Wojtuszkiewicz Anna, Schuurhuis Gerrit J, Kessler Floortje L, Piersma Sander R, Knol Jaco C, Pham Thang V, Jansen Gerrit, Musters René J P, van Meerloo Johan, Assaraf Yehuda G, Kaspers Gertjan J L, Zweegman Sonja, Cloos Jacqueline, Jimenez Connie R
From the ‡Dept. of Pediatric Oncology/Hematology, §Dept. of Hematology.
§Dept. of Hematology.
Mol Cell Proteomics. 2016 Apr;15(4):1281-98. doi: 10.1074/mcp.M115.052944. Epub 2016 Jan 22.
Expression of apoptosis-regulating proteins (B-cell CLL/lymphoma 2 - BCL-2, Myeloid Cell Leukemia 1 - MCL-1, BCL-2 like 1 - BCL-X and BCL-2-associated X protein - BAX) in acute myeloid leukemia (AML) blasts at diagnosis is associated with disease-free survival. We previously found that the initially high apoptosis-resistance of AML cells decreased after therapy, while regaining high levels at relapse. Herein, we further explored this aspect of dynamic apoptosis regulation in AML. First, we showed that the intraindividualex vivoapoptosis-related profiles of normal lymphocytes and AML blasts within the bone marrow of AML patients were highly correlated. The expression values of apoptosis-regulating proteins were far beyond healthy control lymphocytes, which implicates the influence of microenvironmental factors. Second, we demonstrated that apoptosis-resistant primary AML blasts, as opposed to apoptosis-sensitive cells, were able to up-regulate BCL-2 expression in sensitive AML blasts in contact cultures (p= 0.0067 andp= 1.0, respectively). Using secretome proteomics, we identified novel proteins possibly engaged in apoptosis regulation. Intriguingly, this analysis revealed that major functional protein clusters engaged in global gene regulation, including mRNA splicing, protein translation, and chromatin remodeling, were more abundant (p= 4.01E-06) in secretomes of apoptosis-resistant AML. These findings were confirmed by subsequent extracellular vesicle proteomics. Finally, confocal-microscopy-based colocalization studies show that splicing factors-containing vesicles secreted by high AAI cells are taken up by low AAI cells. The current results constitute the first comprehensive analysis of proteins released by apoptosis-resistant and sensitive primary AML cells. Together, the data point to vesicle-mediated release of global gene regulatory protein clusters as a plausible novel mechanism of induction of apoptosis resistance. Deciphering the modes of communication between apoptosis-resistant blasts may in perspective lead to the discovery of prognostic tools and development of novel therapeutic interventions, aimed at limiting or overcoming therapy resistance.
急性髓系白血病（AML）诊断时原始细胞中凋亡调节蛋白（B细胞淋巴瘤/白血病-2 - BCL-2、髓细胞白血病-1 - MCL-1、BCL-2样蛋白1 - BCL-X和BCL-2相关X蛋白 - BAX）的表达与无病生存期相关。我们之前发现，AML细胞最初较高的抗凋亡能力在治疗后降低，而在复发时又恢复到较高水平。在此，我们进一步探讨AML中动态凋亡调节的这一方面。首先，我们表明AML患者骨髓中正常淋巴细胞和AML原始细胞的个体内体外凋亡相关谱高度相关。凋亡调节蛋白的表达值远远超过健康对照淋巴细胞，这暗示了微环境因素的影响。其次，我们证明，与凋亡敏感细胞相反，抗凋亡的原发性AML原始细胞能够在接触培养中上调敏感AML原始细胞中的BCL-2表达（分别为p = 0.0067和p = 1.0）。通过分泌蛋白质组学，我们鉴定出可能参与凋亡调节的新蛋白。有趣的是，该分析表明，参与全局基因调节的主要功能蛋白簇，包括mRNA剪接、蛋白质翻译和染色质重塑，在抗凋亡AML的分泌蛋白组中更为丰富（p = 4.01E-06）。随后的细胞外囊泡蛋白质组学证实了这些发现。最后，基于共聚焦显微镜的共定位研究表明，高凋亡抗性指数（AAI）细胞分泌的含剪接因子的囊泡被低AAI细胞摄取。目前的结果构成了对原发性抗凋亡和敏感AML细胞释放蛋白的首次全面分析。总之，数据表明囊泡介导的全局基因调节蛋白簇释放是诱导抗凋亡的一种合理新机制。解读抗凋亡原始细胞之间的通讯模式可能有望导致发现预后工具并开发新的治疗干预措施，旨在限制或克服治疗抗性。
