Page 130 - Haematologica April 2020

H. Kumar et al.
Introduction
The therapy of chronic myeloid leukemia (CML) has seen tremendous advances following the discovery of imatinib and other BCR-ABL1 tyrosine kinase inhibitors. However, complete molecular response, defined as unde- tectable BCR-ABL1 transcripts, is not achieved in the majority of patients.1 Resistance to tyrosine kinase inhibitors may occur due to BCR-ABL1 mutations; howev- er, in approximately 50% of the cases BCR-ABL1-inde- pendent mechanisms, including tyrosine kinase inhibitor- refractory leukemia stem cells (LSC), contribute to resist- ance and recurrence.1 Therefore therapeutic approaches capable of overcoming resistance to tyrosine kinase inhibitors are needed. Peroxisome proliferator-activated receptor-γ (PPARγ) agonists, pioglitazone in particular, were reported to erode quiescent LSC by targeting signal transducer and activator of transcription 5 (STAT5) expres- sion.1,2 Unfortunately, pioglitazone increases the risk of bladder cancer.3 Although rosiglitazone has not been found to increase the incidence of bladder cancer, it is associated with severe cardiovascular risks.4
To identify new therapeutic strategies we screened 800 Food amd Drug Administration-approved drugs for their anti-CML efficacy in the K562 cell line and identified clo- fazimine as a potent inhibitor of viability. Clofazimine, a riminophenazine leprosy drug, is also effective against multidrug-resistant tuberculosis5 and imparts its anti-bac- terial actions by generating reactive oxygen species (ROS), particularly superoxides and hydrogen peroxide (H2O2).6 Clofazimine also displays anti-inflammatory properties that are important for its suppression of leprosy-associat- ed immune reactions.6 Additionally, clofazimine was shown to be effective against various autoimmune dis- eases, including discoid lupus erythematosus, Crohn dis- ease, ulcerative colitis, psoriasis, Meischer granuloma and graft-versus-host disease.7 Clofazimine is reported to exert its immunomodulatory activities by blocking KV1.3 volt- age-gated potassium channels7 and thereby inhibiting chronic lymphocytic leukemia cells.8,9
Here we report the anti-CML efficacy of clofazimine in cells lacking KV1.3,8,10 and show that clofazimine exerted its effects by binding to PPARγ. Clofazimine not only sup- pressed STAT5 expression by modulating PPARγ tran- scriptional activity but also regulated a novel signaling cas- cade by increasing PPARγ-mediated p65 nuclear factor kappa B (NFκB) degradation, which caused transcriptional downregulation of cellular myeloblastoma oncoprotein (MYB), leading to suppression of peroxiredoxin 1 (PRDX1) expression and consequent induction of ROS-mediated apoptosis and differentiation.
Methods
Cell lines and human primary cells
K562 (CCL-243), HL-60 (CCL-240), U937 (CRL-1593.2), and HEK-293 (CRL-1573) cells were from the American Type Culture Collection (ATCC; Manassas, VA, USA) and were maintained as per ATCC instructions. Peripheral blood samples were obtained from BCR-ABL1+ CML patients (newly diagnosed, imatinib-resis- tant and imatinib responders), and healthy donors from King George’s Medical University (Clinical Hematology and Medical Oncology Division, Lucknow, India) following ethical approval (approval n. 1638/R. Cell-12) as per institutional ethical guidelines
after written consent (patients’ details in Online Supplementary Table S1). Peripheral blood mononuclear cells were isolated on a Percoll (Sigma) density gradient by centrifugation. All analyses of peripheral blood mononuclear cells were conducted on gated mononuclear cells excluding lymphocytes.
Other methods
Chemicals, antibodies, plasmid information and experimental procedures are detailed in the Online Supplementary Methods.
Statistical analysis
Data are expressed as the mean ± standard error of mean of three independent experiments, unless otherwise indicated. Statistical analyses were performed using GraphPad Prism 5.0. An unpaired two-tailed Student t-test or Mann-Whitney U test was used to compare two groups. Equality of variances was assessed by the F-test. Statistical analyses involving more than two groups were performed by one- or two-way analysis of variance followed by the Bonferroni post-test, or Kruskal-Wallis test followed by the Dunn test. For intra-group variances, we used the Levene median test (equal sample size; using XLSTAT) or Bartlett test (unequal sample size). P<0.05 was accepted as statistically significant.
Results
Clofazimine induces apoptosis and differentiation in chronic myeloid leukemia cells
In a screening in K562 cells, we identified clofazimine as a potent inhibitor of viability. Clofazimine has been reported to induce cytotoxicity by targeting KV1.3.7-9 Intriguingly, although K562 does not express KV1.38,10 (Online Supplementary Figure S1A), clofazimine reduced the viability of these cells with a pharmacologically relevant half maximal inhibitory concentration (IC50) of 5.85 μM (Figure 1A). The human plasma Cmax of clofazimine is 0.4- 4 mg/L, equivalent to 0.84-8.4 μM.6,11-13 The loss of viabili- ty was due to apoptosis, as demonstrated by annexin V staining (Figure 1B, Online Supplementary Figure S1B), poly (ADP-ribose) polymerase (PARP) cleavage (Figure 1C) and terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) (Online Supplementary Figure S1C). Clofazimine induced cytochrome C release and activated caspase -3 and -9 but not -8 (Figure 1D), suggesting mito- chondria-mediated apoptosis, which was consistent with decreased B-cell lymphoma 2 (BCL-2) and increased BAX expression (Figure 1D). Clofazimine also induced apopto- sis in peripheral blood mononuclear cells from patients with chronic phase CML (CP-CML cells; one newly diag- nosed patient was in accelerated phase, one imatinib- responder was in blast crisis) with an efficacy similar to that of cells from patients with newly diagnosed CML and imatinib-responders but higher than that of imatinib and dasatinib in imatinib-resistant cells, while it did not affect healthy donor cells (Figure 1E, Online Supplementary Figure S1D). Among the 21 imatinib-resistant patients (Figure 1E), seven harbored the following BCR-ABL1 mutations; M244V (n=1), Y253H (n=2), M351T (n=3) and F359V (n=1); clofazimine showed efficacy in all cases (Figure 1F; upper panel). A separate analysis of apoptosis in imatinib- resistant patients without BCR-ABL1 mutations (from Figure 1E) also showed significant clofazimine-induced apoptosis (n=6: vehicle, imatinib, clofazimine; n=5; dasa- tinib. Figure 1F; lower panel), indicating that clofazimine-
972
haematologica | 2020; 105(4)