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a >2-fold decrease in phosphorylation after short-term tofacitinib treatment, and a rebound in phosphorylation at 24 hours.
This finding both serves to validate our phosphopro- teomic data as well as help us define a signature of tofac- itinib-responsive phosphosites. Remarkably, 336 of the 544 up-regulated phosphosites in untreated coculture vs. monoculture (62%) met the same criteria of being tofaci- tinib-responsive (Figure 5D,E). Furthermore, examination across all measured phosphosites demonstrated that while phosphorylation is broadly increased in the coculture set- ting, noted as a general shift toward positive phosphosite intensities in the untreated sample, treatment with tofaci- tinib largely reverses this finding, recreating a normal dis- tribution around the intensity values found in monocul- ture (Figure 5F). Taken together, these findings demon- strate that tofacitinib broadly reverses the signaling path- ways driving stromal-induced proliferation in MM cells, both at the level of direct JAK targets as well as down- stream proliferative signals, informing our mechanistic
understanding of this treatment beyond targeted Western blots alone.
As a comparator, we also performed unbiased phospho- proteomics of HS5 stromal cells after 1.5 hours tofacitinib treatment. We found many fewer significantly changed phosphosites than identically-treated MM.1S in HS5 coculture (Online Supplementary Figure S3B,C), consistent with the lack of viability change after tofacitinib against HS5 alone (Figure 1B).
Phosphoproteomics does not reveal specific off-target activity for tofacitinib
Another advantage of unbiased phosphoproteomics is potentially detecting additional off-target effects mediat- ing tofacitinib response. We filtered for peptides that appeared unaffected by stromal-induced signaling (less than +/- 50% intensity change in untreated coculture vs. monoculture) that decreased in intensity >4-fold after 1.5 hours tofacitinib treatment (Figure 6A). We identified only 54 peptides that fit this filter, and neither Panther nor
ABC
DEF
Figure 5. Unbiased phosphoproteomics reveals that tofacitinib broadly reverses pro-growth signaling from stroma to MM cells. A. Analysis of 4862 phosphorylation sites quantified by LC-MS/MS on biological replicate samples revealed 544 phosphosites to be upregulated 4-fold in untreated MM.1S in coculture with HS5 vs. monoculture. Of these upregulated phosphopeptides, Panther pathway analysis showed JAK/STAT signaling to be the only significantly enriched pathway (P=0.036). B. Kinase enrichment analysis demonstrated that many of the upregulated phosphopeptides derive from known substrates of other kinases related to proliferation, such as mTOR, CDK1, and CDK2, as well as JAK1. Phosphorylated protein substrates are on the left and enriched kinases across the top. Length of red bar in kinase name is indicative of strength of enrichment. C. Quantitative phosphoproteomic intensity of the JAK-responsive phosphosite Ser727 on STAT3, both in untreated cocul- ture vs. monoculture, and after tofacitinib treatment, is very similar to the pattern found by Western blotting for the other known JAK responsive STAT3 phosphosite Tyr707 (Figure 3A), serving to validate this proteomics approach. D. Dynamics of all phosphosites found to be responsive to tofacitinib based on the criteria: 1) increased 4-fold in coculture vs. monoculture, 2) decreased at least-2 fold from untreated coculture after 1.5 hours of 1 mM tofacitinib treatment, and 3) phosphosite intensity remains below the untreated coculture level after 24 hours of tofacitinib treatment. E. Of 544 upregulated phosphopeptides in coculture, 336 (62%) were defined as being tofacitinib-responsive using the criteria in D. F. Examining the global phosphosite intensity across all quantified phosphopeptides demonstrates a general increase in phosphorylation of MM.1S proteins in untreated coculture with HS5 (“Cocx DMSO”; note shift of distribution maximum to log2-fold change vs. monoculture of ~1) which is then broadly reversed by tofacitinib treatment. Tof: tofacitinib; monocx: monoculture; cocx: coculture.
haematologica | 2018; 103(7)