Decitabine enhances the cytotoxic effect of ruxolitinib in myeloproliferative neoplasms by targeting SR splicing factors Free

Introduction:The Breakpoint Cluster Region–Abelson (BCR-ABL) negative myeloproliferative neoplasms (MPNs) are a group of chronic hematological malignancies characterized by the clonal proliferation of hematopoietic stem cells. Ruxolitinib, a JAK1/2 inhibitor, has demonstrated significant efficacy in alleviating symptom burden and improving the quality of life for MPNs patients. However, the lack of complete responses in most patients treated with ruxolitinib underscores the need for identifying novel therapeutic strategies. Decitabine (DAC), a DNA methyltransferase inhibitor, is currently used as first-line therapy for higher-risk myelodysplastic syndromes (HR-MDS) and elderly patients with acute myeloid leukemia (AML). In this study, we investigated whether decitabine could enhance the efficacy of ruxolitinib in MPNs.

Methods:In vitro, SET-2 and HEL cell lines were used as the main research objects. The effects of combined treatment with DAC and Ruxolitinib on cell viability, apoptosis levels and colony formation capacity were studied using the ATP bioluminescence assay, flow cytometry and soft agar clone formation assay, respectively. Western Blot and RT-qPCR were used to detect the expression level of apoptosis-related proteins and SRSF1, SRSF2. In vivo, NOD.Cg-Prkd^cscid IL2rgtm^1Wjl/SzJ (NSG) mice, injected SET-2-luciferase cells through the tail veins, were used to construct the xenograft murine model.

Results: In both SET-2 and HEL cells, ruxolitinib and decitabine (DAC) each reduced cell viability in a dose- and time-dependent manner. Notably, the combination of ruxolitinib and DAC led to a more pronounced reduction in cell viability compared to either agent alone. Synergy analysis confirmed that the two drugs acted synergistically in both cell lines, with all combination index (CI) values <1. Similar to findings for cell viability, SET2 cells and HEL cells subjected to DAC and ruxolitinib treatment significantly induced apoptosis, and combined treatment presented higher levels of apoptosis compared with monotherapy. Additionally, Cleaved Caspase 3 expression was significantly upregulated, indicating enhanced apoptosis. Cell cycle analysis revealed that ruxolitinib induced G1 phase arrest in both cell lines, whereas DAC had no significant impact on cell cycle progression. In long-term assays, DAC exhibited stronger suppression of clonogenicity than ruxolitinib alone. Strikingly, the combination therapy nearly abolished colony formation. These findings were recapitulated in vivo, where the combination treatment significantly reduced tumor burden in a xenograft murine model. Mechanistic investigations revealed that ruxolitinib monotherapy increased the expression of SRSF1 expression, whereas combination treatment reduced both in protein and transcript levels compared to controls. Additionally, ruxolitinib or DAC alone modestly decreased SRSF2 transcription, with a more pronounced reduction observed under combination therapy. These findings suggest that SRSF1 and SRSF2 may contribute to the observed synergistic effects.

Conclusion：In current study, we demonstrated that the combination of decitabine and ruxolitinib synergistically inhibited cell viability and promoted apoptosis of SET-2 both in vitro and in vivo. Then, we explored the molecular mechanisms behind the synergistic effect, and found SRSF1 and SRSF2 both declined in the combination treatment. These findings provide valuable insights for the development of novel therapeutic strategies for MPNs.

Acknowledgement: This research was funded by Zhejiang Provincial Health High-level Innovative Talent Project (2022-2026).

*Correspondence to: Jian Huang, M.D., Ph.D., Department of Hematology, The First Affiliated Hospital of Zhejiang University School of Medicine, Hangzhou 310003, Zhejiang, China. E-mail: househuang@zju.edu.cn.