靶向afatinib誘導之轉錄反應探索肺癌組合治療

Abstract

小分子通常用於靶向癌症治療，但它們的效果往往受到耐藥性發展的阻礙。最近的證據表明，儘管抗癌藥物具有抑制靶向通路的能力，但治療所誘導的轉錄反應可能作為腫瘤生長的逃逸機制。解決這一障礙的最佳策略是使用同時針對這些轉錄機制的聯合療法。在這項研究中，我們建立了一個系統性的分析，通過分析從 LINCS L1000 平台獲得的小分子擾動圖譜來預測治療誘導的抗性轉錄機制。通過對用約 3,000 種化合物處理的 10 個細胞株的轉錄組數據進行系統分析，發現常用於非小細胞肺癌 (NSCLC) 一線治療的表皮生長因子受體酪氨酸激?抑製劑 afatinib 能誘導絲氨酸生物合成和葉酸介導單碳代謝路徑作為潛在的抗性機制。與我們的預測一致，絲氨酸生物合成基因磷酸甘油酸脫氫? (PHGDH) 和磷酸絲氨酸氨基轉移? 1 (PSAT1) 以及單碳代謝基因亞甲基四氫葉酸脫氫? (MTHFD2)和絲氨酸羥甲基轉移? (SHMT2) 在NSCLC 細胞株 H1299 和 H1975 中處理Afatinib 後表現量升高。西方墨點法分析進一步證實了這些路徑蛋白在 NSCLC 細胞株中會在 afatinib 治療的下被提高。此外，我們藉由分析細胞內的代謝產物絲氨酸以及磷酸絲氨酸在 afatinib 治療的下有被提升。接著我們進行了聚落形成試驗，證明了 afatinib 與此兩種生物路徑的抑製劑對於毒殺非小細胞肺癌細胞具有協同作用。在短期 MTS 細胞存活率分析中我們發現 afatinib 與 NCT-503 的協同性作用，以及其對於 NSCLC 細胞株的細胞週期影響，並發現了兩個藥物合併使用會顯著地提高細胞凋亡的比例。綜上所述，這些研究結果表明，絲氨酸生物合成和單碳代謝的誘導是非小細胞肺癌中 afatinib 耐藥的潛在機制，通過靶向出現在轉錄水平的耐藥機制為癌症治療提供了理論基礎。

Small molecules are commonly used for targeted cancer therapy, but their effects are often hampered by the development of drug resistance. Recent evidence suggests that despite the ability of cancer drugs to inhibit on-target pathways, treatmentinduced transcriptional responses may serve as an escape mechanism for tumor growth.An optimal strategy for addressing this obstacle is the use of combination therapies that simultaneously target these emerging resistance mechanisms. In this study, we established a computational framework for predicting treatment-induced transcriptional mechanisms of resistance by analyzing small-molecule perturbation profiles obtained from the LINCSL1000 platform. Through systematic investigation of transcriptomic data of 10 cell lines treated with ~3,000 compounds, we found that afatinib, an epidermal growth factor receptor (EGFR) tyrosine kinase inhibitor for the first-line treatment of non-small-cell lung cancer (NSCLC), was predicted to induce the serine biosynthesis and the one-carbon pool by folate pathways as a potential resistance mechanism. Consistent with our prediction, the expression of serine biosynthesis genes phosphoglycerate dehydrogenase (PHGDH) and phosphoserine aminotransferase 1 (PSAT1) and one-carbon metabolism genes methylenetetrahydrofolate dehydrogenase (MTHFD2) and serine hydroxymethyltransferase (SHMT2) was elevated in NSCLC cell lines, H1299 and H1975, after afatinib treatment. Immunoblot analysis further confirmed the inducible expression of these pathway proteins in afatinib-treated NSCLC cell lines. We found the content of serine and O-phospho-L-serine was elevated after afatinib treatment in NSCLC cell lines. We then conducted a clonogenic assay to demonstrate that afatinib synergized with the inhibitors of these two pathways in the NSCLC cell lines. In the short-term MTS cell survival analysis, the synergistic effect of afatinib and NCT-503 was found. We examined the effect of the combination of afatinib and NCT-503 on the cell cycle of NSCLC cell lines. Furthermore, the combination of the two drugs was found to significantly increase the proportion of apoptosis. To sum up, these findings suggest that induction of serine biosynthesis and one-carbon metabolism is a potential mechanism of afatinib resistance in NSCLC, providing a rationale for cancer therapy by targeting the resistance mechanisms that emerge at the transcriptional level.