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nisms,10 serving to phosphorylate the JAKs and subse- quently re-activate STAT3. Despite this rebound of STAT3 activation, however, MM growth continues to be inhibited based on the dose-response results of tofacitinib. In INA-6 cells we found similar effects of decreased STAT3 phosphorylation after tofacitinib treatment and a rebound at 24 hours (Online Supplementary Figure S2A).
As tofacitinib is known to potently inhibit JAK3, of additional interest was an increase in JAK3 expression in MM.1S in coculture versus monoculture, found both by RNA-seq (Figure 2A) and Western blot (Figure 3C). However, tofacitinib did not lead to any significant decrease in JAK3 phosphorylation (Figure 3C). We also evaluated signaling through JAK3’s primary pro-prolifera- tive downstream mediator STAT5.31 We found no evi- dence of STAT5 phosphorylation in either monoculture or coculture (Online Supplementary Figure S2B). These results suggest that JAK3/STAT5 signaling is less central to stro- ma-supported MM growth. We further confirmed this result using JAK3i, a newly-described, highly-specific, irre- versible JAK3 inhibitor.31 JAK3i had no effect on MM.1S in mono- or coculture (Figure 4A), nor did it show any synergy with carfilzomib treatment (Online Supplementary Figure S2C,D).
Taken together, these results suggest a mechanism whereby stromal cell-induced MM proliferation is mediat- ed through STAT3 transcriptional effects. These signaling pathways are inhibited by tofacitinib, ultimately leading to the reversal of the proliferation phenotype.
Ruxolinitib has less anti-MM activity than tofacitinib
Given that our results above suggest a more important role for JAK1 and/or JAK2 in stromal-induced MM prolif- eration than JAK3, we turned to an alternate candidate for drug repurposing, ruxolinitib. This agent is FDA-approved for use in myeloproliferative neoplasms and has much
higher affinity for JAK1 and JAK2 over JAK3 or TYK2.32 Ruxolitinib had minimal effects versus RPMI-8226 or JJN-3 cells, and, surprisingly, treatment of MM.1S actually showed a promotion of growth at higher concentrations, both in the monoculture and coculture settings (Figure 4B,C). Western blotting demonstrated that 1 mM ruxoli- tinib was unable to inhibit STAT3 activation in MM.1S in coculture (Figure 4D). In fact, STAT3 phosphorylation increased after two hours of treatment, consistent with the pro-proliferative effect seen in Figure 4B,C. We found that combining ruxolitinib with JAK3i was also insuffi- cient to recapitulate tofacitinib’s effects in mono- or cocul- ture (Figure 4E,F). Taken together, these findings suggest that ruxolitinib is unable to inhibit pro-proliferative STAT3 signaling in MM.1S cells, thereby supporting tofac- itinib as having greater potential as a repurposed anti-MM therapy.
Unbiased phosphoproteomics demonstrates that tofacitinib broadly reverses pro-growth signaling induced by bone marrow stroma
Our targeted investigations above specifically focused on the JAK/STAT pathway. To further elucidate the mech- anism of tofacitinib in this system, as well as gain a broad- er view of stromal-induced proliferation and signaling in MM cells, we pursued unbiased mass spectrometry (MS)- based phosphoproteomics. We studied four samples, per- formed in biological replicate: 1) MM.1S cells in monocul- ture, MM.1S cells in coculture, either 2) untreated (DMSO control), 3) treated with 1 mM tofacitinib for 1.5 hours, or 4) treated with 1 mM tofacitinib for 24 hours. We harvest- ed MM.1S cells in suspension, enriched for phosphorylat- ed peptides using immobilized metal chromatography, and analyzed by liquid chromatography-tandem mass spectrometry (LC/MS-MS) with peptide quantification performed using MaxQuant.33
AB
C
Figure 3. Tofacitinib inhibits JAK/STAT signaling in MM cells in a time-dependent manner. A. Western blotting demonstrates a marked increase in STAT3 phospho- rylation in untreated coculture vs. monoculture in MM.1S cells. After treatment with 1 mM tofacitinib there is a rapid decrease in STAT3 phosphorylation with rebound by 24 hours. B. STAT1 phosphorylation is also increased in response to coculture and rapidly reversed by tofacitinib treatment. C. While coculture increases JAK3 pro- tein expression, there does not seem to be any significant change in signaling via JAK3 after tofacitinib based on phosphorylation status. Western blots are repre- sentative of assays performed in biological duplicate. Monocult: monoculture; Cocult: coculture.
haematologica | 2018; 103(7)