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 SPAG9JAK2 fusion, which was identified in a pediatric patient with Ph-like ALL, to establish molecular targeted therapy. Ba/F3 cells expressing SPAG9JAK2 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 (SPAG9JAK2 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 SPAG9JAK2-mediated cell proliferation. Consistently, STAT1 induced expression of the anti-apoptotic proteins, BCL-2 and MCL-1, as did SPAG9JAK2, but not SPAG9JAK2 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 SPAG9JAK2-related aberrant growth promotion. BCL-2 or MCL-1 inhibition is a potential therapeutic option for B-ALL with SPAG9JAK2 fusion.