靶向性烟酰胺腺嘌呤二核苷酸 CD38的糖水化酶活性在细胞内 NAD 升高的同时发挥抗骨髓瘤作用

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Targeting Nicotinamide Adenine Dinucleotide (NAD) Glycohydrase Activity of CD38 Exerts Anti-Myeloma Effect Accompanying Intracellular NAD Elevation

Introduction. The development of novel agents has improved the outcomes of multiple myeloma (MM) patients. Especially, daratumumab, an anti-CD38 monoclonal antibody which exerts therapeutic effect against MM cells through direct cell damage, antibody dependent cellular cytotoxicity (ADCC) and complement-dependent cytotoxicity (CDC), has shown its high efficacy in clinical practice. CD38 is a transmembrane glycoprotein highly expressed in plasma cells. CD38 is also a major nicotinamide adenine dinucleotide (NAD) glycohydrase in mammalian tissues, which regulate cellular levels of NAD. However, the role of CD38 as a NAD glycohydrase (NADase) in survival of MM cells is not well understood. In the present study, we conducted CD38 enzyme activity inhibition on MM cells using a small molecule compound 78c, a specific inhibitor for NADase enzymatic activity of CD38, in order to study the role of CD38 NADase activity in MM cell survival and to examine whether CD38 enzyme inhibition could be a new therapeutic strategy of MM.

引言。新药物的开发已经改善了多发性骨髓瘤患者的预后。特别是达拉通单抗,这种抗 cd38单克隆抗体通过直接细胞损伤、抗体依赖性细胞毒性(ADCC)和补体依赖性细胞毒性(CDC)对 MM 细胞施加治疗效果,在临床实践中表现出很高的疗效。CD38是一种在浆细胞中高度表达的跨膜糖蛋白。CD38也是哺乳动物组织中一种主要的烟酰胺腺嘌呤二核苷酸,它调节 NAD 的细胞水平。然而,CD38作为 NAD 糖水化酶(NADase)在 MM 细胞存活中的作用还没有得到很好的理解。为了研究 CD38 NADase 活性在 MM 细胞存活中的作用,探讨 CD38 NADase 活性抑制是否可能成为 MM 的一种新的治疗策略。

Materials and methods. MM cell lines (NCI-H929, KMS-12BM, KMS-12PE, U266) were treated with CD38 NADase inhibitor, 78c, in vitro. Viability of MM cell lines and patient-derived MM cells were analyzed by flow cytometry after 7AAD staining. MM cell lines possessing CD38 positive and negative fraction were sorted according to the CD38 expression using CD38 Micro-Beads. CD38 low MM cell lines were treated with All-trans retinoic acid(ATRA)to increase surface CD38 expression. Intracellular NAD and NADH concentrations in MM cells were analyzed using NAD / NADH assay kit. Detection of apoptosis in MM cell lines were examined by Annexin V and PI staining followed by flow cytometry analysis. Caspase inhibitor, Z-VAD-FMK, was used in combination with 78c to study the mechanism of 78c induced MM cell death.

材料和方法。用 CD38型 NADase 抑制剂78c 处理 MM 细胞系(NCI-H929、 KMS-12BM、 KMS-12PE、 U266)。流式细胞仪检测7AAD 染色后 MM 细胞株和患者来源 MM 细胞的活性。利用 CD38微球对 CD38阳性和阴性的 MM 细胞系进行了分类。全反式维甲酸(All-trans retinoic acid,ATRA)处理 CD38低 MM 细胞株,增加细胞表面 CD38的表达。用 NAD/NADH 检测试剂盒测定 MM 细胞内 NAD 和 NADH 浓度。应用 Annexin v 和 PI 染色检测 MM 细胞凋亡,流式细胞仪检测细胞凋亡。应用半胱氨酸天冬氨酸蛋白酶抑制剂 Z-VAD-FMK 与78c 联合应用研究78c 诱导 MM 细胞死亡的机制。

Results. 78c induced cell death in MM cell lines at low concentrations (IC50 10-20 μM). Addition of 78c to patient derived bone marrow cells showed cytotoxicity to MM cells, while toxicity to non-MM cells were limited. CD38 positive fraction of MM cell lines had better sensitivity to 78c compared to CD38 negative fraction. CD38 induction by ATRA in CD38 low MM cell lines showed increased sensitivity to 78c. These results proved that 78c efficacy correlates with surface CD38 expression. Comparison of intracellular NAD and NADH concentrations between CD38 positive and negative fractions of MM cell lines demonstrated a significant increase of NAD in the CD38 negative fraction compared to their positive counterparts, indicating that CD38 is indeed controlling the intracellular NAD concentration. Marked increase of NAD / NADH ratio was observed in 78c treated MM cell lines compared to control, proving that CD38 NADase inhibition affects intracellular NAD concentration in MM cells (Fig. 1). 78c treatment of MM cell lines significantly reduced the number of viable cells in the Annexin / PI region, however, addition of Z-VAD-FMK did not lead to recovery of viable cell numbers, indicating non-apototic cell death induction by CD38 NADase inhibition.

结果。78c 低浓度(ic5010-20m)诱导 MM 细胞死亡。加入78c 的骨髓细胞对 MM 细胞具有细胞毒性,而对非 MM 细胞的毒性较小。多发性骨髓瘤细胞系 CD38阳性分数对78c 的敏感性高于 CD38阴性分数。ATRA 诱导的 CD38低 MM 细胞对78c 的敏感性增加。这些结果表明78c 的疗效与表面 CD38的表达有关。比较 CD38阳性和阴性细胞株与阳性细胞株的细胞内 NAD 和 NADH 浓度,发现 CD38阴性细胞株与阳性细胞株相比,NAD 明显增加,说明 CD38确实控制了细胞内 NAD 浓度。与对照组相比,78c 处理的 MM 细胞 NAD/NADH 比值明显增加,证明 CD38 NADase 抑制对 MM 细胞内 NAD 浓度有影响(图1)。78c 处理可以显著减少 Annexin/PI 区的活细胞数量,但是,加入 Z-VAD-FMK 并不能使活细胞数量恢复,表明 CD38 NADase 抑制可以诱导非切除性细胞死亡。

Conclusions. CD38 is the major NADase in mammalian tissues, and involved in catabolism of NAD. CD38 NADase inhibitor, 78c, inhibited the growth of MM cells at low concentrations. 78c induced cell death was found to be highly specific to MM cells and its cytotoxic effect was associated with surface CD38 expression of MM cells. Increased amount of NAD in MM cells by 78c treatment suggests that NAD elevation is associated with MM cell death induced by CD38 NADase inhibition. Since, daratumumab has limited effect against CD38 NADase activity, modulation of intracellular NAD levels by CD38 NADase inhibition could provide a novel therapeutic strategy for MM (Fig. 2).

结论。CD38是哺乳动物组织中主要的 NADase,参与 NAD 的分解代谢。低浓度 CD38 NADase 抑制剂78c 对 MM 细胞生长有抑制作用。78c 诱导的细胞死亡对 MM 细胞具有高度特异性,其细胞毒作用与 MM 细胞表面 CD38表达有关。78c 处理后 MM 细胞 NAD 含量增加,提示 NAD 含量增加与 CD38 NADase 抑制诱导 MM 细胞死亡有关。由于达拉通单抗对 CD38 NADase 活性的抑制作用有限,通过抑制 CD38 NADase 调节细胞内 NAD 水平可能为治疗多发性骨髓瘤提供一种新的治疗策略。VIEW LARGE 视野宽阔DOWNLOAD SLIDE 下载幻灯片Disclosures 披露

Matsuoka:Kyowa Kirin Co., Ltd.: Research Funding; Bristol-Myers Squibb Corp.: Research Funding; Chugai Pharmaceutical Co., Ltd.: Honoraria.

松冈: 协和麒麟有限公司: 研究基金; 百时美施贵宝公司: 研究基金; 中外制药有限公司: Honoraria。

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