Discovery of JAK2V617F mutant specific allosteric inhibitors for the treatment of myeloproliferative neoplasms Free

The V617F mutation in Janus Kinase 2 (JAK2^V617F) is a recurrent driver of myeloproliferative neoplasms (MPNs), including myelofibrosis (MF), polycythemia vera (PV), and essential thrombocythemia (ET). Current therapies targeting the JAK-STAT pathway such as pan-JAK inhibitors (JAKi) like Ruxolitinib have demonstrated clinical benefit but are limited by a lack of disease-modifying activity. These agents bind the orthosteric site of the JAK2 JH1 domain and fail to selectively inhibit JAK2^V617F over wild-type JAK2, resulting in insufficient suppression of the mutant clone at tolerable doses. Consequently, they do not significantly reduce mutant allele burden, lack disease-modifying effects, and rarely achieve clinical remission. Additionally, inhibition of wild-type JAK2 disrupts normal hematopoiesis, contributing to treatment discontinuation due to adverse events. These limitations underscore the need for mutant-selective, disease-modifying therapies.

Allosteric inhibition of JAK2 represents a promising strategy to selectively target JAK2^V617F through novel mechanisms of action. However, identifying suitable allosteric sites and ligands remains a significant challenge. To address this, Atavistik Bio has developed the AMPS™ (Atavistik Metabolite Proprietary Screening) platform, a scalable and systematic discovery engine for identifying unique ligand-macromolecule interactions. Leveraging this platform, we identified JAK2^V617F selective inhibitors that bind the allosteric pseudokinase JH2 domain, which harbors the V617F mutation. Through structure-based drug design, we optimized initial hits from the AMPS™ screen to develop potent, picomolar-binding compounds with high specificity for the JAK2^V617F JH2 domain. These compounds demonstrated strong binding to JAK2^V617F JH2 and no measurable activity against JAK2 JH1 or the JH1 domains of other JAK family members at concentrations up to 50 µM. They also exhibited >100-fold selectivity over TYK2 and JAK1 JH2 domains.

Compounds with potent and selective binding to the JAK2^V617F JH2 domain also showed strong inhibition of JAK2^V617F dependent signaling and proliferation in multiple human hematopoietic cancer cell lines harboring the mutation. In contrast, these compounds displayed significantly weaker potency against cytokine-stimulated JAK2 signaling and growth in cell lines expressing wild-type JAK2. To further assess selectivity, both copies of the JAK2^V617F allele in UKE-1 cells were CRISPR-edited to wild-type JAK2. Using this isogenic cell pair, we confirmed that multiple compounds consistently exhibited >10-fold selective inhibition of cytokine-independent JAK2^V617F signaling compared to cytokine-dependent wild-type JAK2 signaling. Additional studies in primary MPN patient-derived hematopoietic stem cells to assess mutant selectivity are ongoing.

Select inhibitors were evaluated in mice bearing subcutaneous JAK2^V617F expressing SET2 tumors. Pharmacokinetic and pharmacodynamic analyses in this model demonstrated robust target engagement, as evidenced by decreased phospho-STAT5 levels within xenograft tumors. Compounds with favorable PK profiles, tolerability, and minimal off-target activity were selected for further advancement. Additional studies in preclinical models of JAK2^V617F driven splenomegaly are currently underway.

In summary, we have discovered novel, potent, and selective allosteric inhibitors of JAK2^V617F that effectively suppress pathogenic, cytokine-independent JAK2^V617F driven signaling and cell proliferation while sparing normal cytokine-dependent signaling. These findings support the development of highly mutant selective inhibitors for the treatment of JAK2^V617F driven MPNs.