Journal: Molecular cancer research : MCR

Article Title: EZH2 or HDAC1 Inhibition Reverses Multiple Myeloma-Induced Epigenetic Suppression of Osteoblast Differentiation

doi: 10.1158/1541-7786.MCR-16-0242-T

Figure Lengend Snippet: (A–F) MC4 cells were exposed to 5TGM1 MM cells as diagrammed in Fig 1A in the absence of inhibitors. After MM removal at d0, the MC4 were cultured in OB differentiation media for 2 to 4 days with either DMSO vehicle, MC1294 (10 μM), or GSK126 (5 μM) added as indicated. (A,B) The effects of the inhibitors (A) MC1294 (HDACi) and (B) GSK126 (EZH2i) on global levels of H3K9ac, H3K27me3, H3, HDAC1, EZH2 levels in MC4 cells on day 2 were assessed by Western blot using antibodies as indicated. (C–F) Effects of the inhibitors MC1294 and GSK126 on (C) Runx2, (D) Ocn, (E) Bsp, and (F) Alpl mRNA expression during differentiation of control and 5TGM1 MM-exposed MC4 at day 0 (no inhibitor) or after 4 days of differentiation (d0±MM, d4±MM). Error bars represent SEM for 3 biological replicates. (G) Human MM1.S MM cells in transwells (or empty control transwells) were co-cultured with MC14 cells for 72 h. Following transwell removal, the MC14 cells were cultured in osteogenic media +/- GSK126 (2.5 μM) and mineralization was assessed using Alizarin Red staining at d21; the GSK126 was absent d14–21. Shown is density quantitation for the average of 6 wells with SEM and significance indicated.

Article Snippet: MM1.S in transwells (Corning Inc., 3450) or 5TGM1 cells were carefully removed (FACS analysis demonstrated that ≤ 1% 5TGM1 cells remained).

Techniques: Cell Culture, Western Blot, Expressing, Staining, Quantitation Assay

Journal: bioRxiv

Article Title: Lactoferrin-Derived Peptide Chimera Induces Caspase-independent Cell Death in Multiple Myeloma

doi: 10.1101/2024.10.24.620137

Figure Lengend Snippet: MM cell lines (MM1S, MM1R, RPMI8226) were treated with 3µM of LF or LFch for 48 h. Growth inhibition by LF or LFch was assessed using the CCK-8 assay (A). Ki67 expression (B) and annexin V + PI + apoptotic cells (C) were detected by flow cytometry. (D) Human PBMCs were cultured with 5 µg/ml of anti-human IgM for 48 h, then treated with LFch for indicated times and apoptosis was determined by flow cytometry. Data represent mean plus or minus the standard deviation (± SD) of 3 independent experiments. Statistical analyses were performed using the GraphPad Prism software (GraphPad Software, Inc., La Jolla, CA, USA). (* P < 0.05, ** P < 0.01, versus untreated control). LFch, lactoferrin-derived chimera; MM, multiple myeloma

Article Snippet: Human MM cell lines, MM1S, MM1R, and RPMI8226, were obtained from the American Type Culture Collection (ATCC, Manassas, VA, USA) and the Korean Cell Line Bank (KCLB, Seoul, Korea).

Techniques: Inhibition, CCK-8 Assay, Expressing, Flow Cytometry, Cell Culture, Standard Deviation, Software, Control, Derivative Assay

Journal: Clinical cancer research : an official journal of the American Association for Cancer Research

Article Title: BCMA-specific ADC MEDI2228 and Daratumumab induce synergistic myeloma cytotoxicity via IFN-driven immune responses and enhanced CD38 expression

doi: 10.1158/1078-0432.CCR-21-1621

Figure Lengend Snippet: A - B The transcriptional changes resulting from MEDI2228 (M2, 0.5 μg/ml) treatment were studied in H929 MM cells following 1-day incubation (>85% viable H929 cells). RNA sequencing was performed followed by gene set enrichment analysis (GSEA), showing enrichment of interferon (IFN) pathways and Type I IFN genes. Shown are the top 51 upregulated genes by M2 vs control vehicle (ctrl) ( P < .0002, Supplementary Table S1 ) with IFN-related genes ( A ) and IFN I-upregulated and/or IFN stimulated genes (ISGs) ( B , n = 62, P < .004) in black. Normalized enrichment scores (NES) and false discovery rate (FDR) scores are indicated (FDR = 0.0013 for the comparison). C - F Using real-time qRT-PCR, fold increases in indicated IFN I-related genes are shown for M2- vs ctrl-treated MM cell lines sensitive ( C - D , MM1S; E , H929) and relatively insensitive ( D and F , RPMI8226; F , JJN3) to M2 ( 20 , 21 ). MM1S ( C , D ) and RPMI8226 ( D , F ) cells were treated with 1 and 5 µg/ml of M2, respectively, for 16h (> 90% viability), followed by the examination of upregulated IFN I-related and/or ISG genes ( C , n = 60; D , n = 33). Indicated IFITs were further validated ( E , ctrl (−) vs 0.5 µg/ml M2; F , 0, 1, 10 µg/ml of M2). Transcripts were normalized by geomean of internal controls. Two biological repeats ( A , B ) or three ( C , F ) independent experiments were done for each treatment. Each experiment performed in triplicate at each condition ( C - F ). Data are presented as means ± standard deviations (SDs) (error bars). * P < .05, ** P < .01, *** P < .001, **** P < .0001

Article Snippet: MM1S cells were subcutaneously injected into 6- to 8-week-old NSG (NOD.Cg-Prkdc scid Il2rg tm1Wjl /SzJ) mice lacking functional NK cells (Jackson Labs; Bar Harbor, ME).

Techniques: Incubation, RNA Sequencing, Control, Comparison, Quantitative RT-PCR

Journal: Clinical cancer research : an official journal of the American Association for Cancer Research

Article Title: BCMA-specific ADC MEDI2228 and Daratumumab induce synergistic myeloma cytotoxicity via IFN-driven immune responses and enhanced CD38 expression

doi: 10.1158/1078-0432.CCR-21-1621

Figure Lengend Snippet: NSG mice were s.c. injected with MM1S cells 14 days before start of treatment. Mice with tumor growth were then randomized into 6 groups (6 mice per group except M2+NK+Dara): saline control (ctrl, red), NK (once weekly for 3 times, brown), NK+Dara (8 mg/kg per injection for 3 injections, green), M2 (0.3 mg/kg, only one injection, blue), M2+NK (orange), or M2+NK+Dara (n = 7, purple). A Shown are mean tumor volumes (mm 3 ) ± SDs (error bars) at following days vs start of treatment (d1). Tumor growth was significantly inhibited in the combination-treated group compared with other groups from d7 ( P < .01, Supplementary Fig. 6B ). B Weights of mice were followed. C - D Using Kaplan-Meier and log-rank analysis, the median overall survival of animals was derived (ctrl, 25 days; NK, 25 days; NK+Dara, 48 days; M2, 50 days; M2+NK, 62 days). All 7 mice were tumor-free in M2+NK+Dara group at 150-day follow-up (**** P < .0001). E Tumors were removed from mice 3 days after treatments with M2 vs ctrl, followed by immunoblotting analysis using indicated antibodies (upper left panel) and FC analysis for MICA/B and CD38 surface expression on viable MM1S tumor cells (Aqua-/AnnexinV-) (upper right histograms). Tumor tissue sections were also immunohistochemically analyzed for CD38 (Original magnification, x200) (lower panel).

Article Snippet: MM1S cells were subcutaneously injected into 6- to 8-week-old NSG (NOD.Cg-Prkdc scid Il2rg tm1Wjl /SzJ) mice lacking functional NK cells (Jackson Labs; Bar Harbor, ME).

Techniques: Injection, Saline, Control, Derivative Assay, Western Blot, Expressing