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Repurposing tofacitinib as anti-myeloma therapy
In total, 4862 phosphopeptides had intensity data in all four samples and were used for further analysis (listed in Online Supplementary Dataset S2). >99% of these sites are serine and threonine phosphorylation events, consistent with other phosphoproteomic studies using this enrich- ment method.34 We first evaluated for phosphosites with >4-fold intensity increases in untreated coculture vs. monoculture. Using our RNA-seq data as a proxy for pro- tein-level changes, we verified these phospho-site changes were largely driven by changes in signaling and not pro- tein abundance (Online Supplementary Figure S3A). Panther pathway analysis revealed the only significantly enriched pathway among the 544 upregulated phosphosites to be JAK/STAT signaling (Figure 5A). However, based on
kinase enrichment analysis,35 we found enriched signa- tures not only of JAK1 substrates, but also of other kinases driving proliferation and the cell cycle, such as mTOR, CDK1, and CDK2 (Figure 5B). These findings demonstrate that unbiased phosphoproteomics can uncover broad sig- naling effects of the BM microenvironment even down- stream of JAK/STAT.
Next, for validation of the effects of tofacitinib treat- ment, we first examined Ser727 on STAT3 (Figure 5C), a known JAK-responsive phosphosite.28 Our quantitative MS results were remarkably in line with Western blotting for another JAK-responsive phosphosite on STAT3, Tyr707 (Figure 3A), with a very large increase in both phosphosites in untreated coculture compared to baseline,
AB
C
D
EF
Figure 4. Ruxolitinib demonstrates less anti-MM activity than tofacitinib. A. A highly selective, irreversible inhibitor of JAK3, JAK3i, does not have any effects on MM.1S either in monoculture or in coculture with HS5. B. The JAK1/2 inhibitor ruxolitinib has minimal anti-MM effects in monoculture vs. three MM cell lines, and in fact appears to promote growth of MM.1S at higher concentrations. C. A similar phenomenon is noted in HS5 coculture. D. Ruxolitinib does not inhibit, but in fact increases signaling via STAT3 in MM.1S grown in HS5 coculture. E.-F. Combination of ruxolitinib with JAK3i, to achieve simultaneous JAK1/2/3 inhibition, does not recapitulate effects of tofacitinib in MM.1S. All error bars represent +/- S.D. from CellTiter-Glo assay performed in quadruplicate in 384-well plates. Monocult: mono- culture; Cocult: coculture; PBS: phosphate buffered saline.
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