Abstract
We determined whether the approved myelofibrosis drug ruxolitinib (Jakafi®), an inhibitor of Janus kinases 1/2 (JAK1 and JAK2), could be repurposed as an anti-cancer agent for solid tumors. Ruxolitinib synergistically interacted with dual ERBB1/2/4 inhibitors to kill breast as well as lung, ovarian and brain cancer cells. Knock down of JAK1/2 or of ERBB1/2/3/4 recapitulated on-target drug effects. The combination of (ruxolitinib + ERBB1/2/4 inhibitor) rapidly inactivated AKT, mTORC1, mTORC2, STAT3, and STAT5, and activated eIF2α. In parallel, the drug combination reduced expression of MCL-1, BCL-XL, HSP90, HSP70, and GRP78, and increased expression of Beclin1. Activated forms of STAT3, AKT, or mTOR prevented the drug-induced decline in BCL-XL, MCL-1, HSP90, and HSP70 levels. Over-expression of chaperones maintained AKT/mTOR activity in the presence of drugs and protected tumor cells from the drug combination. Expression of dominant negative eIF2α S51A prevented the increase in Beclin1 expression and protected tumor cells from the drug combination. Loss of mTOR activity was associated with increased ATG13 S318 phosphorylation and with autophagosome formation. Autophagosomes initially co-localized with mitochondria and subsequently with lysosomes. Knock down of Beclin1 suppressed: drug-induced mitophagy; the activation of the toxic BH3 domain proteins BAX and BAK; and tumor cell killing. Knock down of apoptosis-inducing factor (AIF) protected tumor cells from the drug combination, whereas blockade of caspase 9 signaling did not. The drug combination released AIF into the cytosol and increased nuclear AIF: eIF3A co-localization. A 4-day transient exposure of orthotopic tumors to (ruxolitinib + afatinib) profoundly reduced mammary tumor growth over the following 35 days. Re-grown tumors exhibited high levels of BAD S112 phosphorylation and activation of ERK1/2 and NFκB. Our data demonstrate that mitophagy is an essential component of (ruxolitinib + ERBB inhibitor) lethality and that this drug combination should be explored in a phase I trial in solid tumor patients.
Highlights
Immune cell activation in general and during rheumatoid arthritis progression requires signaling by Janus kinases (JAK1, JAK2, JAK3)
Twenty-four hours after transfection, cells were treated with vehicle control or with [ruxolitinib (1.0 μM) + afatinib (1.0 μM)] for 6 h after which cells were fixed in place and permeabilized using 0.5% Triton X100
We discovered that ruxolitinib at clinically relevant free drug concentrations synergized with multiple ERBB1/2/4 inhibitors to kill tumor cells, including those expressing mutated active RAS proteins or lacking the tumor suppressor phosphatase and tensin homolog on chromosome ten (PTEN)
Summary
Immune cell activation in general and during rheumatoid arthritis progression requires signaling by Janus kinases (JAK1, JAK2, JAK3). Pharmaceutical companies, attacking these kinases as drugable targets, have developed several FDAapproved agents in the hope of reducing the negative sequelae of arthritis and of semi-tumorigenic myelo-proliferative disorders: Jakafi and Xeljanz, respectively [1,2,3,4]. Mutated active forms of Janus kinases or the actions of mutated activated growth factor receptors through autocrine loops cause constitutive activation of the STAT1/ STAT3/STAT5 transcription factors that promote the malignant phenotype. Growth factor receptors, such as ERBB1, and c-MET have been shown to phosphorylate STAT factors on tyrosine residues thereby promoting dimerization/activation [11, 12]. Cyto-protective genes activated by STAT transcription factors are many and include those coding for anti-apoptotic genes, such as MCL-1, BCL-XL, BCL-2, survivin, HSP90, and HSP70; proliferation regulatory genes, such as Cyclin D1, Cyclin B, c-Jun, and c-Fos; and angiogenesis promoting genes, such as HIF1α, and growth factors, such as IL-6, FGF, EGF, and VEGF [13,14,15,16,17,18,19,20]
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