探討Akt抑制劑MK-2206對人類急性骨髓性白血病的分子作用機制及其在治療上的潛力

Abstract

PI3K/Akt訊息傳導路徑可以調控細胞的增生、凋亡，對於維持正常的細胞生理功能扮演著很重要的角色。在許多癌症的研究中發現PI3K/Akt訊息傳導路徑的不正常活化，並認為這樣的不正常活化可能是造成癌症的原因之一。在人類急性骨髓性白血病（Acute myeloid leukemia, AML）中，PI3K/Akt訊息傳導路徑亦有不正常活化的情形。為了針對這樣異常活化的訊息傳導路徑，有許多針對PI3K/Akt的抑制劑被研發出來，其中包含Akt抑制劑－MK-2206。為了研究MK-2206是否對於AML細胞具有毒殺作用及其作用機制，一開始先利用細胞抑殺試驗，發現MK-2206可以在低濃度下對AML細胞株達到毒殺作用（IC50 < 3 μM）而不會影響到正常的周邊血單核球細胞（IC50 > 18.8 μM），由此證明MK-2206的安全性。接下來利用流式細胞分析儀，發現在MK-2206處理下，會造成AML細胞停留在細胞週期中的G1期，並引發細胞凋亡。進一步利用西方墨點法分析跟細胞凋亡相關分子的表現，發現Myeloid cell leukemia 1 （Mcl-1）在MK-2206的處理下表現量會減少。Mcl-1是一個可以抵抗細胞凋亡的分子，進一步利用蛋白酶體抑制劑－MG-132及glycogen synthase kinase 3 （GSK3）抑制劑－氯化鋰，發現MK-2206在AML細胞株中所造成的Mcl-1減少的現象可能是經由GSK3引發後續經蛋白酶體降解Mcl-1所致。為了降低MK-2206的使用劑量，我們發現在使用低濃度MK-2206時，能有效降低化療藥物cytarabine的用量。Cytarabine是在臨床上常用來治療AML病人的化療藥品，但常有抗藥性產生或是治療無效的情況，因此，本實驗室建立出對cytarabine具有抗藥性的MV-4-11 AML細胞株（MV-4-11-R）。MV-4-11-R細胞的核質比較大、染色質較不緻密，且CD56表現量高（37.0% v.s. 22.6%），但HLA-DR抗原表現量低（2.3% v.s. 12.6%）。利用流式細胞分析儀，發現MV-4-11-R的細胞增生較慢（34.7% v.s. 48.4%）。使用Human Phospho-Kinase Array與西方墨點法發現MV-4-11-R之Akt磷酸化及ERK之磷酸化較高；進一步，合併使用Akt抑制劑（MK-2206）及MEK抑制劑（CI-1040）確實顯現具協同作用（CI值 < 1），而且無論是原本AML細胞株或是有抗藥性的細胞株都有此一效果。最後，我們以異體移植模式測試MK-2206與CI-1040合併使用的效果是否能有效抑制MV-4-11-P的生長，發現能有效縮減腫瘤的體積，但並不會影響到小鼠的體重，證明了此一用藥策略的可行性。

Phosphoinositide 3-kinase (PI3K)/Akt signaling pathway can regulate cell proliferation, apoptosis and play a critical role in maintaining normal cell function. It has been demonstrated that abnormal PI3K/Akt activation occurred in various cancers, including human acute myeloid leukemia (AML). As a result, many PI3K/Akt inhibitors were created to target this uncontrolled pathway, including MK-2206, an allosteric Akt inhibitor. We used MTS assay (cell proliferation assay) to evaluate the potency of MK-2206 on AML. MK-2206 was more cytotoxic to AML cell at low dose (IC50 < 3 μM) comparing to normal peripheral blood mononuclear cells (IC50 > 18.8 μM), thus demonstrated its biosercurity. Next, treatment of MK-2206 on AML cells resulted in cell cycle G1 arrest and induced apoptosis by using flow cytometry analysis. Further investigation on apoptosis-related molecules demonstrated myeloid cell leukemia 1 (Mcl-1) reduction when AML cells treated with MK-2206. Mcl-1 is an anti-apoptotic molecule. By using proteasome inhibitor MG-132 or glycogen synthase kinase 3 (GSK3) inhibitor lithium chloride, we found that Mcl-1 reduction by MK-2206 on AML cells resulted from proteasome degradation mediated by GSK3. To detect the efficacy of MK-2206 with plasma-reachable dose, 200 nM MK-2206 was used to evaluate the sensitivity of cytarabine. We found that combination of 200 nM and 75 nM cytarabine revealed similar effect of 150 nM cytarabine only. Cytarabine is a chemotherapeutic drug commonly used in AML treatment. However, no response or acquired resistance to cytarabine are frequently shown in AML patients. Thus, we established a cytarabine-resistant MV-4-11 (MV-4-11-R) cell line. The N/C ratio of MV-4-11-R was large and the chromatin of MV-4-11-R was less condensed compared with its parental cell (MV-4-11-P). There were more much more expression of CD56 and less expression of HLA-DR in MV-4-11-R compared with MV-4-11-P by using flow cytometry analysis. MV-4-11-R showed slower proliferation kinetics than MV-4-11-P. There was much more Akt and ERK phosphorylation in MV-4-11-R than MV-4-11-P by using Phospho-kinase Array analysis and western blot analysis. Subsequently, combination of Akt inhibitor MK-2206 and MEK inhibitor CI-1040 demonstrated significantly synergistic effect (CI < 1) in MV-4-11-R and its parental cell. As a result, we demonstrated that combination of MK-2206 and CI-1040 could inhibit proliferation of AML cells more effectively both in vitro and in vivo, representing a novel strategy to treat AML patients.