Multiple myeloma resistance to apoptosis and antitumor immunity contribute to poor therapeutic responses. Pyroptosis, an inflammatory programmed cell death distinct from apoptosis, has potential to be clinically harnessed and to address these problems. Chinese traditional medicine has advanced the care of patients; however, detailed knowledge of the medicine is lacking. Anwulignan is a monomer compound of the Chinese traditional medicine Schisandra sphenanthera lignans. The goal of this study is to determine whether anwulignan could suppress myeloma growth by activating pyroptosis, enhancing anti-tumour immunity.


1. IC50 values of four MM cell lines to anwulignan were detected to determine the effect of anwulignan on myeloma cells. To determine whether pyroptosis was involved during anwulignan treatment, markers of pyroptotic cell death, cleavage of gasdermin (GSDM), caspase 3 and release of HMGB1, IL-18, IL-1β were detected.

2. High-throughput mRNA sequencing was used to screen out the differential genes after anwulignan treatment. Differential genes enrichment analysis based on GO (Gene Ontology Resource), KEGG (Kyoto Encyclopedia of Genes and Genomes) was used to clarify the signaling pathways and biological processes affected by anwulignan.

3. To ascertain the functional contribution of the immune system in anwulignan therapeutic efficacy, we compared tumor responses in immunodeficient mice and immunocompetent mice. Orthotopic MM xenograft models were established in NOD-Prkdcscid Il2rgtm1/Bcgen (NSG) mice with luciferase labelled LP-1 cells. Orthotopic MM allografts models were established in C57Bl/KaLwRij mice with luciferase labelled 5TGM1 cells. Tumor burden was assessed by serial bioluminescence imaging.

4. To determine whether the antitumor immune responses during anwulignan treatment were dependent on mediators of pyroptosis, tumor-infiltrating T lymphocyte populations from control or GSDMD-KO tumors treated with anwulignan were detected. 5. To identify the mechanism and targets underlying the inhibitory anwulignan activity, we performed a high-throughput proteomics platform to screen the potential targets of anwulignan. We synthesized biotinylated anwulignan to lable anwulignan. MM.1S cell lysates were incubated with biotin-anwulignan or free biotin, and the mixtures were precipitated with streptavidin-coated agarose beads, followed by gel electrophoresis and silver staining. Only one band was clearly precipitated by biotin-anwulignan but not by free biotin. The differential band was prepared for LC-MS/MS analysis. Cluster analysis was used to screen out the preferred binding partners of anwulignan.


1. CCK8 assays showed that the IC50 values were found to be 38.29μM, 51.09μM, 70.61μM and 87.16μM at 72 h for LP-1, ARH-77, U266, and MM.1S cells, respectively. In addition to increased cleavage of GSDMD and caspase 3, anwulignan treatment promoted release of HMGB1 and IL-18, indicating that pyroptosis occurred in anwulignan-treated myeloma cells. 2. RNA-seq results revealed that the expression of pyroptosis genes increased significantly in anwulignan treatment group, suggesting that anwulignan induces pyroptotic cell death.

3. Tumors in either immunodeficient mice (Figure 1A) or immunocompetent mice showed robust tumor regression following anwulignan treatment. However, anwulignan induced prolonged tumor regressions in C57Bl/KaLwRij mice, with tumors taking an average of 106.5 days to regrow to 10s pscr compared with short-term regressions averaging 36.2 days in NSG mice. Moreover, we observed more extended overall survival of anwulignan-treated C57Bl/KaLwRij mice compared with NSG mice. These data suggest that an intact immune system significantly contributes to the therapeutic efficacy of anwulignan.

4. Higher levels of CD8+ T cells were detected in control tumors in comparison with the GSDMD-KO counterparts

5. High-throughput proteomics platform detected a series of candidate proteins that may bind to anwulignan (Figure 1B). Cluster analysis was performed using STRING software and showed that anwulignan preferably binds to pyroptosis proteins.


These data implicate the natural product anwulignan-induced pyroptosis in anti-myeloma immune responses and highlight new therapeutic strategies for MM. Our data suggest that pyroptosis may be an indispensable mediator of immune-driven therapeutic response in MM.

No relevant conflicts of interest to declare.

Author notes


Asterisk with author names denotes non-ASH members.

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