Ruxolitinib synergizes with DMF to kill via BIM+BAD-induced mitochondrial dysfunction and via reduced SOD2/TRX expression and ROS

Abstract

We determined whether the myelofibrosis drug ruxolitinib, an inhibitor of Janus kinases 1/2 (JAK1 and JAK2), could interact with the multiple sclerosis drug dimethyl-fumarate (DMF) to kill tumor cells; studies used the in vivo active form of the drug, mono-methyl fumarate (MMF). Ruxolitinib interacted with MMF to kill brain, breast, lung and ovarian cancer cells, and enhanced the lethality of standard of care therapies such as paclitaxel and temozolomide. MMF also interacted with other FDA approved drugs to kill tumor cells including Celebrex® and Gilenya®. The combination of [ruxolitinib + MMF] inactivated ERK1/2, AKT, STAT3 and STAT5; reduced expression of MCL-1, BCL-XL, SOD2 and TRX; increased BIM expression; decreased BAD S112 S136 phosphorylation; and enhanced pro-caspase 3 cleavage. Expression of activated forms of STAT3, MEK1 or AKT each significantly reduced drug combination lethality; prevented BAD S112 S136 dephosphorylation and decreased BIM expression; and preserved TRX, SOD2, MCL-1 and BCL-XL expression. The drug combination increased the levels of reactive oxygen species in cells, and over-expression of TRX or SOD2 prevented drug combination tumor cell killing. Over-expression of BCL-XL or knock down of BAX, BIM, BAD or apoptosis inducing factor (AIF) protected tumor cells. The drug combination increased AIF : HSP70 co-localization in the cytosol but this event did not prevent AIF : eIF3A association in the nucleus.

Multi-kinase inhibitors interact with sildenafil and ERBB1/2/4 inhibitors to kill tumor cellsin vitroandin vivo

“…The protein kinase ULK-1 is considered to be an essential gate-keeper kinase for the regulation of autophagosome formation, and whose activity is negatively regulated by Serine 757 phosphorylation, catalyzed by mTOR [9, 11, 12]. The reduced mTOR S2448 phosphorylation caused by drug exposure and shown in Figure 3 correlated with the drug-induced dephosphorylation of ULK-1 at Serine 757, which collectively in turn correlated with increased phosphorylation of the autophagosome formation regulatory protein ATG13 at Serine 318 seen in Figure 3 (Figure 4A) [20, 21].…”

“…IκB S32A S36A, eIF2α S51A, and all others listed in this manuscript were purchased from Addgene (Cambridge, MA) (Figure 12A; Figure S1A). Commercially available validated short hairpin RNA molecules to knock down RNA/protein levels were from Qiagen (Valencia, CA) or were supplied by collaborators [1–12, 28, 29] (Figure 12A; Figure S1A). …”

“…Other studies then linked the effects of AR-12 on tumor cell biology to the regulation of chaperone proteins [4, 5]. In more recent studies, we have shown that sorafenib, pazopanib, AR-12 and regorafenib can reduce the apparent expression chaperone proteins HSP90, GRP78 and HSP70 using an in-cell western/immuno-fluorescence approach [5–12]. As OSU-03012, sorafenib, pazopanib and regorafenib down-regulate the PERK inhibitory chaperone GRP78, and as the induction of toxic autophagy was PERK dependent, we investigated the role of reduced GRP78 expression caused by these drugs in the regulation of drug toxicity.…”

Monomethyl fumarate inhibits pain behaviors and amygdala activity in a rat arthritis model

“…Target concentrations of MMF were reported to be in the range of 10–30 μM, with 15 μM being tested most commonly although 5 μM was used occasionally [1;3;49;58]. For microdialysis drug application we used 1 mM because a 100 fold higher concertation is needed inside the probe due to the concentration gradient across the microdialysis membrane as indicated by our own work comparing drug effects in brain slices with microdialysis drug application for a variety of compounds [12;31;36;46].…”

“…Concentrations for microdialysis administration were based on the literature that reported target concentrations of MMF in the range of 10–30 μM, with 15 μM being the most commonly tested concentration although 5 μM was used occasionally [1;3;49;58]. Due to the concentration gradient across the microdialysis membrane a 100 fold higher concertation is needed inside the probe [12;31;36;46], and so we used 1 mM.…”