Department of Biomedical Sciences, City University of Hong Kong, Kowloon, Hong Kong SAR, People's Republic of China; Department of Biomedical Sciences, Cornell University, Ithaca, NY 14853, USA.
The Sixth Affiliated Hospital, Sun Yat-sen University, Guangzhou, People's Republic of China.
Cell Signal. 2020 Jul;71:109555. doi: 10.1016/j.cellsig.2020.109555. Epub 2020 Feb 4.
All-trans retinoic acid (ATRA)-based differentiation therapy has been unsuccessful in treating t(15;17) negative acute myeloid leukemia (AML) patients, motivating interest in combination therapies using ATRA plus other agents. Using the t (15, 17) negative HL-60 human myeloblastic leukemia model, we find that the cyclin-dependent kinase (CDK) inhibitor, roscovitine, augments signaling by an ATRA-induced macromolecular signalsome that propels differentiation and enhances ATRA-induced differentiation. Roscovitine co-treatment enhanced ATRA-induced expression of pS259- pS289/296/301- pS621-c-Raf, pS217/221-Mek, Src Family Kinases (SFKs) Lyn and Fgr and SFK Y416 phosphorylation, adaptor proteins c-Cbl and SLP-76, Vav, and acetylated 14-3-3 in the signalsome. Roscovitine enhanced ATRA-induced c-Raf interaction with Lyn, Vav, and c-Cbl. Consistent with signalsome hyper-activation, roscovitine co-treatment enhanced ATRA-induced G1/0 arrest and expression of differentiation markers, CD11b, ROS and p47 Phox. Because roscovitine regulated Lyn expression, activation and partnering, a stably transfected Lyn knockdown was generated from wt-parental cells to investigate its function in ATRA-induced differentiation. Lyn-knockdown enhanced ATRA-induced up-regulation of key signalsome molecules, c-Raf, pS259-c-Raf, pS289/296/301-c-Raf, Vav1, SLP-76, and Fgr, but with essentially total loss of pY416-SFK. Compared to ATRA-treated wt-parental cells, differentiation markers p47 phox, CD11b, G1/G0 arrest and ROS production were enhanced in ATRA-treated Lyn-knockdown stable transfectants, and addition of roscovitine further enhanced these ATRA-inducible markers. The Lyn-knockdown cells expressed slightly higher c-Raf, pS259-c-Raf, pS289/296/301-c-Raf, and SLP-76 than wt-parental cells, and this was associated with enhanced ATRA-induced upregulation of Fgr and cell differentiation, consistent with heightened signaling, suggesting that enhanced Fgr may have compensated for loss of Lyn to enhance differentiation in the Lyn-knockdown cells.
全反式维甲酸（ATRA）为基础的分化疗法在治疗 t（15；17）阴性急性髓系白血病（AML）患者方面不成功，这促使人们对使用 ATRA 加其他药物的联合治疗产生了兴趣。我们使用 t（15，17）阴性 HL-60 人髓样白血病模型，发现细胞周期蛋白依赖性激酶（CDK）抑制剂罗沙替丁增强了 ATRA 诱导的大分子信号体的信号传导，从而推动了分化，并增强了 ATRA 诱导的分化。罗沙替丁共同处理增强了 ATRA 诱导的 pS259-pS289/296/301-pS621-c-Raf、pS217/221-Mek、Src 家族激酶（SFKs）Lyn 和 Fgr 以及 SFK Y416 磷酸化、衔接蛋白 c-Cbl 和 SLP-76、Vav 和乙酰化 14-3-3 在信号体中的表达。罗沙替丁增强了 ATRA 诱导的 c-Raf 与 Lyn、Vav 和 c-Cbl 的相互作用。与信号体的超激活一致，罗沙替丁共同处理增强了 ATRA 诱导的 G1/0 阻滞和分化标志物 CD11b、ROS 和 p47 Phox 的表达。由于罗沙替丁调节 Lyn 的表达、激活和伙伴关系，从 wt-亲本细胞中生成了稳定转染的 Lyn 敲低细胞，以研究其在 ATRA 诱导的分化中的功能。Lyn 敲低增强了 ATRA 诱导的关键信号体分子 c-Raf、pS259-c-Raf、pS289/296/301-c-Raf、Vav1、SLP-76 和 Fgr 的上调，但 SFK 的 pY416 完全缺失。与 ATRA 处理的 wt-亲本细胞相比，ATRA 处理的 Lyn 敲低稳定转染细胞中 p47 phox、CD11b、G1/G0 阻滞和 ROS 产生增强，并且添加罗沙替丁进一步增强了这些 ATRA 诱导的标志物。与 wt-亲本细胞相比，Lyn 敲低细胞表达稍微更高的 c-Raf、pS259-c-Raf、pS289/296/301-c-Raf 和 SLP-76，这与增强的 ATRA 诱导的 Fgr 和细胞分化上调有关，与增强的信号传导一致，表明增强的 Fgr 可能弥补了 Lyn 的缺失，从而增强了 Lyn 敲低细胞的分化。
