Background: Upon prolonged exposure, the therapeutic efficiency of current clinical JAK2 inhibitors (type I inhibitors) can be attenuated, leading to drug resistance both in vitro and in vivo through non-genetic mechanisms. In contrast, type II inhibitors stabilize the inactive conformation of the kinase domain preventing the heterodimerization-mediated JAK2 activation. This confers efficacy to type I inhibitor persistent cells, improved efficacy against myeloproliferative neoplasms and other cancers such as B-cell acute lymphoblastic leukemia. Despite the advantages of type II JAK inhibitors, the discovery of type II inhibitor resistance mutation, L884P, in the kinase domain has hampered the further development of JAK2-targeted type II therapeutics. Methods: Here we use combination of protein-inhibitor binding assays, cell-based assays, X-ray crystallography, and computational tools to elucidate the resistance mechanism of JAK2-L884P mutation, to facilitate the development of type II inhibitors with activity against both wild-type and L884P mutated JAK2. Results: We have determined an atomic resolution crystal structure of JAK2-L884P mutant, revealing that mutation induces local changes in the structure but not in the ATP pocket. Furthermore, we have identified several type II compounds inhibiting. Interestingly, they show no major differences in potency in vitro against wild-type of L884P mutated JAK2. Selected compounds profiled in cell lines carrying wild-type or L884P-mutated JAK2, show differences in their cell viability, indicating that a resistance arises via allosteric effects. Conclusions: L884P mutation impacts the inhibitory potency of type II inhibitors via allosteric mechanism.Crystallographic analysis of our type II shortlisted compounds revealed that they differ in their binding modes and suggests that L884P-insensitive drugs can be developed. Preliminary cell-based experiments results confirm these findings. We will now focus on validating these observations with biomolecular simulations to clarify the molecular basis for the inhibitor resistance via L884P mutation. Our studies will facilitate the development of novel type II drugs against JAK2 in myeloproliferative neoplasms.