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H. Kumar et al.
and combining clofazimine with imatinib caused a further reduction (Figure 7I, Online Supplementary Figure S2C) (although the difference in effect of clofazimine vs. clofaz- imine + imatinib was not statistically insignificant, the P values for the differences in effect between vehicle vs. clo- fazimine and for vehicle vs. clofazimine + imatinib were P<0.01-0.001 and P<0.001, respectively).
We next evaluated whether clofazimine alone or in combination with imatinib could erode quiescent LSC. To that prupose, we labeled CD34+ cells from imatinib-resis- tant patients (one patient in blast crisis) with carboxyfluo- rescein succidimidyl ester (CFSE) and treated them with the indicated drugs for 96 h. While imatinib failed to reduce CFSEbright (non-dividing) cells, clofazimine alone or in combination with imatinib drastically reduced their number and increased the CFSEdim (dividing cell) popula- tion (Figure 7J, K, Online Supplementary Figure S12). Evaluation of apoptosis in these cells revealed that clofaz- imine alone caused apoptosis in both CFSEbright and CFSEdim cells while combining clofazimine with imatinib caused their near obliteration (Figure 7L, Online Supplementary Figure S12). Clofazimine + imatinib did not affect normal CD34+ hematopoietic progenitors from healthy donors as clofazimine alone or in combination with imatinib caused <14% loss of viability (Figure 7M) and did not induce apoptosis in them (Figure 7N, Online Supplementary Figure S2D).
Effect of clofazimine and the combination
of clofazimine and imatinib in K562 xenografts
To assess the effect of clofazimine, imatinib or their combination in vivo, athymic nude (nu/nu) mice harboring K562 xenografts were orally administered vehicle (0.5% methyl cellulose), imatinib (50 mg/kg/day; roughly equiv- alent to a human dose of 200 mg), clofazimine (10 mg/kg/day, human equivalent dose of 50 mg) or a combi- nation of clofazimine and imatinib (10 mg/kg/day and 50 mg/kg/day, respectively) for 12 days. Analysis of tumor volume revealed a decreasing trend in all treatment groups which although not statistically significant (except for the imatinib group in which the tumor volume was statistical- ly significantly reduced on day 13), became so after removal of an outlier (starting tumor volume >150 mm3, marked as red ‘O’, Figure 8A) in the clofazimine + ima- tinib group in which the reduction in tumor volume was statistically significant from day 9 onwards, while that in the imatinib group was statistically significant on day 13 only (Figure 8A, B). Analysis of tumor weight (tumor images in Figure 8C) showed a similar pattern, with a decreasing trend in all treatment groups. However, upon removal of the outlier (marked red in Figure 8D, left panel and corresponding to the tumor shown in Figure 8A) a sta- tistically significant reduction was present only in the clo- fazimine + imatinib group (Figure 8D). Histological analy- sis of the tumors revealed well-defined vasculature and a substantial number of mitotic cells in the group treated with vehicle, which were reduced in treatment groups, especially in the clofazimine + imatinib group (Figure 8E). Furthermore, karyopyknosis, karyorrhexis and degenerat- ing cells were visible in treatment groups, especially in the clofazimine + imatinib group (Figure 8E). Staining of tumor sections for the cellular proliferation marker Ki67 revealed a significant reduction in the clofazimine + ima- tinib group, compared to the groups treated with vehicle or the individual drugs (Figure 8F, G). Determination of
apoptotic cells by TUNEL staining revealed a significant enhancement of signals in all treatment groups (Figure 8H, I). None of the animals in any of the treatment groups showed changes in body, liver or spleen weight (Figure 8J- L). Together, these results indicate that combining clofaz- imine and imatinib causes a more robust anti-tumor activ- ity than any of the drugs alone.
Discussion
Here, we identified clofazimine as an anti-CML agent that was particularly effective in cells from imatinib-resis- tant patients and robustly downregulated LSC including quiescent LSC. Clofazimine exerted its effect through PPARγ. Recent evidence suggests that combining thiazo- lidinediones with tyrosine kinase inhibitors is an effective way to counter drug resistance in CML by eroding quies- cent LSC that do not require BCR-ABL1 for survival.1,45 Thiazolidinediones inhibit quiescent cells by transcrip- tional downregulation of STAT5, which is highly expressed in LSC, while imatinib regulates STAT5 phos- phorylation.1 Combining both drugs thus causes stronger downregulation of STAT5 targets HIF-1α, HIF-2α and CITED2, which are critical for LSC quiescence and main- tenance.1 Here, we show that in addition to STAT5, clo- fazimine also regulated a novel pathway by modulating PPARγ ubiquitin ligase activity, which resulted in ROS- dependent apoptosis via downregulation of PRDX1.
PRDX1, originally cloned from K562 cells,46 was initially described as a tumor suppressor47,48 but was later identified as an oncogene in various types of cancer in which its increased expression is associated with poor clinical out- come.49 PRDX1 mRNA was reported to be elevated in ima- tinib-resistant patients in whom no reduction in BCR- ABL1 was co-related with higher PRDX1 transcript.50 We also observed a higher level of PRDX1 transcripts in cells from CP-CML patients than in those from healthy donor, although the difference was not statistically significant. A significantly higher expression of PRDX1 was also observed in both CD34+38- and CD34+38+ LSC than in non-LSC, with the highest expression in CD34+38+ cells. Interestingly, PRDX1 is a secreted protein that enhances secretion of inflammatory cytokines by interacting with toll-like receptor 4.52,53 Clofazimine suppression of PRDX1 may thus explain its clinically observed anti-inflammatory functions6 and the anti-LSC activities seen here. Although PRDX1 has been well-studied in solid tumors, its function in CML is not clear and our study suggests that a detailed exploration of its role in CML progression will be impor- tant.
A plethora of reports implicate MYB in leukemogenesis which regulates factors such as c-Kit,54 CD34,55 and FLT3,56 which are highly expressed in early progenitor cells and whose aberrant expression or mutations are associated with leukemia and poor clinical outcome.54-56 While MYB overexpression induces transformation in hematopoietic cells,57,58 its depletion inhibits colony growth in cells from CML patients including those in blast crisis.59,60 Here we found that clofazimine suppressed PRDX1 transcription by downregulating MYB expression and, for the first time, identified a specific MYB target sequence on the PRDX1 promoter.
NFκB is one of the important downstream signaling pathways of the BCR-ABL1 oncoprotein.45,61 Abnormal
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