IFN-α Upregulates the Expression of C/EBPβ and Induces Myeloid Differentiation and Exhaustion of CD34⁺ CML Stem Cells

It is widely accepted that chronic myeloid leukemia (CML) stem cells survive independently of BCR-ABL signaling, and maintain their immature and quiescent status in the BM niche. As this population could give rise to relapse after long-term remission with tyrosine kinase inhibitor (TKI) therapy, eradication of CML stem cells is still necessary for the complete cure of the disease. The efficacy of interferon-α (IFN-α), which has been used for the treatment for CML-chronic phase (CP), has recently been re-evaluated. Several clinical trials have shown that combination treatment with IFN-α significantly improves the therapeutic effects of TKI. In some 'Stop TKI' trials, a history of IFN-α therapy was associated with a higher incidence of treatment-free remission. However, the molecular mechanisms underlying the action of IFN-α on CML stem cells have not been fully elucidated yet.

At the annual meeting of ASH last year, we have shown that IFN-α induced differentiation and exhaustion of CML stem cells in a CML mouse model, and that IFN-α significantly upregulated CCAAT/Enhancer Binding Protein β (C/EBPβ), a transcription factor required for stress-induced "on-demand" granulocyte production under hematopoietic stresses, such as infection and cytokine stimulation (Hirai H et al, Nat Immunol 2006; Satake S et al, J Immunol 2012). C/EBPβ was responsible for the IFN-α-mediated differentiation and exhaustion of CML stem cells both in vitro and in vivo . We have also identified the Cebpb 3' distal enhancer region, which was required for efficient upregulation of C/EBPβ in response to IFN-α, and that IFN-α upregulated C/EBPβ at least in part through recruiting STAT5 to this enhancer. Here we investigated the involvement of a major downstream signaling molecule of IFN-α, STAT1, in the effect of IFN-α on CML stem cells, and then verified the relevance of our hypothesis that IFN-α induces exhaustion of CML stem cells through upregulation of C/EBPβ, by using CD34⁺ cells derived from CML patients.

First, we examined whether STAT1 is recruited to the 3' distal Cebpb enhancer region in response to IFN-α. When a mouse hematopoietic stem/progenitor cell line, EML was treated with IFN-α, recruitment of STAT1 to this enhancer was observed by ChIP PCR. IFN-α treatment of BCR-ABL-transduced EML cells also enriched STAT1 strongly in this region, indicating that the STAT1-mediated pathway downstream of IFNα remains intact in cells expressing BCR-ABL. Furthermore, whereas BCR-ABL signaling induced the recruitment of STAT5 to this enhancer region, IFN-α treatment further increased the binding of STAT5. These data suggest that when BCR-ABL expressing CML cells were stimulated with IFN-α, recruitment of STAT1 to the enhancer is responsible for the upregulation of C/EBPβ in addition to the recruitment of STAT5.

Next, we explored the effect of IFN-α on CD34⁺ CML stem cells obtained from CML patients (4 CP and 1 accelerated phase). This protocol was approved by the IRB at Kyoto University, and the written informed consent was obtained from patients prior to this study. Patient-derived lineage^- CD34⁺ cells were treated with 100 or 500 U/mL recombinant human IFN-α in vitro for either 0.5 or 3 hours. IFN-α significantly upregulated CEBPB mRNAexpression in a dose- and time-dependent manner, while the transcript for CEBPA, the transcription factor required for steady-state granulopoiesis, was slightly downregulated. This result suggests that IFN-α directly acts on patient-derived CML stem cells and induces upregulation of C/EBPβ. Then, we performed the colony-forming assay to evaluate the impact of IFN-α on the function of patient-derived CML stem cells. Purified lineage^- CD34⁺ cells were subjected to this assay, and colonies were counted and classified on day 14. The cells harvested from the primary colonies were subjected to another round of colony-forming assay or analyzed by flow cytometry. Notably, IFN-α significantly decreased the total colony number in both first- and second-round of plating. Furthermore, IFN-α strongly promoted differentiation of CML stem cells toward CD66-expressing myeloid cells, and induced their exhaustion in a dose-dependent manner (Figure).

Collectively, these data suggest that STAT1/STAT5-C/EBPβ axis plays an important role in the IFN-α-mediated exhaustion of CML stem cells not only in mouse CML model, but also in patient-derived CML stem cells.

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