Identification of novel, potent, and selective JAK2V617F inhibitors Free

The predominant oncogenic variant driving myeloproliferative neoplasms (MPNs) is the JAK2V617F substitution, where valine is replaced by phenylalanine in Janus kinase 2 (JAK2). Approximately 95% of polycythemia vera (PV) patients, and in over half of primary myelofibrosis (MF) and essential thrombocythemia (ET) cases carry JAK2V617F. Approved therapies that inhibit the JAK2 kinase (JH1) domain offer meaningful clinical improvements but are often associated with substantial on-target side effects, including thrombocytopenia and anemia. Next-generation JAK2 inhibitors that selectively target the JAK2 pseudokinase (JH2) domain and specifically inhibit JAK2V617F, would spare wild-type (WT) JAK2 activity while eliminating mutant cells. Here, we present the discovery and characterization of novel, potent, and selective JAK2V617F inhibitors.

Given the position of V617F in the JH2 domain of JAK2, we sought to discover JH2 ATP pocket binders. Test molecules were triaged with biochemical probe displacement assays to select compounds that bind JH2 and not JH1. In detail, we employed a high-competition probe displacement assay to resolve highly potent compounds binding to JH2. We identified compounds with sub-nanomolar potencies and with >3500-fold selectivity over the JH1 domain. Next, we interrogated cellular selectivity for JAK2V617F against WT JAK2 in the JAK2-dependent human hematological cancer cell lines SET-2 (JAK2V617F) and TF-1 (WT JAK2) using STAT5 transcriptional activity assays and cell proliferation assays. We found molecules with sub-micromolar potencies on STAT5 transcriptional activity and proliferation with >10-fold selectivity over WT JAK2.

Molecules with encouraging cellular selectivity were then assayed for their activity on other signaling pathways driven by heterodimers formed by other JAK-family members, against other kinases, and profiled across a panel of 16 hematological cancer cell lines from myeloid and lymphoid lineages. Compared to ruxolitinib, our molecules were >3-fold less active on other JAK-family members with a similar cell panel profile, which indicate improved selectivity. Taken together, our robust in vitro screening cascade identified potent molecules with the ability to measure selectivity between internal JAK2 domains, JAK2V617F and WT JAK2, other JAK-family members, and JAK2-independent cancer cell lines.

Preliminary pharmacokinetic profiles in mice were determined following single dose oral administration at 1 mg/kg and 30 mg/kg. Compounds exhibited promising exposure covering the IC₅₀ at 30mpk doses with strong bioavailability (>50%). These compounds show promise for assessment in in vivo cellular selectivity models, where pSTAT5 levels could be measured after treatment in tumors derived from SET-2 and UKE-1 (JAK2V617F) as well as TF-1 (WT JAK2) cell lines.

In summary, our comprehensive screening cascades allow for the discovery of compounds with strong selectivity at both biochemical and cellular levels. Using this approach, we identified novel, potent, and selective JAK2V617F inhibitors.