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TGFβ1-mediated inhibition of MDS- and AML-derived MSC
conditions defined a set of 112 deregulated genes (q<0.05) in common, mostly related to general developmental processes and osteogenesis (Figure 1B,C, Online Supplementary Figure S2, and Online Supplementary Tables S3 and S6). Further analysis revealed a remarkable abun- dance of gene signatures associated with inflammatory responses (Figure 1D) as well as altered expression of a variety of cytokines (Figure 1E and Online Supplementary Figure S3). Overall, the expression of 18 cytokines was sig- nificantly altered (11 upregulated, 7 downregulated) with several of them known to be involved in the regulation of hematopoiesis. Collectively, these results are consistent with the malignant phenotypes previously reported in MDS- and AML-related MSC, and further suggest a relat- ed pathomechanism for the MSC from the three entities.
RNA sequencing analysis suggests that transforming growth factor β1 signaling is a common cause of abnormal gene expression patterns in myelodysplastic syndrome- and acute myeloid leukemia-derived mesenchymal stromal cells
Our previous work suggested that the phenotypic abnormalities observed in AML-derived MSC may be triggered by an extrinsic factor.13 We hypothesized that the conserved aberrant gene expression patterns are caused by an extrinsic factor. To identify this molecule, we used ingenuity pathway analysis,29 which enables the prediction of upstream regulators for a given RNA sequencing data set. This computational approach identi- fied the inhibitory cytokines tumor necrosis factor a (TNFa) and TGFβ1 as the two most probable (P values of 2.17x10-11 or lower) secreted factors for the induction of
A
the aberrant gene expression patterns in the MSC derived from RCMD, RAEB and AML patients (Figure 2A). These two molecules also appeared among the most probable secreted factors in analyses of RNA sequencing data sets from pairwise comparisons (Online Supplementary Figure S4). To analyze whether only one of these two molecules could be sufficient to induce the gene expression deregu- lation observed in the MSC from the patients, we used GSEA. The results showed that our gene set was signifi- cantly enriched in the TGFβ1 signature but not in the TNFa signature (Figure 2B). Our RNA sequencing experi- ments therefore suggest that increased TGFβ1 signaling in the bone marrow may lead to the aberrant gene expres- sion patterns observed in the MSC from these myeloid malignancies and, ultimately, to their functional inhibi- tion.
Transforming growth factor β1 induces functional deficits in healthy mesenchymal stromal cells recapitulating the phenotype of these cells in myelodysplastic syndrome and acute myeloid leukemia
Having identified TGFβ1 as a candidate factor for the induction of the observed phenotypic alterations and functional deficits of MSC in MDS and AML, we experi- mentally addressed this hypothesis using an in vitro cul- ture system. For this purpose, we cultured healthy MSC in the presence or absence of TGFβ1 to investigate whether healthy MSC adopt a phenotype that is similar to the phenotype of patient-derived MSC. Furthermore, in this experimental system we also used SD-208, which specifically abrogates the signaling downstream of the TGFβ receptor (Online Supplementary Figure S5).
B
Figure 2. RNA sequencing identified transforming growth factor β1 as the most probable secreted upstream regulator inducing the aberrant gene expres- sion patterns. (A) Ingenuity pathway analysis29 predicted that TNFα and TGFβ1 are the most probable secreted upstream regulators of the gene expression aberrations observed in the MSC from MDS and AML patients. (B) GSEA confirmed that overac- tivation of TGFβ1 signaling, but not TNFa, in the bone marrow caused the aberrant gene expression patterns. For both plots, the normal- ized enriched score (NES), false discovery rate (FDR) and P-values are given.
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