Acriflavine targets oncogenic STAT5 signaling in myeloid leukemia cells

Abstract Acriflavine (ACF) is an antiseptic with anticancer properties, blocking the growth of solid and haematopoietic tumour cells. Moreover, this compound has been also shown to overcome the resistance of cancer cells to chemotherapeutic agents. ACF has been shown to target hypoxia‐inducible factors (HIFs) activity, which are key effectors of hypoxia‐mediated chemoresistance. In this study, we showed that ACF inhibits the growth and survival of chronic myeloid leukaemia (CML) and acute myeloid leukaemia (AML) cell lines in normoxic conditions. We further demonstrated that ACF down‐regulates STAT5 expression in CML and AML cells but activates STAT3 in CML cells in a HIF‐independent manner. In addition, we demonstrated that ACF suppresses the resistance of CML cells to tyrosine kinase inhibitors, such as imatinib. Our data suggest that the dual effect of ACF might be exploited to eradicate de novo or acquired resistance of myeloid leukaemia cells to chemotherapy.

tumours such as osteosarcoma, breast, brain, lung, hepatic and pancreatic cancers. [3][4][5][6][7] Moreover, ACF has also increased the cytotoxic effects of chemotherapeutic compounds such as 5-fluorouracil  or melphalan in cancer cells. 8,9 Other studies have demonstrated that ACF effectively inhibits drug resistance and epithelial-mesenchymal transition (EMT) of pancreatic and hepatic cancer cells. 7 Importantly, long-term administration of ACF to AIDS patients, as an antiviral agent, has not revealed any major side effects suggesting that it could be employed alone or in combination with other drugs to overcome chemotherapy resistance of cancer cells. 10 Mechanistic studies have identified DNA-dependent protein kinases C (DNA-PKcs) and topoisomerase I and II as direct targets of ACF in various cancer cells. 2,9 ACF can also enhance P53 activation in γ-irradiated colorectal cancer cells and, consequently, potentiate radiation-induced cell death. 11 Nowadays, ACF is frequently used as an inhibitor of HIF dimerization and/or protein expression. 12 HIFs are main transcription factors that actively promote the progression of a variety of solid tumours as well as leukaemias. [13][14][15] For instance, HIF-1α has been reported as a crucial driver of chronic myeloid leukaemia (CML) development induced by the BCR-ABL oncogene.
Indeed, in the hypoxic bone marrow (BM) microenvironment,  has been found to support the persistence of CML leukaemic stem cells (LSCs) in a BCR-ABL kinase-independent manner. 16 HIF-1αinduced metabolic reprogramming is also required for imatinib (IM) resistance of CML cells associated with BCR-ABL up-regulation. 17 Cheloni et al have recently shown that ACF inhibits the growth and survival of CML cells as well as the stem cell potential of CML LSCs, while sparing normal haematopoietic stem/progenitor cells. They further demonstrated that ACF blocks leukaemia development and reduced CML LSCs maintenance in a CML mouse model. 18 Based on these studies, ACF has been proposed as a new therapeutic approach to prevent the relapse of CML.
Although HIF proteins are mainly activated by hypoxia, several reports indicated that expression and/or activity of HIF-1α/HIF-2α are also regulated by STAT3 (Signal Transducer and Activator of Transcription 3) and STAT5 in normal and cancer cell types including CML LSCs. [19][20][21] STAT3 and the two closely related STAT5A and STAT5B proteins are key players in the development of solid and haematopoietic cancers. [22][23][24] STAT3 and STAT5 are aberrantly tyrosine-phosphorylated in cancer cells and are crucial effectors of different tyrosine kinase oncogenes (TKO) such as FLT3-ITD in acute myeloid leukaemia (AML) and BCR-ABL in CML. 25,26 Using mouse models of CML, previous studies have demonstrated that, while STAT3 and STAT5 are both necessary for initiation of the disease, only STAT5 is required for the maintenance of leukaemia. 27 In addition, reports have indicated that STAT3 and STAT5 contribute to the resistance of CML cells to tyrosine kinase inhibitors (TKI). 28,29 STAT3 and STAT5 are now well-recognized therapeutic targets in haematologic malignancies and have been the subject of intense investigations for the development of selective pharmacological inhibitors. 30 Some of these inhibitors have been identified by high-throughput screening of drug repurposing libraries. For instance, the antiparasitic pyrimethamine or the antipsychotic pimozide have been shown to, respectively, inhibit the phosphorylation of STAT3 and STAT5 in different haematopoietic cancers, whereas the antidiabetic drug pioglitazone was found to reduce STAT5A and STAT5B gene expression in CML cells. 21,31,32 In all cases, these drugs whether used alone or in combination therapies decreased the growth and survival of solid tumours or leukaemic cells, and resensitized resistant cancer cells to chemotherapy.
Acriflavine effectively inhibits tumour cell growth in both hypoxic and normoxic conditions. Since ACF hampered CML cells development and IM resistance, we investigated whether ACF could regulate STAT signalling in leukaemic cells. Here, we demonstrated that STAT3/5A/5B expression and/or activation were strongly impacted by ACF in CML and AML cells. Thus, ACF-mediated modulation of STAT3/5 signalling might also be responsible for the antileukaemic activity of this compound.

| Cell proliferation assays
Cells were washed and resuspended in culture medium at a concentration of 2 × 10 5 cells/mL. Then, they were dispensed into 96-well culture microplates (Falcon ® ) containing 10 μL/well of serial drug dilutions of ACF. PBS alone was used as control. After treatment at desired times, 10 μL of a 5 mg/mL solution of MTT was added to each well.
Plates were then incubated for another 4 hours before the addition of 10% sodium dodecyl sulphate (SDS) solution with 0.01 M hydrochloric acid to solubilize the formazan crystals. After an overnight incubation at 37°C, the absorbance was measured at λ = 590 nm using a spectrophotometer (CLARIOstar ® Monochromator Microplate Reader; BMG Labtech, Offenburg, Germany). Living cells were also counted using the trypan blue dye exclusion method.

| RNA extraction and real-time reversetranscription quantitative PCR (RT-qPCR)
Total cellular RNA was extracted by TRIzol (Invitrogen) and quantified using NanoDrop Lite spectrophotometer. Five micrograms of RNA were reverse-transcribed using the SuperScript ® VILO TM cDNA synthesis kit (Invitrogen, Paris, France) as recommended by the supplier.
Proteins were then transferred onto 0.

| Statistical analyses
Results are expressed as mean ± SD for 3-6 independent experiments. Statistical significance of differences was determined using two-way ANOVA test for Apoptosis and RT-qPCR analysis, whereas multiple t test was used for cell cycle analysis. The correction of multiple comparisons was done using the Holm-Sidak's method.

| ACF inhibits growth and survival of K562 cells
We first investigated the growth inhibitory effects of ACF on CML cells in normoxic conditions. For this purpose, a CML cell line, K562, was treated with various ACF concentrations. Results showed that ACF reduced CML cell growth and viability, using trypan blue dye exclusion and MTT assays. ACF at a concentration of ~1.3 µmol/L reduced K562 cell viability by 50% (P < 0.05), whereas concentrations above 4 µmol/L caused more than 90% decrease ( Figure 1A). ACF is a mixture of trypaflavine and proflavine with a ratio of 2:1. Proflavine F I G U R E 1 Effect of acriflavine (ACF) on growth and survival of K562 cells. A, K562 cells were treated with various concentrations of ACF or not (PBS) for 72 h. Cell viability was determined by MTT and trypan blue dye exclusion assays (data are presented as mean ± SD of five independent experiments done in triplicates). IC 50 values are indicated. B, Cells were treated with various concentrations of proflavine, and the percentages of viable cells were determined by MTT assays (data are presented as mean ± SD of three independent experiments done in triplicates). Chemical structures of proflavine and trypaflavine are shown. C, Cells cultured without (PBS) or with increasing concentrations of ACF for 72 h were stained with AnnexinV coupled with APC to determine the percentages of apoptotic cells by flow cytometry. One representative experiment is shown (left panel). Data are presented as mean ± SD of six independent experiments done in triplicates. Two-way ANOVA followed by Holm-Sidak's multiple comparison test was used to examine the significance of ACF treatment on apoptosis (*P < 0.05; ***P < 0.0001). D, Protein extracts from K562 cells cultured for 48 h or 72 h with increasing concentrations of ACF were analysed by western blot with the indicated antibodies (n = 3). Actin served as the loading control is required to maintain the stability of ACF. To examine its contribution to the inhibitory effect of ACF, K562 cells were incubated with increasing concentrations of proflavine ( Figure 1B). Results showed that proflavine was three times less efficient than ACF in inhibiting CML cell growth ( Figure 1A vs Figure 1B), indicating that, although both proflavine and trypaflavine are active compound, the latter exhibited a significantly higher toxicity.
In order to determine whether the reduced number of cells is due to ACF-mediated cell death, K562 cells were incubated with ACF for 72 hours prior to Annexin V staining for the detection of apoptotic and necrotic cells. Cell death was strongly induced after treatment with 4 μmol/L ACF (~75%, P < 0.001) but remained low with doses below 2 µmol/L ( Figure 1C). To validate these results, the effects of ACF on markers of apoptosis were determined by western µmol/L ACF, while both caspase 3 and PARP were fully cleaved with 8 μmol/L ACF ( Figure 1D). The induction of apoptosis was also confirmed by the ACF-mediated down-regulation of the anti-apoptotic protein Bcl-X L . ACF is known as an intercalating agent that probably creates DNA damage, thereby triggering apoptosis. 34 We thus anal-  Figure 4B). Overall, these data suggest that ACF blocks STAT5A/5B expression in both CML and AML cells and that inhibition of cell growth induced by ACF may depend on STAT5 down-regulation rather than STAT3 activation.

| ACF overcomes the resistance of K562 cells to IM
We next analysed the effects of the combination of ACF with IM on IM-sensitive K562 (K562S) and IM-resistant K562 (K562R) cells ( Figure 5). The isobologram showed that the combination of various concentrations of ACF and IM resulted in additive rather than synergistic effects on the growth of K562S cells ( Figure 5A). In line with these results, K562S and K562R cells exhibited the same sensitivity to ACF ( Figure 5B). When combined with IM, ACF slightly enhanced the reduction of BCR-ABL phosphorylation and expression in both K562S and K562R cells ( Figure 5C). Finally, the effects of ACF on STAT3 and STAT5 were confirmed in K562R cells ( Figure 5D). ACF suppressed STAT5 phosphorylation and strongly inhibited STAT5A and STAT5B expression, whereas the phosphorylation of STAT3 was strongly stimulated, with no effect on its expression. Together, these results suggest that ACF might be a very useful drug that could be employed in combination with IM in order to eradicate resistant cells. LSCs in the BM of CML patients. 35 Mutations in the IM binding region of BCR-ABL or activation of alternative signalling pathways mainly explained the acquired or de novo resistance to TKI. 36 The BM microenvironment provides a sanctuary for LSCs. Hypoxia, a fundamental microenvironmental determinant, has been associated with the maintenance of CML LSCs through activation of HIFs in a BCR-ABL-independent manner. 16 Similarly, activation of STAT3 or STAT5 via a JAK-dependent but BCR-ABL-independent pathway promotes IM resistance of CML cells in BM microenvironment models. 28,37,38 Moreover, STAT5 has been shown to play a key role in the maintenance of LSCs from CML patients, whereas combining IM with a STAT5 inhibitor triggers the death of CML LSCs. 21 As a HIF inhibitor, ACF also targets CML LSCs while ACF combined with IM was more effective than IM alone in killing CML cells in low oxygen conditions. 18 Targeting both HIF and STAT proteins might therefore help to eradicate CML LSCs and to prevent relapse after TKI combination therapy.

| D ISCUSS I ON
In this work, we demonstrated that ACF affects the growth and survival of CML cells by targeting STAT3 and STAT5 in hypoxic and normoxic conditions. We found that ACF dramatically reduced that targeting STAT5 gene expression might eradicate CML LSCs in vivo. 21 In line with these data, we recently reported that a STAT5 inhibitor associated with IM suppresses the growth of IM-resistant CML cells by blocking STAT5B protein expression. 41 Importantly, we showed that ACF-mediated effects on STAT3/ STAT5 occur via a BCR-ABL-independent mechanism. We found that ACF alone neither affects the phosphorylation nor the expression of this TKO in IM-sensitive cells, even though minor, but significant, changes were observed when ACF was associated with IM. Moreover, we also observed that ACF does not affect the constitutive phosphorylation of the serine/threonine kinase AKT, a crucial downstream effector of BCR-ABL, supporting our findings that suppression of STAT5 expression is responsible for ACF-mediated inhibition of leukaemic cell growth. 42  cell death but also cellular senescence. 43,44 We also found that ACF, as an intercalating agent, induces DNA damage in CML cells at a concentration effective enough to favour apoptosis. At lower concentrations, ACF activates P21 Waf1 protein which is recognized not only as a cyclin-dependent kinase inhibitor but also as an important marker of cellular senescence suggesting that ACF might also induce a senescent phenotype in K562 cells. 45 In summary, our data indicate that the simultaneous inhibition of HIFs and STAT5 might provide new therapeutic opportunities for relapsed CML. This can be achieved by combining different drugs or by employing multitarget inhibitors such as ACF. In addition, blocking both HIFs and STAT5 with ACF, that has been proven to be safe in humans, might help to reduce the side effects of combination therapies.

ACK N OWLED G EM ENTS
This work was supported by funding from the Fondation de France

CO N FLI C T O F I NTE R E S T
The authors declare no conflict of interest.

DATA AVA I L A B I L I T Y S TAT E M E N T
The data that support the findings of this study are available from the corresponding author upon reasonable request.