ABSTRACT
p97/VCP is an endoplasmic reticulum (ER)-associated protein that belongs to the AAA (ATPases associated with diverse cellular activities) ATPase family. It has a variety of cellular functions including ER-associated protein degradation, autophagy, and aggresome formation. Recent studies have shown emerging roles of p97/VCP and its potential as a therapeutic target in several cancer subtypes including multiple myeloma (MM). We conducted a cell-based compound screen to exploit novel small compounds that have cytotoxic activity in myeloma cells. Among approximately 2000 compounds, OSSL_325096 showed relatively strong antiproliferative activity in MM cell lines (IC50 , 100-500 nmol/L). OSSL_325096 induced apoptosis in myeloma cell lines, including a bortezomib-resistant cell line and primary myeloma cells purified from patients. Accumulation of poly-ubiquitinated proteins, PERK, CHOP, and IREα, was observed in MM cell lines treated with OSSL_325096, suggesting that it induces ER stress in MM cells. OSSL_325096 has a similar chemical structure to DBeQ, a known p97/VCP inhibitor. Knockdown of the gene encoding p97/VCP induced apoptosis in myeloma cells, accompanied by accumulation of poly-ubiquitinated protein. IC50 of OSSL_325096 to myeloma cell lines were found to be lower (0.1-0.8 µmol/L) than those of DBeQ (2-5 µmol/L). In silico protein-drug-binding simulation suggested possible binding of OSSL_325096 to the ATP binding site in the D2 domain of p97/VCP. In cell-free ATPase assays, OSSL_325096 showed dose-dependent inhibition of p97/VCP ATPase activity. Finally, OSSL_325096 inhibited the growth of subcutaneous myeloma cell tumors in vivo. The present data suggest that OSSL_325096 exerts anti-myeloma activity, at least in part through p97/VCP inhibition.
Subject(s)
Adenosine Triphosphatases/metabolism , Drug Resistance, Neoplasm/drug effects , Endoplasmic Reticulum/metabolism , Enzyme Inhibitors/administration & dosage , Multiple Myeloma/drug therapy , Nuclear Proteins/metabolism , Small Molecule Libraries/administration & dosage , Adenosine Triphosphatases/antagonists & inhibitors , Adenosine Triphosphatases/chemistry , Animals , Binding Sites , Bortezomib/pharmacology , Cell Line, Tumor , Cell Proliferation/drug effects , Cell Survival/drug effects , Drug Screening Assays, Antitumor , Endoplasmic Reticulum/drug effects , Endoplasmic Reticulum Stress , Endoribonucleases/metabolism , Enzyme Inhibitors/chemistry , Enzyme Inhibitors/pharmacology , Female , Humans , Mice , Models, Molecular , Multiple Myeloma/metabolism , Nuclear Proteins/antagonists & inhibitors , Nuclear Proteins/chemistry , Protein Serine-Threonine Kinases/metabolism , Small Molecule Libraries/chemistry , Small Molecule Libraries/pharmacology , Transcription Factor CHOP/metabolism , Ubiquitination , Xenograft Model Antitumor Assays , eIF-2 Kinase/metabolismABSTRACT
The sialic glycoprotein, MUC1, is known to be involved in the pathogenesis of various types of cancers. KL-6 is one of the surface antigens of MUC1 and also a marker of interstitial pneumonitis. A fraction of patients with myeloma (3.9%) have elevated serum KL-6 levels without any evidence of interstitial pneumonitis and their myeloma cells have high MUC1 expression. We established a myeloma cell line designated EMM1 from a patient with multiple myeloma accompanied with elevated serum KL-6. EMM1 cells expressed high levels of MUC1 compared with other myeloma cell lines. Knockdown of MUC1 in EMM1 cells induced cell cycle arrest during S phase and apoptosis, suggesting that the MUC1 expression is involved in accelerated growth of EMM1 cells. RNA-seq analysis suggests that MUC1 expression activates k-ras and TNFα-induced NFκB pathways in EMM1 cells. We injected EMM1 cells subcutaneously into Rag2-/-Jak3-/- Balb/c mice to establish a mouse xenograft model. These mice had aggressive tumor growth that was accompanied by high serum KL-6 levels. In addition, MUC1 knockdown in EMM1 cells led to inhibited tumor growth. These findings demonstrate that MUC1 serves as a potential target for developing drugs for treatment of patients with KL-6+ myeloma, and EMM1 cells and EMM1-engrafted mice are useful tools for the development of such novel agents.
Subject(s)
Mucin-1/metabolism , Multiple Myeloma/metabolism , Multiple Myeloma/pathology , Animals , Cell Cycle Checkpoints , Cell Line, Tumor , Cell Proliferation , Cell Survival , Female , Gene Knockdown Techniques , Humans , Mice , Middle Aged , Mucin-1/blood , Multiple Myeloma/blood , Neoplasm Invasiveness , S Phase , Xenograft Model Antitumor AssaysABSTRACT
Immunomodulatory drugs (IMiDs) such as thalidomide, lenalidomide, and pomalidomide are efficacious in the treatment of multiple myeloma and significantly prolong their survival. However, the mechanisms of such effects of IMiDs have not been fully elucidated. Recently, cereblon has been identified as a target binding protein of thalidomide. Lenalidomide-resistant myeloma cell lines often lose the expression of cereblon, suggesting that IMiDs act as an anti-myeloma agent through interacting with cereblon. Cereblon binds to damaged DNA-binding protein and functions as a ubiquitin ligase, inducing degradation of IKZF1 and IKZF3 that are essential transcription factors for B and T cell development. Degradation of both IKZF1 and IKZF3 reportedly suppresses myeloma cell growth. Here, we found that IMiDs act as inhibitors of DNA methyltransferases (DMNTs). We previously reported that PU.1, which is an ETS family transcription factor and essential for myeloid and lymphoid development, functions as a tumor suppressor in myeloma cells. PU.1 induces growth arrest and apoptosis of myeloma cell lines. In this study, we found that low-dose lenalidomide and pomalidomide up-regulate PU.1 expression through inducing demethylation of the PU.1 promoter. In addition, IMiDs inhibited DNMT1, DNMT3a, and DNMT3b activities in vitro. Furthermore, lenalidomide and pomalidomide decreased the methylation status of the whole genome in myeloma cells. Collectively, IMiDs exert demethylation activity through inhibiting DNMT1, 3a, and 3b, and up-regulating PU.1 expression, which may be one of the mechanisms of the anti-myeloma activity of IMiDs.
Subject(s)
DNA (Cytosine-5-)-Methyltransferases/antagonists & inhibitors , DNA (Cytosine-5-)-Methyltransferases/metabolism , Immunologic Factors/administration & dosage , Multiple Myeloma/drug therapy , Multiple Myeloma/metabolism , Proto-Oncogene Proteins/metabolism , Trans-Activators/metabolism , Cell Line , Cell Survival/drug effects , Enzyme Activation/drug effects , Humans , Up-Regulation/drug effectsABSTRACT
Although anti-T lymphocyte globulin-Fresenius (ATG-F) is commonly used as prophylaxis for graft-versus-host disease (GVHD), the appropriate dosage of ATG-F in the setting of a reduced-intensity conditioning (RIC) regimen has not been determined. In the present study, we retrospectively analyzed the clinical outcomes of 103 patients after unrelated bone marrow transplant (uBMT) with RIC regimens. RIC regimens consisted of purine analogue plus busulfan with low-dose TBI or ATG-F (5-10 mg/kg in total). Median age was 57 years (range 20-68). The incidence of grade II-IV acute GVHD and chronic GVHD with ATG-F was significantly lower than that with TBI 2 Gy (15 vs. 61 %, P < 0.05; 33 vs. 57 %, P < 0.05). The incidence of 2-year NRM with ATG-F was significantly lower than that with TBI 2 Gy (6 vs. 28 %, P < 0.05). There was no statistically significant difference in the cumulative incidence of 2-year relapse between the ATG-F and TBI 2 Gy groups (37 vs. 20 %, P = 0.13). In conclusion, the addition of low-dose ATG-F to GVHD prophylaxis in patients who received uBMT resulted in decreased incidence of acute and chronic GVHD, which led to a significantly reduced risk of NRM without compromising overall survival. The beneficial effect of low-dose ATG-F should be assessed in a prospective clinical trial.
