Abstract
Introduction: JAK2-rearrangements (JAK2r) are associated with poor prognosis in B-cell acute lymphoblastic leukaemia (B-ALL). Outcomes may be improved by incorporating targeted therapies and a clinical trial is currently assessing the only FDA-approved JAK1/2 inhibitor, ruxolitinib. We predict that a subset of patients will develop resistance to ruxolitinib.
Aim: Investigate mechanisms of acquired JAK inhibitor resistance in JAK2r ALL to inform monitoring and aversion of resistance.
Method: JAK2r B-ALL was modelled in the pro-B cell line, Ba/F3, by expressing a high-risk B-ALL JAK2 fusion gene. Acquired ruxolitinib resistance in three independent cell lines was achieved following dose escalation to a clinically relevant dose (1 µM). Sanger sequencing revealed each resistant line had acquired a different JAK2 kinase domain mutation. Computational modelling of JAK2 mutations and their influence on ruxolitinib binding was performed using ICM-Pro (Molsoft L.C.C.). Therapeutic sensitives were assessed by flow cytometry following staining with Fixable Aqua Dead Cell Stain (Invitrogen) and Annexin V-PE.
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 JAK2 ATP/ruxolitinib binding site. Computational modelling suggested that JAK2 p.L983F sterically hinders ruxolitinib binding, while JAK2 p.Y931C reduces ruxolitinib binding affinity by loss of stacking interactions. 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 all tested type-I JAK inhibitors. JAK2 p.G993A-mutant Ba/F3 cells were also resistant to the typeII JAK inhibitor, CHZ-868, suggesting that sequential use of JAK inhibitors may not overcome resistance.
Conclusion: JAK2r ALL is a high-risk disease that may be amenable to targeted inhibition to improve outcomes. We identified critical residues within the JAK2 ATP-binding site that if mutated may confer resistance to JAK inhibitors anticipated to enter ALL therapeutics
Aim: Investigate mechanisms of acquired JAK inhibitor resistance in JAK2r ALL to inform monitoring and aversion of resistance.
Method: JAK2r B-ALL was modelled in the pro-B cell line, Ba/F3, by expressing a high-risk B-ALL JAK2 fusion gene. Acquired ruxolitinib resistance in three independent cell lines was achieved following dose escalation to a clinically relevant dose (1 µM). Sanger sequencing revealed each resistant line had acquired a different JAK2 kinase domain mutation. Computational modelling of JAK2 mutations and their influence on ruxolitinib binding was performed using ICM-Pro (Molsoft L.C.C.). Therapeutic sensitives were assessed by flow cytometry following staining with Fixable Aqua Dead Cell Stain (Invitrogen) and Annexin V-PE.
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 JAK2 ATP/ruxolitinib binding site. Computational modelling suggested that JAK2 p.L983F sterically hinders ruxolitinib binding, while JAK2 p.Y931C reduces ruxolitinib binding affinity by loss of stacking interactions. 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 all tested type-I JAK inhibitors. JAK2 p.G993A-mutant Ba/F3 cells were also resistant to the typeII JAK inhibitor, CHZ-868, suggesting that sequential use of JAK inhibitors may not overcome resistance.
Conclusion: JAK2r ALL is a high-risk disease that may be amenable to targeted inhibition to improve outcomes. We identified critical residues within the JAK2 ATP-binding site that if mutated may confer resistance to JAK inhibitors anticipated to enter ALL therapeutics
| Original language | English |
|---|---|
| Pages | 58 |
| Number of pages | 1 |
| Publication status | Published - 20 Sept 2021 |
| Externally published | Yes |
| Event | Blood 2021 Annual Scientific Meeting - Adelaide, Australia Duration: 20 Sept 2021 → 23 Sept 2021 |
Conference
| Conference | Blood 2021 Annual Scientific Meeting |
|---|---|
| Country/Territory | Australia |
| City | Adelaide |
| Period | 20/09/21 → 23/09/21 |
Keywords
- JAK2-rearrangements (JAK2r)
- B-cell acute lymphoblastic leukaemia (B-ALL)
- Ruxolitinib
- JAK1/2 inhibitor