以骨髓系特異表現FLT3-ITD轉殖基因斑馬魚作為白血病藥物篩選平台的評估

Abstract

斑馬魚有生長周期快速、胚胎透明易觀察和對小分子化合物穿透性高的優點，且在基因序列及造血過程與哺乳類相似，而常成為研究胚胎造血及相關血液疾病的模型和大規模基因或藥物篩選的動物平台。 急性骨髓性白血病(AML)為一種骨髓系白血球不正常增生及分化的血液疾病 ，在AML的病人中含有FLT3-ITD (為FMS-like tyrosine kinase 3 (FLT3)的juxtamembrane domain突變 )突變的病人占20 ~30%且通常有著較差的預後、低存活率及高復發率；研究指出FLT3和細胞的存活、增生及分化有關，並已在細胞及老鼠實驗中發現FLT3-ITD確實會造成骨髓系白血球不正常增生並抑制其分化。本實驗室之前已成功建立一個可經由骨髓系白血球專一表現之啟動子驅動人類FLT3-ITD表現的基因轉殖斑馬魚模型- Tg(spi1:FLT3-ITD-2A-EGFP)。本論文主要是探討此斑馬魚之胚胎造血特性，並評估此斑馬魚能成為一個AML藥物篩選平台的可能性。 首先使用原位雜交(WISH)來觀察各種血球細胞的標誌，以評估各血球細胞表現狀況。先觀察骨髓系白血球相關標誌spi1、cebpa和mpo在48 hpf的斑馬魚中的表現量，發現在過度表現FLT3-ITD的斑馬魚胚胎中，三者的表現量均增加，也代表著骨髓系白血球的增加，並同時使用蘇丹黑染色再確認此骨髓系白血球增加的結果。另外，在此斑馬魚中可看到48 hpf時，紅血球標誌hbae1表現量減少及96 hpf在胸腺部位淋巴性白血球標誌rag1表現量增加。接著使用Q-PCR分析48 hpf時各造血相關轉錄因子之表現情況，發現血液血管母細胞(scl和lmo2)、淋巴性白血球(rag1)、骨髓系白血球(cebpa和mpo)相關之轉錄因子都有受到FLT3-ITD的影響而有改變；這些改變可能是造成斑馬魚中三種細胞數量都增加的原因。在紅血球中發現轉錄因子gata1的表現量與野生型斑馬魚的並無差異；可是代表血紅素之hbae1的表現量卻有減少的現象，顯示成熟紅血球減少。另外，巨噬細胞相關之轉錄因子則無顯著增加或減少。另一方面，在次級造血hematopoiestic stem cell (HSC)相關之轉錄因子的部分則無顯著增加或減少，推測在骨髓系白血球中專一且過度表現FLT3-ITD的情況下不會影響次級造血。 接著，我們使用常見AML治療藥物以及數種FLT3-ITD的標靶藥物來評估此平台可行性；將6 hpf的斑馬魚胚胎分別浸泡於Cytarabine (AraC)、Midostaurin (PKC412)、Quizartinib (AC220)和Cabozantinib (XL184)或DMSO(對照組)的溶液中，在48 hpf時收集胚胎以原位雜交- mpo及蘇丹黑染色作分析。結果顯示，使用AraC、PKC412和AC220者皆可抑制斑馬魚骨髓系白血球增加的情形，並回復到類似野生型斑馬魚的數量；而在100 nM的XL184中則無抑制效果。雖然將XL184的濃度提高到250 nM可以抑制骨髓系白血球的增加，但卻有20%的魚胚胎發生心臟水腫的現象。 從以上結果中可知，Tg(spi1:FLT3-ITD-2A-EGFP)胚胎雖然只在骨髓系白血球中專一且過度表現FLT3-ITD，卻會造成骨髓系白血球及淋巴性白血球的增加和紅血球的減少；使用AML相關藥物AraC、PKC412和AC220均可達到骨髓系白血球減少的效果。因此我們建議此基因轉殖斑馬魚可以作為白血病藥物篩選平台。

With the advantage of rapid growth, transparent embryos, high permeability for small molecules until 5 dpf and high similarity of hematopoiesis-associated gene sequences to mammals, zebrafish has been as a powerful animal model to study the hematopoiesis and hematologic malignancies, and as a suitable platform for large-scale genetic and drug screening. Acute myeloid leukemia (AML) is a hematologic malignancy characterized by uncontrolled proliferation of myeloid progenitor cells. FLT3-ITD mutation at juxtamembrane domain occurs in 20 ~30% AML patient and is associated with poor prognosis, low survival time and high frequency of relapse. FLT3 can regulate proliferation, differentiation and apoptosis of hematopoietic progenitor cells; in contrast, FLT3-ITD mutation could result in uncontrolled cell proliferation and suppression of myeloid cell maturation both in vitro and in vivo. Previously, we established a transgenic zebrafish- Tg(spi1:FLT3-ITD-2A-EGFP) and characterized its leukemia phenotype. In this study, we want to evaluate the feasibility of this transgenic zebrafish to be an in vivo platform for anti-leukemic drugs screening. At first, we found that the expressions of the myeloid cell markers, spi1, cebpα and mpo, were increased in 48 hpf fish by using whole-mount in situ hybridization (WISH); subsequently, the sudan black (SB) staining revealed similar findings. In addition, we found that the expression of lymphoid cell marker, rag1, was increased in 96 hpf fish, but the expression of erythroid cell marker, hbae1, was decreased in 48 hpf fish. Then, quantitative real-time PCR (Q-PCR) revealed that the expressions of hemangioblast, myeloid cells and lymphoid cell-related transcription factors were increased in this zebrafish. There were no significant differences in the expression of erythroid cell-related transcription factor, gata1; however, significant decreased hbae1 were noted. These results demonstrated decreased mature erythroid cells were decreased in this transgenic zebrafish. Finally, there were no significant differences in the expression of macrophage-related and definitive HSC-related transcription factors. Then, we evaluated the effects of several anti-leukemic drugs on this transgenic zebrafish Tg(spi1:FLT3-ITD-2A-EGFP). Embryos at 6hpf were incubated with Cytarabine (AraC), Midostaurin (PKC412), Quizartinib (AC220), Cabozantinib (XL184) or DMSO. The numbers of mpo+ cells and SB+ cells were calculated at 48 hpf. We found that AraC, PKC412 and AC220 significantly reduced the amount of myeloid cells in this zebrafish, while 100 nM XL184 had no effects on this zebrafish. Although XL184 at dosage of 250 nM could reduce the amount of myeloid cells in this zebrafish, the heart edema was emerged in 20% embryos at the same time. In conclusion, although the Tg(spi1:FLT3-ITD-2A-EGFP) zebrafish only expressed FLT3-ITD in myeloid cells, increased myeloid cells and lymphoid cells, as well as decreased erythroid cells were demonstrated. All compounds we used in this study could decrease the amount of myeloid cells in this zebrafish; therefore, Tg(spi1:FLT3-ITD-2A-EGFP) zebrafish would be an appropriate platform for screening anti-leukemic drugs.