Page 131 - Haematologica April 2020

Clofazimine inhibits chronic myeloid leukemia
induced apoptosis in imatinib-resistant cells is independ- ent of BCR-ABL1 mutations.
We next assessed whether clofazimine inhibited LSC. Increased aldehyde dehydrogenase activity is a hallmark of cancer stem cells14 and clofazimine reduced it in ima- tinib-resistant CP-CML cells at par with the positive con- trol salinomycin (Figure 1G, Online Supplementary Figure S2A). Clofazimine also reduced colony formation in CML CD34+ cells (Figure 1H, Online Supplementary Figure S2C). To further confirm its anti-LSC efficacy we treated puri- fied CML CD34+ cells with clofazimine and analyzed them by CD34 or annexin V staining. Clofazimine reduced the CD34+ population and induced apoptosis in these cells (Figure 1I, Online Supplementary Figure S2B). Treating CML CD34+ cells with clofazimine and analyz- ing them by CD34, CD38 and annexin V co-staining revealed that clofazimine induced apoptosis in both com- mitted CD34+38+ and primitive CD34+38- progenitor cells (Figure 1J). Clofazimine however, caused <5% loss in via- bility and no apoptosis in hematopoietic progenitor cells from healthy donors (Figure 1K, L, Online Supplementary Figure S2D) indicating that it specifically targets LSC. Clofazimine’s effects in CML CD34+ cells were not rout- ed through KV1.3 as KV1.3 transcript was undetectable in CD34+-enriched CP-CML cells (n=7; Online Supplementary Figure S3).
We next investigated whether clofazimine induced dif- ferentiation at sub-lethal concentrations. In K562 cells that
predominantly undergo erythroid or megakaryocytic dif- ferentiation upon various stimuli, clofazimine induced a megakaryocyte-like phenotype characterized by increased cellular size, nuclear to cytoplasmic ratio, vacuolation, and lobulated nuclei (Online Supplementary Figure S4A). Consistently, clofazimine increased megakaryocytic sur- face markers CD61 and CD41 in K562 (Online Supplementary Figure S4B-D) and CML (one newly diag- nosed patient was in accelerated phase, one imatinib- resistant patient was in blast crisis) cells (Figure 1M, Online Supplementary Figure S4E-F). Clofazimine treatment in CML CD34+ cells followed by May-Grünwald-Giemsa staining revealed increased monocyte-like morphology (Figure 1N, Online Supplementary Figure S4G). Concurrently, expression of the monocyte/macrophage differentiation marker CD11b was increased in clofaz- imine-treated CD34+ cells (Figure 1O, Online Supplementary Figure S4H). Furthermore, clofazimine also induced CD61 in CD34+ cells (Figure 1P, Online Supplementary Figure S4H).
Apoptosis- and differentiation-inducing effects
of clofazimine are associated with enhanced reactive oxygen species and decreased PRDX1 expression
We next asked how clofazimine exerts its action and decided to address its differentiation-inducing activity first. Megakaryocytic differentiation in K562 cells is typi- cally associated with prolonged extracellular signal-regu-
ABCD
EFGH
Figure 1. Clofazimine induces apoptosis and differentiation in K562 and chronic phase chronic myeloid leukemia cells and reduces leukemia stem cell load. (A, B) Clofazimine (CFZ) reduces K562 cell viability and induces apoptosis. (A) CFZ dose response, as determined by a CellTiter-Glo assay. (B) Apoptosis (n=3; represen- tative dot plot in Online Supplementary Figure S1B). (C) Poly (ADP-ribose) polymerase cleavage in K562 cells. (D) CFZ induces cytochrome C release, caspase cleav- age, BAX expression and suppresses BCL-2 in K562 cells. (E, F) CFZ (all drugs 5 μM) induces apoptosis in chronic phase chronic myeloid leukemia (CP-CML) cells (annexin/propidium iodide (PI) staining; dot plots in Online Supplementary Figure S1D). (F) Percentage apoptosis in cells from imatinib-resistant patients in Figure 1E, harboring the indicated BCR-ABL1 mutations (upper panel) and CP-CML cells in which no BCR-ABL1 mutations were detected (lower panel). (G) CFZ (or salino- mycin; both at a concentration of 5 μM) reduced aldehyde dehydrogenase activity, determined by percentage aldefluor activity in CP-CML cells (dot plots in Online Supplementary Figure S2A). (H) CFZ reduces the number of colony-forming cells in soft agar (images in Online Supplementary Figure S2C; imatinib 1 μM, CFZ 2.5 μM). (continued on next page)
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