Abstract INTRODUCTION Endoplasmic reticulum stress (ERS) / unfolded protein response (UPR) is essential for multiple myeloma (MM) cell survival. Proteasome inhibitors (PIs) disrupt the protein homeostasis and promote apoptosis of MM cells. Stress granules (SGs) are ribonucleoprotein granules that formed in response to various stress stimuli, and help tumor cells survive under unfavorable microenvironment and treatment. Previously, we have reported that DDX3X promotes MM cell survival and PI resistance through modulation of UPR and SG assembly. However, the underlying molecular mechanisms are unclear. Here, we identified the downstream targets of DDX3X, elucidated how DDX3X regulated MM cell homeostasis in cellular stress response, and provided a molecular rationale for the combinations targeting DDX3X-SGs axis with PIs. METHODS Thapsigargin (TG) was used to induce ERS and trigger SG formation, while anisomycin was used to inhibit SG formation. eCLIP-seq and RIP-qPCR assays were used to identify the target transcripts of RNA-binding proteins. CRISPR/Cas9- or Tet-on shRNA- mediated knockdown was employed to inhibit gene expression. MTS and flow cytometry assays were performed to measure cell viability. Immunoprecipitation-Mass Spectrometry (IP-MS) assay was carried out to identify protein-protein interactions. Calcium was measured using live-cell fluorescent indicators Rhod-2 and X-Rhod-1. RESULTS First, we examined the SG formation in MM patients. Similar to TG-treated MM cell lines, DDX3X also formed SGs in primary MM cells, as indicated by co-localization with cytoplasmic puncta of SG markers G3BP1 and PABPC1. In our previous work, MAPKAPK2 (MK2), which is associated with UPR signaling and plays a role in PI resistance, was identified as a potential downstream target of DDX3X in TG-treated MM cells by ribosome sequencing. Here, we found the direct interaction between DDX3X and MK2 mRNA in MM cells upon TG treatment by eCLIP-seq and RIP-qPCR. Moreover, in TG-treated MM cells, silencing of DDX3X resulted in a substantial decrease in MK2 protein expression, while its mRNA levels and protein stability remained unaffected. Consequently, phosphorylation of Hsp27, a known substrate of MK2, was also downregulated. Collectively, these data suggest that under ERS, DDX3X binds MK2 mRNA and regulates its translation efficiency. We subsequently employed gamcemetinib, an irreversible inhibitor of MK2. Notably, we observed decreased phospho-Hsp27 and enhanced apoptosis in MM cells following treatment with gamcemetinib, either alone or in combination with PIs. Next, by IP-MS we identified interaction between DDX3X and TMCO1, especially upon TG treatment. TMCO1 is an ER calcium-selective channel, which is normally activated in response to ER Ca2+ overloading and maintains calcium homeostasis in the ER. Following treatment with TG, TMCO1 formed granular aggregates and significantly co-localized with DDX3X in the cytoplasm of MM cells, suggesting that TMCO1 could be recruited to SGs by DDX3X. Importantly, TMCO1 also co-localized with DDX3X in primary MM cells derived from patients, exhibiting cytoplasmic aggregation. Furthermore, DDX3X knockdown or inhibition of SG formation by anisomycin elevated the protein expression levels of TMCO1, along with decrease in its protein degradation rate. Moreover, overexpression of TMCO1 in MM cells significantly accelerated the depletion of ER Ca2+ stores, induced cleaved-caspase 3 expression and enhanced apoptosis, particularly under TG-induced stress. These results suggest that under stress conditions, DDX3X affects TMCO1 protein stability by recruiting it into SGs, thereby buffering intracellular calcium imbalances and enhancing cell survival in MM. Finally, using a MM xenograft mouse model, we showed that silencing of DDX3X by doxycycline significantly augmented the anti-tumor efficacy of carfilzomib, as evidenced by greater tumor regression compared to monotherapy controls. Consistent with our in vitro data, elevated protein levels of UPR markers, including ATF4 and CHOP, as well as increased TUNEL staining were observed in tumors from DDX3X knockdown groups. CONCLUSIONS In MM cells, DDX3X modulated UPR and SGs assembly, thereby promoting adaption to proteotoxic stress. Mechanistically, DDX3X supported cell homeostasis by regulating the translation of MK2 and the stability of TMCO1 via SGs. Our findings suggest DDX3X as a promising therapeutic target for improving treatment efficacy in patients with MM.
Ni et al. (Mon,) studied this question.