Dual therapeutic targeting of MYC and JUNB transcriptional programs for enhanced anti-myeloma activity

Abstract

Deregulation of transcription factors (TFs) leading to uncontrolled proliferation of tumor cells within the microenvironment represents a hallmark of cancer. However, the biological and clinical impact of transcriptional interference, particularly in multiple myeloma (MM) cells, remains poorly understood. The present study shows for the first time that MYC and JUNB, two crucial TFs implicated in MM pathogenesis, orchestrate distinct transcriptional programs. Specifically, our data revealed that expression levels of MYC, JUNB, and their respective downstream targets do not correlate and that their global chromatin-binding patterns are not significantly overlapping. Mechanistically, MYC expression was not affected by JUNB knockdown, and conversely, JUNB expression and transcriptional activity were not affected by MYC knockdown. Moreover, suppression of MYC levels in MM cells via targeting the master regulator BRD4 by either siRNA-mediated knockdown or treatment with the novel proteolysis targeting chimera (PROTAC) MZ-1 overcame bone marrow (BM) stroma cell/IL-6-induced MYC- but not MEK-dependent JUNB-upregulation and transcriptional activity. Consequently, targeting of the two non-overlapping MYC- and JUNB-transcriptoms by MZ-1 in combination with genetic or pharmacological JUNB-targeting approaches synergistically enhanced MM cell death, both in 2D and our novel dynamic 3D models of the BM milieu as well as in murine xenografts. In summary, our data emphasize the opportunity to employ MYC and JUNB dual-targeting treatment strategies in MM as another exciting approach to further improve patient outcomes.

Fig. 1. Correlation and ChIP-seq analyses indicate the existence of non-overlapping MYC- and JUNB-transcriptional programs in MM cells.

a Lack of correlation of expression levels of transcription factors MYC and JUNB. Scatterplot of correlation analysis for MYC and JUNB in the CCLE dataset. The Pearson correlation coefficient was calculated to evaluate the correlation between MYC and JUNB. The minimal level of significance was p < 0.05. b Marginal overlaps in MYC and JUNB binding peaks. Venn diagram displaying the overlap of MYC with JUNB peaks (upper panel). Heatmap displaying the percentage of overlap between peaks of MYC and JUNB (lower panel). c Representative ChIP-seq tracks for MYC (upper track) and JUNB (lower track) at the loci of MYC target genes ADSL, CCT2, PIM2, PSME3, and ELAC2, visualized by genome browser Integrative Genomics Viewer (IGV). d Representative ChIP-seq tracks for JUNB (upper track) and MYC (lower track) at the loci of JUNB target genes WT1, DYRK1A, RELA, IRF4, and NFKB1, visualized by genome browser IGV. Peaks considered in this analysis were called significant using MACS with p < 0.001.

Fig. 2. Silencing of MYC but not JUNB abrogates IL-6-induced MYC mRNA and protein levels.

a, b siRNA-mediated silencing of MYC inhibits IL-6-induced upregulation of MYC but not JUNB mRNA and protein levels. MM.1S cells were transiently transfected with siMYC versus control and stimulated with IL-6 (25 ng/ml). After 24 h, MYC and JUNB mRNA (a) or protein levels (b) were determined using RT-qPCR or immunoblotting with indicated antibodies, respectively. ERK2 served as a loading control. c, d Doxycycline-induced inhibition of JUNB inhibits IL-6-induced upregulation of JUNB but not MYC mRNA and protein levels. IL-6-stimulated versus control TetR-shJUNB/ MM.1S cells were treated with doxycycline or left untreated. After 24 h, MYC and JUNB mRNA (c) or protein levels (d) were determined using RT-qPCR or immunoblotting with indicated antibodies, respectively. ERK2 served as a loading control. Data in a and c represent mean ± SD for triplicate samples of three independent experiments. ***p < 0.0001; n.s. not significant, Dox doxycycline.

Fig. 3. Pharmacological inhibition of MYC with MZ-1 abrogates BMSC- and IL-6-induced MYC but not JUNB mRNA and protein levels, as well as transcriptional activity.

a–c MZ-1-mediated silencing of MYC inhibits BMSC- and IL-6-induced upregulation of MYC, but not BRD4 or JUNB mRNA levels. MM.1S cells co-cultured with BMSCs or stimulated with IL-6 (25 ng/ml) were treated with MZ-1 (100 nM) versus control. After 24 h, MYC, BRD4, and JUNB mRNA were determined using RT-qPCR. Data represent mean ± SD for triplicate samples of three independent experiments. ns non-significant; ^*** p = 0,0004; ^** p = 0,002. d MZ-1 downregulates BRD4 and MYC, but not JUNB protein levels in MM cell: stroma cell co-cultures. After 24 h, lysates were immunoblotted with antibodies against BRD4, MYC, and JUNB. ERK2 served as a loading control. e Similar to siBRD4, MZ-1 downregulates MYC protein levels. MM.1S cells were transiently transfected with siBRD4 or treated with MZ-1 versus control and stimulated with IL-6 (25 ng/ml). After 24 h, lysates were immunoblotted with antibodies against BRD4, MYC, and JUNB. ERK2 served as a loading control. f Inhibition of IL-6-induced HBM-Luc activity by MZ-1 but not doxycycline. TetR- shJUNB/ MM.1S cells treated with or without MZ-1 or doxycycline were transiently transfected with the HBM-luc reporter together with the pRL-CMV Renilla luciferase vector. Then, the cells were treated with IL-6 or left untreated. Luciferase activity was measured by a dual-luciferase reporter assay. g Inhibition of IL-6-induced AP-1 activity by JUNB knockdown but not MZ-1. TetR- shJUNB/MM.1S cells treated with or without doxycycline were transiently transfected with the 3× AP-1 reporter together with the pRL-CMV Renilla luciferase vector. Then, the cells were treated with IL-6 or left untreated. At the indicated time points, luciferase activity was measured by dual-luciferase reporter assay. Data in f and g represent the fold change of luciferase activity relative to control cells shown as mean ± SD from three independent experiments.

Fig. 4. Dual targeting of MYC and JUNB enhances ex vivo and in vivo anti-myeloma activity.

a MZ-1 increases anti-MM activity of doxycycline-induced knockdown of JUNB. TetR-shJUNB/ MM.1S cells stimulated with IL-6 (25 ng/ml) were treated with doxycycline alone or in combination with MZ-1 for 24 h. Cell growth was measured by fluorescent induction (CyQUANT™). Data represent mean ± SD for triplicate samples of three independent experiments. b MZ-1 augments cell death in IL-6-stimulated, siJUNB-treated MM cell lines. MM cell line cells stimulated with IL-6 (25 ng/ml) were transiently transfected with siJUNB versus siControl and treated with MZ-1 versus untreated control for 24 h. Cell growth was determined by an MTS assay. * p < 0.01 as compared to control. **p < 0.001; n.s. not significant, Dox doxycycline. c–f MZ-1 and doxycycline-induced knockdown of JUNB results in synergistic inhibition of MM growth in a dynamic 3D model of the MM BM microenvironment. c Dynamic 3D model of the MM BM microenvironment. Qtracker™ 625-stained KM-105 stroma cells were pre-seeded overnight onto poly-ε-caprolactone scaffolds (PCLS). TetR-shJUNB/MM.1S cells were then added in the presence or absence of doxycycline, transferred into the 3D-RCCS™ bioreactor with or without MZ-1, and co-cultured for 72 h in RPMI-1640 media with 2% FBS. d Representative Z-stack confocal images of GFP+ TetR-shJUNB/MM.1S cells (green) and Qtracker™ 625-stained KM-105 stroma cells (yellow) cultured alone or together. Scale bars = 100 μm. e Quantification of GFP+ TetR-shJUNB/MM.1S cells in Z-stack confocal images of the 3D cultures. f Quantification of cleaved caspase-3 TetR-shJUNB/MM.1 S cells in Z-stack confocal images of the 3D cultures. In e, f, image processing, and analyses were performed with FiJi ImageJ. g–i MZ-1- and doxycycline-induced knockdown of JUNB results in synergistic inhibition of MM growth in the NSG™ xenograft model of MM. g Immunodeficient NSG™ mice were injected subcutaneously with TetR-shJUNB/ MM.1S together with human-derived BMSCs and Matrigel. They were then fed with or without doxycycline in their drinking water and treated with or without MZ-1 (5 mg/kg) i.p. (5×/week). The cartoon was created with BioRender.com. h Tumor sizes of TetR-shJUNB/ MM.1S xenografts. i Survival curves of mice carrying TetR-shJUNB/ MM.1S xenografts. * p < 0.01, ** p < 0.001, *** p < 0.0001.

Fig. 5. Dual targeting of MYC and MEK1/2 enhances anti-myeloma activity.

a In contrast to the MEK inhibitor trametinib, MZ-1 does not inhibit the phosphorylation of ERK2. MM.1S cells were treated with MZ-1 or trametinib alone or in combination and stimulated with IL-6 (25 ng/ml). After 24 h, lysates were immunoblotted with antibodies against MYC, pERK2. ERK2 served as a loading control. b MZ-1 but not trametinib inhibits IL-6-induced HBM-Luc activity. IL-6 (25 ng/ml)-stimulated TetR- shJUNB/ MM.1S cells treated with MZ-1 or trametinib versus control were transiently transfected with the HBM-luc reporter together with the pRL-CMV Renilla luciferase vector. Then, cells were treated with IL-6 or left untreated. Luciferase activity was measured by a dual-luciferase reporter assay. c Trametinib but not MZ-1 inhibits IL-6-induced AP-1 activity. TetR- shJUNB/ MM.1S cells treated with or without trametinib were transiently transfected with the 3× AP-1 reporter together with the pRL-CMV Renilla luciferase vector. Then, cells were treated with IL-6 or left untreated. Luciferase activity was measured by dual-luciferase reporter assay. Data in b and c represent the fold change of luciferase activity relative to control cells shown as mean ± SD from three independent experiments. **p < 0.001 as compared with control. d–g Synergistic increase of MZ-1-induced anti-MM activity by trametinib. Heat maps represent the percentage of inhibition achieved by MZ-1 and trametinib separately and in combination in MM.1 S (d), NCI-H929 (e), and RPMI8226 (f) MM cell lines. Cells were incubated with increasing concentrations of MZ-1 and trametinib for 24 h and inhibition of cell growth was determined by MTS assay (left panels). Synergy scores were determined by the HSA method (right panels). An HSA synergy score less than −10 is considered to indicate antagonistic, a synergy score between −10 and +10 additive, and a synergy score >10 synergistic anti-MM effects. The percentage of cell growth inhibition is depicted in a colorimetric scale from red (high) to green (low) normalized to DMSO (control). HSA [51], highest single agent. Data represent mean ± SD for triplicate samples of three independent experiments. g–i MZ-1-mediated degradation of MYC and trametinib-induced JUNB inhibition result in synergistic anti-MM growth in an NSG™ xenograft MM model. g Immunodeficient NSG™ mice were injected subcutaneously with TetR-shJUNB/ MM.1S together with human-derived BMSCs and Matrigel. They were then fed with or without trametinib (1 mg/kg) in their drinking water and treated with or without MZ-1 (5 mg/kg) i.p. (5×/week). h Tumor sizes of TetR-shJUNB/MM.1S xenografts. i Survival curves of mice carrying TetR-shJUNB/ MM.1S xenografts. Tra trametinib.