喹唑啉衍生物作為人類神經膠質母細胞瘤抗癌藥物之評估

Abstract

神經膠質母細胞瘤（GBM）是最常見也最具侵襲能力的成人原發性腦腫瘤。根據世界衛生組織（WHO）分級，神經膠質母細胞瘤被分類為第四級的成人型神經膠質細胞瘤，同時神經膠質母細胞瘤的浸潤和遷移也是導致高侵略性和難以治療的關鍵，因其具有浸潤並擴散到周圍腦組織的能力，經腫瘤經手術切除後，剩餘癌細胞仍可以遷移至附近的健康腦組織，即便進行合併放療和化療等積極的治療方法，患者的預後仍然不佳。因此迫切需要一種新的治療方法來治療神經膠質母細胞瘤。 盤狀結構域受體1（Discoidin Domain Receptor 1, DDR1）是以膠原蛋白作為配體的酪氨酸激酶。在多種癌症中，其異常表現增加會活化細胞內多個下游訊息傳遞，促進癌細胞的存活、增殖、貼附、遷移和細胞外基質重塑。在資料庫分析中，我們發現DDR1的表現量在神經膠質母細胞瘤顯著增加，為了研究DDR1在神經膠質母細胞瘤中扮演的角色，我們利用CRISPR/Cas9將神經膠質母細胞瘤細胞株的DDR1基因進行剔除，發現經DDR1剔除的細胞株顯著抑制了細胞增生與腫瘤生長的能力，同時我們使用RNA-seq分析DDR1剔除後所造成的基因富集差異，發現DDR1 影響細胞貼附及遷移的相關路徑，因此抑制DDR1可能是治療神經膠質母細胞瘤的新策略。 本論文中，我們設計並合成出喹唑啉衍生物作為新穎性DDR1抑制劑且於神經膠質母細胞瘤驗證其療效並詳細探討其相關作用機轉，其中PANDD083對於 DDR1 的抑制活性IC50為3.3 nM。在細胞試驗中，該化合物顯著抑制DDR1的作用並降低了癌細胞的增生、存活及遷移能力。另外在動物實驗中PANDD083也有效抑制了腫瘤細胞的生長，展現其抗腫瘤效果。作用機轉發現PANDD083 可藉由抑制 collagen I誘發的DDR1磷酸化並影響下游蛋白，減少Src及AKT/mTOR的活化，造成細胞週期停滯在G2/M並誘導細胞凋亡；此外，PANDD083會抑制HTRA1和HDAC6的表現量，進而影響癌細胞存活、生長及遷移。綜合以上結果，我們研究說明了DDR1可作為神經膠質母細胞瘤的一個新治療標的，也證實PANDD083在神經膠質母細胞瘤細胞的抗癌效果。

Glioblastoma (GBM) is the most common and aggressive primary brain tumor in adults. According to the World Health Organization (WHO), GBM is categorized as Grade IV adult-type gliomas. The invasive and migratory capabilities of GBM cells are key factors contributing to their aggressiveness and treatment resistance, as remaining cancer cells can migrate even after surgical resection into surrounding healthy brain tissue. Despite aggressive treatments such as surgery, radiation therapy, and chemotherapy, the prognosis for patients remains poor. Therefore, there is an urgent need for a new treatment approach for GBM. Discoidin Domain Receptor 1 (DDR1) is a type of receptor tyrosine kinase (RTK) binding to collagens as its ligand. The abnormal expression of DDR1 is implicated in various cancers, leading to the activation of several downstream signaling pathways that participate in cell survival, proliferation, adhesion, migration, and extracellular matrix remodeling. Database analysis has shown a significant increase in DDR1 expression in GBM. To investigate the role of DDR1 in GBM, we utilized CRISPR/Cas9 to knock out the DDR1 gene in the GBM cell line. We also found that DDR1 knockout significantly inhibited cell proliferation and tumor growth in animal models. Using NGS RNA-seq analysis, we identified significant gene enrichment differences caused by DDR1 knockout, particularly affecting cell adhesion and migration pathways. Therefore, inhibition of DDR1 may represent a novel strategy for treating GBM. In this study, we designed and synthesized a novel DDR1 inhibitor named PANDD083, a quinazoline derivative with an IC50 value of 3.3 nM. PANDD083 also significantly reduced DDR1 activity and inhibited cancer cell proliferation, survival, and migration. Additionally, in animal experiments, PANDD083 suppressed tumor cell growth, demonstrating its anti-tumor effects. Mechanistic studies revealed that PANDD083 inhibits collagen I-induced phosphorylation of DDR1 and affects its downstream proteins, reducing the activation of Src and AKT/mTOR pathways. This inhibition caused cell cycle arrest at the G2/M phase and induced apoptosis. Furthermore, PANDD083 inhibited the expression of HTRA1 and HDAC6, thereby impacting cancer cell survival, growth, and migration. In conclusion, we have developed a novel DDR1 inhibitor for treating GBM and investigated its relevant mechanisms of action. Collectively, our study suggests DDR1 as a novel therapeutic target for GBM and confirms the anti-cancer effects of PANDD083 in GBM cells.