG2-STAT5 complex must be identified to effectively target the infiltrative and resistant glioma stem cells. We hypothesize that activation of STAT5-OLIG2 signaling mediated by oncogenic RTKs, such as EGFRvIII, promote increased invasion, therapy resistance, and stemness properties in GBM. Aim 1 will determine the mechanistic role of STAT5 isoform signaling in the context of EGFR variants in GBM cells. Aim 2 will determine the molecular mechanism(s) involved in OLIG2-mediated regulation of STAT5 activation in GSCs. Aim 3 will assess the functional and clinical significance of STAT5-OLIG2 signaling axis in GBM. Success of the proposal will identify, validate, and place into a clinically meaningful context the STAT5/OLIG2 signaling pathway as a therapeutic target for infiltrating cells that commonly underlie GBM fatality.