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INTRODUCTION ETV6::JAK2 is a fusion known to drive Acute Lymphoblastic Leukaemia (ALL) in the presence of other genomic lesions which define the JAK/STAT class of Philadelphia-like Acute Lymphoblastic Leukaemia (Ph-like ALL). Ph-like ALL comprises approximately 15% of ALL. Patients with mutations or gene fusions signaling through the JAK/STAT pathway have particularly poor prognosis. Emerging treatments targeting JAK2 fusions and mutations are promising and phase 3 clinical trials are in progress. However, with widespread use of JAK2 inhibitors, it is important to anticipate and manage resistance mechanisms. The JAK2 p.G993A mutation confers resistance in vitro, even to high dose JAK2 inhibitors such as ruxolitinib. We postulated that direct inhibition of STAT3 and STAT5, downstream from JAK2, may overcome resistance. METHODS Murine derived IL-3 dependent Ba/F3 cells transfected with ETV6::JAK2 containing a p.G993A mutation were used in this study. These cells were confirmed to demonstrate IL-3 independence and ruxolitinib resistance prior to use in experiments. An inhibitor-response assay was conducted using differing concentrations of SH-4-54 and pimozide (STAT3/5 inhibitors) applied to ETV6::JAK2 p.G993A cells and two control cell lines. RESULTS SH-4-54 and pimozide were effective against ETV6::JAK2 p.G993A cells with median lethal doses (LD50) of 296nM for SH-4-54 and 455nM for pimozide. Both drugs demonstrated a lesser effect on empty vector Ba/F3 cells, with an LD50 of 371nM for SH-4-54 and 596nM for pimozide. Neither drug demonstrated significant effect on non-JAK/STAT activated KG-1a myeloid cells at doses near the LD50. CONCLUSION SH-4-54 and pimozide both overcame treatment resistance in our in-vitro model of JAK/STAT driven Ph-like ALL with a mutation conferring JAK2 inhibitor resistance. While SH-4-54 demonstrates greater potency than pimozide, pimozide may be a more promising option given its demonstrated safety profile in humans. Direct STAT3 and STAT5 inhibition may be an effective approach for overcoming inevitable JAK2 inhibitor resistance-conferring mutations in patients with the poor prognostic subtype of JAK/STAT class Ph-like ALL., (The Author(s). Published by S. Karger AG, Basel.)