Abstract
Even in the era of tyrosine kinase inhibitors, eradication of chronic myeloid leukemia (CML) stem cells still remains to be a prerequisite for the complete cure of the disease. Interferon-α (IFNα), which has been used long for the treatment for chronic phase (CP)-CML, are now being re-evaluated since recent clinical trials showed that IFNα significantly improved the clinical outcome of patients treated with imatinib. IFNα promotes progression of cell cycle and differentiation of normal hematopoietic stem/progenitor cells (HSPCs) (Essers MA et al, Nature 2009; Sato T et al, Nat Med2009). However, its effect and mechanism of action on CML stem cells have not been investigated yet.
CCAAT/enhancer binding protein β (C/EBPβ) is a transcription factor playing an essential role for emergency granulopoiesis. Under stressed conditions including infections and cytokine stimulations, prompt and sufficient supply of granulocytes is evoked by C/EBPβ, which promotes both proliferation and myeloid differentiation of HSPCs (Hirai H et al, Nat Immunol 2006; Satake S et al, J Immunol2012). In addition, we have previously shown that (1) C/EBPβ is upregulated in HSPCs obtained from patients with CP-CML, (2) STAT5 is responsible for the upregulation, and (3) C/EBPβ promotes myeloid differentiation of CML stem cells (Hayashi Y et al, Leukemia2013). IFNα is one of the inflammatory cytokines, thus we hypothesized that C/EBPβ is involved in the effect of IFNα on CML stem cells, through promoting their differentiation and exhaustion. In this study, we investigated the molecular interplay between IFNα and C/EBPβ in CML stem cells.
First, we searched for regulatory elements of Cebpb, which is responsible for C/EBPβ upregulation induced by BCR-ABL-STAT5 signaling. By ChIP-seq analysis of a HSC line, EML cells, we identified novel BCR-ABL-responsive STAT5 binding sites in 3' distal region of Cebpb. Interestingly, two STAT5 consensus motifs (TTCNNNGAA) are tandemly located in this region, which is highly conserved among various species including human. Enrichment of H3K27Ac histone mark within this region in BCR-ABL-expressing EML cells was more evident than in empty vector-transduced cells. These results suggest that this region is a critical enhancer required for BCR-ABL-dependent expression of C/EBPβ.
Next, we explored whether IFNα induces C/EBPβ expression in EML cells and found that IFNα rapidly phosphorylated STAT5 as well as STAT1 and STAT3, and increased C/EBPβ expression irrespective of the presence of BCR-ABL. Notably, IFNα also recruited STAT5 to the 3' distal enhancer region of Cebpb mentioned above, suggesting that this enhancer might also regulate cytokine-responsive C/EBPβ expression.
In order to clarify involvement of C/EBPβ in the action of IFNα, KSL cells from WT or C/EBPβ KO mouse bone marrow (BM) cells were retrovirally transduced with BCR-ABL, and subjected to serial replating assay. IFNα promoted differentiation of WT CML stem cells toward myeloid cells, and reduced their replating ability. In contrast, C/EBPβ-deficient CML stem cells retained their immature status and colony-forming ability even in the presence of IFNα.
Finally, we evaluated the in vivo effect of IFNα on CML stem cells by serial BM transplantation experiments. The first recipients of BCR-ABL-transduced BM cells were administrated with PolyI:C, which induces IFNα production in vivo. The frequencies and numbers of WT CML stem cells in BM of the first recipients were significantly decreased by administration of PolyI:C, and this effect was severely abolished when C/EBPβ was absent in CML stem cells (Figure). Notably, PolyI:C treatment prolonged the survival of the secondary recipient mice only in the presence of C/EBPβ in CML stem cells, suggesting that IFNα promotes exhaustion of CML stem cells through C/EBPβ.
Collectively, these data demonstrate that IFNα upregulates C/EBPβ at least in part through recruiting STAT5 to 3' distal enhancer and that C/EBPβ is the critical regulator of differentiation and exhaustion of CML stem cells induced by IFNα. Our basic study presented here will shed light on the molecular mechanisms involved in the clinical efficacy of IFNα on CML.
Hirai:Kyowa Hakko Kirin: Research Funding; Novartis Pharma: Research Funding. Maekawa:Bristol-Myers K.K.: Research Funding.
