Several lines of evidence suggest the importance of the IRE1α-XBP1 pathway in tumor progression, adaptation to the hypoxic tumor microenvironment, as a prognostic marker and a potential therapeutic target in both solid tumors and MM. Here, we identified toyocamycin as an inhibitor of both ER stress-induced and constitutive activation of the IRE1α-XBP1 pathway in MM cells, and showed that it exerted synergistic or at least additive anti-tumor effects with BTZ. Furthermore, it induced marked apoptosis of primary MM cells as well as MM cell lines without showing any cytotoxicity to PBMCs from healthy donors. This anti-tumor effect was also confirmed in a mouse model in vivo.
Previously, toyocamycin was reported to inhibit RNA synthesis and ribozyme function.31, 32, 44
This raised the possibility that it inhibited ER stress-induced XBP1 mRNA splicing and transcription of EDEM
genes through RNA synthesis inhibition. However, comparing the IC50
values of toyocamycin on RNA synthesis, ER stress-induced XBP1 mRNA splicing and transcription of UPR target genes revealed that its inhibitory activity on ER stress-induced XBP1 mRNA splicing and transcription of EDEM
genes was much stronger than the RNA synthesis blockade effect. Furthermore, although 100
actinomycin D completely inhibited both RNA synthesis () and ER stress-induced transcription of GRP78 (Supplementary Figure 1
), it did not inhibit ER stress-induced XBP1 mRNA splicing (). These results support the notion that the inhibitory activity of toyocamycin on RNA synthesis is not responsible for inhibition of XBP1 mRNA splicing and transcription of EDEM
genes. On the other hand, toyocamycin has also been reported to inhibit kinase activities, such as PKC,45
Considering the structure of toyocamycin and its analogs, it was also predicted that it would inhibit IRE1 auto-phosphorylation. However, it inhibited IRE1 phosphorylation on Ser724 not only in IRE1-overexpressing 293T cells () but also in MM cell lines ( and ). Recent studies have suggested that the trigger for IRE1 endoribonuclease activity is not phosphorylation but a conformational change in the kinase domain induced by cofactor (ATP or ADP) binding. Therefore, it is likely that toyocamycin inhibits IRE1α-induced XBP1 mRNA cleavage through a cofactor-induced conformational change of IRE1α rather than inhibition of IRE1α auto-phosphorylation.
Previous studies have suggested that the IRE1α-XBP1 pathway has a critical role in ER stress-induced cytoprotection. 1NM-PP1, a small molecule selectively activating the IRE1α(I642G) mutant,18
protected cells from tunicamycin- or thapsigargin-induced cell death.48, 49
In contrast, overexpression of dominant-negative XBP1 or knockdown of XBP1 has been reported to enhance tunicamycin-induced apoptosis.50
Consistent with previous reports, we also found that toyocamycin synergistically induced cell death in ER-stressed HeLa, HT29 and HCT116 cells (Supplementary Figure 2
and data not shown). More recently, as with toyocamycin, STF-083010 was shown to inhibit IRE1 ndonuclease activity without affecting its kinase activity in vitro
However, these compounds mediate their inhibitory activity at
, and show little MM cell apoptosis induction as single agents. STF-083010 also shows anti-tumor activity in human MM xenograft models. However, while toyocamycin suppressed tumor volume to around 50% at 1
mg/kg by once-weekly injection, the STF-083010 dose needed to be 30
mg/kg by once-weekly injection.14
In our study, adenosine analogs showed potent inhibition of ER stress-induced IRE1α-XBP1 activation at the nanomolar level. Compared with these, toyocamycin induced marked apoptosis in ER-stressed tumor cells and MM cells at much lower concentrations. In addition, it also inhibited the constitutive activation of XBP1 in MM cells even at suboptimal concentrations such as 10
. However, the mechanism by which toyocamycin mediates dose-dependent apoptosis remains unknown. Although three MM cell lines with low active XBP1 expression showed lower sensitivity to toyocamycin treatment than other seven MM cells with high active XBP1 expression, these three cell lines still demonstrated sensitivity to the drug at the nM concentration. It may be speculated that toyocamycin also triggers another stress-inducing factor at higher concentrations, which may induce strong apoptosis under IRE1α-XBP1-suppressed conditions. Further analysis needs be conducted to elucidate the whole picture of its mechanisms of action on MM cells.
An earlier phase I toyocamycin single-agent study also testing possible anti-tumor effects in patients with advanced solid tumors has been reported.51
However, because no apparent clinical responses were observed in that study, further clinical evaluation was not planned. In that study, toyocamycin showed no systemic side effects, such as organ dysfunction and cytopenia, and only local necrosis at the site of infusion was reported to occur when the drug was delivered into the soft tissues. This suggests that toyocamycin adverse events could be manageable if it is infused through central venous catheters. In addition, this study does not exclude potential clinical efficacy of toyocamycin against solid tumors, because it was a phase I trial lacking evaluation of stable disease often applied in more recent clinical trials of molecular-targeting therapies.
In conclusion, we demonstrated that the adenosine analog toyocamycin has a potent IRE1-XBP1 inhibitory effect on ER-stressed tumors and MM cells, as well as triggering dose-dependent apoptosis in these cells. These results provide a preclinical rationale for clinical trials of toyocamycin and other adenosine analogs alone and in combination with BTZ for treating MM.