Elucidation of the mechanism of disease of VEXAS Syndrome

项目摘要 阐明一种新的特征性克隆骨髓和自身炎性疾病，VEXAS综合征， 提示重新检查泛素化的主调节器，UBA 1。VEXAS代表了E1，X- 连锁、自身炎症、躯体综合征，并伴有不同的炎症（如复发性 多发性骨髓炎、血管炎）和血液学（例如全血细胞减少症、骨髓增生异常综合征）病症。的 50岁以上的男性发病率为1：4000，死亡率为40%。目前的治疗 仅限于高剂量皮质类固醇。在受影响的患者中，UBA 1基因的获得性或体细胞变异是 在造血干细胞（HPSC）中存在并成为仅限于骨髓细胞的谱系。Vexas介绍 主要在老年男性中，因为UBA 1位于X染色体上。UBA 1编码初级泛素（Ub） 活化酶（E1），占所有下游泛素化的97%以上。蛋白质泛素化发生 通过严格调节的酶级联反应，其中依次激活E1至Ub-缀合酶（E2）， 泛素连接酶（E3）最终导致底物泛素化。以前的研究主要集中在 E3连接酶在疾病进展中的作用，因为它们在泛素化中具有蛋白特异性靶向作用。少得多 关于UBA 1在疾病发病机制中的作用是已知的。值得注意的是，VEXAS强调了以下方面的重要性： UBA 1在维持免疫稳态和驱动克隆性血液病中的作用通过转录组和细胞因子 通过分析方法，我们的研究小组已经确定了突变的骨髓细胞是炎症的主要驱动因素。 尽管潜在的机制仍不清楚。在这里，我建议确定的机制VEXAS 使用无偏的方法。尽管HSPC中存在UBA 1突变，但该突变仅限于 髓样细胞和淋巴细胞中缺失。我们利用温度敏感细胞系的初步结果 （中国人卵巢[CHO]品系ts 20）表明，VEXAS突变体UBA 1不能对卵巢上皮细胞的同源E2进行充电。 内质网相关降解途径（ERAD），UBE 2 J1和UBE 2G 2，可能上调 未折叠蛋白反应（Unfolded protein response，UPR）这些发现表明UBA 1的不适当表达可能 在VEXAS中驱动疾病，并允许促炎性骨髓谱系的选择性克隆扩增， 通过可能与ERAD和UPR相关的特定下游效应物增强。在本研究中，我建议1） 对驱动UBA 1依赖性炎症通路的新型效应物进行无偏描绘，以及2） 通过模型系统和无偏方法， 小分子、生物和转基因方法。我的综合研究不仅对 阐明VEXAS的疾病和治疗机制，但也可能适用于更广泛的理解， 关键的细胞泛素化过程、自身炎性疾病和骨髓源性疾病。

PROJECT SUMMARY Elucidation of a newly characterized clonal bone marrow and autoinflammatory disease, VEXAS Syndrome, prompts re-examination of the master regulator of ubiquitylation, UBA1. VEXAS stands for Vacuoles, E1, X- linked, Autoinflammatory, Somatic syndrome and presents with disparate inflammatory (e.g. relapsing polychondritis, vasculitides) and hematologic (e.g. pancytopenia, myelodysplastic syndrome) conditions. The disease is present in 1:4000 men over the age of 50 and has a 40% mortality rate. Current therapeutic treatment is limited to high dose corticosteroids. In affected patients, acquired or somatic variants in the UBA1 gene are present in hematopoietic stem cells (HPSCs) and become lineage restricted to myeloid cells. VEXAS presents primarily in elderly males as UBA1 is located on the X-chromosome. UBA1 encodes the primary ubiquitin (Ub) activating enzyme (E1), accounting for over 97% of all downstream ubiquitylation. Protein ubiquitylation occurs via a tightly regulated enzymatic cascade where sequential activation of E1 to Ub-conjugating enzyme (E2) to Ub-ligase (E3) enzymes ultimately leads to substrate ubiquitylation. Prior studies have predominantly focused on the role of E3 ligases in disease progression due to their protein-specific targeting in ubiquitylation. Far less is known regarding the role of UBA1 in disease pathogenesis. Notably, VEXAS highlights the importance of UBA1 in maintaining immune homeostasis and driving clonal blood disease. Through transcriptomic and cytokine profiling approaches, our group has identified mutant myeloid cells as the primary drivers of inflammation although the underlying mechanism remains unclear. Here I propose to determine the mechanism of VEXAS using unbiased approaches. Despite UBA1 mutation present in HSPCs, the mutation is lineage restricted to myeloid cells and absent in lymphocytes. Our preliminary results utilizing a temperature sensitive cell line (Chinese Hamster Ovary [CHO] line ts20) suggests VEXAS mutant UBA1 fails to charge the cognate E2s of the endoplasmic reticulum associated degradation pathway (ERAD), UBE2J1 and UBE2G2, potentially upregulating the unfolded protein response (UPR). These findings suggest that inappropriate UBA1 expression may both drive disease in VEXAS and allow selective clonal expansion of a pro-inflammatory myeloid lineage as potentiated by specific downstream effectors possibly linked to ERAD and UPR. In this study I propose to 1) perform an unbiased delineation of novel effectors driving UBA1-dependent inflammation pathways and 2) functionally validate effectors identified through model systems and unbiased approaches through a combination of small molecule, biologic, and transgenic approaches. My combined studies will not only be important for elucidating the mechanism of disease and treatment in VEXAS but may also apply to a wider understanding of key cellular ubiquitylation processes, autoinflammatory disease, and bone marrow-derived diseases.