Autophagy inhibition potentiates ruxolitinib-induced apoptosis in JAK2V617F cells

Summary

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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Acknowledgments

The authors would like to thank Dr. Nicola Conran for English revision.

Funding

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).

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Correspondence to Fabiola Traina.

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ESM 1

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

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Keywords

  • Autophagy
  • Ruxolitinib
  • Myeloproliferative neoplasms
  • Chloroquine
  • Apoptosis