E-Poster Presentation 33rd Lorne Cancer Conference 2021

Acquired mutations within the JAK2 kinase domain confer resistance to JAK inhibitors in in vitro models of acute lymphoblastic leukaemia driven by high-risk JAK2 fusion genes (#104)

Charlotte EJ Downes 1 2 , Barbara J McClure 1 3 , John B Bruning 4 , Jimmy Breen 3 5 6 , Jacqueline Rehn 1 3 , David T Yeung 1 3 7 , Deborah L White 1 2 3 8
  1. Cancer Program, Precision Medicine Theme, South Australian Health & Medical Research Institute (SAHMRI), Adelaide, South Australia, Australia
  2. School of Biological Sciences, University of Adelaide, Adelaide, South Australia, Australia
  3. Adelaide Medical School, University of Adelaide, Adelaide, South Australia, Australia
  4. Institute of Photonics and Advanced Sensing, School of Biological Sciences, University of Adelaide, Adelaide, South Australia, Australia
  5. Computational and Systems Biology Program, South Australian Health & Medical Research Institute (SAHMRI), Adelaide, South Australia, Australia
  6. Robinson Research Institute, University of Adelaide, Adelaide, South Australia, Australia
  7. Department of Haematology, Royal Adelaide Hospital and SA Pathology, Adelaide, South Australia, Australia
  8. Australian and New Zealand Children's Oncology Group (ANZCHOG), Clayton, Victoria, Australia

Introduction

JAK2 rearrangements (JAK2r) occur in approximately 5% of paediatric B-cell acute lymphoblastic leukaemia (B-ALL) cases and are associated with poor prognosis. A clinical trial is currently assessing the only FDA-approved JAK1/2 inhibitor, ruxolitinib in high-risk B-ALL cases harbouring JAK2 alterations. Elucidating mechanisms of ruxolitinib resistance in JAK2r B-ALL will inform approaches to monitor the emergence of resistance in ongoing clinical trials and enable the development of new therapeutic strategies to overcome or avert resistance.

 

Methods

JAK2r B-ALL was modelled in the pro-B cell line, Ba/F3, by expressing the high-risk B-ALL fusion, ATF7IP-JAK2. Ruxolitinib resistance in three independent ATF7IP-JAK2 Ba/F3 cell lines was achieved following dose escalation to a clinically relevant dose of 1 µM. Sanger sequencing of the RT-PCR amplified JAK2 fusion revealed each resistant line had acquired a different mutation within the JAK2 kinase domain. Computational modelling of acquired JAK2 mutations and their influence on ruxolitinib binding was performed using ICM-Pro (Molsoft L.C.C.). Therapeutic sensitives were assessed by staining with Fixable Aqua Dead Cell Stain (Invitrogen) and Annexin V-PE, and analysis by flow cytometry.

 

Results

In addition to the identification of two known ruxolitinib resistant mutations, JAK2 p.Y931C and p.L983F, a novel p.G993A mutation was identified. All mutations localised to the ATP/ruxolitinib binding site. Computational modelling suggested that JAK2 p.L983F sterically hinders ruxolitinib binding, while JAK2 p.Y931C may reduce ruxolitinib binding affinity by disruption of a critical hydrogen-bond. The novel JAK2 p.G993A mutation is predicted to alter DFG-loop dynamics by stabilising the JAK2 activation loop. All three ruxolitinib-resistant mutations conferred resistance to multiple type-I JAK inhibitors. JAK2 p.G993A ATF7IP-JAK2 Ba/F3 cells were also resistant to the type-II JAK inhibitor, CHZ-868.

 

Conclusion

The JAK2 ATP-binding site is susceptible to JAK inhibitor resistant mutations following ruxolitinib exposure, highlighting the importance of monitoring emergence of mutations within this region. Understanding mechanisms of ruxolitinib resistance has the potential to inform future drug design and development of therapeutic strategies for this high-risk cohort.