Haertle Larissa, Barrio Santiago, Munawar Umair, Han Seungbin, Zhou Xiang, Simicek Michal, Vogt Cornelia, Truger Marietta, Fernandez Rafael Alonso, Steinhardt Maximilian, Weingart Julia, Snaurova Renata, Nerreter Silvia, Teufel Eva, Garitano-Trojaola Andoni, Da Viá Matteo, Ruiz-Heredia Yanira, Rosenwald Andreas, Bolli Niccolò, Hajek Roman, Raab Peter, Raab Marc S, Weinhold Niels, Haferlach Claudia, Haaf Thomas, Martinez-Lopez Joaquin, Einsele Hermann, Rasche Leo, Kortüm K Martin
Department of Internal Medicine II, University Hospital Würzburg, Würzburg, Germany.
Department of Hematology, Hospital Universitario 12 de Octubre, Spanish National Cancer Research Center, Complutense University Madrid, Madrid, Spain.
Clin Cancer Res. 2023 Jan 4;29(1):279-288. doi: 10.1158/1078-0432.CCR-22-1161.
Proteasome inhibitors (PI) are the backbone of various treatment regimens in multiple myeloma. We recently described the first in-patient point mutations affecting the 20S subunit PSMB5 underlying PI resistance. Notably, in vivo, the incidence of mutations in PSMB5 and other proteasome encoding genes is too low to explain the development of resistance in most of the affected patients. Thus, additional genetic and epigenetic alterations need to be explored.
We performed DNA methylation profiling by Deep Bisulfite Sequencing in PSMB5, PSMC2, PSMC5, PSMC6, PSMD1, and PSMD5, a subset of proteasome subunits that have hitherto been associated with PI resistance, recruited from our own previous research, the literature, or a meta-analysis on the frequency of somatic mutations. Methylation was followed up on gene expression level and by dual-luciferase reporter assay. The KMS11 cell line served as a model to functionally test the impact of demethylating agents.
We identified PSMD5 promoter hypermethylation and subsequent epigenetic gene silencing in 24% of PI refractory patients. Hypermethylation correlated with decreased expression and the regulatory impact of this region was functionally confirmed. In contrast, patients with newly diagnosed multiple myeloma, along with peripheral blood mononuclear cells and CD138+ plasma cells from healthy donors, generally show unmethylated profiles.
Under the selective pressure of PI treatment, multiple myeloma cells acquire methylation of the PSMD5 promoter silencing the PSMD5 gene expression. PSMD5 acts as a key orchestrator of proteasome assembly and its downregulation was described to increase the cell's proteolytic capacity. PSMD5 hypermethylation, therefore, represents a novel mechanism of PI tolerance in multiple myeloma.
蛋白酶体抑制剂（PI）是多种骨髓瘤治疗方案的核心。我们最近报道了首例影响20S亚基PSMB5的住院患者点突变，该突变是PI耐药的基础。值得注意的是，在体内，PSMB5和其他蛋白酶体编码基因突变的发生率过低，无法解释大多数受影响患者耐药性的产生。因此，需要探索其他遗传和表观遗传改变。
我们通过深度亚硫酸氢盐测序对PSMB5、PSMC2、PSMC5、PSMC6、PSMD1和PSMD5进行DNA甲基化分析，这些蛋白酶体亚基是从我们之前的研究、文献或体细胞突变频率的荟萃分析中筛选出来的，此前已发现它们与PI耐药有关。对甲基化情况进行基因表达水平跟踪，并通过双荧光素酶报告基因检测进行验证。使用KMS11细胞系作为功能测试去甲基化剂影响的模型。
我们在24%的PI难治性患者中发现了PSMD5启动子高甲基化及随后的表观遗传基因沉默。高甲基化与表达降低相关，并且该区域的调控作用在功能上得到了证实。相比之下，新诊断的骨髓瘤患者以及健康供体的外周血单个核细胞和CD138 +浆细胞通常表现为非甲基化状态。
在PI治疗的选择压力下，骨髓瘤细胞获得PSMD5启动子甲基化，使PSMD5基因表达沉默。PSMD5是蛋白酶体组装的关键协调因子，其下调被认为可增加细胞的蛋白水解能力。因此，PSMD5高甲基化代表了骨髓瘤中PI耐受的一种新机制。
