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tant in adipogenesis is consistent with other studies from our group and another group that found AML-MSC are primed toward osteoblastic differentiation and not adipocytic differentiation.9,40,41 We reported that AML-MSC express higher levels of osteogenic markers, including Tissue Non-specific Alkaline Phosphatase (TNAP), RUNX2, Osterix, and Ostepontin, compared to MSC from age- matched heathy donors.40 In addition, in that study we found that AML-MSC readily differentiate along the osteogenic lineage pathway but are unable to differentiate into adipocytes. This differentiation potential of the MSC may be influenced by the leukemia cells themselves, as exposure of healthy donor MSC to AML cell lines such as OCI-AML3 induces gene expression of RUNX2, TNAP, and other osteogenic genes, and induces osteogenic differentia- tion of the stromal cells.40 The protein networks prevalent in NL-MSC that we identify here are consistent with signaling that skews toward adipocytic differentiation in the normal cells. Diaz de la Guardia et al. found that MSC from a high- risk AML group failed to differentiate into adipcocytes.41 The IPA data on canonical pathways in the AML-MSC compared at first diagnosis with refractory and relapse sam- ples suggests the importance of MSC of the osteoblastic lin- eage in the AML niche, as many of these proteins are asso- ciated with osteoblast survival and differentiation.
PI3K/AKT is very prevalent in group 1 proteins which are associated with Class 4 MSC by IPA. We have previ- ously reported that leukemia cells in co-culture with MCS induce activation of AKT and other survival kinases.42 It is plausible that the observed activation of AKT signaling in AML-MSC is due to the presence of leukemia cells in the niche or at least that the malignant cells may contribute to activation. In MSC, AKT has been implicated in positive regulation of Cyclin D1 (CCND1) and CDK4.43,44 The pres- ence of the PP2A B55 a subunit in group 1 suggests that either the protein phosphatase is not active against AKT in those cells or that AKT is less active in the AML-Like MSC compared to Normal-Like MSC. Despite its tumor sup- pressor role in suppression of AKT signaling, the PP2A subunit does support β-catenin expression by dephospho- rylating serine and threonine residues that are required for destruction of the transcription factor (e.g. serine 33, thre- onine 41).45-47 It is interesting that when expression of the 151 proteins surveyed by RPPA are correlated in the nor- mal MSC and in the AML-MSC, β-catenin exhibits the greatest difference in correlation pattern between normal MSC and AML-MSC compared to the other proteins. It is plausible that the PP2A B55 a subunit may be a factor in this phenomenon, though further investigation will be required to verify this potential mechanism. It is interest- ing to note that suppression of PP2A (albeit via the catalyt- ic core subunit Ca) in MSC cell line or pre-adipocyte cells
results in differentiation to adipocytes via a mechanism involving loss of β-catenin.48 As pathway analysis of pro- teins associated with normal MSC (i.e. Constellation 3) pathways identifies adipogenesis as a key pathway, it will be important to determine if AML-MSC would be less likely to skew toward adipocyte differentiation because of a PP2A B55 a subunit/β-catenin axis.
BCL-XL is necessary for MSC survival during differenti- ation.27 AML-MSC expressed higher BCL-XL and Cyclin D1 protein. These findings may be attributed to STAT5 as this transcription factor is a regulator of both BCL-XL and Cyclin D1.49 We found by qRT-PCR that gene expression of both BCL-XL and Cyclin D1 was higher in AML-MSC (n=9) compared to normal MSC (n=10) (Online Supplementary Figure S9). These findings suggest that ele- vation of BCL-XL and Cyclin D1 protein in AML-MSC can be attributed at least in part to a transcriptional mecha- nism possibly involving STAT5.
Two prominent proteins identified as elevated in the AML-MSC group are p21 and p53 (Figure 1B). Elevated expression of p21 and increased senescence of MSC is consistent with the study on MDS-MSC that showed a role for p21 in IL-6 and TGF-β production.25 However, a recent study from Desbourdes et al. found p21 and p53 levels were similar between AML-MSC and healthy donor-derived MSC.50 The reason for the difference between our results and their results is not clear. It should be noted that the Desbourdes et al. study used less than 5 samples each of AML-derived MSC and healthy donor- derived MSC to determine p21 and p53 levels, so perhaps Class 1 or Class 2 MSC (which would have lower levels of p21 and p53) are over-represented in their samples.50 In addition, the average age of the AML patients in the Desbourdes et al. study was 49 y while the average age of the healthy donors was near 60 y, so perhaps the p21 and p53 levels in the healthy donor MSC are skewed higher as the donors are older than the patients. For p21 and p53 expression, an age match comparison of p21 and p53 in the AML-MSC and NL-MSC shows levels of these pro- teins are higher in the AML-MSC in at least 2 ages for each (Online Supplementary Table S2).
In conclusion, proteomic analysis identified a distinct set of proteins that distinguish normal MSC from AML- MSC. Our RPPA studies identified four major signatures of MSC in AML patients that may impact their function in the tumor microenvironment.
Funding
This work was supported in part by the research funding from the National Institutes of Health (P01CA49639 and MD Anderson Cancer Center Support Grant CA016672) and the Paul and Mary Haas Chair in Genetics (to MA).
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