Page 134 - Haematologica April 2020

H. Kumar et al.
clofazimine altered the expression of factors that modu- late cellular ROS or impart protection against them. Evaluation of peroxiredoxin thioperoxidases, which cat- alyze reduction of peroxynitrite, H2O2 and organic hydroperoxides24,28 revealed that clofazimine reduced PRDX1 expression at 12 h (Figure 2J) which coincided with clofazimine-induced ROS production (Figure 2C). Clofazimine also reduced PRDX3 (24 h), PRDX2 and PRDX5 (48 h) but not PRDX4 and PRDX6 (Figure 2J). Clofazimine suppressed cytosolic superoxide dismutase SOD1 (24 h onwards) but not mitochondrial SOD2 expression (Figure 2J). Nuclear factor erythroid 2 like 2 (NFE2L2), which regulates expression of various cytopro- tective and multidrug resistant proteins,29 was also down- regulated by clofazimine (24 h onwards; Figure 2J). Clofazimine did not alter catalase expression (Figure 2J).
Since suppression of PRDX1 expression was the most proximal event observed (Figure 2J), we studied it in detail. Clofazimine suppressed PRDX1 mRNA in K562 cells as early as 6 h (Figure 2K; quantitative real-time poly- merase chain reaction primer sequences are listed in Online Supplementary Table S2) indicating that clofazimine may regulate the PRDX1 promoter. We thus assessed clo- fazimine’s effect in HEK-293 cells transfected with a PRDX1 promoter-driven luciferase reporter (PRDX1-luc; - 1065-+83) or an empty reporter and found that clofaz- imine specifically repressed the PRDX1-luc (Figure 2L), confirming that it modulates the PRDX1 promoter. Figure 2L also indicates that factor(s) responsible for clofazimine- mediated downregulation of the PRDX1 promoter is(are) endogenously expressed in HEK-293. Clofazimine also reduced PRDX1 protein in CML cells (Figure 2M).
Introduction of exogenous PRDX1 ameliorates clofazimine-induced generation of cellular reactive oxygen species, differentiation and apoptosis
We next asked whether clofazimine’s actions were medi- ated by PRDX1 and thus conducted rescue experiments with exogenous PRDX1. PRDX1 overexpression in K562 cells abrogated clofazimine-induced ROS production (Figure 3A), caspase cleavage and BAX expression (Figure 3B). Clofazimine-induced K562 apoptosis and differentia- tion were also blocked in PRDX1-transfected cells (Figure 3C, D and Online Supplementary Figure S6A,B). Consistently, clofazimine failed to induce ROS and apoptosis in PRDX1- transfected CML CD34+ cells (Figure 3E-H). Given its observed protective function we investigated whether PRDX1 expression is elevated in CML cells and found that there was a trend to increased PRDX1 mRNA (albeit statis- tically insignificant) in CP-CML cells compared to healthy donor cells (Figure 3I). We also compared PRDX1 levels in LSC versus non-LSC. Both CD34+38+ and CD34+38- cells expressed significantly higher levels of PRDX1 transcript than did CD34-38+ cells (which were primarily gated mono- cytic cells), with the highest expression observed in the CD34+38+ population (Figure 3J, Online Supplementary Figure S6C). These results indicate that clofazimine-mediated transcriptional repression of PRDX1 expression plays a key role in imparting clofazimine’s actions.
Clofazimine-mediated suppression of PRDX1 expression is achieved through downregulation of MYB expression
Clofazimine decreased PRDX1 mRNA at 6 h (Figure 2K) and suppressed a PRDX1 promoter reporter (Figure
2L), indicating that it may regulate transcription factors that modulate the PRDX1 promoter. A literature search revealed predicted binding sites for MYB, E2F transcrip- tion factor 1 (E2F1), glucocorticoid receptor (GR), CCAAT/enhancer binding protein alpha (CEBPα), cAMP response element binding protein (CREB), activating transcription factor 4 (ATF-4) and activator protein 1 (AP-1) on the PRDX1 promoter.30 In K562 cells, clofaz- imine treatment decreased MYB expression from 3 h and CREB from 12 h onwards but had only a modest or no effect on GR, E2F1, C-Jun, C-Fos and ATF-4 (Figure 4A, Online Supplementary Figure S7).
Since MYB downregulation was the most proximal event observed which preceded PRDX1 downregulation and that MYB is endogenously expressed in HEK-293 cells,31 we studied it in detail. Clofazimine reduced MYB transcripts in K562 cells from 1 h onwards (Figure 4B). Consistently, clofazimine suppressed a canonical MYB response element-driven reporter (3X MRE), while transfection of exogenous MYB rescued it (Figure 4C). We next assessed whether MYB could regulate PRDX1 promoter, and introduction of exogenous MYB did indeed activate PRDX1-luc (-1065-+83) (Figure 4D). Furthermore, clofazimine downregulated PRDX1-luc in vector-transfected cells, and introduction of exogenous MYB dampened it (Figure 4D). These results indicate that MYB regulated the PRDX1 promoter probably by directly binding to it, and clofazimine suppressed PRDX1 expression by downregulating endogenous MYB expression. We thus attempted to identify the MYB- responsive element on the PRDX1 promoter. PRDX1 promoter deletion-mapping revealed that MYB activat- ed the -11-+83 but not the +9-+83 construct (Figure 4E). Analysis of the -11-+9 region revealed a sequence resembling the consensus MYB binding site “PyAACG/TG”32,33 in reverse and complementary orien- tation; “CCGTTC”, at position -8--3. Mutation of this sequence in the -11-+83 promoter reporter to “CCGggC”, led to complete loss of MYB-responsiveness (Figure 4F). To further confirm that this sequence is indeed the MYB-responsive region on the PRDX1 pro- moter, we constructed a reporter containing three copies of the -11-+9 sequence (PRDX-MYB-RE) and co-trans- fected it with MYB or empty vector and found that MYB did indeed specifically activate PRDX-MYB-RE (Figure 4G). Chromatin immunoprecipitation confirmed that MYB was recruited on the PRDX1 promoter and that clofazimine reduced its recruitment, and consequently reduced histone H3 acetylation (indicating reduced tran- scription) (Figure 4H). Consistently, clofazimine reduced MYB protein in CP-CML (Figure 4I-J), and MYB tran- script in CML CD34+ cells (Figure 4K).
We next assessed whether the introduction of exoge- nous MYB could compromise clofazimine’s actions and found that MYB overexpression in K562 cells did indeed mitigate the clofazimine-mediated decrease in PRDX1 expression and increase in caspase-3 cleavage, apopto- sis, differentiation and ROS (Figure 4L-O, Online Supplementary Figure S8A,B). MYB mRNA expression in both imatinib-resistant and -responsive CP-CML cells was significantly higher than in control cells (Figure 4P). These results indicate that MYB binds to the PRDX1 promoter and regulates its expression and clofazimine- mediated cellular functions are achieved through down- regulation of MYB.
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