Kubicki Tadeusz, Bednarek Kinga, Kostrzewska-Poczekaj Magdalena, Luczak Magdalena, Lewandowski Krzysztof, Gil Lidia, Jarmuz-Szymczak Malgorzata, Dytfeld Dominik
Department of Hematology and Bone Marrow Transplantation, Poznan University of Medical Sciences Poznan, Poland.
Institute of Human Genetics, Polish Academy of Sciences Poznan, Poland.
Am J Cancer Res. 2022 Jul 15;12(7):3280-3293. eCollection 2022.
Proteasome inhibitors are among the most potent classes of drugs in multiple myeloma treatment. One of the main challenges in myeloma therapy is acquired resistance to drugs. Several theories have been proposed to describe the mechanisms responsible for resistance to the most commonly used proteasome inhibitors bortezomib and carfilzomib. This study aimed to describe functional differences between sensitive myeloma cells (MM1S WT) and their daughter cell lines resistant to either bortezomib (MM1S/R BTZ) or carfilzomib (MM1S/R CFZ), as well as between both resistant cell lines. Bortezomib- and carfilzomib-resistant cell lines were successfully generated by continuous exposure to the drugs. When exposed to different drugs than during the resistance generation period, MM1S/R BTZ cells showed cross-resistance to carfilzomib, whereas MM1S/R CFZ cells were similarly sensitive to bortezomib as MM1S WT cells. Following proteomic profiling, unsupervised principal component analysis revealed that the MM1S/R BTZ and MM1S/R CFZ cell lines differed significantly from the MM1S WT cell line and from each other. Canonical pathway analysis showed similar pathways enriched in both comparisons - MM1S WT vs. MM1S/R CFZ and MM1S WT vs. MM1S/R BTZ. However, important differences were present in the statistical significance of particular pathways. Key alterations included the ubiquitin-proteasome system, metabolic pathways responsible for redox homeostasis and the unfolded protein response. In functional studies, both drugs continued to reduce chymotrypsin-like proteasome activity in resistant cells. However, the baseline activity of all three catalytic domains of the proteasome was higher in the resistant cells. Differences in generation of reactive oxygen species were identified in MM1S/R BTZ (decreased) and MM1S/CFZ cells (increased) in comparison to MM1S WT cells. Both baseline and drug-induced activity of the unfolded protein response were higher in resistant cells than in MM1S WT cells and included all three arms of this pathway: IRE1α/XBP1s, ATF6 and EIF2α/ATF4 (downstream effectors of PERK). In conclusion, contrary to some previous reports, resistant MM1S cells show upregulation of unfolded protein response activity, reflecting the heterogeneity of multiple myeloma and prompting further studies on the role of this pathway in resistance to proteasome inhibitors.
蛋白酶体抑制剂是多发性骨髓瘤治疗中最有效的药物类别之一。骨髓瘤治疗的主要挑战之一是获得性耐药。已经提出了几种理论来描述对最常用的蛋白酶体抑制剂硼替佐米和卡非佐米产生耐药性的机制。本研究旨在描述敏感骨髓瘤细胞（MM1S WT）与其对硼替佐米（MM1S/R BTZ）或卡非佐米（MM1S/R CFZ）耐药的子代细胞系之间的功能差异，以及两种耐药细胞系之间的功能差异。通过持续暴露于药物成功产生了对硼替佐米和卡非佐米耐药的细胞系。当暴露于与耐药产生期不同的药物时，MM1S/R BTZ细胞对卡非佐米表现出交叉耐药，而MM1S/R CFZ细胞对硼替佐米的敏感性与MM1S WT细胞相似。在进行蛋白质组学分析后，无监督主成分分析显示MM1S/R BTZ和MM1S/R CFZ细胞系与MM1S WT细胞系以及它们彼此之间存在显著差异。典型通路分析表明，在MM1S WT与MM1S/R CFZ以及MM1S WT与MM1S/R BTZ这两个比较中富集的通路相似。然而，特定通路的统计学显著性存在重要差异。关键改变包括泛素 - 蛋白酶体系统、负责氧化还原稳态的代谢通路以及未折叠蛋白反应。在功能研究中，两种药物继续降低耐药细胞中的糜蛋白酶样蛋白酶体活性。然而，蛋白酶体所有三个催化结构域的基线活性在耐药细胞中更高。与MM1S WT细胞相比，在MM1S/R BTZ细胞（降低）和MM1S/CFZ细胞（升高）中发现了活性氧生成的差异。耐药细胞中未折叠蛋白反应的基线活性和药物诱导活性均高于MM1S WT细胞，并且包括该通路的所有三个分支：IRE1α/XBP1s、ATF6和EIF2α/ATF4（PERK的下游效应器）。总之，与一些先前的报道相反，耐药的MM1S细胞显示未折叠蛋白反应活性上调，这反映了多发性骨髓瘤的异质性，并促使对该通路在蛋白酶体抑制剂耐药中的作用进行进一步研究。
