Receptor activator of NF-κB ligand (RANKL), a critical mediator of osteoclastogenesis, is upregulated in multiple myeloma (MM). The xanthine oxidase inhibitor febuxostat, clinically used for prevention of tumor lysis syndrome, has been demonstrated to effectively inhibit not only the generation of uric acid but also the formation of reactive oxygen species (ROS). ROS has been demonstrated to mediate RANKL-mediated osteoclastogenesis. In the present study, we therefore explored the role of cancer-treatment-induced ROS in RANKL-mediated osteoclastogenesis and the suppressive effects of febuxostat on ROS generation and osteoclastogenesis. RANKL dose-dependently induced ROS production in RAW264.7 preosteoclastic cells; however, febuxostat inhibited the RANKL-induced ROS production and osteoclast (OC) formation. Interestingly, doxorubicin (Dox) further enhanced RANKL-induced osteoclastogenesis through upregulation of ROS production, which was mostly abolished by addition of febuxostat. Febuxostat also inhibited osteoclastogenesis enhanced in cocultures of bone marrow cells with MM cells. Importantly, febuxostat rather suppressed MM cell viability and did not compromise Dox’s anti-MM activity. In addition, febuxostat was able to alleviate pathological osteoclastic activity and bone loss in ovariectomized mice. Collectively, these results suggest that excessive ROS production by aberrant RANKL overexpression and/or anticancer treatment disadvantageously impacts bone, and that febuxostat can prevent the ROS-mediated osteoclastic bone damage.

Although chimeric antigen receptor (CAR)–modified T cells have shown great success in treating B cell malignancies, they have demonstrated only limited efficacy against solid tumors. Here, we designed a CAR by integrating an antigen-independent OX40 that showed superior antitumor efficacy against multiple solid tumors. We unexpectedly found, through a CRISPR-Cas9–based whole-genome screen, that heparan sulfate is a ligand for OX40. We found that heparan sulfate can directly bind OX40 at the biochemical and cellular levels and that the interaction of heparan sulfate and OX40 activated the AKT, MAPK, and NF-κB signaling pathways. Functionally, the heparan sulfate–OX40 interaction enhanced cell adhesion and CAR T cell functional binding avidity to tumor cells. In vivo, OX40-expressing CAR T cells exhibited increased solid tumor infiltration and persistence dependent on the OX40–heparan sulfate interaction. Our findings provide insights into a glycan-costimulation interaction that is capable of regulating T cell immunity and has potential application in CAR T cell optimization.

Multiple myeloma (MM) is a heterogeneous clonal plasma cell proliferative disorder with CRAB features. Although survival of MM patients has been greatly prolonged by recent implementation of various combinatory treatments with novel anti-MM agents, MM still remains incurable. MM cells preferentially grow and expand in the bone marrow to elicit the alteration of gene expression and thereby drug resistance. To improve the therapeutic efficacy, we urgently need to develop novel treatment strategies targeting the BM microenvironment-mediated drug resistance. The serine/threonine kinase Pim-2 is constitutively over-expressed and acts as a pro-survival mediator in MM cells. We have reported that cocultures with bone marrow stromal cells (BMSCs) or osteoclasts (OCs) further up-regulate Pim-2 expression in MM cells to confer drug resistance (Leukemia 2011, 2015). Therefore, Pim-2 appears to be an important therapeutic target to impair the BM microenvironment-mediated drug resistance in MM. Histone deacetylases (HDACs) are generally accepted to be therapeutic targets for MM treatment. However, clinical application of currently available pan-HDAC inhibitors is limited with their adverse effects induced by a non-selective HDAC inhibition. To develop safe and effective HDAC inhibitor-based treatment, the therapeutic roles of HDAC isoform-specific inhibition should be elucidated. In this regard, we have recently reported therapeutic impacts on MM cells of inhibition of class-I HDACs, especially HDAC1 and HDAC3. HDAC3-selective inhibitor BG45 induces anti-MM activity in combination with DNA methyltransferase (DNMT) inhibitor azacytidine (Leukemia 2017). In the present study, we aimed to clarify the underlying mechanisms for impairment of MM cell growth and survival by HDAC1 inhibition. We first referenced the expression of class-I HDACs using a publicly available GSE6691 data set. Among class-I HDACs, HDAC1 and HDAC3 were highly expressed in MM cells. We then knockdowned HDAC1 gene using lentiviral shRNA system in MM cell lines. The HDAC1 gene silencing induced MM cell death with caspase-3 activation, indicating the critical role of HDAC1 in MM cell growth and survival. To determine target molecules of HDAC1, we carried out RNA-sequencing with and without the HDAC1 gene silencing in RPMI 8226 cells. Among genes whose expression significantly changed by the HDAC1 knockdown (adjusted P values < 0.05, log fold change > 0.5), we focused on IRF4 together with PIM2, because MM cell has been demonstrated to addict to aberrant IRF4-c-Myc regulatory network (Nature 2008). Downregulation of IRF4 and Pim-2 by the HDAC1 knockdown was further confirmed by quantitative PCR (Q-PCR) and immunoblotting in RPMI 8226 and MM.1S cells. Treatment with the class I HDAC-selective inhibitor MS-275 (entinostat) also induced MM cell death along with reduction of IRF4 and Pim-2 expression. Since previous study has shown that IRF4 binds to PIM2 promoter in MM cells (Nature 2008), we examined whether IRF4 regulates PIM2 expression. We found that IRF4 binds to the PIM2 promoter region by analyzing ChIP-Seq data in KMS-12 cells (GSE22901). We further confirmed the binding of IRF4 on PIM2 promoter by ChIP-Q-PCR. Indeed, the IRF4 knockdown downregulated Pim-2 expression in RPMI 8226 cells. These results suggest that HDAC1 inhibition downregulates IRF4 expression, thereby transcriptionally reducing PIM2 expression in MM cells. Pim-2 expression can also be augmented by multiple signaling pathways, including HIF-1a, JAK-STAT and NF-kB-mediated ones in MM cells through the interaction with BM microenvironment. Interestingly, the Pim inhibitor SMI-16a and MS-275 cooperatively induced apoptotic cell death in MM cell lines and CD138-positive primary MM cells even in the presence of BMSCs. Taken together, our results demonstrate the critical role of the HDAC1-IRF4-Pim-2 axis in MM cell growth and survival, and provoke the novel treatment strategy targeting the HDAC1-IRF4-Pim-2 axis in MM cells.

Objective: To develop a herbal preparation for respiratory ailments from plants used in Mongolian medicine. Method: TLC, HPLC, chemical and spectrophotometric analysis were used for the quality and quantitative determination of biologically active compounds in plant raw materials, extracts and granules. The total and internal absorption coefficients of the plant raw materials, tapped density and flowability of the granules were determined using the method of I.A. Muravyev and V.D. Ponomarev. The quality parameters of the granules (appearance, particle size, moisture content, dispersion) were determined by the Chinese pharmacopoeia methods. Results: The highest amount of extractive matters was determined when extracting 2 mm powdered roots of Glycyrrhiza uralensis Fisch.ex DC. using 0.25% ammonia (34.46±3.01), 2 mm powdered roots of Inula helenium L. with 50% ethanol (56.69±2.83), and 2 mm powdered fruits of Rosa acicularis Lindl. with 40% ethanol (32.15±1.62). The glycyrrhizic acid content in the root of Glycyrrhiza uralensis Fisch.ex DC. was measured at 3.51±1.73%. Similarly, the inulin content in the root of Inula helenium L. was found to be 5.56±0.003%, and the ascorbic acid content in the fruits of Rosa acicularis Lindl. was also identified as 1.91±0.1%. After determining the extractive matters, particle size, total and internal absorption coefficients, and the content of majn biologically active compounds in the plant materials, liquid extracts were obtained from the plant raw materials, and thick extracts were prepared from these liquid extracts. The main substances in the liquid and thick extracts were identified and quantified. The glycyrrhizic acid content was 1.04±0.01% in the liquid extract and 2.87±0.08% in the thick extract; the ascorbic acid content was 0.47 ± 0.06% in the liquid extract and 0.73 ±0.06% in the thick extract; and the inulin content was 4.72±0.002% in the liquid extract and 15.07±0.002% in the thick extract. Five different models of granules were prepared from thick extracts using the wet granulation method. Model 1 granules were excluded from the study due to their irregular texture, clamminess, and inability to be sieved. The quality and technological parameters of the granule F2 model were superior to those of the other models (P<0.001). Granule model F2 contains 0.092±0.002% glycyrrhizic acid, 2.153±0.049% inulin, and 0.27±0.03% ascorbic acid. The prepared granules met the criteria for the microbiological test. Conclusion: It is suitable to extract the roots of Glycyrrhiza uralensis Fisch.ex DC., the root of Inula helenium L., and the fruits of Rosa acicularis Lindl. by powdering them into 2 mm particle sizes. Of the five models of granules prepared by wet granulation from plant extracts, model 2 was superior to the others in terms of technological and quality parameters. Sucrose was not suitable for granulation as an excipient.

Herbal products are considered important medicinal sources in both traditional medicine and modern pharmacology. Our study examined the lung-protective and anti-inflammatory properties of the herbal formulation in alipopolysaccharide (LPS)induced model of acute lung injury. Non-cytotoxic concentrations in cells were evaluated in vitrousing the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay and analysis of nitric oxide (NO) production. In our invivo study, acute lung inflammation was induced in experimental animals by LPS. Treatment with the herbal formulation notably decreased NO production, increased superoxide dismutase (SOD) levels, and reduced the protein levels of tumor necrosis factor-alpha (TNF-α) and high-mobility group box 1 (HMGB1). Furthermore, histopathological studies confirmed its protective effects against LPS-induced lung inflammation. Molecular docking revealed that glycyrrhizic acid and inulin interact strongly with TNF-α and HMGB1 proteins, supporting their potential to mediate the observed anti-inflammatory effects.In conclusion, the herbal formulation demonstrates anti-inflammatory and lung-protective properties, suggesting its potential efficacy in mitigating LPS-induced acute lung injury.

CAR-T cell therapy is a cutting-edge form of immunotherapy that involves extracting T cells from a patient, genetically modifying them into CART cells, and then reinfusing them to the target and to eliminate cancer cells. Lentiviruses are commonly used in this process to deliver the chimeric antigen receptor (CAR) gene into T cells, enabling them to recognize and attack cancer cells. Recombinant lentiviral vectors are especially advantageous because they can transduce both dividing and non-dividing cells, are generally considered safe, and provide long-term transgene expression. This is due to the integration of the viral genome known as the provirus into the host cell’s DNA, allowing it to be passed on to daughter cells. These properties are particularly important for CAR-T cell therapy, where stable and sustained expression of the CAR is essential. Lentiviral vectors are often used to deliver protein-encoding cDNAs, such as CAR constructs or reporter genes, as well as noncoding sequences for RNA interference or genome editing, including shRNA or gRNA. This study aimed to construct and validate lentiviral vectors for use in CAR-T cell therapy. Four essential plasmids—pMDLg/pRRE, pRSV-Rev, pCMV-VSV-G, and a CAR-encoding plasmid—were successfully amplified using competent cells. The plasmids were then purified, quantified, and transfected into HEK293T cells using PEI STAR. Lentiviral particles were harvested post-transfection and confirmed by RT-qPCR. In HEK293T cells, transduction efficiency reached 24.5%, while transfection efficiency was measured at 80%, as determined by fluorescence microscopy and flow cytometry. These results confirm the successful construction of functional lentiviral vectors suitable for downstream CAR-T cell applications. This establishes a foundational platform for CAR-T cell applications development in Mongolia.