Changes in gene expression profiles of multiple myeloma cells induced by arsenic trioxide (ATO): possible mechanisms to explain ATO resistance in vivo

Abstract Introduction The development of new treatments for high-risk multiple myeloma (HRMM) are needed. The PD-1/PD-L1 axis is one of the chief inhibitory immune checkpoints in antitumor immunity. Despite the success of PD-1 (PDCD1) / PD-L1 (CD274) blockade in some neoplasms, use of it as a monotherapy has failed to improve outcome in RRMM. We have previously demonstrated that the cell-cycle-regulated serine-threonine kinase, NEK2 is elevated in HRMM and that inhibition of NEK2 can overcome drug-resistance and prolong survival of xenografted MM cells. Here, we aimed to investigate the possible role of NEK2 in regulating the immune checkpoint response in MM and development of possible anti-PD1/PDL1 combination therapies. Methods Gene expression profiles and pathway enrichment analyses were conducted on oligonucleotide microarray gene expression profiles from over 1000 primary MM samples to evaluate the correlation of NEK2 and immune checkpoint expression levels. To elucidate the underlying mechanism, we used Nek2 -/- mice crossed with EμMyc mice to generate B cell tumor mouse model with NEK2 deficiency. RNA-sequencing analyses of premalignant B cells was compared between EμMyc/Nek2 WT and EμMyc/Nek2 -/- mice. The hub molecular regulators in the NEK2 correlated pathways were further determined by western blot using NEK2 overexpressing and knockdown cell lines and then verified by co-immunoprecipitation with a NEK2 antibody. Lastly, to establish its clinic significance, the efficacy of INH1 (small compound NEK2 inhibitor), (D)-PPA 1 (peptide-based PD-1/PD-L1 interaction inhibitor) or a PD-L1 (monoclonal antibody) was tested in bone marrow BM mononuclear cells from primary MM patients in-vitro as well as in MM xenografts. Tumor burden and T cell immune responses were monitored by M-spike and mass cytometry. Results Gene expression profiles demonstrated that CD274 expression was significantly higher in the non-proliferative hyperdiploid (HY) subtype of MM, representing between 25-35% of all MM. NEK2 was negatively correlated with CD274 gene expression across all 7 MM subtypes. Gene set enrichment analysis showed that the IFN-γ signaling pathway, which can induce CD274 expression, was significantly enriched in the HY subtype as well as premalignant B cells from EμMyc/Nek2 -/- mice. Elevated expression of EZH2, a histone methyltransferase gene, is also highly correlated wirth NEK2 levels in primary MM. We found that NEK2 inhibition increases CD274 expression as well as reduced EZH2 expression and H3K27me3 levels in MM cell lines. In contrarst, myeloma cells overexpressing NEK2 showed increased expression and activity of EZH2 and H3K27me3 levels. Thus, NEK2 appears to regulate CD274/PD-L1 expression through EZH2-mediated histone methylation. Next we demonstrated that NEK2 and EZH2 directly interact and that overexpression of NEK2 leads to increased methylation of the CD274/PD-L1 gene. We treated BM mononuclear cells from primary MM with PD-1/PD-L1 inhibitor with and without a NEK2 inhibitor. The combination was most effective at eliminating CD138 + myeloma cells while having no effects on T, B and myeloid cell populations. Conclusion Our study showed that expression of CD274/PD-L1 is suppressed in primary HRMM and that CD274/PD-L1 expression is negatively regulated by NEK2 via EZH2-mediated methylation. Inhibition of NEK2 sensitizes myeloma cells to PD-1/PD-L1 blockade, showing either a synergistic or an additive effect in MM cell cytotoxicity. Disclosures No relevant conflicts of interest to declare.

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Effect of Imids on Gene Expression Profiles of Fresh Human Myeloma Cells

Blood ◽

10.1182/blood.v116.21.5013.5013 ◽

2010 ◽

Vol 116 (21) ◽

pp. 5013-5013

Author(s):

Ines Tagoug ◽

Adriana Plesa ◽

Julie Vendrell ◽

Charles Dumontet

Keyword(s):

Gene Expression ◽

Multiple Myeloma ◽

Family Member ◽

Magnetic Beads ◽

Expression Profiles ◽

Calf Serum ◽

Gene Expression Profiles ◽

Differentially Expressed ◽

Myeloma Cells ◽

Human Myeloma Cells

Abstract Abstract 5013 Immunomodulatory drugs represent a major therapeutic advance in the treatment of patients with multiple myeloma. While these agents appear to exert various effects on the microenvironment, including effect on immune cells and angiogenesis, a direct effect on the tumor cells themselves is also likely. To describe and compare the effect of the three clinically available agents (thalidomide, lenalidomide, pomalidomide) we analyzed the gene expression profiles of fresh human myeloma cells exposed to thalidomide, lenalidomide or pomalidomide, using high density DNA arrays. Fresh human myeloma samples were obtained from bone marrow aspirates of patients with myeloma, and myeloma cells were immunopurified using anti CD138 magnetic beads. Purified myeloma cells (1.106 cells/ml) were incubated for 24 hours in RPMI 1640 medium supplemented with 10% fetal calf serum under each of the four following conditions: 1) DMSO; 2) thalidomide 40 microM; 3) lenalidomide 1 microM; 4) pomalidomide 100 nM. These levels are achievable in the plasma of MM pts. Pangenomic array experiments were performed usingWhole Human Genome 4 × 44K Agilent one-color microarrays. Data were normalized using the quantile normalization method. Samples were analysed for differentially expressed genes, taking into account both the level of significance and the fold-change. Ten evaluable samples were processed. Exposure to thalidomide, lenalidomide and pomalidomide induced differential expression of 36, 50 and 75 genes, respectively, in comparison to DMSO-exposed controls, the total list including 101 genes. Twelve of these were found to be differentially expressed after exposure to all of the three agents, including trophoblast glycoprotein, WAS protein family member 1, dickkopf homolog 1, pentraxin-related gene, CD28, interleukin 12B, tissue factor pathway inhibitor 2, phospholipase A2, dehydrogenase/reductase (SDR family) member 9, hypothetical LOC145788 and betacellulin. These commonly altered genes could be mechanistically involved in themultiple activities of these agents in multiple myeloma or may represent epiphenoma mechanistically unrelated to drug-induced cell death. Genes differentially expressed between the treatment with each of these agents could be indicative of the different and non-overlapping actions these agents have in multiple myeloma. An example of this is the recent demonstration that pomalidomide is clinically active in lenalidomide refractory patients. These results suggest that exposure to IMIDs induce various intracellular signalization pathways in myeloma cells which might be involved in the cytotoxic activity of these compounds. Disclosures: Dumontet: Celgene: Research Funding.

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Cryopreservation Preserves Cell-Type Composition and Gene Expression Profiles in Bone Marrow Aspirates From Multiple Myeloma Patients

Frontiers in Genetics ◽

10.3389/fgene.2021.663487 ◽

2021 ◽

Vol 12 ◽

Author(s):

Duojiao Chen ◽

Mohammad I. Abu Zaid ◽

Jill L. Reiter ◽

Magdalena Czader ◽

Lin Wang ◽

...

Keyword(s):

Gene Expression ◽

Multiple Myeloma ◽

Bone Marrow ◽

Single Cell ◽

Expression Profiles ◽

Gene Expression Profiles ◽

Cell Type ◽

Myeloma Cells ◽

Freeze Thaw ◽

Single Cell Rna Sequencing

Single-cell RNA sequencing reveals gene expression differences between individual cells and also identifies different cell populations that are present in the bulk starting material. To obtain an accurate assessment of patient samples, single-cell suspensions need to be generated as soon as possible once the tissue or sample has been collected. However, this requirement poses logistical challenges for experimental designs involving multiple samples from the same subject since these samples would ideally be processed at the same time to minimize technical variation in data analysis. Although cryopreservation has been shown to largely preserve the transcriptome, it is unclear whether the freeze-thaw process might alter gene expression profiles in a cell-type specific manner or whether changes in cell-type proportions might also occur. To address these questions in the context of multiple myeloma clinical studies, we performed single-cell RNA sequencing (scRNA-seq) to compare fresh and frozen cells isolated from bone marrow aspirates of six multiple myeloma patients, analyzing both myeloma cells (CD138+) and cells constituting the microenvironment (CD138−). We found that cryopreservation using 90% fetal calf serum and 10% dimethyl sulfoxide resulted in highly consistent gene expression profiles when comparing fresh and frozen samples from the same patient for both CD138+ myeloma cells (R ≥ 0.96) and for CD138– cells (R ≥ 0.9). We also demonstrate that CD138– cell-type proportions showed minimal alterations, which were mainly related to small differences in immune cell subtype sensitivity to the freeze-thaw procedures. Therefore, when processing fresh multiple myeloma samples is not feasible, cryopreservation is a useful option in single-cell profiling studies.

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Transcriptome analysis of gravitational effects on mouse skeletal muscles under microgravity and artificial 1 g onboard environment

Scientific Reports ◽

10.1038/s41598-021-88392-4 ◽

2021 ◽

Vol 11 (1) ◽

Author(s):

Risa Okada ◽

Shin-ichiro Fujita ◽

Riku Suzuki ◽

Takuto Hayashi ◽

Hirona Tsubouchi ◽

...

Keyword(s):

Gene Expression ◽

Muscle Mass ◽

Muscle Atrophy ◽

Transcriptome Analysis ◽

Fiber Type ◽

Expression Profiles ◽

Space Station ◽

Gene Expression Profiles

AbstractSpaceflight causes a decrease in skeletal muscle mass and strength. We set two murine experimental groups in orbit for 35 days aboard the International Space Station, under artificial earth-gravity (artificial 1 g; AG) and microgravity (μg; MG), to investigate whether artificial 1 g exposure prevents muscle atrophy at the molecular level. Our main findings indicated that AG onboard environment prevented changes under microgravity in soleus muscle not only in muscle mass and fiber type composition but also in the alteration of gene expression profiles. In particular, transcriptome analysis suggested that AG condition could prevent the alterations of some atrophy-related genes. We further screened novel candidate genes to reveal the muscle atrophy mechanism from these gene expression profiles. We suggest the potential role of Cacng1 in the atrophy of myotubes using in vitro and in vivo gene transductions. This critical project may accelerate the elucidation of muscle atrophy mechanisms.

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Gene Expression Profiles of Cultured Rat Cardiomyocytes (H9C2 Cells) in Response to Arsenic Trioxide at Subcytotoxic Level and Oxidative Stress

JOURNAL OF HEALTH SCIENCE ◽

10.1248/jhs.52.512 ◽

2006 ◽

Vol 52 (5) ◽

pp. 512-521 ◽

Cited By ~ 8

Author(s):

Eun-Jung Park ◽

Kwangsik Park

Keyword(s):

Oxidative Stress ◽

Gene Expression ◽

Arsenic Trioxide ◽

Expression Profiles ◽

Gene Expression Profiles ◽

H9c2 Cells ◽

Rat Cardiomyocytes ◽

And Oxidative Stress

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Classify Hyperdiploidy Status of Multiple Myeloma Patients Using Gene Expression Profiles

PLoS ONE ◽

10.1371/journal.pone.0058809 ◽

2013 ◽

Vol 8 (3) ◽

pp. e58809 ◽

Cited By ~ 9

Author(s):

Yingxiang Li ◽

Xujun Wang ◽

Haiyang Zheng ◽

Chengyang Wang ◽

Stéphane Minvielle ◽

...

Keyword(s):

Gene Expression ◽

Multiple Myeloma ◽

Expression Profiles ◽

Gene Expression Profiles

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Multiple Myeloma Cells Alter Adipogenesis, Increase Senescence-Related and Inflammatory Gene Transcript Expression, and Alter Metabolism in Preadipocytes

Frontiers in Oncology ◽

10.3389/fonc.2020.584683 ◽

2021 ◽

Vol 10 ◽

Author(s):

Heather Fairfield ◽

Samantha Costa ◽

Carolyne Falank ◽

Mariah Farrell ◽

Connor S. Murphy ◽

...

Keyword(s):

Gene Expression ◽

Multiple Myeloma ◽

Bone Marrow ◽

Lipid Accumulation ◽

Expression Profiles ◽

Adipogenic Differentiation ◽

Gene Expression Profiles ◽

Mouse Bone Marrow ◽

Gene Transcript ◽

Bone Marrow Mscs

Within the bone marrow microenvironment, mesenchymal stromal cells (MSCs) are an essential precursor to bone marrow adipocytes and osteoblasts. The balance between this progenitor pool and mature cells (adipocytes and osteoblasts) is often skewed by disease and aging. In multiple myeloma (MM), a cancer of the plasma cell that predominantly grows within the bone marrow, as well as other cancers, MSCs, preadipocytes, and adipocytes have been shown to directly support tumor cell survival and proliferation. Increasing evidence supports the idea that MM-associated MSCs are distinct from healthy MSCs, and their gene expression profiles may be predictive of myeloma patient outcomes. Here we directly investigate how MM cells affect the differentiation capacity and gene expression profiles of preadipocytes and bone marrow MSCs. Our studies reveal that MM.1S cells cause a marked decrease in lipid accumulation in differentiating 3T3-L1 cells. Also, MM.1S cells or MM.1S-conditioned media altered gene expression profiles of both 3T3-L1 and mouse bone marrow MSCs. 3T3-L1 cells exposed to MM.1S cells before adipogenic differentiation displayed gene expression changes leading to significantly altered pathways involved in steroid biosynthesis, the cell cycle, and metabolism (oxidative phosphorylation and glycolysis) after adipogenesis. MM.1S cells induced a marked increase in 3T3-L1 expression of MM-supportive genes including Il-6 and Cxcl12 (SDF1), which was confirmed in mouse MSCs by qRT-PCR, suggesting a forward-feedback mechanism. In vitro experiments revealed that indirect MM exposure prior to differentiation drives a senescent-like phenotype in differentiating MSCs, and this trend was confirmed in MM-associated MSCs compared to MSCs from normal donors. In direct co-culture, human mesenchymal stem cells (hMSCs) exposed to MM.1S, RPMI-8226, and OPM-2 prior to and during differentiation, exhibited different levels of lipid accumulation as well as secreted cytokines. Combined, our results suggest that MM cells can inhibit adipogenic differentiation while stimulating expression of the senescence associated secretory phenotype (SASP) and other pro-myeloma molecules. This study provides insight into a novel way in which MM cells manipulate their microenvironment by altering the expression of supportive cytokines and skewing the cellular diversity of the marrow.

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Abstract 268: Strain-specific Cardiac Fibroblast Response to Isoproterenol-induced Cardiac Fibrosis

Circulation Research ◽

10.1161/res.121.suppl_1.268 ◽

2017 ◽

Vol 121 (suppl_1) ◽

Author(s):

Shuin Park ◽

Sara Ranjbarvaziri ◽

Fides Lay ◽

Peng Zhao ◽

Aldons J Lusis ◽

...

Keyword(s):

Gene Expression ◽

Cardiac Fibrosis ◽

Cardiac Fibroblasts ◽

Expression Profiles ◽

Inbred Mouse ◽

Gene Expression Profiles ◽

Mouse Strains ◽

Sequencing Analysis ◽

Different Strains

Fibroblasts are a heterogeneous population of cells that function within the injury response mechanisms across various tissues. Despite their importance in pathophysiology, the effects of different genetic backgrounds on fibroblast contribution to the development of disease has yet to be addressed. It has previously been shown that mice in the Hybrid Mouse Diversity Panel, which consists of 110 inbred mouse strains, display a spectrum in severity of cardiac fibrosis in response to chronic treatment of isoproterenol (ISO). Here, we characterized cardiac fibroblasts (CFbs) from three different mouse strains (C57BL/6J, C3H/HeJ, and KK/HIJ) which exhibited varying degrees of fibrosis after ISO treatment. The select strains of mice underwent sham or ISO treatment via intraperitoneally-implanted osmotic pumps for 21 days. Masson’s Trichrome staining showed significant differences in fibrosis in response to ISO, with KK/HIJ mice demonstrating the highest levels, C3H/HeJ exhibiting milder levels, and C57BL/6J demonstrating little to no fibrosis. When CFbs were isolated and cultured from each strain, the cells demonstrated similar traits at the basal level but responded to ISO stimuli in a strain-specific manner. Likewise, CFbs demonstrated differential behavior and gene expression in vivo in response to ISO. ISO treatment caused CFbs to proliferate similarly across all strains, however, immunofluorescence staining showed differential levels of CFb activation. Additionally, RNA-sequencing analysis revealed unique gene expression profiles of all three strains upon ISO treatment. Our study depicts the phenotypic heterogeneity of CFbs across different strains of mice and our results suggest that ISO-induced cardiac fibrosis is a complex process that is independent of fibroblast proliferation and is mainly driven by the activation/inhibition of genes involved in pro-fibrotic pathways.

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