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Repurposing tofacitinib as anti-myeloma therapy
kinase enrichment analysis identified any enriched signa- tures (data not shown). Given the small number of peptides and lack of any biological signatures, it appears most likely these 54 peptides are background noise in the data and do not reveal any specific off-target effects of tofacitinib.
To further investigate the possibility of any off-target effects of tofacitinib, we downloaded available data from the LINCS KINOMEscan database and plotted versus the human kinase phylogenetic tree (Online Supplementary Figure S4). These results demonstrate that, at least in cell- free assays, tofacitinib shows much greater specificity for JAK-family kinases compared to ruxolitinib. Together, these findings suggest that tofacitinib’s effects in this coculture model appear to be due to on-target activity. However, these results do not rule out off-target effects of tofacitinib not detected by these analyses herein.
Tofacitinib synergizes strongly with venetoclax only in the coculture setting
To begin to identify potential rational combinations with myeloma therapies, we first studied tofacitinib in combination with carfilzomib in MM.1S cells. Based on zero-inflated Poisson regression (ZIP) model scoring,30,36
we found very mild synergy with this proteasome inhibitor in both monoculture or HS5 coculture (Online Supplementary Figure S5). Subsequently, inspired by recent findings in acute myeloid leukemia (AML) primary sam- ples,30 we tested the combination of tofacitinib and the Bcl-2 inhibitor venetoclax, a promising investigational agent in MM.37 Intriguingly, similar to the findings in AML, we found strong synergy of these two agents only in the coculture context but not in monoculture (Figure 6).
Tofacitinib has anti-MM activity in the BM microenvironment in vivo
Toward the goal of repurposing tofacitinib as an anti- MM therapy in patients, we next examined the efficacy of tofacitinib in vivo. For this orthotopic disseminated xenograft model, we used a luciferase-labeled MM.1S cell line, which specifically homes to the murine BM after intravenous implantation in NOD scid g (NSG) mice. Treatment was initiated after two weeks of tumor growth and continued for four weeks at ~2/3 of the maximal tol- erated dose of tofacitinib (21.5 mg/kg/day by subcuta- neous infusion).38 Encouragingly, we found significantly increased murine survival in this cell line model (Figure
A
B
CD
Figure 6. Tofacitinib shows synergy with venetoclax only in the coculture setting. A-B. Treatment was performed sequentially with 24 hours of tofacitinib followed by 24 hours of venetoclax at specified doses. C-D. For combination matrices, the interaction landscapes are shown in 2D plots. The ZIP method is used to calculate syn- ergy across the landscape (red = positive score, synergistic; green = negative score, antagonistic) as well as to calculate an overall synergy score d, the difference in percentage inhibition compared with the expected additive compound effect. The coculture results demonstrate synergy across all combinations with the strongest synergy at high doses of venetoclax, as well as an overall very high synergy score of ~10.5 across the landscape. The monoculture results demonstrate mild antag- onism at high venetoclax doses and overall antagonistic score of ~-1.5. Note different scale bars on each 2D landscape output from ZIP. Error bars represent +/- S.D. from CellTiter-Glo assay performed in quadruplicate in 384-well plates. ZIP: zero-inflated Poisson regression.
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