胞内PSF通过调控PC增强糖异生作用诱导髓系白血病细胞干性和耐药性的分子机制

Multidrug resistance is the root cause of relapse and refractory of acute myeloid leukemia (AML). We reported for the first time that abnormal localization of nucleoprotein PSF outside nucleus was closely related to sensitivity of leukemia chemotherapy: PSF highly expressed on cell membrane in sensitive cell line while accumulated in the cytoplasm in resistant cell line. In the youth project, we have confirmed that PSF can mediate drug resistance and clarified the mechanism of PSF-membrane translocation. Meanwhile, we found that the higher expression level of membrane translocation leads to the better clinical prognosis in patients. However, the mechanism of intracellular PSF mediated drug resistance is still unknown. In the previous stage, we preliminary verified that the function of intracellular PSF mediated resistance was related to pyruvate carboxylase (PC), a rate limiting enzyme of gluconeogenesis. Based on this finding, we hypothesized that PSF enhances gluconeogenesis by regulating PC, which leads to a high glucose microenvironment and further activates downstream factors to induce stem cell characteristics and drug resistance. This study will explore the mechanisms of how PSF to regulated PC, which further promotes gluconeogenesis according to the aspects of inducing PC expression and enhancing PC stability, and the molecular mechanisms of downstream factors activated by high glucose microenvironment inside and outside the cells induce stem cell and multidrug resistance characteristics. We will also verify the functions to the drug resistance mechanisms in cells, animals, and clinical patients. This study will elaborate the mechanism of multidrug resistance mediated by abnormally PSF-cytoplasmic localization, and reveal the relationship among drug resistance, tumor microenvironment, and tumor stem cells, which will provide experimental basis for developing clinical diagnostic and therapeutic target of AML, especially of refractory and relapsed AML.

耐药是急性髓系白血病AML难治复发的根源。我们首次报道细胞核蛋白PSF的核外异常定位和白血病化疗敏感性密切相关：敏感株高度膜易位，耐药株积累在胞浆。青年课题中我们已确证PSF能介导耐药并明确其膜易位机制，发现患者PSF膜易位水平越高临床预后越好；但PSF胞内介导耐药的机理仍不明。我们前期初步验证PSF在胞内介导耐药的作用和糖异生限速酶丙酮酸羧化酶PC相关，推测PSF通过调控PC增强糖异生作用导致细胞内外高糖微环境，高糖激活下游因子诱导干性和耐药性。本研究将从诱导表达和增强稳定性角度探究PSF调控PC促进糖异生的机制；和该机制致细胞内外高糖微环境激活下游因子诱导干性和耐药性的分子机制；在细胞动物和临床水平对上述机制进行功能验证。该研究将深入揭示PSF胞内异常定位介导耐药的详细机制和耐药与微环境及肿瘤干细胞间的关系，为开发PSF评估AML尤其是难治复发AML的临床标志物和治疗靶点提供实验基础。

前期项目中我们通过对PSF细胞膜易位机制的研究发现，在敏感的血液肿瘤细胞中，K18介导PSF向细胞膜转运和定位，过表达PSF后，细胞膜PSF水平持续升高。耐药细胞中缺乏上述机制，PSF积累在细胞内介导耐药性。我们发现野生型的敏感株膜PSF表达水平低于耐药株，仅细胞膜易位水平高于耐药株。这些工作将PSF介导髓系白血病耐药性的机制指向胞内。.敲减PSF，三种髓系白血病细胞系的IC50值显著降低，提示PSF是介导耐药性的原因。对细胞系的功能鉴定发现，PSF敲减后能抑制细胞增殖，使细胞处于严重的生长停滞状态；促进细胞凋亡，在IC20的DOX剂量下，细胞晚期凋亡和坏死比例显著增加。. PSF有维持细胞干性和微弱的调控HIF1α的作用，但这些并非维持耐药性的直接原因。利用患者样本研究中发现，敏感和难治复发患者的差异基因仅富集到蛋白错误折叠内质网应激（UPR/ERS）这一条信号通路上。敲减PSF，BIP和PERK水平显著降低；在敲减PSF的克隆中再过表达PSF，BIP和PERK水平回复；在功能上PERK抑制剂有协同PSF基因敲减的作用，提示PSF直接调控ERS通路且通过PERK发挥作用，PERK表达水平和功能都与PSF正相关。.在临床研究方面我们发现PSF是AML患者的预后不良的独立因子。难治复发(耐药)患者PSF的转录水平高于敏感患者，化疗前检测PSF可精准判断患者化疗结局。多因素回归分析表明，PSF是独立于患者年龄、染色体、融合/突变基因、初诊骨髓象和血象以及化疗方案的预后因子。我们建立了判定难治复发不良事件的基因阈值和PSF预测AML化疗耐药的单基因模型（AUC=0.89）。该模型还可为AML患者做预后分层，尤其对现行NCCN和ELN分层体系不能精细分层的中危组患者也有很高的分层效能。HIF1α具有和PSF类似的作用，但预后评估效能不及PSF。用HIF1α和PSF做联合诊断也不能提高PSF的诊断效能。