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Repurposing tofacitinib as anti-myeloma therapy
opment times and associated costs can be drastically reduced, thus accelerating the potential benefits to patients.6,7 To identify agents which may reverse the tumor-promoting effects of the MM BM microenviron- ment, we recently reported a repurposing screen of 2684 compounds against three MM cell lines, either grown alone (monoculture) or in coculture with MM patient- derived BM mesenchymal stromal cells.8 From that screen, we identified tofacitinib citrate, an FDA-approved small molecule for the treatment of rheumatoid arthritis (RA), as an agent which may reverse stromal-induced growth proliferation of malignant plasma cells.
Tofacitinib citrate is a potent inhibitor of all four mem- bers of the Janus kinase (JAK) family, with preferential inhibition of JAK1 and JAK3 over JAK2 and TYK2 in cellu- lar assays.9 JAK signaling, mediated by downstream STAT transcription factors, is necessary for lymphocyte stimula- tion in response to encountered antigens.10 Therefore, JAK inhibition holds promise for the treatment of autoimmune diseases like RA.11 In parallel, the JAKs have gained inter- est as therapeutic targets in MM as they mediate signaling via interleukin-6 (IL-6). IL-6 is secreted by many cell types within the BM microenvironment, as well as by malignant plasma cells themselves, and it is thought that prolifera- tion of malignant plasma cells within the human BM is dependent on this cytokine.12 This dependence on IL-6 was underscored by the recent development of a patient- derived xenograft model of MM, where primary plasma cell growth only occurred in immunocompromised mouse BM after knock-in of human IL-6.13
In fact, a number of groups have variously targeted JAK1/JAK2,14,15 JAK2,16-18 or all four JAKs19 with reported preclinical therapeutic efficacy in MM. However, per the registry at clinicaltrials.gov, none of these experimental JAK inhibitors have entered into MM clinical trials. Therefore, all of these agents are very far from use in MM patients, if they ever become available. Herein, we demonstrate that the already FDA-approved agent tofac- itinib has robust preclinical activity in MM models. We further use ribonucleic acid sequencing (RNA-seq) and unbiased mass spectrometry-based phosphoproteomics to delineate pro-proliferative signals from the BM stroma and show that they are largely reversed by tofacitinib treatment. Furthermore, we find that an alternate repur- posing candidate, the FDA-approved JAK1/2 inhibitor ruxolitinib, surprisingly does not show the same anti- myeloma properties. Therefore, our results support the rapid repurposing of tofacitinib as an anti-myeloma ther- apeutic to reverse the pro-growth effects of the BM microenvironment and potentiate the effects of existing myeloma therapies.
Methods
Cell culture conditions
All cell lines were authenticated by DNA genotyping at ATCC. All cells, including patient BM mononuclear cells, were main- tained in complete media with roswell park memorial institute- 1640 (RPMI-1640; Gibco) supplemented with 10% fetal bone serum (FBS; Gemini), 1% penicillin-streptomycin University of California San Francisco (UCSF), and 2 mM L-Glutamine (UCSF) with 5% CO2. INA-6 media was supplemented with 50 ng/mL recombinant human IL-6 (ProSpec). Additional details are provid- ed in the Online Supplementary Methods.
MM and bone marrow stromal cells (BMSC) coculture and viability testing
Cocultures were seeded into 384 well plates (Corning) with the Multidrop Combi (Thermo Scientific). 800 stromal cells were seeded and incubated overnight. 17 hours later, 700 myeloma cells were added on top of the stromal cells. On the third day, 24 hours after the addition of myeloma cells, cocultures were treated with tofacitinib (LC Laboratories), ruxolitinib (Selleck Chemicals), JAK3i,20 or IL-6 blocking antibody (R&D Systems). For drug com- bination studies, on the fourth day, melphalan (Sigma Aldrich), carfilzomib (Selleck Chemicals), or venetoclax (Selleck Chemicals) were additionally added to cocultures. On the fifth day, myeloma cell viability was detected with the addition of luciferin (Gold Biotechnology) and read for luminescence on Glomax Explorer plate reader (Promega) as previously described.21 For monoculture studies cell viability was measured using CellTiter-Glo reagent (Promega). All measurements were performed in quadruplicate. All viability data are reported as normalized to dimethyl sulfoxide (DMSO)-treated cell line in monoculture.
RNA-seq
For coculture RNA-seq, 5x106 MM.1S cells were grown with 3x106 HS5 cells for 24 hours. CD138+ enrichment to >95% was verified by flow cytometry for mCherry expression (Online Supplementary Figure S1A,B). MM.1S harvested from coculture, as well as MM.1S and HS5 grown in monoculture, were then processed for RNA-seq as previously described.22 Significantly upregulated transcripts were identified by DESeq23 and bioinfor- matic analysis was performed using Enrichr.24 Raw sequencing data are available at the Gene Expression Omnibus (GEO) reposi- tory (Accession number GSE99293). Additional details are provid- ed in the Online Supplementary Methods.
Western Blot Analysis
Described in the Online Supplementary Methods.
Liquid chromatography–tandem mass spectrometry phosphoproteomics
For coculture experiments, 5x106 HS5 cells were seeded into a T75 flask. Seventeen hours later, cultures were washed with phos- phate buffered saline (PBS), before the addition of 107 MM.1S mC/Luc cells. Twenty-four hours later, cocultures were treated with 1 mM tofacitinib for 1.5 hours and 24 hours. MM.1S cells in suspension were harvested by aspiration, centrifuged, washed with PBS, and flash-frozen prior to analysis. For untreated MM.1S monoculture or HS5 monoculture experiments, 107 cells were used. For sample preparation, frozen cell pellets were lysed in 8 M urea. 1 mg of total protein was then reduced in tris(2-car- boxyethyl)phosphine (TCEP) and free cysteines alkylated with iodoacetamide. Proteins were then digested at room temperature for 18 hours with trypsin. Peptides were desalted, lyophilized, and enriched for phosphopeptides using immobilized-metal affinity column (IMAC) with Fe-NTA loaded beads.25 Phosphopeptides were analyzed on a Thermo Q-Exactive Plus mass spectrometer coupled to a Dionex Ultimate 3000 NanoRSLC liquid chromatog- raphy instrument with 3.5 hour linear gradient. Raw proteomic data files are available at the ProteomXchange PRIDE repository (Accession number PXD006581). Additional details are provided in the Online Supplementary Methods.
Xenograft mouse model
NOD.Cg-Prkdcscid Il2rgtm1Wjl/SzJ (NSG) mice were obtained from the Jackson laboratory. 106 MM.1S mC/Luc cells, stably expressing luciferase, were transplanted via tail vein injection into each mouse. Tumor burden was assessed through weekly biolumines-
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