Study On The Mechanism Of Synergistic Inhibition Of HEL Cells Transwell Co-culture With HS-5 Cells By Ruxolitinib And BGB324

Objective Myeloproliferative neoplasms is hematopoietic stem cell clonal proliferative tumor.The use of JAK2 inhibitor Ruxolitinib can reduce the symptom load,improve the quality of life and prolong the overall survival in MPNs patients,but some patients still have poor response or intolerance to Ruxolitinib treatment.In recent years,studies have found that the Gas6/Axl signaling pathway is related to the proliferation and drug resistance of a variety of tumors,while Axl is up-regulated and overactivated in AK2 related MPNs,and inhibition of Axl is preferred to kill tumor cells in PV patients,but the specific mechanism remains unclear.BGB324 is a highly selective inhibitor targeting Axl.Ruxolitinib and BGB324 were used separately or in combination to study the inhibitory effect on HEL cells transwell co-cultured with bone marrow stromal cells HS-5,verify the feasibility of Axl inhibitor intervention in MPNs,and explore the possible mechanism.Methods The levels of IL-6 and Gas6 in the peripheral venous blood supernatant of newly diagnosed MPNs patients and healthy donors were detected by ELISA.HS-5cells（1×10⁶/ml）and HEL cells（1×10⁶/ml）in logarithmic growth phase were transwell co-cultured as the experimental group,and separately cultured HEL cells（1×10⁶/ml）or HS-5 cells（1×10⁶/ml）as the control group.After treatment with Ruxolitinib and BGB324 alone or in combination for 48 hours,the IC₅₀value of each group was detected by CCK8 assay,the apoptosis rate of each group was detected by Annexin V-FITC/PI flow cytometry,and the levels of IL-6 and Gas6 were detected by ELISA.The expression levels of IL-6 and Gas6 genes in each group were detected by RT-PCR,and the protein expressions of Axl,p-Axl,p-Stat3,p-Stat5,p-Akt and Bcl-2in each group were detected by Western Blot.Results The average contents of IL-6 and Gas6 in the peripheral venous blood supernatant of MPNs patients were significantly higher than those of healthy donors（P<0.05）.After treated with different concentrations of Ruxolitinib or BGB324 for48h,the proliferation of HEL cells cultured alone and in the transwell group was inhibited,and the sensitivity of HEL cells in the transwell group to drugs was reduced.The combination of drugs promoted the apoptosis of HEL cells more obviously than that of single drugs,and the apoptosis rate of HEL cells in transwell group was lower than that in the corresponding single culture group（P<0.05）.The levels of IL-6 and Gas6 in supernatant of the transwell group were higher than those of the single culture group,and the decrease of IL-6 and Gas6 was more obvious with the combination drug than with the single drug（P<0.05）.The expression levels of IL-6 and Gas6 in HEL cells in the transwell group were not significantly changed compared with that in the HEL cell culture alone group.The expression levels of IL-6 and Gas6 in HS-5cells in the transwell co-culture group were significantly higher than those in the HS-5alone culture group（P<0.05）.The expression levels of IL-6 in HEL cells were decreased 48 hours after the treatment of Ruxolitinib and BGB324 alone or in combination with the transwell group.The expression level of Gas6 gene did not change significantly,and the expression levels of IL-6 and Gas6 gene in HS-5 cells were significantly decreased（P<0.05）.The expressions of Bcl-2,p-Axl,p-Akt,p-Stat5 and p-Stat3 in transwell co-cultured HEL cells increased gradually after 12,24and 48h,compared with those in HEL cells cultured alone.The expression levels of Bcl-2,p-Akt,p-Stat5 and p-Stat3 in HEL cells were decreased when treated with Ruxolitinib alone,while the expression levels of p-Axl and Axl were not significantly changed.The expression levels of p-Axl,Axl,Bcl-2,p-Akt and p-Stat5 in HEL cells were decreased by BGB324 alone,while the expression levels of p-Stat3 were not significantly changed,and the decrease of Bcl-2,p-Akt,p-Stat5 and p-Stat3 in HEL cells was more significant by combination therapy than by single therapy.Conclusion HS-5 cells in transwell co-culture system supported the survival of HEL cells and resisted the pro-apoptotic effect of Ruxolitinib.Ruxolitinib combined with BGBG324 can reverse the resistance of Ruxolitinib to HEL cells and promote the apoptosis of HEL cells by down-regulating the expression of IL-6 and Gas6,inhibiting the activation of Gas6/Axl,JAK/STAT and PI3K/Akt signaling pathways,and decreasing the expression of Bcl-2.