建立跳躍子突變庫來篩選造成BRAF抑制劑抗藥性之基因

Abstract

在相對應的跳躍酶協助下，跳躍子有兩種主要的特徵，一是擁有簡單插入DNA的機轉，二是有承載很大段DNA序列於跳躍子內的能力. 飛蛾跳躍系統在哺乳類細胞中相當有活性，因此對於基因交付，與藉由插入性突變而來的基因發掘，皆是很有用的工具。為了改善跳躍效率，前人努力地嘗試增進跳躍酶的活性。而我們藉由將核仁優先的訊號胜肽，接在經哺乳類密碼子優化過的飛蛾跳躍酶，製造了一種核仁優先型的飛蛾跳躍酶，可以在胚胎幹細胞與癌細胞中，增進跳躍的效率約三倍。這種跳躍酶的主要分布在核仁上。其所協助的插入點位，則如同原本的飛蛾跳躍酶一般，散布於整個基因體之中，此特色適合拿來作基因發掘之用。 BRAF突變是黑色素瘤最主要的驅動突變，BRAF抑製劑已被證明對攜帶BRAF V600E突變的晚期黑色素瘤有效。但是，大多數患者的腫瘤最終還是產生了抗藥性。為了尋找逆轉抗藥性的關鍵，高通量篩選是很有價值的實驗方法。在這個CRISPR-Cas9的時代，可藉由破壞基因而有效地作一失去活性的篩選，另外，增加活性的篩選則可以由帶有活化子的CRISPR-Cas9系統及病毒cDNA庫所執行。這些是屬於反向型基因篩選的方式，需要一些既有的知識去設計sgRNA、cDNA病毒，或是RNA干擾，才能去鎖定特定基因，因此，很難保證這類篩選的全面性與表達的平等性。我們提出另一種篩選方式，以增加活性的跳躍子製作突變庫，成為一種正向基因篩選的方法，來篩選BRAF抑制劑的抗藥基因，我們使用對BRAF抑制劑敏感的SK-MEL-28黑色素瘤細胞株，來建立跳躍子突變庫，建立後用BRAF抑製劑vemurafenib與encorafenib各別篩選後，直到對照組的敏感細胞已無明顯細胞集落，而篩選後仍發現突變庫有抗藥性細胞集落存活，接著對原始突變庫與篩選後存活的細胞作架設型聚合酶連鎖反應(splinkerette PCR)並做次世代定序。由於發現encorafenib的篩選比vemurafenib更完整，因此主要選擇encorafenib篩選組的結果做驗證，經由方向性導向的KC-RBM研究方式來分析常見插入點的位置，我們發現存活細胞的跳躍子插入點，特別集中於MITF, USP47, MAP3K4等候選基因的位置。為了驗證候選基因的重要性，我們經過6個月的BRAF抑制劑處理，建立了黑色素瘤SK-MEL-28與A375的抗藥株，兩者皆發現MAP3K4蛋白量的上升，RNA-Seq合併GSEA分析，發現JNK訊息路徑在抗藥的情況下是被激活的。西方點墨法實驗在抗藥細胞發現了JNK與p38的活化。而MAP3K4是JNK與p38的已知共同調控者，我們進一步在原本帶有敏感性的黑色素瘤細胞中高表達MAP3K4，結果細胞的抗藥性明顯增加。反之，在抗藥細胞中，以RNA干擾技術抑制MAP3K4的表達則可以減少抗藥性，這個抗藥性的減少，主要是因為細胞凋亡的增加。另外，在原本敏感的黑色素瘤細胞中去抑制MAP3K4的表現，則對藥物敏感性與細胞凋亡沒有太大的影響。這些結果顯示BRAF抑制劑的抗藥性是複雜的，而MAP3K4與JNK及p38訊息路徑的協同，在抗藥性中是重要的，因此是一個有潛力去反轉BRAF抑制劑抗藥性的標靶。

With the corresponding transposase, the transposon has the features of simple DNA integration machinery and large cargo capacity. The piggyBac transposition system, which is active in the mammalian cells, is a useful tool for gene delivery as well as gene discovery by insertional mutagenesis. To improve the efficiency of transposition, several efforts were made to improve the activity of transposase. By fusing nucleolus-predominant (NP) peptide to mammalian codon-optimized PBase (mPB), we created NP-mPB. NP-mPB increased the DNA integration efficiency by about 3 folds in embryonic stem cells and cancer cells. The subcellular localization of NP-mPB was mainly distributed in the nucleolus. The insertional mutagenesis mediated by NP-mPB was found across the whole genome, like that mediated by mPB. This feature was suitable for gene discovery. BRAF mutation is the major melanoma driver mutation and BRAF inhibitor was proved effective for late-stage melanoma carrying BRAF V600E mutation. However, most patients finally developed tumor resistance. To find the key to reverse the resistance, high-throughput screening was valuable. In the era of CRISPR-Cas9, loss-of-function screening can be done efficiently by directly disrupting the genes. Gain-of-function screening can also be done by the CRISPR-Cas9 system carrying gene activator as well as lentiviral cDNA library. These screening methods were of reverse genetics, requiring a priori knowledge to design sgRNA, cDNA virus, or RNA interference, to specifically target the genes. Therefore, these systems are difficult to assure the comprehensiveness and equal expression. We proposed an alternative screen method by gain-of-function transposon mutagenesis, a forward genetic approach, to screen for genes offering resistance to BRAF inhibitor. The transposon mutagenesis library was established in the sensitive SK-MEL-28 melanoma cell line and then treated with BRAF inhibitors, vemurafenib and encorafenib, respectively until the original sensitive cells, as the control, had no visible surviving colony. At the selection endpoint, there were still surviving colonies in the mutagenesis library. After next-generation sequencing of the splinkerette PCR products from the baseline mutagenesis library and post-selection cells, insertion sites were mapped. Because the encorafenib selection was more complete than the vemurafenib selection, we primarily chose the results from encorafenib selection for further validation. The analysis for common insertion sites by orientation-directed Kernel Convolved Rule-Based Mapping (KC-RBM) method identified targeted genes in the surviving cells were MITF, USP47, MAP3K4 and other genes. To validate the screening results and the resistance to BRAF inhibitor, we established resistant melanoma cell lines by treating SK-MEL-28 and A375 melanoma cells with vemurafenib for 6 months. The resistant cell lines were proved to have increased MAP3K4 protein expression. RNA-Seq with GSEA was performed in resistant cells and found that JNK signaling pathway was activated in the resistant cell. Western blot for MAPK pathway showed that the resistant cells had activated JNK pathway and p38 pathway. Overexpression of their common upstream mediator, MAP3K4, offered resistance in the sensitive melanoma cells. Suppression of their common upstream mediator, MAP3K4, by RNAi reverse the resistance of both resistant cell lines. The reversal of the resistance was mainly due to the markedly increase apoptosis. Suppression of MAP3K4 in the parental melanoma cells did not significantly alter the drug sensitivity or the level of apoptosis. The results suggest that the development of resistance to the BRAF inhibitor is complex. MAP3K4 with coordination of JNK and p38 pathways is important in the resistance, which is a potential target to reverse the resistance to BRAF inhibitor.