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S.M. Kornblau et al.
and the proteins in this constellation are elevated in Class 3 and Class 4 while they are lower in Class 1 and Class 2. Perhaps the integrin/GSK3 axis is also important in AML- MSC.
Finally, there were statistically significant biases with different cytogenetic groups associating with different MSC protein signatures (P=0.026) (Table 1). Specifically, although few patients had favorable cytogenetics in this dataset, these were found exclusively in the Class 1 and Class 2 signatures. Conversely, patients with unfavor- able cytogenetics were not observed in the Class 2 sig- nature, but they were found in relatively equal propor- tions in the three AML specific signatures (i.e. Classes 1, 3, and 4).
Having determined that leukemic MSC divide AML patients into discrete protein signatures, we next exam- ined whether signature membership affected outcomes. There was no significant difference in median overall sur- vival between the four MSC populations, although the median survival of 105 weeks (wk) in the Class 3 signature was longer than the other three groups at 25, 48 and 58 wk (Figure 2A); this suggested that patients with Class 3 MSC may have a better disease prognosis. Despite the small sample size, relapse rates among the newly diag- nosed cases were different, with 4 of 5 Class 1 MSC patients and 13 of 16 Class 4 MSC patients relapsing com- pared to only 2 of 7 Class 3 MSC cases (P=0.076). Furthermore the median remission duration was different between patients with Class 3 MSC and the three other signatures (101 wk for Class 3 MSC vs. 42.2 wk for Class 4 MSC; P=0.05) (Figure 2B). Other comparisons were not made due to the small sample size. These findings suggest that MSC can influence patient survival, although at pres- ent the mechanism is unknown.
Effect of age on protein expression in NL-MSC and AML-MSC
As AML is a disease of the elderly, many of the AML- MSC samples were obtained from individuals aged over 60 years (y). Of the 106 AML-MSC samples, 50 samples were from individuals under 60 y. Of the 71 MSC samples from healthy donors, 68 samples were from individuals under 60 y. To assess the impact of age on the differences in protein expression between the AML-MSC and NL- MSC groups, the protein expression of 24 of the differen- tially-expressed proteins was reassessed in age-matched sets. The three groups were: a) under 30 y (AML n=13; NL n=37); b) 30-49 y (AML n=17; NL n=25); and c) 50 to 59 y (AML n=20; NL n=6). As shown in Online Supplementary Table S2 and Online Supplementary Figure S4A, of the 19 proteins elevated in AML-MSC compared to NL-MSC, only two (i.e. BCL-XL and PPP2R2A/B/C/D) were signifi- cantly higher in all age groups. This suggests that increased expression of these 2 proteins is dependent on the disease state and not on age. Six other proteins includ- ing p53 and CDKN1A (p21) were also elevated in two of the three age groups suggesting expression of these pro- teins is also dependent on the disease state rather than on age (Online Supplementary Table S2).
Analysis of p53 and CDKN1A (p21) protein expression was performed by western blot analysis on age-matched AML and NL samples (age 40-49 y; n=3 for both groups). Protein expression of both p53 and CDKN1A (p21) was at least 2- to 3-fold higher in AML-MSC compared to the healthy donor samples (Online Supplementary Figure 4B).
These findings suggest that the disease state dictates expression of at least some of the identified proteins in MSC.
Evidence for dysregulated signaling in AML-MSC
The observed differences in protein expression between AML-MSC and NL-MSC suggest that pathway utilization may be dysregulated or non-canonical in the AML-MSC. To look for evidence of abnormal utilization, we searched for statistically significant (R>0.2; P< 0.0001) protein-pro- tein correlations that were reversed in the direction of cor- relation in the AML-MSC compared to the pattern in the NL-MSC. A representative analysis of STAT5 expression with the other proteins in NL-MSC revealed that this tran- scription factor is negatively correlated with hnRPK and positively correlated with STAT1 (Online Supplementary Figure S5B). However, in AML-MSC the correlations are reversed (Online Supplementary Figure S5B).
A list of all protein correlations that are reversed in AML-MSC compared to NL-MSC is provided in Table 2. Of note, there are significant changes in protein correla- tions involving β-catenin. As shown in Table 2, BCL2, BCL2L11, FOXO1.3 p24.p32, FOXO3, KIT, ITGAL, PSMD9 p10, RPS6KB1 p389, SFN, SRC, and SRC p416 are negatively correlated with β-catenin in NL-MSC but
Figure 2. Survival and remission duration differ in patients based on mesenchy- mal stromal cell (MSC) population. Kaplan-Meir curves showing overall survival (A) and remission duration (B). N: number.
A
B
P=0.26
P=0.6
P=0.05
P=0.12
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