Type II mode of JAK2 inhibition and destabilization are potential therapeutic approaches against the ruxolitinib resistance driven myeloproliferative neoplasms

Sivahari P Gorantla; Gerin Prince; Jasmin Osius; Dhurvas Chandrasekaran Dinesh; Vijay Boddu; Justus Duyster; Nikolas von Bubnoff

doi:10.3389/fonc.2024.1430833

Type II mode of JAK2 inhibition and destabilization are potential therapeutic approaches against the ruxolitinib resistance driven myeloproliferative neoplasms

Front Oncol. 2024 Jul 18:14:1430833. doi: 10.3389/fonc.2024.1430833. eCollection 2024.

Authors

Sivahari P Gorantla^{1

2}, Gerin Prince¹, Jasmin Osius¹, Dhurvas Chandrasekaran Dinesh³, Vijay Boddu¹, Justus Duyster², Nikolas von Bubnoff¹

Affiliations

¹ Department of Hematology and Oncology, Medical Center, University of Schleswig-Holstein, Lübeck, Germany.
² Department of Internal Medicine I, University Medical Center Freiburg, Freiburg, Germany.
³ Department of Biochemistry and Molecular Biology, Institute of Organic Chemistry and Biochemistry of the Czech Academy of Sciences, Prague, Czechia.

Abstract

Background: Ruxolitinib has been approved by the US FDA for the treatment of myeloproliferative neoplasms such as polycythemia vera and primary myelofibrosis. Ruxolitinib will remain a main stay in the treatment of MPN patients due to its effective therapeutic benefits. However, there have been instances of ruxolitinib resistance in MPN patients. As JAK2 is a direct target of ruxolitinib, we generated ruxolitinib-resistant clones to find out the mechanism of resistance.

Methods: Cell-based screening strategy was used to detect the ruxolitinib-resistant mutations in JAK2. The Sanger sequencing method was used to detect the point mutations in JAK2. Mutations were re-introduced using the site-directed mutagenesis method and stably expressed in Ba/F3 cells. Drug sensitivities against the JAK2 inhibitors were measured using an MTS-based assay. JAK2 and STAT5 activation levels and total proteins were measured using immunoblotting. Computational docking studies were performed using the Glide module of Schrodinger Maestro software.

Results: In this study, we have recovered seven residues in the kinase domain of JAK2 that affect ruxolitinib sensitivity. All these mutations confer cross-resistance across the panel of JAK2 kinase inhibitors except JAK2-L983F. JAK2-L983F reduces the sensitivity towards ruxolitinib. However, it is sensitive towards fedratinib indicating that our screen identifies the drug-specific resistance profiles. All the ruxolitinib-resistant JAK2 variants displayed sensitivity towards type II JAK2 inhibitor CHZ-868. In this study, we also found that JAK1-L1010F (homologous JAK2-L983F) is highly resistant towards ruxolitinib suggesting the possibility of JAK1 escape mutations in JAK2-driven MPNs and JAK1 mutated ALL. Finally, our study also shows that HSP90 inhibitors are potent against ruxolitinib-resistant variants through the JAK2 degradation and provides the rationale for clinical evaluation of potent HSP90 inhibitors in genetic resistance driven by JAK2 inhibitors.

Conclusion: Our study identifies JAK1 and JAK2 resistance variants against the type I JAK2 inhibitors ruxolitinib, fedratinib, and lestaurtinib. The sensitivity of these resistant variants towards the type II JAK2 inhibitor CHZ-868 indicates that this mode of type II JAK2 inhibition is a potential therapeutic approach against ruxolitinib refractory leukemia. This also proposes the development of potent and specific type II JAK2 inhibitors using ruxolitinib-resistance variants as a prototype.

Keywords: Hsp90 inhibitior; JAK2-V617F; myeloproliferative neoplasm; ruxolitinib resistance; type I JAK2 inhibition.

Grants and funding

The author(s) declare financial support was received for the research, authorship, and/or publication of this article. The authors would like to thank their funding agencies. SG, JO and NB are supported by DFG (3554/1-3). GP was funded by the DAAD fellowship. DD was supported by the National Institute of Virology and Bacteriology (Programme EXCELES, Project No. LX22NPO5103) - Funded by the European Union - Next Generation EU.