Page 139 - Haematologica April 2020

Clofazimine inhibits chronic myeloid leukemia
that clofazimine causes p65 proteasomal degradation by ubiquitinating it, which leads to sequential MYB and PRDX1 downregulation, ultimately resulting in the cellu- lar effects imparted by clofazimine.
Clofazimine functions through a direct interaction with PPARγ.
Since clofazimine induced p65 ubiquitination (Figure 5F), we next investigated whether clofazimine modulated any of the E3 ubiquitin ligases that are reported to ubiqui- tinate p65. PDZ and LIM domain protein 2 (PDLIM2), inhibitor of growth family member 4 (ING4), cullin 5 (CUL5), copper metabolism domain containing 1 (COMMD1; also called MURR1) and PPARγ ubiquitinate p65 and cause its proteasomal degradation.40,41 We thus assessed whether clofazimine acted through any of these factors. While RNAi-mediated depletion of PDLIM2, ING4, CUL5 and COMMD1 failed to affect clofazimine- induced p65 degradation (Figure 6A), PPARγ depletion mitigated clofazimine-mediated decrease in p65, MYB and PRDX1 and increase in caspase-3 cleavage, apoptosis, differentiation and ROS (Figure 6B-E, Online Supplementary Figure S10A, B) indicating that clofazimine may modulate PPARγ activity (it is important to note here that HEK-293 cells also express endogenous PPARγ42,43).
We therefore evaluated whether clofazimine interacts with PPARγ and assessed its interaction with purified PPARγ protein in a cell-free, time-resolved fluorescence resonance energy transfer assay. Clofazimine successfully competed with a fluorophore-labeled PPARγ agonist for binding to purified PPARγ-LBD with an IC50 of 0.1 μM (Figure 6F). Since PPARγ is also a transcription factor, we assessed whether clofazimine also modulated its tran- scriptional activity in a heterologous system, in which the cells were transfected with pM-PPARγ [Gal4-DNA-bind- ing-domain (DBD) fused to PPARγ-ligand-binding domain] or an empty pM vector containing Gal4-DBD, and a Gal-UAS-luc reporter containing binding sites for GAL4-DBD. Clofazimine concentration-dependently increased the GAL-UAS reporter activity in the presence of pM-PPARγ but not pM alone (Figure 6G). To confirm clofazimine-mediated transcriptional activation of PPARγ we also studied it on a direct repeat-1 (DR-1; 3-copy) PPAR response element-driven reporter (PPRE-luc) using full-length PPARγ and, in this case too, clofazimine increased PPRE-Luc activity in the presence of transfected PPARγ (a modest response was also seen in vector-trans- fected cells; due to endogenous PPARγ) (Figure 6J). To fur- ther probe the interaction of clofazimine with PPARγ, we titrated clofazimine with PPARγ-LBD (the purification and characterization of PPARγ-LBD are illustrated in Online Supplementary Figure S11A-D) by isothermal titration calorimetry. The titration curve of clofazimine with PPARγ-LBD shows a series of endothermic reactions fol- lowed by exothermic isotherms (Figure 6H). Stoichiometry calculated by integrating isotherms was one: i.e., one molecule of clofazimine bound to one macromolecule of PPARγ-LBD. The equilibrium rate dis- sociation constant (KD) value was 0.2178 nM (Figure 6H). PPARγ also interacted with clofazimine in a surface plas- mon resonance experiment (Figure 6I; the KD calculated by this method was 79 nM). Clofazimine did not alter the activities of pM-PPARα or pM-PPARδ, indicating its speci- ficity for PPARγ (Figure 6K).
We next assessed whether there is any difference in
PPARγ mRNA expression between healthy control and CP-CML cells. A remarkably lower level of PPARγ tran- scripts was observed in the CP-CML cells than in healthy donors’ cells, with the difference being more pronounced in cells from imatinib-resistant patients (Figure 6L). Together, these results demonstrate that clofazimine binds to PPARγ and modulates its transcriptional as well as E3 ubiquitin ligase activity and via its increased ubiquitin lig- ase activity PPARγ induces proteasomal degradation of p65 which in turn results in sequential transcriptional downregulation of MYB and PRDX1 leading to the cellular effects of clofazimine.
Clofazimine shows superior cytotoxic activity compared to thiazolidinediones, acts in synergy with imatinib and drastically reduces quiescent CD34+ cells
The PPARγ agonist pioglitazone synergizes with ima- tinib in eroding LSC by transcriptional downregulation but not dephosphorylation of STAT5.1 Since clofazimine modulated PPARγ transcriptional activity, we determined whether clofazimine also regulated STAT5 expression. As expected, clofazimine suppressed STAT5 protein (Figure 7A) and mRNA (Figure 7C) expression without altering its phosphorylation in K562 cells (Figure 7B). Consistent with the ability of PPARγ agonists to suppress BCL-2 expression in CML cells44 clofazimine decreased BCL-2 mRNA (Figure 7C) and protein (Figure 1D). Clofazimine did not alter CrkL or BCR-ABL1 phosphorylation (Figure 7D) indicat- ing that it is not a BCR-ABL1 inhibitor per se. Furthermore, like pioglitazone,1 clofazimine also reduced STAT5B and other LSC maintenance factors such as HIF-1α, HIF-2α and CITED2 transcripts in CD34+ cells from imatinib- resistant patients (Figure 7E).
We next compared the anti-CML efficacy of clofazimine with that of other PPARγ agonists. In a cell viability assay, clofazimine was found to be the most potent among all the PPARγ ligands tested. While the IC50 of clofazimine was 6.08 μM, those of rosiglitazone, troglitazone and pioglitazone were 32.28 μM, 50.01 μM, and 37.39 μM, respectively (Figure 7F).
Since pioglitazone and imatinib synergistically inhibit CML cells,1 we assessed whether the same was true with clofazimine. In a K562 viability assay imatinib, dasatinib and clofazimine displayed IC50 values of 0.95 μM, 0.64 μM and 4.13 μM respectively. However, combining 1.56 μM clofazimine, which is close to the average human plasma level of clofazimine (0.7 mg/L) following daily oral admin- istration of 100 mg clofazimine,6 with imatinib reduced the IC50 of imatinib to 36.4 pM (Figure 7G). The combina- tion index (CI) calculated using the Compusyn program revealed CI values <1 (Online Supplementary Table S3), indi- cating a synergistic effect. Compared to clofazimine, pioglitazone (48 h) showed a rather modest effect, which although synergistic (Online Supplementary Table S4), only reduced the IC50 of imatinib from 0.399 μM (alone) to 0.052 μM (with 5 μM pioglitazone) or 0.032 μM (with 10 μM pioglitazone) (Figure 7H). Clofazimine also displayed synergism with dasatinib, where the IC50 of dasatinib of 0.64 μM (alone) was reduced to 0.0124 μM in the presence of clofazimine (Figure 7G) and the calculated CI was <1 (Online Supplementary Table S5). We next assessed whether the combination of imatinib and clofazimine, like pioglita- zone,1 also eroded LSC. First, we performed a colony- forming assay in which clofazimine alone drastically reduced colony number compared to vehicle or imatinib,
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