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Effect of PBX1 overexpression in PCM6 and MM1S myeloma cell lines

ABSTRACT: Multiple Myeloma is an incurable plasma cell malignancy with a poor survival rate that is usually treated with immunomodulatory drugs (iMiDs) and proteosome inhibitors (PIs). The malignant plasma cells quickly become resistant to these agents causing relapse and uncontrolled growth of resistant clones. From whole genome sequencing (WGS) and RNA sequencing (RNA-seq) studies, different high-risk translocation, copy number, mutational, and transcriptional markers have been identified. One of these markers, PHF19, epigenetically regulates cell cycle and other processes and has already been studied using RNA-seq. In this study a massive (325,025 cells and 49 patients) single cell multiomic dataset was generated with jointly quantified ATAC- and RNA-seq for each cell and matched genomic profiles for each patient. We identified an association between one plasma cell subtype with myeloma progression that we have called relapsed/refractory plasma cells (RRPCs). These cells are associated with 1q alterations, TP53 mutations, and higher expression of PHF19. We also identified downstream regulation of cell cycle inhibitors in these cells, possible regulation of the transcription factor (TF) PBX1 on 1q, and determined that PHF19 may be acting primarily through this subset of cells.

ORGANISM(S): Homo sapiens

PROVIDER: GSE254307 | GEO | 2024/01/31

REPOSITORIES: GEO

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Project description:As multiple myeloma tumors universally dysregulate cyclin D genes we conducted high-throughput chemical library screens for compounds that induce suppression of cyclin D2. The top-ranked compound was a natural triterpenoid, pristimerin. We used gene expression microarray studies to identify co-regulated pristimerin-response genes and to deduce the compoundâ??s direct molecular target(s), utilizing pattern-matching algorithms available at the Connectivity Map (Cmap). The early transcriptional response of cells treated with pristimerin closely resembles cellular responses elicited by proteosome inhibitors, with rapid induction of heat shock proteins, activating transcription factor (ATF) 3 and CHOP. Enzymatic assays and immunoblotting confirm that pristimerin rapidly (<90min) and specifically inhibits chymotrypsin-like proteosome activity at low concentrations (<100nM) and causes accumulation of cellular ubiquitinated proteins. Notably, cytotoxic triterpenoids including pristimerin inhibit NF-kB activation via inhibition of IKKa or IKKbï?¬ while proteosome inhibitors instead suppress NF-kB function by impairing degradation of ubiquitinated-IkB. By inhibiting both IKK and the proteosome pristimerin causes overt suppression of constitutive NF-kB activity in myeloma cells that may mediate its suppression of cyclin D. Multiple myeloma is exquisitely sensitive to proteosome or NF-kB pathway inhibition. Consistent with this, pristimerin is potently and selectively lethal to primary myeloma cells (IC50<100nM), inhibits xenografted plasmacytoma tumors in mice and is synergistically cytotoxic with bortezomib â?? providing the rationale for pharmaceutical development of triterpenoid dual-function proteosome/NF-kB inhibitors as therapeutics for human multiple myeloma and related malignancies. Experiment Overall Design: H929 and U266 human myeloma cell lines were treated with pristimerin 500nM (~2x IC50) or DMSO vehicle and harvested at 4h, prior to the appearance of any macroscopic evidence of perturbed viability. RNA was isolated using Trizol, column purified (Qiagen, Valencia, CA) and hybridized to Hg_U133_plus_2 microarrays (Affymetrix, Santa Clara, CA) according to the manufacturerâ??s instructions. Probe-set signal intensities were PLIER16-normalized using Affymetrix Expression Console software and flagged using Affymetrix Microarray Suite 5 (MAS5) Present/Absent/Marginal detection calls. Expression data was analyzed with GeneSpring 7 (Agilent Technologies, Santa Clara CA) software, with drug-treated samples normalized per cell line to DMSO-treated controls. Gene lists were filtered to exclude non-expressed probe-sets with MAS5 â??Absentâ?? detection calls across all samples tested (treated and untreated). Probe-sets upregulated or suppressed by pristimerin in each cell line were delineated by volcano plot filter (>1.5 fold change, FDR<0.25) and the Venn intersection of probe-sets modulated by drug in both cell lines was used in deriving a pristimerin signature.

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Effect of PBX1 overexpression in PCM6 and MM1S myeloma cell lines

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