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B. de Boer et al.
tors whereby the majority forms a dimer with the IL1RAP co-receptor.15 Activation of the IL1 receptor com- plex activates the MyD88/IRAK1/IRAK4/TRAF6/TAK1 signaling pathway, which in turn results in activation of NFkB and mitogen-activated protein kinases (MAPK), including p38.16 Many proteins in this pathway are often upregulated in myelodysplastic syndromes (MDS) and AML, which suggests an important role for this pathway in leukemogenesis.17-19 The IL1 signaling route can induce a variety of inflammatory cytokines and chemokines, which has been shown to be an important factor for development and maintenance of MDS.20 In AML, IL1 has been proposed to enhance proliferation and sur- vival.21-23
Here, we studied the IL1–IL1RAP signaling axis in pri- mary AML patients in the context of the BM niche and revealed that the IL1b-induced secretome impacts on leukemogenesis and most notably on normal hematopoiesis.
Methods
Extensive details on the methods used can be found in the
Online Supplementary Appendix.
Primary samples
Neonatal cord blood (CB), mobilized peripheral blood stem cells (PBSC), normal bone marrow (NBM), mesenchymal stro- mal cells (MSC), MDS, and AML patient material was obtained as described in the Online Supplementary Methods. All healthy individuals and AML patients gave an informed consent in accordance with the Declaration of Helsinki at the University Medical Center Groningen (UMCG) and Martini Hospital Groningen, the Netherlands. All protocols were approved by the Medical Ethical Committee of the UMCG. Details of AML char- acteristics used in this study can be found in the Online Supplementary Table S1.
Cell (co-)cultures
MSC co-cultures/triple-cultures with CB CD34+, PBSC CD34+ or AML CD34+ cells were performed in Gartner’s medium with the addition of 20 ng/mL granulocyte colony-stimulating factor (G-CSF), N-plate and IL3. Inhibition of the IL1-signaling path- way was established by the addition of 500 ng/mL Anakinra (Swedish Orphan Biovitrum BVBA). In case of triple co-cultures, CB CD34+ cells were transduced with pLKO eGFP to distinguish them from AML cells. Co-cultures were grown at 37˚C and 5% CO2 and demi-populated regularly, replacing 50-80% of the vol- ume with fresh or conditioned Gartner’s medium. Suspension cells were used for further analysis.
Colony-forming cell assay
The colony-forming capacity of CB CD34+ cells was evaluat- ed in methylcellulose (1,600 mL) mixed with CM (900 mL) from MSC-AML co-cultures treated for 7 days with or without IL1b and Anakinra.
Lentiviral transfection
For knockdown of IL1RAP, cells were transduced with a pLKO eGFP construct, containing short hairpins against IL1RAP sh1: 5’-TGGCCTTACTCTGATCTGGTATTGGACTA-3’, sh2: 5’-CGGGCATTAATTGATTTCCTACTATATTC-3’,10 or scram- bled control 5’-TTCTCCGAACGTGTCACGTT-3’.
Results
IL1RAP is upregulated in acute myeloid leukemia patients and correlates with a leukemic granulocyte-monocyte progenitor cell signature
IL1RAP expression was evaluated in NBM CD34+ cells (n=11) and blasts of AML, acute promyelocytic leukemia (APL) and MDS patients (CD34+ or SSClowCD45mid in case of NPM1 mutant AML with CD34 expression <1%). De novo AML patients (n=110), patients that developed AML from an MDS (s-AML) (n=27), APL patients (n=4), and MDS/MDS-excess blasts (MDS-EB) patients (n=13), all showed heterogeneous but on average significantly upregulated expression of IL1RAP (Online Supplementary Figure S1A and B). Immunofluorescent staining showed clear IL1RAP expression on the PM in AML cells repre- senting different genetic subtypes with high IL1RAP expression (Figure 1A).
For 42 AML patients, quantitative proteome data was generated previously7 and we evaluated cellular processes that were enriched in patients with either high or low IL1RAP expression. For 31 of these patients IL1RAP expression was also measured by flow cytometry inde- pendently and a significant correlation with our quantita- tive proteome data was observed (Online Supplementary Figure 1C). Gene ontology (GO) and gene set enrichment analysis (GSEA) was performed on a ranked list based on Pearson correlations of IL1RAP protein expression with the complete quantitative proteome and showed that high expression of IL1RAP was associated with the terms “mitochondrial translation elongation and termination”, “energy production via oxidative phosphorylation” and a “leukemic granulocyte-monocyte progenitor (L-GMP) sig- nature”, whereas AML patients with low IL1RAP expres- sion were associated with “regulation of RNA metabolic processes”, “gene expression” and a glycolysis-enriched HSC-like signature (Figure 1B to C). While IL1A and IL1B were expressed by AML cells at varying levels, no correla- tions were seen with IL1RAP expression, neither in our quantitative proteome data nor in various published tran- scriptome datasets (data not shown).24,25 Neither did we find positive correlations of IL1RAP high-expressing AML with RELA, encoding the NFkB transcription factor, MyD88, IRAK1, and TRAF6, all associated with the IL1-IL1RAP downstream pathway (data not shown).
IL1-induced IL1RAP signaling is associated with an inflammatory secretome
Data generated in previous studies comparing gene expression profiles of primary AML and NBM CD34+ cells suggested that the IL1RAP pathway might be actively used in many AML patients since components of the IL1RAP-TAK1 signaling pathway were significantly upregulated in AML CD34+ cells while negative feedback proteins such as IL1R2, IL1RN and MARCH8 were signif- icantly downregulated (Online Supplementary Figure S2A).24 In order to investigate the repertoire of targets down- stream of the IL1-IL1RAP axis we performed genome- wide transcriptome studies in CD34+ of AML#1 (for details of all AML used in this study, see the Online Supplementary Table S1), THP1, and K562 cells that were stimulated with IL1b for 1 hour (Online Supplementary Table S2). While the IL1RAP receptor can be activated by multiple different cytokines including IL1α, IL1b, IL33,
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