Kavitha Gnanasambandan

Key Points Using WES, we designed an extended thrombophilia panel consisting of 55 genes of significance to thrombosis. The extended thrombophilia panel identified multiple novel genetic variants with predicted roles in thrombosis or thrombophilia.

Somatic mutations in the Jak2 allele that lead to constitutive kinase activation of the protein have been identified in human disease conditions such as the myeloproliferative neoplasms (MPNs). The most common mutation in these patients is a V617F substitution mutation, which is believed to play a causative role in the MPN pathogenesis. As such, identifying the molecular basis for the constitutive activation of Jak2-V617F is important for understanding its clinical implications and potential treatment. Here, we hypothesized that conversion of residue 617 from Val to Phe resulted in the formation of novel π stacking interactions with neighboring Phe residues. To test this, we first examined the Jak2 structure via molecular modeling and identified a potential π stacking interaction between F594, F595, and F617. Disruption of this interaction through site-directed mutagenesis impaired Jak2 autophosphorylation, Jak2-dependent gene transcription, and in vitro kinase activity of the Jak2-V617F protein. Further, substitution of F594 and F595 with Trp did not affect Jak2 function significantly, but replacement with charged residues did, showing the importance of aromaticity and hydropathy index conservation at these positions. Using molecular dynamics (MD) simulations, we found that the π stacking interaction between residues 595 and 617 in the Jak2-V617F protein was of much greater energy and conformed to the properties of π stacking, relative to the Jak2-WT or Jak2-V617F/F594A/F595A. In summary, we have identified a π stacking interaction between F595 and F617 that is specific to and is critical for the constitutive activation of Jak2-V617F.

Jak2 is a non-receptor tyrosine kinase that is involved in the control of cellular growth and proliferation. Due to its significant role in hematopoiesis, Jak2 is a frequent target for mutations in cancer, especially myeloid leukemia, lymphoid leukemia and the myeloproliferative neoplasms (MPN). These mutations are common amongst different populations all over the world and there is a great deal of effort to develop therapeutic drugs for the affected patients. Jak2 mutations, whether they are point, deletion, or gene fusion, most commonly result in constitutive kinase activation. Here, we explore the structure-function relation of various Jak2 mutations identified in cancer and understand how they disrupt Jak2 regulation. Current Jak2 inhibitors target the highly conserved active site in the kinase domain and therefore, these inhibitors may lack specificity. Based on our knowledge regarding structure-function correlations as they pertain to regulation of Jak2 kinase activity, an alternative approach for specific Jak2 targeting could be via allosteric inhibitor design. Successful reports of allosteric inhibitors developed against other kinases provide precedent for the development of Jak2 allosteric inhibitors. Here, we suggest plausible target sites in the Jak2 structure for allosteric inhibition. Such targets include the type II inhibitor pocket and substrate binding site in the kinase domain, the kinase-pseudokinase domain interface, SH2-JH2 linker region and the FERM domain. Thus, future Jak2 inhibitors that target these sites via allosteric mechanisms may provide alternative therapeutic strategies to existing ATP competitive inhibitors.