PO.ET03.07 · 实验与分子治疗
果糖转运蛋白的过表达增强促存活信号并赋予Ph样B细胞急性淋巴细胞白血病对venetoclax的耐药
Overexpression of fructose transporter increases pro-survival signaling and confers venetoclax resistance in Ph-like B-cell acute lymphoblastic leukemia
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作者与单位 Authors & Affiliations
摘要 Abstract
中文摘要
费城染色体样（Ph样）B细胞急性淋巴细胞白血病（B-ALL）是一种高危亚型，其特征为治疗耐药和与Ph+ ALL相似的不良临床特征。近期证据凸显了代谢重编程在B-ALL耐药中日益增长的重要性。与其他B-ALL亚群相比，我们在Ph样和Ph+原代样本及细胞系中鉴定出果糖转运蛋白GLUT5表达增加。本研究中，我们旨在探讨GLUT5在Ph样B-ALL促存活信号和耐药中的新型机制作用。首先，为研究GLUT5和果糖可用性在细胞增殖中的作用，将稳定表达GLUT5或空载体对照的Ph样B-ALL细胞系（MHH-cALL4和MUT5Z）培养于富含葡萄糖或果糖的标准培养基中。与富含葡萄糖培养基中的对照相比，富含葡萄糖或果糖培养基中的GLUT5过表达B-ALL细胞系显示出增殖增加（通过CFSE定量）。GLUT5过表达细胞在富含葡萄糖或果糖的培养基中显示出集落形成增加，而对照细胞仅在富含果糖培养基中（与富含葡萄糖培养基中相比）显示出相同效应，表明GLUT5介导的果糖摄取提供了增殖优势。为更好地了解GLUT5表达在高危B-ALL中的意义，对富含葡萄糖或果糖培养基中的对照或GLUT5过表达Ph样B-ALL细胞系进行了bulk RNA-Seq。促存活信号通路（如PI3K/AKT、MAPK、NFKB、HIF-alpha等）在富含葡萄糖或果糖条件下的GLUT5过表达细胞系以及富含果糖条件下的对照细胞中（对比富含葡萄糖条件下的对照）显著上调。我们通过免疫荧光和免疫印迹证实，富含葡萄糖或果糖条件下的GLUT5过表达Ph样B-ALL细胞系中p-AKT/AKT、MYC和NFKB表达增加。此外，仅在富含果糖条件下的GLUT5过表达Ph样细胞系中观察到BCL-2表达增加，提示GLUT5和果糖可用性在赋予venetoclax耐药中的潜在作用。与此一致，在富含果糖培养基中用venetoclax处理的GLUT5过表达B-ALL细胞系较对照显示出显著增加的耐药。venetoclax与丝氨酸合成通路（SSP）抑制剂联合可部分逆转venetoclax耐药。这提示GLUT5表达和果糖可用性可能将糖酵解通路中间产物转向SSP，作为一种替代的细胞存活机制。总之，GLUT5在高危B-ALL中可能具有临床相关作用，其调节促存活信号并赋予治疗耐药。
AD和VP为同等贡献的资深作者
查看英文原文 English abstract
Philadelphia chromosome-like (Ph-like) B-cell acute lymphoblastic leukemia (B-ALL) represents a high-risk subtype characterized by therapy resistance and an adverse clinical profile similar to Ph+ ALL. Recent evidence highlights the growing significance of metabolic reprogramming in drug resistance in B-ALL. We have identified increased fructose transporter GLUT5 expression in Ph-like and Ph+ primary samples and cell lines compared to other B-ALL subsets. In this study, we aim to investigate the novel mechanistic roles of GLUT5 in pro-survival signaling and drug resistance in Ph-like B-ALL. Firstly, to study the role of GLUT5 and fructose availability in cell proliferation, Ph-like B-ALL cell lines (MHH-cALL4 and MUT5Z) stably expressing GLUT5 or empty vector control, were cultured in glucose- or fructose-rich standard media. GLUT5-overexpressing B-ALL cell lines in glucose- or fructose-rich media showed increased proliferation (quantified by CFSE) when compared to control in glucose-rich media. GLUT5-overexpressing cells showed increased colony formation in glucose- or fructose-rich media, while control cells showed the same effect only in fructose-rich media when compared to those in glucose-rich media, indicating that GLUT5-mediated fructose uptake provided a proliferation advantage. To gain better insights on the significance of GLUT5 expression in high-risk B-ALL, bulk RNA-Seq was performed on control or GLUT5-overexpressing Ph-like B-ALL cell lines in glucose- or fructose-rich media. Pro-survival signaling pathways (such as PI3K/AKT, MAPK, NFKB, HIF-alpha and others) were significantly upregulated in GLUT5-overexpressing cell lines in glucose- or fructose-rich and control cells in fructose-rich conditions (vs. control in glucose-rich condition). We confirmed increased expression of p-AKT/AKT, MYC and NFKB in GLUT5-overexpressing Ph-like B-ALL cell lines in glucose- or fructose-rich conditions by immunofluorescence and immunoblotting. Further, increased BCL-2 expression was observed in GLUT5-overexpressing Ph-like cell lines in fructose-rich conditions only, indicative of the potential role of GLUT5 and fructose availability in conferring venetoclax resistance. Consistent with this, GLUT5-overexpressing B-ALL cell lines treated with venetoclax in fructose-rich media showed significantly increased resistance vs. control. A combination of venetoclax with serine synthesis pathway (SSP) inhibitor partially reversed venetoclax resistance. This suggests that GLUT5 expression and fructose availability may divert glycolytic pathway intermediates to SSP as an alternative cell survival mechanism. In summary, GLUT5 may have clinically relevant role in high-risk B-ALL where it modulates pro-survival signaling and confers therapy resistance.
AD and VP - equally contributing senior authors
利益披露 Disclosure
S. Xavier, None..
S. Rodriguez, None..
Z. Gu, None..
L. Ghoda, None..
S. Blakemore, None..
L. Frenzel, None..
A. Danilov, None..
V. Pullarkat, None.