Identification of ruxolitinib resistant mutations in Pro-B cells driven by a high-risk JAK2 fusion in B-cell acute lymphoblastic leukaemia

JAK2 rearrangements (JAK2r) occur in approximately 5% of paediatric B-cell acute lymphoblastic leukemia (B-ALL) patients and are associated with poor prognosis. The resultant gene fusions drive leukaemogenesis through constitutive kinase activation, which may be amenable to Jak inhibitor therapy. A Phase II clinical trial is currently assessing the only FDA approved Jak1/2 inhibitor, ruxolitinib (rux) in high-risk B-ALL cases harbouring JAK2 pathway alterations. Given the development of treatment resistance to targeted inhibitors in other diseases such as CML, elucidating mechanisms of rux resistance in JAK2r B-ALL will enable the development of therapeutic strategies to overcome or avert resistance.

The ATF7IP-JAK2 fusion gene was identified in the bone marrow cells of a male with paediatric BALL and expressed in the pro-B cell line, Ba/F3. Rux resistance was generated following dose escalation to a clinically relevant dose of 1 µM rux in three independent experiments. Acquired mutations within JAK2 were detected by Sanger sequencing of RT-PCR amplified JAK2 fusion transcript. Alterations in signalling were determined in comparison to untreated ATF7IP-JAK2 Ba/F3’s (rux-naïve). Computational modelling of acquired mutations in the Jak2 kinase domain and their influence on rux binding was performed using ICM-Pro (Molsoft L.C.C.).

All three independently derived rux resistant ATF7IP-JAK2 Ba/F3 lines demonstrated significantly increased survival as measured by Annexin-V in rux concentrations of 500 nM (p
This study demonstrates that the JAK2 kinase domain ATP-binding site is susceptible to mutations following rux exposure and a novel JAK2 G993A mutation was identified in addition to previously reported mutations. This mutation is postulated to modulate the DFG loop stability and is in a region commonly affected by gatekeeper mutations in other kinases. Understanding mechanisms of rux resistance has the potential to inform combinational therapeutic strategies and importantly inform future drug design in this high-risk patient cohort.