Department of Hematology and Immunology-Myeloma Center Brussels, Vrije Universiteit Brussel, Laarbeeklaan 103, 1090, Jette, Brussels, Belgium.
Department of Clinical Hematology, Ghent University Hospital, Gent, Belgium.
J Exp Clin Cancer Res. 2022 Feb 1;41(1):45. doi: 10.1186/s13046-022-02250-3.
Multiple myeloma (MM) remains an incurable cancer despite advances in therapy. Therefore, the search for new targets is still essential to uncover potential treatment strategies. Metabolic changes, induced by the hypoxic bone marrow, contribute to both MM cell survival and drug resistance. Pyrroline-5-carboxylate reductase 1 and 2 (PYCR1 and PYCR2) are two mitochondrial enzymes that facilitate the last step in the glutamine-to-proline conversion. Overexpression of PYCR1 is involved in progression of several cancers, however, its' role in hematological cancers is unknown. In this study, we investigated whether PYCR affects MM viability, proliferation and response to bortezomib.
Correlation of PYCR1/2 with overall survival was investigated in the MMRF CoMMpass trial (653 patients). OPM-2 and RPMI-8226 MM cell lines were used to perform in vitro experiments. RPMI-8226 cells were supplemented with C-glutamine for 48 h in both normoxia and hypoxia (< 1% O, by chamber) to perform a tracer study. PYCR1 was inhibited by siRNA or the small molecule inhibitor pargyline. Apoptosis was measured using Annexin V and 7-AAD staining, viability by CellTiterGlo assay and proliferation by BrdU incorporation. Differential protein expression was evaluated using Western Blot. The SUnSET method was used to measure protein synthesis. All in vitro experiments were performed in hypoxic conditions.
We found that PYCR1 and PYCR2 mRNA expression correlated with an inferior overall survival. MM cells from relapsed/refractory patients express significantly higher levels of PYCR1 mRNA. In line with the strong expression of PYCR1, we performed a tracer study in RPMI-8226 cells, which revealed an increased conversion of C-glutamine to proline in hypoxia. PYCR1 inhibition reduced MM viability and proliferation and increased apoptosis. Mechanistically, we found that PYCR1 silencing reduced protein levels of p-PRAS40, p-mTOR, p-p70, p-S6, p-4EBP1 and p-eIF4E levels, suggesting a decrease in protein synthesis, which we also confirmed in vitro. Pargyline and siPYCR1 increased bortezomib-mediated apoptosis. Finally, combination therapy of pargyline with bortezomib reduced viability in CD138 MM cells and reduced tumor burden in the murine 5TGM1 model compared to single agents.
This study identifies PYCR1 as a novel target in bortezomib-based combination therapies for MM.
尽管在治疗方面取得了进展，但多发性骨髓瘤（MM）仍然是一种无法治愈的癌症。因此，寻找新的靶点对于发现潜在的治疗策略仍然至关重要。低氧骨髓引起的代谢变化有助于 MM 细胞的存活和耐药性。吡咯啉-5-羧酸还原酶 1 和 2（PYCR1 和 PYCR2）是两种线粒体酶，可促进谷氨酰胺到脯氨酸转化的最后一步。PYCR1 的过表达与多种癌症的进展有关，然而，其在血液系统癌症中的作用尚不清楚。在这项研究中，我们研究了 PYCR 是否影响 MM 的活力、增殖和对硼替佐米的反应。
在 MMRF CoMMpass 试验（653 例患者）中研究了 PYCR1/2 与总生存期的相关性。使用 OPM-2 和 RPMI-8226 MM 细胞系进行体外实验。RPMI-8226 细胞在常氧和低氧（<1% O，通过室）下用 C-谷氨酰胺补充 48 小时，以进行示踪研究。用 siRNA 或小分子抑制剂帕吉林抑制 PYCR1。用 Annexin V 和 7-AAD 染色测量细胞凋亡，用 CellTiterGlo 测定法测量细胞活力，用 BrdU 掺入法测量增殖。使用 Western Blot 评估差异蛋白表达。使用 SUnSET 法测量蛋白质合成。所有体外实验均在低氧条件下进行。
我们发现 PYCR1 和 PYCR2 mRNA 表达与总体生存不良相关。复发/难治性患者的 MM 细胞表达明显更高水平的 PYCR1 mRNA。与 PYCR1 的强烈表达一致，我们在 RPMI-8226 细胞中进行了示踪研究，结果表明在低氧条件下 C-谷氨酰胺向脯氨酸的转化率增加。PYCR1 抑制降低了 MM 的活力和增殖，并增加了细胞凋亡。在机制上，我们发现 PYCR1 沉默降低了 p-PRAS40、p-mTOR、p-p70、p-S6、p-4EBP1 和 p-eIF4E 水平的蛋白水平，表明蛋白质合成减少，这也在体外得到了证实。帕吉林和 siPYCR1 增加了硼替佐米介导的细胞凋亡。最后，与单药治疗相比，帕吉林与硼替佐米联合治疗可降低 CD138 MM 细胞的活力并减少 5TGM1 小鼠模型中的肿瘤负担。
这项研究确定了 PYCR1 是硼替佐米为基础的 MM 联合治疗的新靶点。
