Page 136 - Haematologica April 2020

H. Kumar et al.
Furthermore, consequent to its downregulation of p65, clofazimine suppressed an NFκB-RE reporter (Figure 5D). We next investigated whether the clofazimine-mediated rapid decrease in p65 was due to proteasomal degrada- tion, and found that clofazimine failed to reduce p65 in the presence of the proteasomal inhibitors MG132 and
ABC
lactacystin (Figure 5E). p65 degradation was associated with its increased ubiquitination by clofazimine (Figure 5F). These results indicate that clofazimine causes p65 ubiquitination leading to its proteasomal degradation. Clofazimine also significantly decreased p65 protein in CP-CML cells (Figure 5G).
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Figure 4. Clofazimine modulates PRXD1 expression via transcrip- tional regulation of MYB. (A, B) Clofazimine (CFZ) (5 μM) reduces myeloblastoma oncoprotein (MYB) protein (A), mRNA (B) expression in K562 cells. (C) CFZ (5 μM, 24 h) reduces a three copy MYB consen- sus response element-containing reporter activity in K562 cells and overexpression of MYB, mitigates this repression. (D) CFZ (5 μM, 24 h) represses the peroxiredoxin 1 (PRDX1) promoter-luc and overex- pression of MYB activates this reporter and ameliorates its CFZ- mediated repression. (E) Deletion mapping of the ~1 kb PRDX1 pro- moter in HEK-293 cells reveals MYB response between the -11 to +9 region in the MYB promoter. (F) MYB activates a reporter contain- ing -11 to +83 wildtype (Wt) but not mutated (mt; mutated bases in lower case letters) PRDX1 promot- er sequence in HEK-293 cells. (G) MYB activates a reporter contain- ing the -11 to +9 PRDX1 promoter sequence in three tandem repeats in HEK-293 cells. (H) MYB is recruited to the endogenous PRDX1 promoter in K562 cells and CFZ (24 h) reduces its recruitment and concomitantly reduces histone 3 (K9) acetylation. (I, J) CFZ (5 μM, 24 h) reduces MYB protein in ima- tinib-resistant chronic phase chron- ic myeloid leukemia (CP-CML) cells (I; graphical representation, J; rep- resentative histogram). (K). CFZ (5 μM, 24 h) suppresses MYB expres- sion in CD34+ cells. (L-O; treatment concentration and duration same as Figure 3A-D) Overexpression of MYB in K562 cells mitigates CFZ- mediated downregulation of PRDX1 protein level, and induction of caspase-3 cleavage (L), enhanced K562 apoptosis (M; dot plots in Online Supplementary Figure S8A), CD61 expression (N; histograms in Online
LMNO Supplementary Figure S8B) and cellular reactive oxygen species (O). (P) MYB mRNA expression in CP-CML cells. Graphs, except (P), show the mean ± standard error of mean of three independent experi- ments. Immunoblots are one repre- sentative of three independent experiments. *P<0.05, **P<0.01, ***P<0.001. (B, H) one-way, (M-O) two-way ANOVA followed by the Bonferroni post-test. (I, K); unpaired two-tailed Student t-test. (P) Kruskal-Wallis test followed by P the Dunn test. E2F1; E2F transcrip- tion factor ATF-4: activating tran- scription factor 4; RLU: relative light unit; 3X MRE: three copy MYB consensus response element; V; vehicle; DCFDA: 2',7'-dichlorofluo- rescein diacetate; GAPDH: glycer- aldehyde 3-phosphate dehydroge-
nase.
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haematologica | 2020; 105(4)