JAK3 Mutants Transform Hematopoietic Cells through JAK1 Activation Causing T-Cell Acute Lymphoblastic Leukemia in a Bone Marrow Transplant Mouse Model
Authors: Sandrine Degryse , de Bock Charles E , Vicente Carmen , Cox Luk , Gielen Olga , Mentens Nicole , Jacobs Kris , Geerdens Ellen , Tousseyn Thomas , Cools Jan 
Published: October 2019

Abstract

JAK3 is a cytosolic tyrosine kinase that associates with the common gamma chain in different cytokine receptors, in which the JAK1 kinase is another essential signaling protein. Large scale sequencing efforts recently identified mutations in the IL7R, JAK1 or JAK3 genes in about 25% of T-cell acute lymphoblastic leukemia (T-ALL) cases, with JAK3 being the most frequently (15% of T-ALL cases) mutated gene in this pathway. To determine if all mutations in JAK3 are true oncogenic mutations, we generated expression plasmids for 16 JAK3 mutants (M511I, A572T, A573V, R657Q, R657W, V674A, V678M, V678L, R775C, L857Q, Q865E, L875H, P906S, R925S, E958K, E1106G) and determined the in vitro and in vivo transforming properties, as well as their sensitivity to JAK kinase inhibitors.
For 12 of 16 mutants expression in the IL3-dependent Ba/F3 cell line resulted in transformation to IL3 independent growth. Similarly, expression of the transforming mutants in IL7-receptor reconstituted 293T cells confirmed their ligand independent activation, while this was not observed for the non-transforming mutants. These 4 non-transforming mutants are likely to be passenger mutations, illustrating that results from sequencing always need to be confirmed by functional assays to distinguish driver mutations from passenger mutations. Most JAK3 mutants, except JAK3 L857Q and JAK3 L875H, were dependent on JAK1 kinase activity for their transforming capacities. In agreement with this, we observed that Ba/F3 cells transformed by the JAK1 dependent JAK3 mutants could be inhibited by ruxolitinib, a JAK1/JAK2 selective inhibitor, while the JAK3 L857Q and JAK3 L875H transformed cells were significantly less sensitive to ruxolitinib treatment. As expected, all JAK3 mutants were sensitive to the JAK3 selective inhibitor tofacitinib, except for JAK3 L875H, which showed resistance to all inhibitors tested.
To determine the in vivo oncogenic properties of the JAK3 mutants, we expressed selected JAK3 mutants (M511I, A573V, L857Q, V674A and R657Q) in mouse hematopoietic cells through viral transduction. Mice transplanted with cells expressing JAK3 M511I, A573V or V674A showed a gradual increase of the WBC count and developed a T-ALL like disease within 120 to 200 days. In contrast, mice transplanted with cells expressing JAK3 L857Q or R657Q showed a lower increase in WBC count, and did present with severe splenomegaly and lymphadenopathy. Expression of JAK3 L857Q caused severe thymus hyperplasia, while the JAK3 R657Q mutant caused B-cell leukemia, illustrating that different JAK3 mutants seem to have variable oncogenic characteristics.
Mice transplanted with cells expressing JAK3 M511I were treated with the JAK3 selective inhibitor tofacitinib and disease progression was followed by white blood cell count measurements. Treatment of the animals for 5 weeks with tofacitinib (oral gavage, 40 mg/kg/day) significantly decreased the disease progression compared to placebo treated mice. Moreover, we observed severe apoptosis of the leukemia cells in spleen and thymus in tofacitinib treated animals and not in placebo treated mice. However, tofacitnib treatment could not eradicate all leukemia cells, and the mice progressed when treatment was stopped.
In conclusion, JAK3 is recurrently mutated in T-ALL patients, and we demonstrate that most JAK3 mutants are transforming proteins using In vitro and in vivo experiments. Our results show that JAK1 is an essential kinase for most JAK3 mutants, and that the majority of JAK3 mutants are sensitive to JAK3 and JAK1 specific inhibitors such as tofacitinib and ruxolitinib. However, some JAK3 mutants do show resistance to these inhibitors, which will need to be taken into account when trials are initiated for the treatment of ALL patients with JAK specific inhibitors.

Disclosures
No relevant conflicts of interest to declare.