Page 126 - 2020_08-Haematologica-web

S. von Palffy et al.
cells, we performed RNA-sequencing following 3-h and 24-h culture of CD34+CD38low cells with or without MSTNpp (Figure 4A). After 3 h, 147 genes were signifi- cantly up-regulated and 115 genes were significantly down-regulated in the MSTNpp stimulated cells com- pared to the non-stimulated control cells (FDR<0.01) (Online Supplementary Table S3). After 24 h, there were fewer differentially expressed genes, with 56 up-regulat- ed genes and only 14 genes down-regulated (FDR<0.01) (Online Supplementary Table S3). To explore whether MSTNpp stimulation activates specific downstream pathways in CML cells, we performed GSEA. Following 3 h stimulation with MSTNpp, an enrichment of STAT5 target genes was observed (Figure 4B). In addition, MSTNpp, which is a TGF-b superfamily member, induced a significant enrichment of TGF-b1 target genes both after 3 h and after 24 h (Figure 4B and Online Supplementary Figure S3A). After 24 h, genes associated with cell cycle and DNA-replication were also enriched (Online Supplementary Figure S3A), consistent with the increased proliferative response seen after stimulation by MSTNpp (Figures 1B and C and 2A and Online Supplementary Figure S2A).
Given that MSTNpp stimulation resulted in enrichment of STAT5 and the TGF-b1 target genes, we performed phospho-flow cytometry of STAT5 and SMAD2/3 in CD34+ chronic phase CML cells. Already 15 min follow- ing MSTNpp stimulation, we observed an increase in the phosphorylation levels of STAT5 and SMAD2/3 (Figure 4C and D and Online Supplementary Figures S3B and S4). Interestingly, the MSTNpp-induced activation of SMAD2/3 was similar to that of TGF-b1 (Online Supplementary Figure S3C), one of the well-known activa- tors of SMAD2 and SMAD3.29 However, culturing CD34+ CML cells with TGF-b1 reduced cell numbers (Online Supplementary Figure S3D), whereas MSTNpp stimulation resulted in growth-promoting effects (Figure 2A and Online Supplementary Figure S2A). When an additional panel of phospho-antibodies was tested to evaluate changes in MAPK-pathways (pP38, pJNK and pERK1/2),30 IL-1 signaling (NF-kB and pAkt)31,32 and general phospho- tyrosine activation (pTyr)33 (Online Supplementary Figure S4), signaling pathways associated with cell proliferation and cancer,8,32,34 no changes were detected for any of these markers.
Discussion
Deregulation of cytokines in the BM niche is thought to play a major role in leukemic disease progression, with altered cytokine levels promoting the growth of LSC while suppressing normal HSC.7,35,36 In CML, a transgenic BCR-ABL1 mouse model revealed extensive remodeling of the BM along with altered cytokine and chemokine levels, secreted both by proximal stroma cells and leukemic cells.37 CML cells also produce and respond to autocrine stimulation by IL-3 and granulocyte-colony stimulating factor (G-CSF),13 and recent work by our group and others have highlighted IL-1 as a positive reg- ulator of CML LSC.8,9 Hence, increased understanding of the cytokine-receptor interactions and signaling path- ways activated in the immature cell population of both CML and normal BM cells might reveal disease depend- encies that could translate into new treatment opportuni-
ties in CML and other malignancies.
We here conducted a high-content cytokine screen
with the purpose of finding novel positive regulators of primitive CML cells. Previous studies have mainly inves- tigated the effects on CML cells of single or a restricted set of cytokines.8,9,13,38,39 With a library of 313 cytokines screened, we identified 11 cytokines that at least doubled the cell number over seven days compared to no cytokine control. The screen confirmed the growth-promoting effect of IL-3,12,13 IL-1a/b,8 GM-CSF,14 IL-6,15,16 and IFN-γ17 for CD34+CD38low PB and BM chronic phase CML cells. The screen also identified five cytokines not previously reported to be important for CML; MSTNpp, sCD14, IL- 21 and IL-13v, and CCL-28. Out of these novel cytokines, the TGF-b superfamily member MSTNpp was the most potent in promoting the growth of primitive CML cells.
Previously, most studies of MSTNpp had been carried out in the context of muscle physiology where it regu- lates the muscle inhibiting myokine myostatin.22,40 By binding free myostatin in the blood, MSTNpp hinders myostatin from binding to activin receptors and activat- ing muscle wasting programs in the cell.22,41 Recently, it was also shown that MSTNpp can bind directly to activin receptors on muscle cells, blocking access by myostatin, and thus providing another mechanism of myostatin inhibition.40 No studies describing MSTNpp effects inde- pendent of myostatin have been previously reported. However, our data strongly suggest that MSTNpp has a direct effect on hematopoietic cells, by binding to a recep- tor on the cell surface independently of myostatin. This conclusion is based on the following observations; the in vitro screen was performed in serum-free media with no myostatin present, and addition of myostatin had no adverse effects on the growth of CML cells. Therefore, the mechanism by which MSTNpp acts in normal and malignant hematopoiesis seems to differ from what has previously been described in muscle cells. Further, we confirmed that MSTNpp is present in the plasma of CML patients and normal individuals, and demonstrate for the first time that MSC, MNC and CD34+ cells from CML patients express MSTN. Out of these cell types, MSC expressed the highest levels of MSTN. Importantly, even though there was no difference in MSTNpp plasma con- centration between the two groups, primitive CML cells are more responsive to MSTNpp stimulation than corre- sponding normal cells.
MSTNpp stimulation of chronic phase CML CD34+CD38low cells greatly increased the number of cells in culture without loss of CD34-expression, suggesting that MSTNpp expands the primitive cells while keeping them in an immature state. This finding was further strengthened by colony-forming assays, where MSTNpp pre-stimulation increased the number of colonies of both CML and normal BM cells. This indicates that the growth promoting effect of MSTNpp is not restricted to CML cells, but also applies to normal hematopoietic stem and progenitor cells (HSPC). Whether MSTNpp would elicit a differential regulatory effect on primary CML LSC com- pared to normal HSC in vivo is difficult to assess, given that primary CML cells engraft poorly in immunodefi- cient mice.
The observation that MSTNpp stimulation expanded primitive CML cells and resulted in the activation of both STAT5 and SMAD2/3 is intriguing, as these signaling pathways are associated with different cellular effects. By
2102
haematologica | 2020; 105(8)