Synergy of Ruxolitinib and Carfilzomib in Targeting the PAX5::JAK2 fusion I n Vitro: Potential Therapeutic Advantage for a Subset of Ph-like Acute Lymphoblastic Leukemia

• Published in: Blood 2023-11, Vol.142 (Supplement 1), p.5806-5806
• Main Authors: Thompson, Jane Frances, Yeung, David T, Grose, Randall Hilton, White, Deborah L
• Format: Article
• Language: English
• ISSN: 0006-4971; 1528-0020
• DOI: 10.1182/blood-2023-174423

INTRODUCTION PAX5::JAK2 is a gene fusion that drives a subset of Philadelphia chromosome-like acute lymphoblastic leukemia (Ph-like ALL) and confers aggressive disease, poor prognosis and poor response to treatment. PAX5::JAK2 is present in approximately 12% of Ph-like ALL cases. Targeted therapy is urgently required to improve outcomes. We evaluated the efficacy of JAK inhibitor ruxolitinib (RUX) and proteosome inhibitors carfilzomib (CFZ) and bortezomib (BTZ) as monotherapy and in combination against PAX5::JAK2 transfected Ba/F3 cells in vitro. Downregulation of proteosome genes via reduction in STAT3 phosphorylation is a shared mechanism of RUX and proteosome inhibitors. Given that the PAX5::JAK2 fusion results in increased STAT3 phosphorylation driving proliferation, it was proposed that the combination of proteosome inhibitors and RUX would demonstrate synergy in inhibiting proliferation of PAX5::JAK2 driven leukemic cells. METHODS Mouse derived Ba/F3 cells, an IL-3 dependent B-ALL model, were transfected with the PAX5::JAK2 fusion and demonstrated IL-3 independence. We applied, as single treatments, differing concentrations of RUX and the proteosome inhibitors CFZ and BTZ to PAX5::JAK2 transfected Ba/F3 cells and two control cell lines - IL-3 dependent empty vector Ba/F3 cells and an IL-3 independent KG1a myeloid cell line. Differing concentrations of CFZ and 200nM of RUX were then applied in combination. 200nM of RUX was selected because it is a dose at which the drug has minimal effect as a single agent. Inhibitors were applied to cells for 72 hours then percentage of live and dead cells at each concentration was ascertained by flow cytometry using Annexin V and LIVE/DEAD fixable aqua dead cell stain. Downstream STAT5 and STAT3 activation in basal state, and as a response to treatment, was assessed using phosphoflow. We demonstrated mechanisms of growth inhibition via reduction in STAT5 and STAT3 phosphorylation by the inhibitors. RESULTS RUX, CFZ and BTZ were all effective against PAX5::JAK2 transfected Ba/F3 cells with a median lethal dose (LD50) of 514nM (CI 498-530nM) for RUX, 38nM (CI 36-39nM) for CFZ and 19nM (CI 18-20nM) for BTZ. RUX had a variable effect on IL-3 dependent empty vector control cells which also demonstrate JAK/STAT activation, but no effect on the IL-3 independent KG1a cells. CFZ had a lesser effect on empty vector control cells with a LD50 of 59nM (CI 58-60nM) and no effect on KG1a control cells. BTZ had a significant effect