Pereira Marcelo S F, Sorathia Kinnari, Sezgin Yasemin, Thakkar Aarohi, Maguire Colin, Collins Patrick L, Mundy-Bosse Bethany L, Lee Dean A, Naeimi Kararoudi Meisam
Center for Childhood Cancer and Blood Disease, Abigail Wexner Research Institute, Nationwide Children's Hospital, Columbus, OH 43205, USA.
Department of Microbial Infection and Immunity, The Ohio State University, Columbus, OH 43210, USA.
Cancers (Basel). 2023 Jan 24;15(3):705. doi: 10.3390/cancers15030705.
Loss of cytotoxicity and defective metabolism are linked to glycogen synthase kinase 3 beta (GSK3β) overexpression in natural killer (NK) cells from patients with acute myeloid leukemia or from healthy donors after expansion ex vivo with IL-15. Drug inhibition of GSK3β in these NK cells improves their maturation and cytotoxic activity, but the mechanisms of GSK3β-mediated dysfunction have not been well studied. Here, we show that expansion of NK cells with feeder cells expressing membrane-bound IL-21 maintained normal GSK3β levels, allowing us to study GSK3β function using CRISPR gene editing. We deleted and expanded paired-donor knockout and wild-type (WT) NK cells and then assessed transcriptional and functional alterations induced by loss of GSK3β. Surprisingly, our data showed that deletion of did not alter cytotoxicity, cytokine production, or maturation (as determined by CD57 expression). However, -KO cells demonstrated significant changes in expression of genes related to rRNA processing, cell proliferation, and metabolic function, suggesting possible metabolic reprogramming. Next, we found that key genes downregulated in -KO NK cells were upregulated in GSK3β-overexpressing NK cells from AML patients, confirming this correlation in a clinical setting. Lastly, we measured cellular energetics and observed that -KO NK cells exhibited 150% higher spare respiratory capacity, a marker of metabolic fitness. These findings suggest a role for GSK3β in regulating NK cell metabolism.
细胞毒性丧失和代谢缺陷与急性髓系白血病患者或健康供体经白细胞介素-15体外扩增后的自然杀伤（NK）细胞中糖原合酶激酶3β（GSK3β）的过表达有关。对这些NK细胞中GSK3β的药物抑制可改善其成熟度和细胞毒性活性，但GSK3β介导的功能障碍机制尚未得到充分研究。在这里，我们表明，用表达膜结合白细胞介素-21的饲养细胞扩增NK细胞可维持正常的GSK3β水平，从而使我们能够使用CRISPR基因编辑来研究GSK3β的功能。我们删除并扩增了配对供体的基因敲除和野生型（WT）NK细胞，然后评估了因GSK3β缺失而诱导的转录和功能改变。令人惊讶的是，我们的数据表明，删除 并未改变细胞毒性、细胞因子产生或成熟度（通过CD57表达确定）。然而， -KO细胞在与核糖体RNA加工、细胞增殖和代谢功能相关的基因表达上表现出显著变化，提示可能存在代谢重编程。接下来，我们发现，在 -KO NK细胞中下调的关键基因在急性髓系白血病患者过表达GSK3β的NK细胞中上调，在临床环境中证实了这种相关性。最后，我们测量了细胞能量代谢，观察到 -KO NK细胞的备用呼吸能力高出150%，这是代谢适应性的一个标志。这些发现表明GSK3β在调节NK细胞代谢中发挥作用。
