EP4 influences bortezomib resistance in multiple myeloma by modulating endoplasmic reticulum stress via the phosphatidylinositol 3-kinase/protein kinase B pathway.

Background: Nowadays, microRNAs (miRNAs) attract much attention in regulating anticancer drug resistance in cancers including multiple myeloma (MM). Bortezomib is the first-line choice in MM treatment, and bortezomib resistance caused by aberrant DNA repair leads to the recurrence and therapeutic failure of MM. Objective: Our study aims to identify a miRNA that overcomes bortezomib resistance in MM. Methods: We established bortezomib-resistant MM cell lines, and screened several miRNAs that have aberrant expressions in MM cell lines. The expression of DNA-repair-related proteins were assessed by western blot, and cell viability was determined by the MTT assay in bortezomib-resistant cell lines. The binding between miRNAs and 3ʹ-UTR of APE1 mRNA was confirmed by luciferase reporter assay. The mouse bortezomib-resistant xenograft was established to verify the therapeutic effect of miRNA overexpression. Results: miR-520g and miR-520h were significantly downregulated in bortezomib-resistant MM cell lines, and overexpression of miR-520g and miR-520h together inhibited expression of homologous recombination-related protein Rad51 and cell viability of bortezomib-resistant MM cells in vitro by binding with 3ʹ-UTR of APE1 mRNA. Combined overexpression of miR-520g and miR-520h inhibited bortezomib-resistant MM tumor growth in vivo. Conclusion: Our findings demonstrated that combined overexpression of miR-520g and miR-520h overcomes bortezomib resistance in MM through inhibition of DNA repair, offering a promising therapeutic target for MM treatment.

Bortezomib is an effective agent for treating multiple myeloma (MM). To investigate the underlying mechanisms associated with acquired resistance to this agent, we established two bortezomib-resistant MM cell lines, KMS-11/BTZ and OPM-2/BTZ, the 50% inhibitory concentration values of which were respectively 24.7- and 16.6-fold higher than their parental cell lines. No activation of caspase and BH3-only proteins such as Noxa was noted in bortezomib-resistant cells after exposure to the drug. The accumulation of polyubiquitinated proteins was reduced in bortezomib-resistant cells compared with the parental cells, associated with avoidance of catastrophic ER stress as assessed by downregulation of CHOP expression. These resistant MM cells have a unique point mutation, G322A, in the gene encoding the proteasome beta5 subunit (PSMB5), likely resulting in conformational changes to the bortezomib-binding pocket of this subunit. KMS-11 parental cells transfected to express mutated PSMB5 also showed reduced bortezomib-induced apoptosis compared with those expressing wild-type PSMB5 or the parental cells. Expression of mutated PSMB5 was associated with the prevention of the accumulation of unfolded proteins. Thus, a fraction of MM cells may acquire bortezomib resistance by suppressing apoptotic signals through the inhibition of unfolded protein accumulation and subsequent excessive ER stress by a mutation of the PSMB5 gene.

目的探索细胞分裂周期蛋白37（Cdc37）通过调控细胞自噬参与多发性骨髓瘤（MM）对硼替佐米（BTZ）耐药的机制，为MM的治疗提供新的思路。方法应用实时荧光定量PCR（qRT-PCR）和Western blot（WB）技术测定BTZ耐药细胞株ANBL-6.BR中Cdc37及自噬相关基因LC3b的表达水平。采用慢病毒转染技术上调ANBL-6.BR细胞中Cdc37的表达水平，WB技术检测LC3b的表达，流式细胞术检测BTZ诱导的细胞凋亡。采用shRNA干扰技术下调MM细胞株NCI-H929中Cdc37的表达水平，CCK-8法进一步验证Cdc37低表达与BTZ耐药相关，WB技术检测AKT/mTOR信号通路及自噬相关蛋白的表达变化；通过流式细胞术检测自噬抑制剂氯喹（CQ）是否可以增加耐药MM细胞对BTZ的敏感性。结果Cdc37在BTZ耐药细胞株ANBL-6.BR中低表达，而自噬相关基因LC3b在耐药株中高表达。上调ANBL-6.BR细胞中Cdc37的表达水平可抑制LC3b的表达，且增加了MM细胞对BTZ的敏感性。在MM细胞系NCI-H929中抑制Cdc37的表达导致BTZ耐药，同时引起自噬水平升高。而自噬抑制剂CQ可逆转Cdc37下调所引起的BTZ耐药。结论Cdc37可能通过调控细胞自噬参与MM细胞对BTZ的耐药过程。

Multiple myeloma (MM), the seco nd most common haematological malignancy, is currently incurable because patients often develop multiple drug resistance and experience subsequent relapse of the disease. This study aims to identify a potential therapeutic agent that can counter bortezomib (BTZ) resistance in MM. DCZ0358, a novel alkaloid compound, is found to exert potent cytotoxic effects against BTZ-resistant MM cellsin vivo andin vitro. The anti-myeloma activity of DCZ0358 is associated with inhibition of cell proliferation, promotion of cell apoptosis via caspase-mediated apoptotic pathways, and induction of G0/G1 phase arrest via downregulation of cyclin D1, CDK4, and CDK6. Further investigation of the molecular mechanism shows that DCZ0358 suppresses the JAK2/STAT3 signaling pathway. In conclusion, DCZ0358 can successfully counter BTZ resistance in MM cells. This study provides evidence that warrants future preclinical assessments of DCZ0358 as a therapeutic agent against BTZ resistance in MM.

MiR-197-3p reduces bortezomib resistance in multiple myeloma by inhibiting IL-6 expression in a MEAF6-dependent manner

Cinobufagin overcomes bortezomib resistance in multiple myeloma strains by targeting SEC62/TRPM4-mediated NECSO.

Autophagy plays an important role in multiple myeloma (MM) for homeostasis, survival and drug resistance, but which genes participate in this process is unclear. We identified several cytoskeleton genes upregulated in MM patients by gene expression profiling (GEP) datasets; in particular, patients with high profilin 1 (PFN1) expression had poor prognosis in MM. In vitro, overexpressed PFN1 promotes proliferation and bortezomib (BTZ) resistance in MM cells. Further study indicated overexpression of PFN1 significantly promoted the process of autophagy and induced BTZ resistance in MM. Otherwise, knockdown of PFN1 blocked autophagy and sensitized MM to BTZ. Co‐immunoprecipitation in MM cells indicated that PFN1 could bind Beclin1 complex and promote the initiation of autophagy. Inhibition of autophagy by blocking the formation of Beclin1 complex could reverse the phenotype of BTZ resistance in MM. Our findings suggested that PFN1 could promote autophagy through taking part in Beclin1 complex and contribute to BTZ resistance, which may become a novel molecular target in the therapy of MM.

TM-233 Exerts Anti-Myeloma Activity and Overcomes Bortezomib-Resistance In Human Multiple Myeloma Cells By Targeting NF-κB and JAK/STAT Dual-Signaling Pathways

BackgroundAlthough current chemotherapy using bortezomib (Velcade) against multiple myeloma in adults has achieved significant responses and even remission, a majority of patients will develop acquired resistance to bortezomib. Increased thioredoxin level has been reported to be associated with carcinogenesis; however, the role of thioredoxin in bortezomib drug resistance of myeloma remains unclear.MethodsWe generated several bortezomib-resistant myeloma cell lines by serially passaging with increased concentrations of bortezomib over a period of 1.5 years. Thioredoxin expression was measured by real-time PCR and western blot.ResultsThe role of thioredoxin in the survival of bortezomib-resistant myeloma cells was determined by specific shRNA knockdown in vitro and in vivo. Thioredoxin inhibitor (PX12) was used to determine the effectiveness of thioredoxin inhibition in the treatment of bortezomib-resistant myeloma cells. The effect of thioredoxin inhibition on mitophagy induction was examined. The correlation of thioredoxin expression with patient overall survival was interrogated. Thioredoxin expression was significantly upregulated in bortezomib-resistant myeloma cells and the change correlated with the increase of bortezomib concentration. Thioredoxin gene knockdown using specific shRNA sensitized bortezomib-resistant myeloma cells to bortezomib efficiency in vitro and in vivo. Similarly, pharmacological inhibition with PX12 inhibited the growth of bortezomib-resistant myeloma cells and overcame bortezomib resistance in vitro and in vivo. Furthermore, inhibition of thioredoxin resulted in the activation of mitophagy and blockage of mitophagy prevented the effects of PX12 on bortezomib-resistant myeloma cells, indicating that mitophagy is the important molecular mechanism for the induction of cell death in bortezomib-resistant myeloma cells by PX12. Moreover, inhibition of thioredoxin resulted in downregulation of phosphorylated mTOR and ERK1/2. Finally, thioredoxin was overexpressed in primary myeloma cells isolated from bortezomib-resistant myeloma patients and overexpression of thioredoxin correlated with poor overall survival in patients with multiple myeloma.ConclusionsOur findings demonstrated that increased thioredoxin plays a critical role in bortezomib resistance in multiple myeloma through mitophagy inactivation and increased mTOR and ERK1/2 phosphorylation. Thioredoxin provides a potential target for clinical therapeutics against multiple myeloma, particularly for bortezomib-resistant/refractory myeloma patients.

A Small Molecule, Shikonin, Dually Functions As a Proteasome Inhibitor and a Necroptosis Inducer In Multiple Myeloma Cells

Studies have shown that bortezomib resistance in multiple myeloma (MM) is mediated by the abnormalities of various molecules and microenvironments. Exploring these resistance mechanisms will improve the therapeutic efficacy of bortezomib. In this study, bone marrow tissues from three patients with MM, both sensitive and resistant to bortezomib, were collected for circRNA high-throughput sequencing analysis. The relationship between circ_0000337, miR-98-5p, and target gene DNA2 was analyzed by luciferase detection and verified by RT-qPCR. We first found that circ_0000337 was significantly upregulated in bortezomib-resistant MM tissues and cells, and overexpression of circ_0000337 could promote bortezomib resistance in MM cells. circ_0000337 may act as a miR-98-5p sponge to upregulate DNA2 expression, regulate DNA damage repair, and induce bortezomib resistance. Furthermore, it was determined that the increased circ_0000337 level in bortezomib-resistant cells was due to an increased N6-methyladenosine (m6A) level, resulting in enhanced RNA stability. In conclusion, the m6A level of circ_0000337 and its regulation may be a new and potential therapeutic target for overcoming bortezomib resistance in MM.

The Establishment of Bortezomib Resistant Myeloma Cell Line KM3/BTZ and Explore the Resistance Mechanism

Clinical success of the proteasome inhibitor established bortezomib as one of the most effective drugs in treatment of multiple myeloma (MM). While survival benefit of bortezomib generated new treatment strategies, the primary and secondary resistance of MM cells to bortezomib remains a clinical concern. This study aimed to highlight the role of p53-induced RING-H2 (Pirh2) in the acquisition of bortezomib resistance in MM and to clarify the function and mechanism of action of Pirh2 in MM cell growth and resistance, thereby providing the basis for new therapeutic targets for MM. The proteasome inhibitor bortezomib has been established as one of the most effective drugs for treating MM. We demonstrated that bortezomib resistance in MM cells resulted from a reduction in Pirh2 protein levels. Pirh2 overexpression overcame bortezomib resistance and restored the sensitivity of myeloma cells to bortezomib, while a reduction in Pirh2 levels was correlated with bortezomib resistance. The levels of nuclear factor-kappaB (NF-κB) p65, pp65, pIKBa, and IKKa were higher in bortezomib-resistant cells than those in parental cells. Pirh2 overexpression reduced the levels of pIKBa and IKKa, while the knockdown of Pirh2 via short hairpin RNAs increased the expression of NF-κB p65, pIKBa, and IKKa. Therefore, Pirh2 suppressed the canonical NF-κB signaling pathway by inhibiting the phosphorylation and subsequent degradation of IKBa to overcome acquired bortezomib resistance in MM cells.

Although bortezomib (BTZ) displays efficacy in treating multiple myeloma (MM), BTZ resistance in MM patients has been reported. Meanwhile, treating BTZ resistant MM cells with β-catenin inhibitors have demonstrated the ability to reserve BTZ resistance. Thus, the present study aimed to investigate the synergistic effect of the β-catenin inhibitors, ICG-001 and pyrvinium (PP), with BTZ in the treatment of BTZ-resistant MM cells. Different concentrations of ICG-001 (0–32 µM) or PP (0–32 nM) were used to treat the BTZ-resistant RPMI-8226 (RPMI-8226BR) and BTZ-resistant KMS-11 (KMS-11BR) cell lines, followed by a BTZ combination treatment. Subsequently, cell viability and apoptosis in these two cell lines were determined by CCK-8 assay and flow cytometry, respectively. The proteins involved in the Wnt/β-catenin signaling pathway were detected using western blotting. The Wnt/β-catenin signaling pathway was activated in the RPMI-8226BR and the KMS-11BR cells. In addition, the cell viability of RPMI-8226BR and KMS-11BR cells were decreased following β-catenin inhibitor (ICG-001 and PP) treatment alone. Furthermore, the β-catenin inhibitors, ICG-001 and PP, plus BTZ combination treatment revealed a notable decrease in cell viability and a marked increase in cell apoptosis rate, compared with that in cells treated with ICG-001, PP or BTZ alone in the RPMI-8226BR and KMS-11BR cell lines. In conclusion, the β-catenin inhibitors, ICG-001 and PP not only increased apoptosis, but also sensitized BTZ-resistant MM cells to BTZ, indicating their potential therapeutic application in MM.

Lnc NEAT1/miR-29b-3p/Sp1 form a positive feedback loop and modulate bortezomib resistance in human multiple myeloma cells