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The combination of ruxolitinib and Bcl-2/Mcl-1 inhibitors has a synergistic effect on leukemic cells carrying a SPAG9::JAK2 fusion

Abstract

JAK2 rearrangements can occur in Philadelphia chromosome-like acute lymphoblastic leukemia (Ph-like ALL). Here, we performed functional analysis of the SPAG9::JAK2 fusion, which was identified in a pediatric patient with Ph-like ALL, to establish molecular targeted therapy. Ba/F3 cells expressing SPAG9::JAK2 generated by retroviral transduction (Ba/F3-SPAG9-JAK2), proliferated in the absence of IL-3, and exhibited constitutive phosphorylation of the tyrosine residues in the JAK2 kinase domain of the fusion protein and STAT3/STAT5. Mutation of tyrosine residues in the JAK2 kinase domain (SPAG9::JAK2 mut) abolished IL-3 independence, but had no influence on STAT3/STAT5 phosphorylation levels. Gene expression analysis revealed that Stat1 was significantly upregulated in Ba/F3-SPAG9-JAK2 cells. STAT1 was also phosphorylated in Ba/F3-SPAG9-JAK2 but not SPAG9-JAK2 mut cells, suggesting that STAT1 is key for SPAG9::JAK2-mediated cell proliferation. Consistently, STAT1 induced expression of the anti-apoptotic proteins, BCL-2 and MCL-1, as did SPAG9::JAK2, but not SPAG9::JAK2 mut. Ruxolitinib abrogated Ba/F3-SPAG9-JAK2-mediated proliferation in vitro, but was insufficient in vivo. Venetoclax (a BCL-2 inhibitor) or AZD5991 (an MCL-1 inhibitor) enhanced the effects of ruxolitinib on Ba/F3-SPAG9-JAK2 in vitro. These findings suggest that activation of the JAK2-STAT1-BCL-2/MCL-1 axis contributes to SPAG9::JAK2-related aberrant growth promotion. BCL-2 or MCL-1 inhibition is a potential therapeutic option for B-ALL with SPAG9::JAK2 fusion.

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Fig. 1: SPAG9::JAK2 induces IL-3-independent proliferation of Ba/F3 cells with constitutive phosphorylation of SPAG9::JAK2 tyrosine residues and STAT3/STAT5.
Fig. 2: Effect of mutating tyrosine residues (Y1007F and Y1008F) in the JAK2 kinase domain to phenylalanine.
Fig. 3: Analysis of gene expression in Ba/F-SPAG9-JAK2 versus mock Ba/F3 cells revealed that Ba/F3-SPAG9-JAK2 cells depend on STAT1 for survival.
Fig. 4: BCL-2 and MCL-1 expression is upregulated in Ba/F3-SPAG9-JAK2 cells.
Fig. 5: Treatment with ruxolitinib leads to death of Ba/F3-SPAG9-JAK2 cells and prolongs the survival time of a SPAG9::JAK2 xenograft mouse model.
Fig. 6: Venetoclax (a BCL-2 inhibitor) reduced viability of Ba/F3-SPAG9-JAK2 cells.
Fig. 7: AZD5991 (an MCL-1 inhibitor) reduced Ba/F3-SPAG9-JAK2 cell viability.

Data availability

The datasets generated during the current study are available from the corresponding author on reasonable request.

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Acknowledgements

This study was supported by grants-in-aid for scientific research from the Japanese Ministry of Education, Culture, Sports, Science and Technology (17K10124), and by a Grant-in-Aid for Practical Research for Innovative Cancer Control (17ck0106253h0001, 18ck0106253h0002, and 19ck0106253h0003).

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AM designed the research, performed the experiments, analyzed the data, generated the figures, and wrote the manuscript. TI designed the research, analyzed the data, and wrote the manuscript. TT, TK, TM, KT, and HU performed the experiments and analyzed the data. HY, IK, MK, TN, JT, and HH supervised the research and participated in editing the manuscript. All authors approved the final version of the manuscript.

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Correspondence to Toshihiko Imamura.

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Mayumi, A., Tomii, T., Kanayama, T. et al. The combination of ruxolitinib and Bcl-2/Mcl-1 inhibitors has a synergistic effect on leukemic cells carrying a SPAG9::JAK2 fusion. Cancer Gene Ther (2022). https://doi.org/10.1038/s41417-022-00511-z

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