探討具抗藥性之FLT3-ITD急性骨髓性白血病代謝機制的改變

Abstract

急性骨髓性白血病（Acute myeloid leukemia, AML）是一種骨髓性造血前驅細胞異常增生及分化所導致的血液惡性疾病，在AML患者中，以FLT3-ITD（Fms-like tyrosine kinase 3-internal tandem duplication) 基因突變最為常見，發生率約25-30%，並且與預後不良有關。因此，FLT3被認為是在AML治療上具有潛力的目標分子。 Cytarabine (Ara-C)是目前常用於治療AML病人的化療藥物之一，約60-80%病患經治療後得到完全緩解（complete remission），然而許多病患在於緩解後再復發或無法達到緩解，主要原因是患者體內的癌細胞對於化療藥物產生抗藥性。另外，在實驗室先前的研究中，發現低劑量的酪胺酸激酶抑制劑cabozantinib (XL-184)能夠抑制具有FLT3-ITD突變的AML細胞株MV4-11以及Molm-13的生長，可是，目前發現酪胺酸激酶抑制劑在臨床應用上同樣面臨著抗藥性問題的阻礙。因此，針對臨床上AML病人使用化療藥物和標靶藥物後可能所面臨的抗藥性問題，本實驗室先前利用MV4-11以及Molm-13兩株人類AML細胞株分別建立出三株具抗藥性的細胞株：MV4-11-R、MV4-11-XR以及Molm-13-XR。 結果顯示，三株抗藥性細胞株的生長速度明顯較母株增加。另外，我們分析三株抗藥性細胞株在FLT3及其下游訊息傳遞路徑中的蛋白表現，結果顯示三株具抗藥性的細胞株的AKT磷酸化程度都明顯高於母株細胞；AKT的活化可以調控細胞的能量代謝，影響細胞對於葡萄糖的攝取與利用。 我們利用RNA次世代定序（RNA-seq）分析抗藥性細胞與母株細胞在mRNA表達之差異。我們發現MV4-11-R中具差異性顯著上升與下降的基因分別與代謝路徑和p53訊息路徑有關，而MV4-11-XR和Molm-13-XR中具差異性顯著上升的基因同樣與代謝機制有關。因此，我們針對三株抗藥細胞株的代謝路徑進行探討。我們分別進行了葡萄糖攝取試驗、GAPDH活性試驗、乳酸產量試驗、ATP測定以及海馬生物能量代謝分析等。結果顯示，MV4-11-R、MV4-11-XR與Molm-13-XR三株抗藥細胞的代謝路徑有別於母株細胞，在三株抗藥細胞中都有發現他們對糖解作用的依賴性有增加的現象。接著，我們進行了代謝體分析，再合併先前RNA次世代定序的結果，最終在MV4-11-R、MV4-11-XR、Molm-13-XR中分別找出了6個、5個和1個有機會作為藥物標的的候選基因。 總體而言，實驗室所建立的三株抗藥性細胞株在代謝機制上有別於母株細胞。另外，在先前實驗室的研究中，已經發現MV4-11-R在p53基因序列上除了有原本母株細胞的p53 R248W突變以外，同時還增加了p53 D281G突變，而p53基因又與代謝路徑的調控有關，因此，p53 D281G突變可能是造成代謝路徑異常並誘導細胞抗藥性的發生的原因。

Acute myeloid leukemia (AML) is a malignant disease representing abnormal prolif-eration and differentiation of myeloid progenitor cells. An internal tandem duplication in the FLT3 gene (FLT3-ITD) is the most common genetic alterations in AML patients, with an incidence of about 25-30%, associated with poor prognosis. Therefore, FLT3 is con-sidered as a potential target molecule in AML treatment. Cytarabine (Ara-C) is one of the chemotherapeutic drugs for the treatment of AML. The complete remission (CR) rate reaches 60-80% for AML patients after treatment. However, many patients relapse or unable to achieve remission and relapses results from the existence of chemoresistant cells, which is a major obstacle in cancer therapy. Our previous studies demonstrated that low dose of cabozantinib (XL-184) was able to inhibit the growth of FLT3-ITD AML cell lines MV4-11 and Molm-13. It has been known that the clinical use of Tyrosine kinase inhibitor (TKI) for the cancer treatment might be hindered by drug resistance among some patients. To better elucidate the drug resistance mechanisms of cytarabine and cabozantinib, we established three drug-resistant cell lines from MV4-11 and Molm-13 by increasing exposure to cytarabine or cabozantinib, including cytarabine-resistant MV4-11(MV4-11-R), cabozantinib-resistant MV4-11(MV4-11-XR) and cabozantin-ib-resistant-Molm-13(Molm-13-XR), respectively. In our current study, we found that three drug-resistant cells proliferated more rapid-ly compared with the parental cells. Furthermore, we examined the downstream signaling pathways of FLT3. Western blotting analysis demonstrated the high expression of total FLT3, p-FLT3 and p-AKT among three resistant cell lines. Of them, PI3K/AKT/mTOR signaling is known to play a role in cell proliferation, survival and metabolism. Next, we performed RNA-sequencing (RNA-seq) analysis to investigate the differ-ential expression genes (DEGs) between drug-resistant cell lines and parental cells. Up-regulated and down-regulated DEGs with significant difference in MV4-11-R are associ-ated with metabolic pathways and p53 signaling pathway, respectively. In addition, upreg-ulated DEGs with significances in MV4-11-XR and Molm-13-XR are also related to the metabolic pathways. Thus, we investigated the metabolic alterations of the three drug-resistant cell lines. We performed glycose uptake, GAPDH activity, lactate produc-tion, ATP content and Seahorse bioenergy metabolism analysis, etc. The results showed that MV4-11-R, MV4-11-XR and Molm-13-XR highly relied on glycolysis. Furthermore, we measured the alterations in metabolites between drug-resistant cells and parental cells. Herein, we combined both transcriptome and metabolome data, and found out 6, 5 and 1 candidate gene(s) in MV4-11-R, MV4-11-XR, and Molm-13-XR, respectively. In general, the metabolism of three drug-resistant cell lines are different from parental cells. Moreover, our previous studies showed that MV4-11-R harbored p53 R248W and D281G mutations as MV4-11-P harbored p53 R248W mutation only. Therefore, the p53 D281G mutation may contribute to the cytarabine-resistance of MV4-11-R and induce drug resistance.