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in liquid culture conditions nor when grown on MSC (Figure 5D-E). Possibly, this relates to the high levels of TGFb, BMP and angiopoietins that are known to be secreted by stromal cells. These factors negatively impact on the cell cycle and can preserve stemness, which would coincide with the observation that CD34+ cell populations were better maintained under stromal coculture condi- tions (Online Supplementary Figure 5C and D). CM harvest- ed from MSC, only marginally reduced CB cell growth in liquid culture and not when grown on MSC (Figure 5D to E). Strikingly, the CM harvested from AML#1 grown on MSC negatively impacted on normal CB proliferation, both in liquid cultures and on MSC, which was even fur- ther aggravated when the AML cultures were treated with IL1b (Figure 5 D and E; Online Supplementary Figure S5A and B). The percentage of DAPI-positive CB cells was slightly increased upon treatment with CM from AML cells, compared to the addition of Gartner’s medium (Online Supplementary Figure 5B). Similar results on cell proliferation and survival were obtained using adult PB CD34+ cells that were cultured in presence of CM from a different AML (AML#18; Figure 5F; Online Supplementary Figure 5E). Importantly, the negative phenotype could be partially rescued by addition of Anakinra, an FDA- approved inhibitor of the IL1 receptor, and consequently, the IL1RAP pathway (Figure 5F and Online Supplementary Figure S5F to H). The CB CD34+ percentage, but not absolute CD34+ cell counts, were increased 3 days after addition of CM of AML co-cultures (Online Supplementary Figure S5C and D). We observed no clear differences in the CD34+CD38+/CD34+CD38– distribution after addition of CM to CB cells grown in liquid culture or on MSC, respec- tively (data not shown). For a more functional HSPC analy- sis, we performed a CFC assay with CB CD34+ cells sup- plemented with CM of AML-MSC co-cultures. Two high-expressing IL1RAP (AML#9 and #19) and two low-expressing IL1RAP (AML#20 and #21) AMLs were grown for 7 days on a stromal layer of MSCs with or without IL1b/Anakinra (Online Supplementary Figure S5I). IL8 was strongly upregulated by IL1b in the IL1RAP+ AML cells (AML#19), but not in the IL1RAP– AML cells (AML #20), and Anakinra abrogated this effect (Online Supplementary Figure S5F). At day 7, CM was harvested and added to the methylcellulose mixture in a 1:2 v/v ratio. Only the addition of CM from AMLs with a high IL1RAP expression resulted in a significant reduction of colony-forming potential of CB CD34+ cells, which was completely reversed by the addition of Anakinra (Figure 5G; Online Supplementary Figure 5J).
Likely, the stromal cells play an important role in medi- ating the negative effects of IL1b on the proliferation of healthy CD34+ HSPCs. Surprisingly, MSCs expressed high levels of IL1RAP and were responsive to IL1b comparable to what was seen for IL1RAP+ AML cells (Figure 5H-I). Both the CM of AML cells as well as MSCs treated with IL1b affected cell proliferation of healthy HSPCs, which was further enhanced when CM from AML + MSC + IL1b was used (Figure 5J, Online Supplementary Figure 5H). This would argue that both the stromal cells as well as AML cells participate in generating an inflammatory environ- ment. In addition, two triple co-cultures were performed with MSC, AML CD34+ cells of two IL1RAP+ AML patients (AML#16 and #22), and CB CD34+ cells (Figure 5J to K; Online Supplementary Figure S5K to L). We observed reduced CB proliferation in the presence of AML#16,
which was further reduced by the addition of IL1b (Figure 5J). CB in co-culture with AML#22 without IL1b did not result in reduced CB proliferation, but this was the case when cultured in the presence of IL1b (Online Supplementary Figure S5K). We observed a slight increase of Annexin V+ cells at day 16, which might not account fully for the reduced normal hematopoiesis. These data propose a model in which interplay between AML cells and MSC results in an inflammatory secretome that impairs normal hematopoiesis (Figure 5K). This negative phenotype of normal hematopoiesis is partly IL1-induced and the addition of exogenous IL1b can aggravate this observed phenotype.
Discussion
The fate of normal and leukemic stem cells critically depends on signals arising from the BM niche.30-32 Many of them initiate signal transduction in target cells by binding to PM receptors. The identification and functional analy- ses of such AML-specific PM proteins will help our under- standing of leukemia initiation, progression and mainte- nance. Here, we studied IL1RAP, which was associated with a L-GMP like signature suggesting that cells from IL1RAP+ AML patients differ significantly in their origin, metabolic state, and cell cycle state, compared IL1RAP–/low patients.33,34 High IL1RAP expression in nor- mal karyotype AML patients showed worsened overall survival suggesting that indeed these are different AML subtypes.35 Also at the subclonal level, IL1RAP expression is associated with different biology, as an IL1RAP+ NRAS- mutated subclone differed significantly from an IL1RAP- WT1-mutated subclone, while both subclones contained similar founder mutations.7 The IL1RAP+ NRAS-mutated subclone was strongly enriched for L-GMP and inflamma- tory gene signatures. The connection between dysregula- tion of the Ras-pathway and IL1 signaling was previously investigated in non-small cell lung cancer in the context of GATA2 dependency.36 Combined, these data indicate that IL1RAP expression can be used to distinguish distinct bio- logical characteristics of leukemic clones.
A recent study indicated that IL1RAP can also co-dimer- ize with CD117 and CD135 receptors, which resulted in an amplification of survival and proliferation signaling in AML.28 In our dataset, FLT3 and CD123, but not CD117 expression, correlated well with IL1RAP expression. Stimulation of these receptors with FLT3L, IL3 and SCF showed heterogeneous downstream activation between different AML but no clear synergism was observed when FLT3, CD123 and CD117 were co-stimulated with IL1b. While the hypothesis that IL1RAP receptors can interact with other receptors and thereby affect signal transduc- tion is certainly intriguing, further studies are required to resolve these issues.
We showed that the IL1RAP pathway was functional in primary AML CD34+ cells and could be activated by IL1b. Various pathways were activated downstream, including canonical NFkB signaling. Muto et al. showed that MDS HSPC could switch to non-canonical NFkB signaling in response to inflammation, resulting in a competitive advantage over normal HSPC.29 In general, we observed rather low levels of proteins involved in active non-canon- ical NFkB signaling, which was not further increased upon stimulation with IL1b suggesting that this switch might be
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haematologica | 2021; 106(12)