Project description:Here we report the use of high-throughput sequencing technologies (RNA-seq, ChIP-seq) to identify the molecular programme of PBX1 and FOXM1 in multiple myeloma cells (MM1S, U266 cell lines). We performed Chromatin Immunoprecipitation followed by sequencing (ChIP-seq) against PBX1 in MM1S and U266 cells (n=2). In addition, we identifed the transcriptome of PBX1-depleted and FOXM1-depleted myeloma cells 3 days after transduction with shRNA-expressing lentiviral vectors. Molecular characterization revealed PBX1 as a novel epigenetic regulator of myeloma cell survival and proliferation. PBX1 directly and unilaterally controls the FOXM1 transcriptional programme and, together,they regulate the high-risk transcriptional signature of chr1q-amplified cells. Pharmacological inhibition of the unified PBX1-FOXM1 axis with thiostrepton showed selectivity against chr1q-amplified MM. Altogether, these data reveal PBX1-FOXM1 axis as a novel therapeutic avenue against chr1q-amplified MM.
Project description:Forkhead Box M1 (FOXM1) is a promising molecular target for high-risk multiple myeloma and relapse and refractory myeloma, but whether FOXM1 is essential in myeloma has not yet been established. To address this knowledge gap, we used Western blotting to measure FOXM1 protein levels in 11 human myeloma cell lines (HMCLs) and then chose the two lines expressing the largest amount of the transcription factor, OPM2 and Delta47, for gene editing using CRISPR-Cas9. In both cases, two independent FOXM1-deficient daughter lines, designated KO-1 and KO-2, were generated. They contained 2 different 10-bp deletions at the target site of their respective guide RNA in case of OPM2 and a 10-bp deletion and 1-bp insertion in case of Delta47. Western analysis confirmed the lack of FOXM1 in all knockout clones. FOXM1-deficient myeloma cells proliferated more slowly than their parental counterparts containing normal levels of FOXM1. Moreover, we added back FOXM1 to FOXM1-KO cells by transfection of a constitutively expressed FOXM1c cDNA gene. The reconstituted OPM2 and Delta47 cells, designated FOXM1 KO-R contained high amounts of FOXM1 protein. Here, we used these cell lines to investigate the role of FOXM1 in regulating the gene expression in multiple myeloma.
Project description:We report the genomic localization of 5-hydroxymethylcytosines in multiple myeloma patients through the use of selective chemical labeling (SCL) and exonuclease digestion. Data highlighted super-enhancers and enhancers specifically undergoing active DNA demethylation in multiple myeloma cells.
Project description:In multiple myeloma (MM), endothelial progenitor cells (EPCs) regulate tumor angiogenesis and disease progression. They share a common bone marrow microenvironment with myeloma tumor cells. CD138+ tumor plasma cells from 12 newly diagnosed patients with advanced MM were examined for genomic instability by RNA microarrays to assess changes in gene expression.
Project description:In multiple myeloma (MM), endothelial progenitor cells (EPCs) regulate tumor angiogenesis and disease progression. They share a common bone marrow microenvironment with myeloma tumor cells. CD138+ tumor plasma cells from 12 newly diagnosed patients with advanced MM were examined for genomic instability by RNA microarrays to assess changes in gene expression. Tumor cells were derived from single-cell suspensions of bone marrow (BM) aspirates from newly diagnosed MM patients, and RNA was extracted for microarray hybridization.
