PBX1 directly regulates FOXM1- and E2F1/2-associated transcriptional programs in chr1q-amp MM cells. (A) Regulatory connections between PBX1 and its downstream targets FOXM1, E2F1/2, and NEK2 in chr1q-amp MM cells as derived from Figure 3E-F. (B) IGV snapshots display the epigenomic features of prominent genetic loci: PBX1 promoter and enhancer, E2F1 promoter, E2F2 promoter and enhancer, FOXM1 enhancer, NEK2 promoter and enhancer. From top tobottom: PBX1 ChIP-seq in MM.1S and U266 cells, ChromHMM maps in MM.1S and U266 cells (color code same as in Figure 3A), and SEs as identified in chr1q-amp MMCL and primary samples. (C) Flow cytometry–based analysis of MM.1S cell survival (n = 3) upon transduction with anti-FOXM1 shRNAs (O1, O4) and scrbl lentiviral vectors. Statistical analysis was performed using 2-way analysis of variance (ANOVA) with a post hoc multiple-comparisons test. (D) Analysis of PBX1, FOXM1, and NEK2 expression levels by RT-qPCR after lentiviral transduction with anti-FOXM1 and scrbl shRNA in MM.1S cells (n = 3). Statistical analysis was performed using 1-way ANOVA with a post hoc multiple-comparisons test. (E) Heat map representation of differentially expressed genes after FOXM1 depletion with O1 and O4 shRNAs in comparison with scrbl (RNA-seq, n = 2). (F) Overrepresentation analysis of significantly upregulated (upper panel) and downregulated (lower panel) genes upon FOXM1 knockdown in MM.1S cells. (G) Intracellular staining was followed by flow cytometric analysis of MM.1S (upper panels) and NCU.MM1 (lower panels) cells transduced with control (MIGR-EV) or PBX1-overexpressing (MIGR-PBX1) vectors using anti-PBX1 or isotype control antibodies (mean fluorescence intensity ratio between antibodies is shown). (H) RT-qPCR analysis of NEK2, E2F2, and FOXM1 mRNA expression in PBX1-overexpressing vs control MM.1S (upper panel) and NCUMM1 (lower panel) cells (n = 4). Data were analyzed using 1-way ANOVA with a post hoc multiple-comparisons test. (I) Drug-sensitivity assays in MIGR-EV and MIGR-PBX1 transduced MM.1S (upper panel) and NCU.MM1 (lower panel) cells 48 hours after treatment with the FOXM1 inhibitor thiostrepton (n = 3). Fifty percent inhibitory concentration (IC₅₀) values were calculated for each cell line using a nonlinear fitting model (fitting line represented here). Error bars show standard errors of the mean. *P < .05; **P < .01; ***P < .001; ****P < .0001. FDR, false discovery rate; miRNA, microRNA; ns, not significant; Regul., regulation; Resp., response.