热激对砷剂耐药 PML-RARα 蛋白降解作用及参与蛋白的发现

Arsenic trioxide (As2O3) has been world widely used to treat acute promyelocytic leukemia (APL), and PML-RARα onco-fusion protein solubility changes, followed by SUMOylation and degradation by arsenic are considered to be the key process for APL treatment. Unfortunately, As2O3-resistance patients are frequently found in relapsed APL after consolidation therapy, which are considered to be associated with missense mutations in B-box2 domain of PML-RARα (P/R) fusion protein. In this project, we for the first time identified that heat treatment (43°C) not only induce wild type P/R fusion protein degradation, but also induce missense mutants P/R fusion protein degradation. Additionally, we found that ligand binding domain (LBD) of RARα portion is responsible for PML-RARα fusion protein degradation by heat treatment. More interestingly, although arsenic alone is unable to induce the mutants P/R fusion protein degradations, combination of heat treatment with As2O3 significantly increase the P/R fusion protein SUMOylation and ubiquitination as well as facilitate its protein degradation. The extraordinary characteristics of heat treatment in both wild type and missense mutants P/R fusion protein solubility changes and/or degradations makes us enthusiastic to identify the probable mechanism of action of heat treatment in APL therapy. Therefore, in the current project we are trying to resolve the following issues, i.e., (1) to clarify the exact action site of P/R protein that responsible for protein degradation by heat treatment; (2) to identify what kind of nuclear proteins are involved in P/R protein solubility changes and degradation as well as its functional and physiological significances; (3) how combination of heat treatment with As2O3 could facilitate its mutant P/R protein degradation or solubility changes? Thus, this project will helpful to improve therapeutic strategies for newly diagnosed and arsenic-resistance APL in near future.

砷剂已被广泛应用于治疗急性早幼粒白血病（APL）。然而，在治疗过程中部分患者出现砷剂耐药现象，其原因由于PML蛋白的B-box2结构域上发生点突变，导致砷剂无法诱导上述PML/RARα(P/R)突变体的溶性改变，使其无法进一步降解，故而产生耐药。我们首次发现单用热激（43°C）不仅能够诱导野生型P/R蛋白的降解，还能够使得砷剂耐药的所有P/R突变体的降解。我们还发现RARα部分的LBD结构域是热激诱导P/R融合蛋白溶性转变及降解的关键性区域。有趣的是，虽然砷剂不能诱导P/R突变体的溶性改变，但是热激与砷剂联用可增强P/R突变体的SUMO化及Ub修饰，加速其降解。本项目通过研究热激降解砷剂耐药P/R融合蛋白，明确回答以下三个问题：明确热激诱导P/R蛋白的降解的作用位点；确定参与热激反应的关键蛋白及其功能和生理学意义；揭示热激与砷剂协同作用的分子机理，有望用于治疗APL患者（包括砷剂耐药）。

PML/RARα融合蛋白是急性早幼粒细胞白血病（APL）的致癌融合蛋白，被认为驱动癌症发生。尽管大多数APL患者通过PML/RARα靶向治疗药物如三氧化二砷、全反式维甲酸等能够被治愈，但部分患者由于融合蛋白发生耐药突变，导致复发和耐药，对于这部分患者，目前并无有效治疗手段。在本课题的研究中，我们发现热激能够使PML/RARα蛋白发生稳定性和可溶性改变，并且对临床上鉴定的三氧化二砷和全反式维甲酸的耐药突变体也具有同样的作用。通过对其机制的探究，我们发现温和的高温刺激PML/RARα与核受体协同抑制因子NCoR/SMART复合物发生聚集，并且在SIAH2 E3泛素连接酶介导下发生泛素化修饰，最终通过自噬-溶酶体和泛素-蛋白酶体途径降解。由于热激和砷治疗通过不同的机制破坏PML/RARα的稳定性从而使其降解，我们发现热激和砷具有较好的协同作用，并在原发性患者样本中得到验证。进一步的，我们通过热激和砷联合治疗三例难治性APL病例，均获得了不同程度的缓解。此外，我们发现热激对AML1/ETO和TEL/AML1融合蛋白具有类似的作用。总之，我们的结果表明，通过利用PML/RARα的生物物理脆弱性，热激疗法可以改善耐药或难治性APL的预后。本课题的研究为临床热激治疗复发难治急性早幼粒白血病提供了理论依据。