抑制DOCK1基因藉由減少Notch訊息傳遞而抑制白血病生成

Abstract

此研究探討DOCK1基因在血液生成中的角色及其於急性骨髓性白血病的臨床與生物意義。DOCK家族的基因轉錄並轉譯一群作用於Rho鳥苷三磷酸酶之演化上保守的鳥糞嘌呤核苷酸交換因子，與多種生理程序的調節有關。DOCK1基因屬於DOCK家族基因的一員，所轉錄並轉譯的DOCK1蛋白已知與乳癌及神經膠母細胞瘤的轉移和侵襲性有關。雖然某些DOCK家族的蛋白可能調控血液生成，而它們的異常可能與白血病生成有關，但是它們在急性骨髓性白血病實際的角色仍不清楚。先前一項研究發現DOCK1基因表現量在急性骨髓性白血病患有獨立的預後意義，且其在急性骨髓性白血病預後評估方面表現更佳，預測能力優於多個已發表的基因標誌。DOCK1基因主要在造血幹細胞表現，且基因富集分析顯示與造血幹細胞及白血病幹細胞相關的基因在DOCK1基因表現高的病患表現也較高，因此我們假設DOCK1基因藉由對細胞幹性、增殖、運動與趨化性的調節而影響白血病病患的預後。我們檢測了多個急性骨髓性白血病細胞株中DOCK1基因的表現量，發現TF1細胞株和HEL92.1.7細胞株(以下簡稱HEL細胞)有較高的DOCK1基因表現。這兩種細胞株在使用CRISPR-Cas9系統剔除DOCK1基因後存活率都降低，而且HEL細胞的凋亡率顯著升高。使用shRNA降低HEL細胞以及DOCK1低表達之HL-60細胞的DOCK1基因表現後也獲得了一致的結果。在異種移植模型中，DOCK1基因表現受抑制的HEL細胞在受體小鼠中移植成功的速度較慢，而接受DOCK1基因表現受抑制的HEL細胞移植的小鼠比接受相同數量對照細胞移植的小鼠存活期顯著延長。小鼠骨髓細胞在剔除造血細胞之Dock1基因後，在5-FU注射試驗與競爭性移植試驗中呈現的造血能力較未剔除Dock1基因之細胞差。藉由反轉錄病毒轉導MN1基因，我們發現剔除Dock1基因的小鼠骨髓細胞在MN1基因過度表現下的增殖與遷移能力較過度表現MN1基因之Dock1基因野生型骨髓細胞差。接受致死劑量輻射照射的小鼠移植了這些骨髓細胞後，其存活期明顯長於接受相同數量之過度表現MN1基因的Dock1基因野生型骨髓細胞移植的小鼠，且血液採樣顯示接受剔除Dock1基因之骨髓細胞移植的小鼠血液中MN1基因過度表現之細胞所佔的百分比亦較低。這些結果顯示DOCK1基因對造血細胞和白血病細胞存活與增殖的重要性。轉錄組分析和免疫化學染色證明DOCK1基因表現與Notch訊號上調存在關聯，而細胞株實驗則顯示DOCK1基因表現與Notch訊號上調之間有因果關係。對MN1基因過度表現的Dock1基因條件性剔除小鼠和Dock1基因野生型小鼠骨髓細胞進行單細胞定序分析，發現一群獨特的細胞群集幾乎只存在於Dock1基因野生型小鼠的樣本中。此群集的幹細胞標誌訊號上調，同時，身為Notch訊號傳導主要目標的Hes1基因表現也增加。這些研究結果突顯了DOCK1基因在急性骨髓性白血病中臨床與致病機轉上的重要性，以及其與Notch訊號傳導的關係，顯示其是潛在的治療標的。

In this study, we focus on the roles of Dedicator of cytokinesis 1 (DOCK1) gene in hematopoiesis and its clinical and biological significance in acute myeloid leukemia (AML). DOCK family genes encode evolutionarily conserved guanine nucleotide exchange factors (GEFs) for Rho GTPases involving in the regulation of various physiological processes. The DOCK1 gene, a member of DOCK family genes, encodes the DOCK1 protein, which has been known to be involved in the migration and invasion of breast cancer cells and glioblastoma cells. Although some of the DOCK family proteins possibly regulate hematopoiesis, and their deregulation might contribute to leukemogenesis, little is known about their roles in AML. A previous study revealed that the mRNA levels of DOCK1 gene have independent prognostic significance in AML cohorts, and its predictive power outperformed several published gene signatures in AML prognostication. In addition, DOCK1 expression appears to be exclusively upregulated in hematopoietic stem cells, and Gene Set Enrichment Analysis (GSEA) also showed concordant upregulation of hematopoietic stem cell- and leukemic stem cell-associated genes in patients with higher DOCK1 expression in the analysis, implying its role in stem cell biology. Therefore, we hypothesize that the DOCK1 influences the prognosis of AML possibly through its correlation with stemness, cell proliferation, motility and chemotaxis. We tested the expression of DOCK1 in various myeloid leukemia cell lines and found TF1 and HEL 92.1.7 (shortened as HEL) cell lines exhibit higher DOCK1 expression. We knocked out the DOCK1 gene in these two cell lines using the CRISPR-Cas9 system. This resulted in decreased viability in both cell lines, with HEL cells showing a higher apoptosis rate after DOCK1 knockout. Consistent findings were obtained following shRNA-mediated DOCK1 knockdown in both HEL cells and DOCK1-low-expressing HL-60 cells. In a xenograft model, the DOCK1-knockdown HEL cells exhibited delayed engraftment in the recipient mice, and the mice transplanted with DOCK1-knockdown HEL cells showed a significantly prolonged survival than those transplanted with the same number of control cells. In the Dock1-conditional knockout (Dock1-cKO) mouse model, the bone marrow cells of Dock1-cKO mice tended to have worse regeneration ability than those of Dock1-wild type (Dock1-WT) mice in the 5-fluorouracil survival assays. In the competitive repopulating units assay, recipient mice transplanted with the bone marrow cells from Dock1-cKO mice showed a significantly inferior capacity to reconstitute their hematopoietic system compared with those transplanted with the bone marrow cells from gender- and age-matched Dock1-WT mice. By means of retroviral transduction, we demonstrated that MN1-overexpressing bone marrow cells from Dock1-cKO mice tend to have a reduced ability to maintain populations, aggregate, and migrate. The lethally irradiated mice transplanted with bone marrow cells from MN1-overexpressing Dock1-cKO mice had a significantly longer median overall survival than those transplanted with the same number of bone marrow cells from MN1-overexpressing Dock1-WT mice, with blood sampling revealing a lower percentage of circulating MN1-overexpressing cells in the recipient mice. These findings support the importance of DOCK1 expression in the survival and proliferation of hematopoietic stem cells and leukemia cells. Transcriptome analyses and immunochemistry stain demonstrated an association between DOCK1 expression and upregulated Notch signaling, and the causal relationship was supported by cell line experiments. Single-cell sequencing analysis of MN1-overexpressing bone marrow cells from Dock1-cKO and Dock1-WT mice identified a unique cell cluster that was present almost exclusively in the Dock1 wildtype background. This cluster was defined by the concomitant upregulation of both a stem cell signature and Hes1 expression, a primary effector of Notch signaling. These findings highlight the clinical and pathogenic significance of DOCK1 in AML and its relationship with Notch signaling, thereby supporting its potential as a therapeutic target.