SRP326092 PRJNA741841 Acquired mutations within the JAK2 kinase domain confer resistance to type-I and type-II JAK inhibitors in in vitro models of a high-risk acute lymphoblastic leukemia Ruxolitinib (rux) Phase II clinical trials are underway for the treatment of high-risk JAK2-rearranged (JAK2r) B-cell acute lymphoblastic leukemia (B-ALL). Treatment resistance to targeted inhibitors in other settings is common, this elucidating potential mechanisms of rux resistance in JAK2r B-ALL will enable development of therapeutic strategies to overcome or avert resistance. We generated a murine pro-B cell model of ATF7IP-JAK2 with acquired resistance to multiple type-I JAK inhibitors. Resistance was associated with mutations within the JAK2 ATP/rux binding site, including a novel JAK2 p.G993A mutation. Using in vitro models of JAK2r B-ALL, JAK2 p.G993A conferred resistance to six type-I JAK inhibitors and the type-II JAK inhibitor, CHZ-868. Using computational modelling, we postulate that JAK2 p.G993A enabled JAK2 activation in the presence of drug binding through a novel resistance mechanism that modulates the mobility of the conserved JAK2 activation loop. This study highlights the importance of monitoring mutation emergence and may inform future drug design and the development of therapeutic strategies for this high-risk patient cohort.