DDX3X Promotes Multiple Myeloma Cell Survival and Proteasome Inhibitor Resistance through Modulation of Stress Granule Assembly and MAPKAPK2 Translation
Authors: Jiang Shijun , Fan Fengjuan , Podar Klaus , Sun Chunyan , Hu Yu 
Published: December 2024

INTRODUCTION

Multiple myeloma (MM) is a malignant disease characterized by the abnormal proliferation of clonal plasma cells in the bone marrow. Relapse and drug resistance during treatment remain challenges in the management of MM. c-Myc is a key regulator of MM progression and contributes to drug resistance in MM. Targeting c-Myc directly or indirectly, such as its accessibility to downstream genes, may provide new treatment strategies for MM. In the present study, we analyzed the published c-Myc ChIP-seq data and identified DDX3X as a potential c-Myc downstream target in MM. DDX3X plays a crucial role in proliferation and survival of various types of cancers. Specifically, DDX3X is a key component of stress granules and an important regulatory molecule in the cellular stress response. However, its function in MM is unknown. Endoplasmic reticulum stress/ unfolded protein response (ERS/UPR) is significantly upregulated in MM cells and essential for their survival. Conversely, the proteasome inhibitor bortezomib disrupts the ER-associated degradation (ERAD) process and protein homeostasis of MM, thus promoting apoptosis. Here, we report for the first time our investigations on the role of DDX3X in MM, particularly its involvement in mediating resistance to proteasome inhibitors.

METHODS

Following analysis of published c-Myc ChIP-seq data from GEO datasets (GEO Series ID GSE36354), ChIP-qPCR was used to verify the binding of c-Myc to DDX3X DNA sequences. Dual-luciferase reporter assays were conducted to assess the effect of c-Myc on DDX3X gene promoter activity. Gene expression and survival analyses were conducted using publicly available MM patient datasets (GSE13591 and GSE24080). Western blot and qPCR assays were employed to detect the expression levels of proteins and mRNAs, respectively. DDX3X inhibition was induced either genetically by CRISPR/Cas9- mediated knockdown or pharmacologically by the DDX3X inhibitor RK-33. MTS assays were performed to evaluate cell viability. Immunofluorescence was used to detect the localization of proteins within cells. Immunoprecipitation-Mass Spectrometry (IP-MS) was carried out to identify proteins that interact with DDX3X. Ribosome sequencing (Ribo-seq) combined with RNA-seq was utilized to identify downstream targets of DDX3X. NCG mice were subcutaneously inoculated with DDX3X knockdown MM cells or the control cells into the flanks to establish the MM xenograft mouse model, and then the mice were treated with or without bortezomib by intraperitoneal injection.

RESULTS

Our data show that c-Myc binds to the promoter region of DDX3X and significantly enhance its transcriptional activity. Furthermore, pharmacological inhibition of c-Myc with JQ-1 markedly reduced DDX3X mRNA and protein levels in MM cells.

Kaplan-Meier survival curves indicate that high expression of DDX3X in MM patients is associated with a poor prognosis. Consequently, inhibition of DDX3X by gene knockdown or its inhibitor RK-33 in MM cells significantly increased the expression of the UPR signaling pathway components p-PERK, p-eIF2α, ATF4, and CHOP, as well as induced Caspase-3 cleavage. Moreover, DDX3X inhibition enhanced bortezomib or the ERS inducer thapsigargin (TG) -triggered apoptosis in MM cells.

Importantly, following treatment with bortezomib or TG, DDX3X formed stress granules in MM cells, showing colocalization with stress granules markers G3BP1 as well as PABPC1. Knockdown of DDX3X abrogated the formation of stress granules. IP-MS and co-IP results confirmed the interaction between DDX3X and PABPC1 in MM cells. Moreover, the translation efficiency of MAPKAPK2, which has been reported to be upregulated in MM and involved in bortezomib resistance, was significantly reduced in TG- treated MM cells following DDX3X knockdown. Consistently, we observed a notable downregulation of MAPKAPK2 protein levels when knockdown of DDX3X, particularly after TG treatment.

Finally, the functional role of DDX3X in MM was verified in vivo using a MM xenograft mouse model. Our results show that knockdown of DDX3X, in combination with bortezomib treatment, significantly decreases tumor growth compared to the control groups.

CONCLUSION

In MM cells, DDX3X promotes cell survival and proteasome inhibitor resistance through modulation of stress granule assembly and MAPKAPK2 translation. Our findings suggest DDX3X as a promising therapeutic target in MM.