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

项目摘要 胶质母细胞瘤（GBM）是最致命的人类癌症之一，具有广泛的遗传和细胞异质性。 GBM中的遗传异质性通过多种受体酪氨酸激酶（RTK）的激活来观察，包括 EGFR对大约50-60%的GBM肿瘤具有EGFR基因扩增，包括EGFR基因扩增。 组成型活性变体EGFRvIII和EGFR胞外结构域内的点突变。临床努力 靶向EGFR本身并不能增加生存率，部分原因是下游信号的冗余 通过EGFR或其他致癌RTK触发的途径。我们的数据显示，EGFRvIII和可能的热 点突变通过致癌STAT 5信号传导的下游激活促进治疗抗性。 因此，了解STAT 5如何作为致癌RTK（如EGFR）的汇聚点， 为GBM患者的治疗干预提供了新的机会。STAT 5的作用 在GBM中，信号传导被低估是因为长期培养的GBM细胞经常丢失EGFRvIII。 表达并且不显示STAT 5激活。然而，保留EGFRvIII表达的原代GBM 表现出活性STAT 5信号传导。我们的数据表明，EGFRvIII信号通过JAK 1/2激活STAT 5， 独立的机制，并代表了一个有吸引力的RTK模型，了解STAT 5的作用， GBM。我们还发现，保留EGFRvIII表达的治疗抗性胶质瘤干细胞（GSC）也 增加STAT 5的激活。在GSC中，EGFR参与了一个前馈回路， 和促有丝分裂转录因子OLIG 2。耗尽OLIG 2的GSC显示磷酸化水平降低， STAT 5及其下游靶标（例如Fn 14）的表达。STAT 5和OLIG 2均作为二聚体发挥功能 在GSC中我们发现STAT 5与OLIG 2相互作用。此外，STAT 5抑制降低了OLIG 2- 依赖性GSC入侵然而，涉及OLIG 2依赖性的上游分子机制， 必须鉴定STAT 5的活化和由OLIG 2-STAT 5复合物调节的信号通路 以有效靶向浸润性和抗性胶质瘤干细胞。我们假设， 由致癌RTK如EGFRvIII介导的STAT 5-OLIG 2信号传导促进侵袭增加， 治疗抗性和GBM中的干性性质。目的1将确定STAT 5的机制作用 在GBM细胞中EGFR变体背景下的同种型信号传导。目标2将确定分子 GSC中的STAT 5活化的0 LIG 2介导的调节中涉及的一种或多种机制。目标3将评估 STAT 5-OLIG 2信号轴在GBM中的功能和临床意义。该提案的成功将 鉴定、验证并将STAT 5/OLIG 2信号通路置于临床有意义的背景下， 浸润细胞通常是GBM致死的基础。

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.