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Bone Marrow Stroma-induced Transcriptome and Regulome Signatures of Multiple Myeloma [ATAC-seq]

ABSTRACT: Multiple myeloma (MM) is a hematological cancer with inevitable drug resistance. MM cells interacting with bone marrow stromal cells (BMSCs) undergo substantial changes in transcriptome and develop de novo drug resistance. As a critical component in transcriptional regulation, how chromatin landscape is transformed in MM cells and regulates the transcriptional response to BMSCs remains elusive. We profiled transcriptome and regulome for MM cells using a transwell coculture system with BMSCs. The transcriptome and regulome of MM cells from the upper transwell resembled MM cells coexisting with BMSCs from the lower chamber but were distinctive to monoculture. BMSC-induced genes were enriched in JAK2/STAT3 signaling pathway, unfolded protein stress, signatures of early plasma cells, and response to proteasome inhibitors. Genes with accessibility increased at multiple regulatory sites were preferentially induced by BMSCs and enriched in response to drug and unfavorable prognostic markers from several MM patient cohorts. We proposed JUNB and ATF4::CEBPβ as candidate transcription factors (TFs) that modulated the BMSC-induced transformation of regulome linked to the response to external stimuli. Together, we characterized BMSC-induced transcriptome and regulome signatures of MM cells and prioritized TFs for future study to depict epigenetic mechanisms of BMSC-induced drug resistance in MM.

ORGANISM(S): Homo sapiens

PROVIDER: GSE193657 | GEO | 2022/02/16

REPOSITORIES: GEO

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Project description:miRNA expression profiling was performed on MM.1S MM cells cultured 8 hours in control media or 50nM RGB-286638, with or without BMSCs. The emerging role of miRNAs in the pathogenesis of multiple myeloma (MM) led us to hypothesize that the miRNA network might be among the inducible transcriptional alterations consequent to MM-bone marrow stromal cell (BMSC) interactions. Our data suggests that BMSC induced MM transcription led to aberrant miRNA expression. We therefore hypothesized that agents interfering with RNAPII transcription might inhibit aberrant miRNA expression in MM. To test this hypothesis we used RGB-286638, a novel protein kinase inhibitor, which works primarily via RNAPII inhibition followed by transcriptional arrest in MM cells. miRNA profiling of RGB-286638-exposed MM cells resulted in RNAPII arrest associated with reduced miRNA levels. RGB-286638 abrogated BMSCs-induced miRNAs, which correlated with growth arrest in MM cells. Analysis of RGB-286638-induced differentially-expressed miRNAs in MM cells, in the presence or absence of BMSCs, revealed RNAPII regulation of expression of BMSC-inducible miRNAs with established oncogenic functions in MM Our findings demonstrate the role of RNAPII in regulating miRNA network, suggesting a new rationale for using agents interfering with RNAPII transcription in the treatment of MM. TaqMan Low-Density Array (TLDA) using human miRNA version 2.0A and version 3.0B cards (Applied Biosystems) were applied to examine the global change of miRNA expression levels in MM.1S cells when co-cultured with BMSCs, with or without RGB-286638 treatment. A total of 756 mature miRNA updated in the Sanger miRBase v.15.0 were quantified according to the manufacturer's instructions as previously described. miRNAs with Ct values higher than 37 were excluded from the analysis. Normalization was carried out with the mean of RNU44 and RNU48. Relative quantification of miRNA expression was calculated with the 2M-bM-^HM-^RM-NM-^TM-NM-^TCt Ct method using the ddCt program (Shannon McCormack Advanced Molecular Diagnostics Laboratory Research Services). The data was presented as log10 of the relative quantity of each miRNA.

Project description:Bone marrow derived stromal cells (BMSCs) are a multipotent population that supports angiogenesis, wound healing, immunomodulation and plays an active role in the hematopoietic niche. On the other hand, they are also involved in the nurturing of bone marrow tumors and metastasis, showing a pro-tumorigenic behavior. BMSCs secrete a wide range of cytokines, growth factors and matrix proteins that are likely responsible for many of these effects. However, it is not clear whether this pro-tumorigenic behavior of BMSCs is induced by the tumor cells, neither in what extent the tumor cells affect the type and quantity of factors produced by BMSCs. To determine how tumor cells that arise from bone marrow affect the BMSCs, we selected three myeloid leukemia cell lines (TF-1, TF-1alpha and K562) and co-cultured them with BMSCs from healthy donors. We found that, under co-culture condition, the gene expression profiling of BMSCs revealed up-regulation of many pro-inflammatory signaling related genes, mainly IL-17 signaling-related genes. Moreover, IL-17 signaling-related cytokines CCL2 and IL8, were increased in co-culture supernatants. We conclude that BMSCs react to the presence of leukemia cells undergoing changes in the cytokine and chemokine secretion profile. Thus, BMSCs and leukemia cells both contribute to the creation of a competitive niche more favorable to leukemia stem cells. BMSCs from healthy donors were transwell co-cultured with three different myeloid leukemia cell lines: TF-1 (n=3), TF-1alpha (n=3) and K562 (n=3). A 1-um Transwell system (BDBiosciences, San Jose, CA USA) was used to maintain the cultured BMSC and leukemia cell populations separate from each other. As a control BMSCs were also transwell co-cultured under the same conditions with CD34+ cells (n=9) isolated from G-CSF-mobilized peripheral blood stem cells from healthy donors. An alternative co-culture method was used to analyze BMSCs and leukemia cells in direct contact: TF-1 (n=3), TF-alpha (n=3) and K562 (n=3). The two populations were cultured together in the same well without any membrane separation. BMSCs (n=18), TF-1 (n=3), TF-1alpha (n=3), K562 (n=3) and CD34+ (n=9) cells cultured alone (mono-cultures) were used as controls. Cells from both mono- and co-culture conditions were harvested at 4h, 10h, and 24h.

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Bone Marrow Stroma-induced Transcriptome and Regulome Signatures of Multiple Myeloma [ATAC-seq]

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