JAK2V617F can mimic growth factor signaling, leading to PI3K/AKT/mTOR activation and inhibition of autophagy. We hypothesized that selective inhibition of JAK1/2 by ruxolitinib could induce autophagy and limit drug efficacy in myeloproliferative neoplasms (MPN). Therefore, we investigated the effects of ruxolitinib treatment on autophagy-related genes and cellular processes, to determine the potential benefit of autophagy inhibitors plus ruxolitinib in JAK2V617F cells, and to verify the frequency and clinical impact of autophagy-related gene mutations in patients with MPNs. In SET2 JAK2V617F cells, ruxolitinib treatment induced autophagy and modulated 26 out of 79 autophagy-related genes. Ruxolitinib treatment reduced the expressions of important autophagy regulators, including mTOR/p70S6K/4EBP1 and the STAT/BCL2 axis, in a dose- and time-dependent manner. Pharmacological inhibition of autophagy was able to significantly suppress ruxolitinib-induced autophagy and increased ruxolitinib-induced apoptosis. Mutations in autophagy-related genes were found in 15.5% of MPN patients and were associated with increased age and a trend towards worse survival. In conclusion, ruxolitinib induces autophagy in JAK2V617F cells, potentially by modulation of mTOR-, STAT- and BCL2-mediated signaling. This may lead to inhibition of apoptosis. Our results suggest that the combination of ruxolitinib with pharmacological inhibitors of autophagy, such as chloroquine, may be a promising strategy to treat patients with JAK2V617F-mutated MPNs.
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The authors would like to thank Dr. Nicola Conran for English revision.
This study was supported by grants #2014/23092–0, #2017/19864–6, #2014/50947–7, and #2013/08135–2, São Paulo Research Foundation (FAPESP) and grant #402587/2016–2, Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq).
Informed consent was obtained from all individual participants included in the study prior to sample collection and the study was approved by the Institutional Review Board.
The authors declare that they have no competing interests.
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Electronic supplementary material
Supplementary Fig. 1. Whole gel images of Western blotting analysis. Western blot analysis for protein phosphorylation and expression in total cell extracts from SET2 upon treatment with ruxolitinib and/or 3-methyladenine (3-MA), Bafilomycin A1 (Baf-A1), and chloroquine, as indicated; membranes were reprobed with the antibody for the detection of the respective total protein or actin, and developed with the SuperSignal™ West Dura Extended Duration Substrate system and a Gel Doc XR+ imaging system. Antibodies, merged and unmerged images are indicated. (PDF 3883 kb)
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Machado-Neto, J.A., Coelho-Silva, J.L., Santos, F.P.d. et al. Autophagy inhibition potentiates ruxolitinib-induced apoptosis in JAK2V617F cells. Invest New Drugs 38, 733–745 (2020). https://doi.org/10.1007/s10637-019-00812-5
- Myeloproliferative neoplasms