探討去乙醯基酶抑制劑Trichostatin A及LBH589對於白血病細胞之分子機制：著重在對cabozantinib具感受性及抗藥性之MV4-11細胞株

Abstract

在急性骨髓性白血病(Acute myeloid leukemia, AML)患者中，約有20~30%帶有FLT3基因突變，其中以FLT3之內部串聯重複(FLT3-ITD)最為常見，通常會使用酪胺酸激酶抑制劑治療，但最終因產生抗藥性而導致治療失敗；實驗室先前發現一個FDA核准用於固態腫瘤的酪胺酸激酶抑制劑cabozantinib，可選擇性抑制具有FLT3-ITD白血病細胞株。為了預防未來用於臨床白血病治療產生抗藥性的問題，實驗室建立具有cabozantinib抗性之MV4-11與Molm-13細胞，並研究產生抗藥性的機制。近年來文獻指出表觀遺傳的異常也是造成AML的原因，所以未來針對表觀遺傳的藥物可能是AML的替代用藥。因此，我們利用表觀遺傳調控物質的藥庫（SCREEN-WELL® Epigenetics library : BML-2836）篩選具發展潛力的藥物。 經藥庫篩選後，我們選擇出Trischosatin A (TSA, 廣泛型組蛋白去乙醯酶抑制劑)，及與TSA同為廣泛型組蛋白去乙醯酶抑制劑LBH589 (Panobinostat, FDA核准藥物)。TSA與LBH589對白血病細胞(MV4-11)及具有cabozantinib抗性之白血病細胞(MV4-11-XR)有良好的抑制效果，TSA之IC50在MV4-11與 MV4-11-XR細胞分別為59.93±15.9 nM、108.9±40.4 nM；而LBH589之IC50在MV4-11與MV4-11-XR細胞分別為1.80±0.24 nM、5.03±1.50 nM。我們發現TSA與LBH589可以停滯白血病細胞的細胞週期於G0/G1期，並抑制PI3K及MAPK訊息傳遞路徑且引發細胞凋亡以及造成DNA損傷。另外，TSA與LBH589可以抑制白血病細胞形成細胞群落的能力，並抑制自我更新相關基因及促進腫瘤抑制基因表現。最後評估TSA與LBH589合併使用cabozantinib的效果，發現TSA合併使用cabozantinib於MV4-11及MV4-11-XR細胞效果為加成、協同效果；LBH589合併使用cabozantinib於MV4-11及MV4-11-XR細胞效果均為協同效果。 總體而言，廣泛型組蛋白去乙醯酶抑制劑對MV4-11與MV4-11-XR細胞有良好的毒殺效果，因此未來可以評估使用組蛋白去乙醯酶抑制劑治療對具對TKI有抗藥性的AML，以期提高AML病人的生存率。

A total of 20-30% acute myeloid leukemia (AML) patients have mutations in the FLT3 (Fms-like tyrosine kinase 3) gene; among those, FLT3-internal tandem repeats (FLT3-ITD) were the most common. Patients with FLT3-ITD usually treated with tyrosine kinase inhibitors, but eventually the treatment fails due to drug resistance. In our laboratory previous studies, we found that the tyrosine kinase inhibitor cabozantinib approved by FDA for solid tumor treatment could selectively inhibit the proliferation of FLT3-ITD mutant AML cell lines. To evaluate the possibility of drug resistance in clinical leukemia treatment by using cabozantinib, we established cabozantinib-resistant cell lines MV4-11-XR and Molm13-XR from MV4-11 cells and Molm13 cells, respectively, to figure out the drug resistance mechanism. Many studies demonstrate that epigenetic changes participate in the pathogenesis of AML. As a consequence, epigenetically targeted therapies may be an ideal alternative strategy for AML treatment. In this study, we used a drug library (BML-2836) to select potential drugs for AML treatment. Following drug screening, two pan-HDAC inhibitors, trichostain A(TSA) and LBH589 were selected for further investigation. We found that TSA and LBH589 had good antileukemic effects on cabozantinib-sensitive leukemia cell MV4-11 cells and cabozantinib-resistant leukemia cell MV4-11-XR cells. IC50 for TSA against MV4-11 cells and MV4-11-XR cells were 59.93±15.9 nM and 108.9±40.4 nM, respectively; and IC50 for LBH589 against MV4-11 cells and MV4-11-XR cells were 1.80±0.24 nM and 5.03±1.50 nM, respectively. We also found that either TSA or LBH589 treatment could trigger G0/G1 cell cycle arrest and inhibit the PI3K and MAPK signaling pathways, subsequently accompanied by cell apoptosis and DNA damage. Moreover, both TSA and LBH589 inhibited colony forming ability and reduced the expression of self-renewal related genes and promoted the expression of tumor suppressor gene in both MV4-11 cells and MV4-11-XR cells. Finally, we evaluated the pan-HDAC inhibitor in combination with cabozantinib in MV4-11 cells and MV4-11-XR cells. Combination treatment of TSA and cabozantinib showed synergistic effect on MV4-11-XR cells and additive effect on MV4-11 cells. LBH589 plus cabozantinib showed synergistic effect on MV4-11 cells and MV4-11-XR cells. In this study, we demonstrated that pan-HDAC inhibitor exhibited promising antileukemic activity againt both MV4-11 cells and MV4-11-XR cells. Therefore, pan-HDAC inhibitor may be a clinically poteinal therapeutic agent in treating drug-resistance AML, improving the survival rates of patients.