Anti-β2 Microglobulin Monoclonal Antibodies Overcome Bortezomib-Induced Drug Resistance In Multiple Myeloma By Inhibition Of Autophagy
Authors: Zhang Mingjun , He Jin , Lu Yong , Zheng Yuhuan , Hong Bangxing , Li Haiyan , Liu Zhiqiang , Xu Jingda , Liu Huan , Qian Jianfei , Yi Qing , Yang Jing 
Published: October 2019

Abstract
Chemotherapy is the most effective treatment for multiple myeloma (MM). Several new drugs have been developed to prolong MM patient survival. However, application of these drugs, such as bortezomib (BTZ), usually induces drug resistance, and patients are prone to quick relapse. It is known that cell death or proliferation is regulated by the crosstalk between apoptosis and autophagy, and autophagy activation inhibits apoptosis by reducing caspase cleavage. Recent studies have shown that autophagy activation plays a role in chemotherapy drug resistance in cancer patients, in particular, BTZ treatment activates autophagy in MM, indicating that inhibition of autophagy could overcome BTZ-induced drug resistance. We have recently reported the generation of monoclonal antibodies (mAbs) against human beta2 microglobuline (b2M), and have found that high dose mAbs have strong apoptotic effects on MM. In this study, using a BTZ treatment-setting, we hypothesized that anti-β2M mAbs could overcome drug resistance and enhance chemotherapy efficacy in MM by inhibiting autophagy. To investigate the combined effects of mAbs and BTZ, MM cells were cultured with addition of BTZ (5 nM) or mAbs (clone D1; 10 mg/ml) or both for 24 hours. Results from Annexin-V binding assay indicated that combined treatment significantly enhanced apoptosis in MM cell lines, such as ARK, ARP-1, MM.1S, and U266, and in primary MM cells as compared with individual treatment, respectively. Next, BTZ-sensitive or -resistant MM cells KAS-6 WT/V10R or OPM-2 WT/V10R were used to determine the importance of BTZ in the synergistic effects, and such synergistic effects was only shown in apoptosis of BTZ-sensitive cells, but not in BTZ-resistant cells. Furthermore, combined treatment significantly increased apoptosis in β2M-overexpressing, but decreased in β2M-knocking down MM cells, which indicated that the synergistic effects is dependent on the surface β2M expression on MM cells. Mechanistic studies further showed that BTZ treatment resulted in an accumulation of cleaved-caspase 9/3 and PARP cleavage, suggesting that combined treatment enhanced caspase activation. In addition, Western blot analysis showed that BTZ treatment upregulated the expression of autophagy proteins, such as LAMP-1, Beclin 1, and LC3B, in a dose dependent manner, whereas combined treatment decreased their expression. Beclin 1, which is a key protein required for autophagy, has been reported to have a potential NF-kB p65 binding site on its promoter. Therefore, we checked if mAbs inhibited BTZ-induced autophagy via NF-kB signaling pathway. Our data showed that BTZ treatment increased p65 nuclear translocation and the levels of phosphorylated p65 in MM cells, while combined treatment significantly reduced both. ChIP assay has further verified that mAb treatment inhibited p65 binding to Beclin 1 promoter. To examine our hypothesis in vivo, MM cells ARP-1 or MM.1S were subcutaneously injected into SCID mice. The mice were then subcutaneously injected with mAbs (0.6 mg/kg) or intraperitoneally injected with BTZ (0.1 mg/kg) individually or in combination. Tumor burdens were assessed by measuring tumor volumes and serum M-protein levels by ELISA. The combination of anti-β2M mAb and BTZ treatment repressed tumor growth and prolonged the survival of tumor-bearing mice as compared with individual treatment. In conclusion, our study for the first time demonstrated that anti-β2M mAbs prevent BTZ resistance and enhance its anti-MM efficacy by reducing the expression of autophagy proteins via NF-kB signaling. Thus, our studies provide a new insight into clinical development of anti-β2M mAbs to overcome chemotherapy drug resistance and improve MM patient survival.

Disclosures:
No relevant conflicts of interest to declare.