以小分子藥物調節p53功能以克服急性骨髓性白血病對Cabozantinib的抗藥性

Abstract

急性骨髓性白血病(AML)是由於骨髓系造血前驅細胞異常增生且無法分化所導致的血液惡性腫瘤。FLT3是一種酪胺酸激酶受體(receptor tyrosine kinase, RTK)，大約有20%-30%的AML病人帶有FLT3基因突變，其中又以FLT3內部串聯重複(FLT3-ITD)最常見，並且具有FLT3-ITD突變的病人通常預後不佳。過去實驗室的研究發現，FLT3標靶藥物Cabozantinib (CBZ)能夠選擇性的抑制FLT3-ITD突變的AML細胞株MV4-11和Molm13之增生，在動物實驗中也發現CBZ能有效抑制其腫瘤的生長。目前臨床上使用FLT3標靶藥物最大的困境就是產生抗藥性問題。對於產生抗藥性的原因至今仍不清楚，因此發展一個新的治療策略是必要的。 為探討CBZ的抗藥性，實驗室先前分別建立了對於CBZ具有抗藥性的細胞株MV4-11-XR和Molm13-XR，我們利用RNA次世代定序(RNA-seq)分析母株細胞及抗藥性細胞株間mRNA表達量的差異。我們發現MV4-11-XR和Molm13-XR共同具有87個差異性下調的基因，經Metascape做基因富集路徑分析發現這些差異性下調的基因和p53訊號傳遞路徑最有相關。同時，以即時監控聚合酶鏈鎖反應(Real-time quantitative PCR)驗證這兩株對CBZ具有抗藥性的細胞株之p53訊號傳遞路徑下游相關基因表達量相較於母株細胞皆明顯下調。 p53訊號傳遞路徑功能失活在多種癌症中都有被發現，但p53功能喪失和癌症抗藥性之間的關係，並未被研究地很透徹。因此，我們以西方墨點法進一步探討MV4-11-XR和Molm13-XR細胞之p53訊號傳遞路徑下調的原因，結果顯示MV4-11-XR細胞之p53功能喪失的可能原因是野生型的TP53發生基因座缺失所致。而Molm13-XR細胞p53功能喪失的原因則可能和p53負調節蛋白質MDM2堆積有關。 依據上述實驗結果，我們針對其p53功能喪失的原因分別予以不同藥物激活p53活性以期能克服抗藥性細胞株對CBZ的抗藥性。過去文獻指出PRIMA-1及RITA可以透過和p53結合，使突變型的p53重新獲得野生型p53的功能，並得以正常活化下游相關基因。於是，我們將PRIMA-1及RITA分別合併CBZ處理MV4-11-XR細胞，結果顯示RITA合併CBZ可以達到協同作用，使CBZ用藥濃度從1µM降低至400nM即可達到半致死效果。 RG7388是第二代的MDM2抑制劑，我們以RG7388合併CBZ處理Molm13-XR細胞，結果顯示RG7388合併CBZ可以達到協同作用，使CBZ用藥濃度從400nM降低至100nM即可達到半致死效果。 透過小分子藥物重新活化p53合併FLT3抑制劑的用藥策略，在兩株抗藥性細胞中，都可顯著提升細胞內p53訊息路徑中之下游相關基因的表達及蛋白質總量，誘導細胞週期停滯且抑制細胞自我更新及增生能力；而在Molm13-XR細胞中更發現合併藥物使用可抑制DNA修補機制相關蛋白RAD51的總量，使細胞一直處於DNA損傷狀態，誘導細胞凋亡。此外，針對Molm13-XR細胞之合併藥物策略在斑馬魚動物實驗中也有顯著抑制效果。 整體來說，對CBZ具有抗藥性的MV4-11-XR和Molm13-XR細胞株都因不同原因而發生p53路徑功能喪失；而透過小分子藥物針對不同原因重新激活p53，可以有效增強抗藥性細胞株對CBZ的敏感性。上述實驗結果也揭示了重新活化p53並同時標靶FLT3，可能是未來臨床上有潛力的治療策略。

Acute myeloid leukemia (AML) is a hematological malignancy caused by abnormal proliferation of bone marrow-derived hematopoietic precursor cells and differentiation arrest. FLT3 is a receptor tyrosine kinase (RTK), with approximately 20%-30% of AML patients with FLT3 gene mutations, among which FLT3 internal tandem repeats (FLT3-ITD) are the most common one and patients with FLT3-ITD mutations usually showed a poor prognosis. In the past, our laboratory studies have found that the FLT3 target drug Cabozantinib (CBZ) can selectively inhibit the proliferation of FLT3-ITD mutant AML cell lines MV4-11 and Molm13. It has also been found that CBZ can effectively inhibit tumor growth in animal experiments. Currently, the resistance of leukemia cells to target therapy drugs becomes the main obstacle in the treatment of AML. The reasons for drug resistance are still unclear, so developing a new treatment strategy is necessary. In order to study the drug resistance mechanism, we established CBZ -resistant cell lines from MV4-11 and Molm13 cells, named MV4-11 XR and Molm-13 XR, respectively.We used RNA next generation sequencing (RNA-seq) to analyze differences in mRNA expression between parental and drug-resistant cell lines. We found that 87 differentially down-regulated genes between MV4-11-XR and Molm13-XR. Then, we used Metascape for gene enrichment pathway analysis to find that these differentially down-regulated genes are most relevant to the p53 signaling pathway. We also used real-time quantitative PCR to verify that the expression of the downstream gene related to the p53 signal pathway was significantly down-regulated in the two cell lines resistant to CBZ. To explore the reasons for the loss of p53 pathway function in cell lines resistant to CBZ.We used western blot analysis, IGV analysis and microsatellite analysis to find that the loss of p53 function in MV4-11-XR cells may be due to mutations in the TP53 sequence, and further confirmed by microsatellite PCR analysis that the wild type TP53 is loss of heterozygosity in MV4-11-XR cells. On the other hand, the loss of p53 function in Molm13-XR cells may be related to the accumulation of p53 negative regulatory protein MDM2. Based on the above experimental results, we applied different drugs to activate p53 activity for the reason of its loss of p53 function in order to enhance the sensitivity of drug-resistant cell lines to CBZ. In the past, it was pointed out that PRIMA-1 and RITA can bind to p53, and the mutant p53 can regain the function of wild-type p53 and activate the downstream related genes. Therefore, we combined PRIMA-1 and RITA with CBZ to treat MV4-11-XR cells, respectively. The results showed that RITA combined with CBZ can achieve synergistic effect, and the CBZ concentration can be reduced from 1μM to 400nM to achieve semi-lethal effect. RG7388 is a second-generation MDM2 inhibitor. We combined RG7388 with CBZ to treat Molm13-XR cells. The results showed that RG7388 combined with CBZ can achieve synergistic effect, and the CBZ concentration can be reduced from 400nM to 100nM to achieve semi-lethal effect. The strategy of re-activating p53 combined with FLT3 inhibitors can significantly increase the expression of downstream related genes and total protein level in the p53 signaling pathway, induce cell cycle arrest and inhibit cell self-renewal and proliferation. In Molm13-XR cells, it was found that the combined use of drugs inhibited the total protein level of RAD51, DNA repair related protein, and the cells were always in DNA damage state then induced apoptosis. In addition, the combined drug strategy for Molm13-XR cells also showed significant inhibitory effects in zebrafish in vivo experiments. Overall, the causes of loss of p53 pathway function in MV4-11-XR and Molm13-XR cell lines are different. Reactivation of p53 by small molecule drugs for different reasons can effectively enhance the sensitivity of drug-resistant cell lines to CBZ. The above results also revealed that reactivation of p53 and simultaneous targeting of FLT3 may be a clinically potential therapeutic strategy in the future.