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In vivo impact of JAK3 A573V mutation revealed using zebrafish
Faiza Basheer1, Vilasha Bulleeraz1, Viet Q T Ngo1
1School of Medicine, Deakin University, Pigdons Road, Geelong, VIC, 3216, Australia.
Background:
Janus kinase 3 (JAK3) acts downstream of the interleukin-2 (IL-2) receptor family to play a pivotal role in the regulation of lymphoid cell development. Activating JAK3 mutations are associated with a number of lymphoid and other malignancies, with mutations within the regulatory pseudokinase domain common.
Methods:
The pseudokinase domain mutations A572V and A573V were separately introduced into the highly conserved zebrafish Jak3 and transiently expressed in cell lines and zebrafish embryos to examine their activity and impact on early T cells. Genome editing was subsequently used to introduce the A573V mutation into the zebrafish genome to study the effects of JAK3 activation on lymphoid cells in a physiologically relevant context throughout the life-course.
Results:
Zebrafish Jak3 A573V produced the strongest activation of downstream STAT5 in vitro and elicited a significant increase in T cells in zebrafish embryos. Zebrafish carrying just a single copy of the Jak3 A573V allele displayed elevated embryonic T cells, which continued into adulthood. Hematopoietic precursors and NK cells were also increased, but not B cells. The lymphoproliferative effects of Jak3 A573V in embryos was shown to be dependent on zebrafish IL-2Rγc, JAK1 and STAT5B equivalents, and could be suppressed with the JAK3 inhibitor Tofacitinib.
Conclusions:
This study demonstrates that a single JAK3 A573V allele expressed from the endogenous locus was able to enhance lymphopoiesis throughout the life-course, which was mediated via an IL-2Rγc/JAK1/JAK3/STAT5 signaling pathway and was sensitive to Tofacitinib. This extends our understanding of oncogenic JAK3 mutations and creates a novel model to underpin further translational investigations.
Insights
A single Janus kinase 3 (JAK3) mutation, A573V, enhances T cell production throughout life. This lymphoproliferative effect is mediated by the IL-2Rγc/JAK1/JAK3/STAT5 pathway and can be blocked by Tofacitinib.
Area of Science:
- Immunology
- Molecular Biology
- Genetics
Background:
- Janus kinase 3 (JAK3) is crucial for lymphoid cell development, functioning downstream of the interleukin-2 (IL-2) receptor family.
- Activating mutations in JAK3, particularly in its pseudokinase domain, are linked to various lymphoid malignancies.
Purpose of the Study:
- To investigate the functional impact of specific JAK3 pseudokinase domain mutations (A572V and A573V) on lymphoid cell development.
- To establish a zebrafish model for studying the long-term effects of oncogenic JAK3 activation in a physiological context.
Main Methods:
- Introduced JAK3 pseudokinase domain mutations (A572V, A573V) into zebrafish Jak3 and expressed them in cell lines and embryos.
- Utilized genome editing to integrate the A573V mutation into the zebrafish genome for life-course studies.
- Analyzed downstream signaling (STAT5 activation) and cell populations (T cells, hematopoietic precursors, NK cells, B cells).
Main Results:
- Zebrafish Jak3 A573V significantly increased T cell numbers in embryos and adults, even with a single allele.
- Elevated levels of hematopoietic precursors and NK cells were observed, but not B cells.
- The lymphoproliferative effect was dependent on IL-2Rγc, JAK1, and STAT5B signaling and was inhibited by Tofacitinib.
Conclusions:
- A single endogenous JAK3 A573V allele enhances lymphopoiesis throughout the life-course via the IL-2Rγc/JAK1/JAK3/STAT5 pathway.
- This JAK3 activation is sensitive to the JAK3 inhibitor Tofacitinib.
- The study provides a novel zebrafish model for investigating oncogenic JAK3 mutations and their therapeutic targeting.
