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Repurposing tofacitinib as anti-myeloma therapy
ture setting was SOCS3, part of a well-characterized neg- ative feedback mechanism strongly induced by JAK-STAT activation10 (Figure 2A). We further examined all 67 tran- scripts significantly upregulated in MM.1S in the coculture vs. monoculture setting (P< 0.005 per DESeq tool;23 listed in Online Supplementary Dataset S1). Using the Enrichr tool,24 ChIP-X enrichment analysis (ChEA) of chromatin immunoprecipitation sequence (ChIP-seq) datasets26 found the most significant enrichment of STAT3-binding sites at the promoter of these upregulated transcripts, among all transcription factors (Figure 2B). Furthermore, Protein ANalysis THrough Evolutionary Relationships (Panther)27 pathway analysis found the only two signifi- cantly enriched pathways to be related to JAK/STAT sig- naling and interleukin signaling (Figure 2C). Taken togeth- er, these RNA-seq findings suggest that factors secreted from stromal cells mediate proliferation by activating JAK/STAT signaling, with STAT3 playing a central role.28 Notably, similar activation of SOCS3 and related JAK/STAT genes were previously found after exposure to IL-6 in the INA-6 cell line.29
However, using recombinant cytokines, even at high concentrations of IL-6, we did not observe the same degree of growth promotion as coculture with stromal cells (Figure 2D). A recent study identified other cytokines besides IL-6 highly secreted from HS5 stromal cells.30 We
tested four of these: macrophage inflammatory protein 3A (MIP-3A), interleukin-8 (IL-8), granulocyte colony stimu- lating factor (G-CSF), granulocyte-macrophage colony stimulating factor (GM-CSF), and none showed any growth promotion (Figure 2D). Furthermore, an IL-6 blocking antibody could only partially reverse stromal cell effects at the highest achievable concentration (Figure 2E). These findings support the role of IL-6 in this system, but also indicate that other factors likely play a role in MM growth promotion.
Tofacitinib inhibits JAK/STAT signaling
Given our results above, we chose to further evaluate downstream effects of tofacitinib inhibition of the JAK/STAT pathway. Two of the primary downstream mediators of IL-6 receptor and JAK activation are thought to be pro-proliferation signaling by STAT3 and inhibitory signaling by STAT1.28 We found that STAT3 and STAT1 phosphorylation in MM.1S dramatically increases when in coculture with HS5 (Figure 3A,B). 1 mM tofacitinib inhibits STAT3 phosphorylation in MM.1S cells in cocul- ture almost to monoculture level by two hours of treat- ment. In addition, phosphorylation of JAK1, JAK2, and TYK2, which can also activate STAT3, were studied. We found evidence of a “rebound” effect by 24 hours of treat- ment, mediated by well-characterized feedback mecha-
ABC
DE
Figure 2. Stromal-induced signatures in MM.1S identified by transcriptome analysis. A. Examples of significantly upregulated genes in MM.1S cells cocultured with HS5 stromal cells in comparison to MM.1S grown in monoculture. B. ChEA analysis of 67 significantly upregulated transcripts from untreated MM.1S in HS5 coculture vs. monoculture (P<0.005 based on DESeq analysis) demonstrates a significant enrichment of STAT3 transcription-factor binding sites based on ChIP-seq data. C. Panther pathway analysis of this gene list demonstrates significant upregulation of interleukin signaling and JAK-STAT signaling. D. Recombinant cytokines known to be secreted from HS5 stromal cells30 were tested for their ability to promote MM.1S proliferation. Up to the maximum achievable concentration, neither IL-6 alone nor a combination of all tested cytokines could recapitulate the growth promotion induced by BMSC. E. An IL-6 neutralizing antibody could partially reverse stromal- induced proliferation of MM.1S. All error bars represent +/- S.D. from CellTiter-Glo assay performed in quadruplicate in 384-well plates. PBS: phosphate buffered saline; RNA-seq: ribonucleic acid sequencing; ChIP-seq: chromatin immunoprecipitation sequencing.
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