A novel enhancer RNA sustains NRF1 transcription. (A) Digital polymerase chain reaction (PCR) results showing DNA concentration of NRF1 (copies per microliter × 100) of CD138⁺ PCs isolated from the BM of 11 patients with MGUS and 16 patients with NDMM. Student t test ∗P < .05. (B) Western blot (WB) analysis of NRF1 of total cellular extracts (TCEs) of CD138⁺ PCs purified as above for comparing expression between 5 MGUS and 5 NDMM (the percentage of mPC is showed in the corresponding table) (left). Densitometric analysis of NRF1 protein expression from the WB previously described (right). Each dot represents an individual patient. Student t test ∗∗∗P < .005. (C) Quantitative reverse transcription PCR (qRT-PCR) (left) and WB (middle) analyses of NRF1 and cell proliferation assay (right) in Kms18 MM cell line transiently transfected with siNRF1 or siControl for 72 hours. Data are presented as the mean ± standard deviation (SD) of 3 independent experiments, with values normalized to actin expression. Error bars represent the SD. Student t test ∗P < .05; ∗∗∗∗P < .001. (D) qRT-PCR (left) and WB (middle) analyses of NRF1 and cell proliferation assay (right) in Kms18 MM cells stably expressing the dCas9-KRAB transcriptional repressor complex and transiently transfected with control (scramble) or NRF1 targeting sgRNAs for 72 hours. Data are presented as the mean ± SD of 3 independent experiments, with values normalized to actin expression. Error bars represent the SD. Student t test ∗∗∗P < .005; ∗∗∗∗P < .001. (E) Kaplan-Meier survival curve showing OS in the CoMMpass cohort (n = 460). Patients were stratified into 2 groups based on NRF1 expression: NRF1 high (n = 243, orange) and NRF1 low (n = 217, light blue), using the median expression value as the threshold. Patients have a maximum survival time of 60 months. (F) Box plot displaying the normalized read counts (transcripts per million [TPM]) for sample groups with low (top) and high (bottom) NRF1 expression, organized by disease stages (ISS I, ISS II, and ISS III). Statistical significance was calculated using a Kruskal-Wallis and Jonckheere-Terpstra trend tests (P value depicted in the figure). (G) Snapshots reporting genomic regions containing NRF1 promoter and its enhancer regulator. From top to bottom: gene annotation based on hg19 reference; overlapped profile of ATAC-seq from 4 NDMM samples (purple) and 4 MGUS samples (light blue); ChromHMM tracks in 2 primary MM cell lines MM196 and MM217; track showing the open chromatin sites from our in-house MM cohort (dark purple); and H3K27ac HiChIP interaction loops in MM1S (green loops). (H) Combinatorial pattern of histone marks derived from a ChromHMM model with 6 states. Heat maps display the frequency of histone modifications found in each state (left) and the probability of a state being located near another state (right). (I) qRT-PCR analysis showing the levels of eNRF1 in patients with MGUS (n = 10) and NDMM (n = 15). qRT-PCR was preceded by amplification using nested PCR. For graphical representation, the y-axis of the plot depicts the –ΔCt values with the addition of a constant (k = 15). Values were normalized to actin expression. Student t test, P value depicted in the figure. (J) Comparison of NRF1 enhancer expression levels in NDMM and matched treated samples (n = 11). Each dot represents the log-transformed normalized expression level of the NRF1 enhancer for a sample, with dashed lines connecting paired pretreated and posttreated measurements. A statistically significant reduction in NRF1 enhancer expression was observed after treatment, as determined by the Wilcoxon signed-rank test (P = .0068). Data points are color coded by treatment status, with red indicating pretreated and yellow indicating posttreated samples (left). Table showing the respective mPC percentages for each patient analyzed (right). (K) Expression levels of NRF1 and eNRF1 transcript (qRT-PCR) in Kms18 and Kms27 transiently transfected with si-eNRF1 or siControl (left). Data are presented as the mean ± SD of 3 independent experiments, with values normalized to actin expression. Error bars represent the SD. Student t test ∗∗P < .01; ∗∗∗P < .005; ∗∗∗∗P < .001. WB analysis of NRF1 (middle) and cell proliferation assay (right) of Kms18 and Kms27 transiently transfected as previously described. Data are presented as the mean ± SD of 3 independent experiments and error bars represent the SD. Student t test ∗P < .05; ∗∗∗P < .005. Chr7, chromosome 7; H3K27ac, histone H3 lysine 27 acetylation; mPC, malignant plasma cell; mRNA, messenger RNA; N°, number of cells; sgControl, single-guide control; sgRNA, single-guide RNA; si-eNRF1, small interfering enhancer nuclear respiratory factor 1; siControl, small interfering control; TMM, trimmed mean of M-values; TSS, transcription starting site.