CHARACTERISATION OF JAK3 KINASE DOMAIN MUTANTS
(Abstract release date: 05/19/16)
EHA Library. Degryse S. 06/09/16; 132391; E842
Ms. Sandrine Degryse
Contributions
Contributions
Abstract
Abstract: E842
Type: Eposter Presentation
Background
Janus kinase 1 (JAK1) and JAK3 are cytosolic tyrosine kinases that are required for cytokine receptor signaling. Recent studies have identified both JAK1 and JAK3 mutations in ALL, AML and lymphoma. JAK3 is mutated in 16% of T-ALL cases. The maojority of these mutations are found within the pseudokinase and kinase domains of JAK3.
Aims
To determine the transforming mechanism of newly identified JAK3 kinase domain mutants L875H, P906S and E958K and to characterize their sensitivity to various JAK inhibitors.
Methods
We studied six JAK3 mutations, two pseudokinase (M511I and R657Q) and four kinase domain (L857Q, L875H, P906S and E958K) mutants. We expressed these proteins in Ba/F3 (proB-cell) and ex vivo cultured primary mouse T-cells. CRISPR/Cas9 genome editing was used to generate common gamma chain (Il2rg) and Jak1 knock out cells.
Results
All six JAK3 mutants were able to transform Ba/F3 cells to growth factor independent growth. The JAK3 L857Q and L875H mutants were able to transform the Ba/F3 cells in the absence of Jak1 or Il2rg, indicating that these two kinase domain mutants do not require binding to a cytokine receptor and do not require Jak1 as a signaling partner. In contrast, the JAK3 M511I, R657Q, P906S and E958K mutants required the presence of Jak1 and Il2rg. Moreover, the growth of these four JAK3 mutants was decreased by overexpression of wild type JAK3 showing that the JAK3 mutants compete with wild type Jak3 for binding to the receptor. Using a proT-cell model, the two JAK3 kinase domain mutants L857Q and L875H showed increased transformation to cytokine independent growth compared to the remaining JAK3 mutants.We next determined the sensitivity of the cells transformed by the JAK3 mutants to either JAK3 selective inhibitors (tofacitinib and decernotinib) or JAK1 selective inhibitors (ruxolitinib and baricitinib). Decernotinib was able to inhibit all JAK3 mutants. While tofacitinib was able to inhibit most JAK3 mutants, the L875H mutant was resistant to this compound. Whilst the JAK1 selective inhibitors were able to inhibit the JAK3 M511I, R657Q, P906S and E958K, they were not able to inhibit the JAK3 L857Q and L875H mutants that were shown to transform cells independently of JAK1.
Conclusion
The majority of JAK3 mutant proteins require binding to a cytokine receptor complex for full activation and transformation of cells to cytokine independent growth and are sensitive to JAK1 selective inhibition. All data indicated that the transforming mechanism of the two kinase domain mutants L857Q and L875H differ from the other JAK3 mutants. They can activate downstream signaling independent of the receptor complex and are not sensitive to JAK1 selective inhibition. In addition, the L857H mutants show a high tolerance for the JAK inhibitor tofacitinib.
Session topic: E-poster
Keyword(s): Tyrosine kinase, Tyrosine kinase inhibitor
Type: Eposter Presentation
Background
Janus kinase 1 (JAK1) and JAK3 are cytosolic tyrosine kinases that are required for cytokine receptor signaling. Recent studies have identified both JAK1 and JAK3 mutations in ALL, AML and lymphoma. JAK3 is mutated in 16% of T-ALL cases. The maojority of these mutations are found within the pseudokinase and kinase domains of JAK3.
Aims
To determine the transforming mechanism of newly identified JAK3 kinase domain mutants L875H, P906S and E958K and to characterize their sensitivity to various JAK inhibitors.
Methods
We studied six JAK3 mutations, two pseudokinase (M511I and R657Q) and four kinase domain (L857Q, L875H, P906S and E958K) mutants. We expressed these proteins in Ba/F3 (proB-cell) and ex vivo cultured primary mouse T-cells. CRISPR/Cas9 genome editing was used to generate common gamma chain (Il2rg) and Jak1 knock out cells.
Results
All six JAK3 mutants were able to transform Ba/F3 cells to growth factor independent growth. The JAK3 L857Q and L875H mutants were able to transform the Ba/F3 cells in the absence of Jak1 or Il2rg, indicating that these two kinase domain mutants do not require binding to a cytokine receptor and do not require Jak1 as a signaling partner. In contrast, the JAK3 M511I, R657Q, P906S and E958K mutants required the presence of Jak1 and Il2rg. Moreover, the growth of these four JAK3 mutants was decreased by overexpression of wild type JAK3 showing that the JAK3 mutants compete with wild type Jak3 for binding to the receptor. Using a proT-cell model, the two JAK3 kinase domain mutants L857Q and L875H showed increased transformation to cytokine independent growth compared to the remaining JAK3 mutants.We next determined the sensitivity of the cells transformed by the JAK3 mutants to either JAK3 selective inhibitors (tofacitinib and decernotinib) or JAK1 selective inhibitors (ruxolitinib and baricitinib). Decernotinib was able to inhibit all JAK3 mutants. While tofacitinib was able to inhibit most JAK3 mutants, the L875H mutant was resistant to this compound. Whilst the JAK1 selective inhibitors were able to inhibit the JAK3 M511I, R657Q, P906S and E958K, they were not able to inhibit the JAK3 L857Q and L875H mutants that were shown to transform cells independently of JAK1.
Conclusion
The majority of JAK3 mutant proteins require binding to a cytokine receptor complex for full activation and transformation of cells to cytokine independent growth and are sensitive to JAK1 selective inhibition. All data indicated that the transforming mechanism of the two kinase domain mutants L857Q and L875H differ from the other JAK3 mutants. They can activate downstream signaling independent of the receptor complex and are not sensitive to JAK1 selective inhibition. In addition, the L857H mutants show a high tolerance for the JAK inhibitor tofacitinib.
Session topic: E-poster
Keyword(s): Tyrosine kinase, Tyrosine kinase inhibitor
Abstract: E842
Type: Eposter Presentation
Background
Janus kinase 1 (JAK1) and JAK3 are cytosolic tyrosine kinases that are required for cytokine receptor signaling. Recent studies have identified both JAK1 and JAK3 mutations in ALL, AML and lymphoma. JAK3 is mutated in 16% of T-ALL cases. The maojority of these mutations are found within the pseudokinase and kinase domains of JAK3.
Aims
To determine the transforming mechanism of newly identified JAK3 kinase domain mutants L875H, P906S and E958K and to characterize their sensitivity to various JAK inhibitors.
Methods
We studied six JAK3 mutations, two pseudokinase (M511I and R657Q) and four kinase domain (L857Q, L875H, P906S and E958K) mutants. We expressed these proteins in Ba/F3 (proB-cell) and ex vivo cultured primary mouse T-cells. CRISPR/Cas9 genome editing was used to generate common gamma chain (Il2rg) and Jak1 knock out cells.
Results
All six JAK3 mutants were able to transform Ba/F3 cells to growth factor independent growth. The JAK3 L857Q and L875H mutants were able to transform the Ba/F3 cells in the absence of Jak1 or Il2rg, indicating that these two kinase domain mutants do not require binding to a cytokine receptor and do not require Jak1 as a signaling partner. In contrast, the JAK3 M511I, R657Q, P906S and E958K mutants required the presence of Jak1 and Il2rg. Moreover, the growth of these four JAK3 mutants was decreased by overexpression of wild type JAK3 showing that the JAK3 mutants compete with wild type Jak3 for binding to the receptor. Using a proT-cell model, the two JAK3 kinase domain mutants L857Q and L875H showed increased transformation to cytokine independent growth compared to the remaining JAK3 mutants.We next determined the sensitivity of the cells transformed by the JAK3 mutants to either JAK3 selective inhibitors (tofacitinib and decernotinib) or JAK1 selective inhibitors (ruxolitinib and baricitinib). Decernotinib was able to inhibit all JAK3 mutants. While tofacitinib was able to inhibit most JAK3 mutants, the L875H mutant was resistant to this compound. Whilst the JAK1 selective inhibitors were able to inhibit the JAK3 M511I, R657Q, P906S and E958K, they were not able to inhibit the JAK3 L857Q and L875H mutants that were shown to transform cells independently of JAK1.
Conclusion
The majority of JAK3 mutant proteins require binding to a cytokine receptor complex for full activation and transformation of cells to cytokine independent growth and are sensitive to JAK1 selective inhibition. All data indicated that the transforming mechanism of the two kinase domain mutants L857Q and L875H differ from the other JAK3 mutants. They can activate downstream signaling independent of the receptor complex and are not sensitive to JAK1 selective inhibition. In addition, the L857H mutants show a high tolerance for the JAK inhibitor tofacitinib.
Session topic: E-poster
Keyword(s): Tyrosine kinase, Tyrosine kinase inhibitor
Type: Eposter Presentation
Background
Janus kinase 1 (JAK1) and JAK3 are cytosolic tyrosine kinases that are required for cytokine receptor signaling. Recent studies have identified both JAK1 and JAK3 mutations in ALL, AML and lymphoma. JAK3 is mutated in 16% of T-ALL cases. The maojority of these mutations are found within the pseudokinase and kinase domains of JAK3.
Aims
To determine the transforming mechanism of newly identified JAK3 kinase domain mutants L875H, P906S and E958K and to characterize their sensitivity to various JAK inhibitors.
Methods
We studied six JAK3 mutations, two pseudokinase (M511I and R657Q) and four kinase domain (L857Q, L875H, P906S and E958K) mutants. We expressed these proteins in Ba/F3 (proB-cell) and ex vivo cultured primary mouse T-cells. CRISPR/Cas9 genome editing was used to generate common gamma chain (Il2rg) and Jak1 knock out cells.
Results
All six JAK3 mutants were able to transform Ba/F3 cells to growth factor independent growth. The JAK3 L857Q and L875H mutants were able to transform the Ba/F3 cells in the absence of Jak1 or Il2rg, indicating that these two kinase domain mutants do not require binding to a cytokine receptor and do not require Jak1 as a signaling partner. In contrast, the JAK3 M511I, R657Q, P906S and E958K mutants required the presence of Jak1 and Il2rg. Moreover, the growth of these four JAK3 mutants was decreased by overexpression of wild type JAK3 showing that the JAK3 mutants compete with wild type Jak3 for binding to the receptor. Using a proT-cell model, the two JAK3 kinase domain mutants L857Q and L875H showed increased transformation to cytokine independent growth compared to the remaining JAK3 mutants.We next determined the sensitivity of the cells transformed by the JAK3 mutants to either JAK3 selective inhibitors (tofacitinib and decernotinib) or JAK1 selective inhibitors (ruxolitinib and baricitinib). Decernotinib was able to inhibit all JAK3 mutants. While tofacitinib was able to inhibit most JAK3 mutants, the L875H mutant was resistant to this compound. Whilst the JAK1 selective inhibitors were able to inhibit the JAK3 M511I, R657Q, P906S and E958K, they were not able to inhibit the JAK3 L857Q and L875H mutants that were shown to transform cells independently of JAK1.
Conclusion
The majority of JAK3 mutant proteins require binding to a cytokine receptor complex for full activation and transformation of cells to cytokine independent growth and are sensitive to JAK1 selective inhibition. All data indicated that the transforming mechanism of the two kinase domain mutants L857Q and L875H differ from the other JAK3 mutants. They can activate downstream signaling independent of the receptor complex and are not sensitive to JAK1 selective inhibition. In addition, the L857H mutants show a high tolerance for the JAK inhibitor tofacitinib.
Session topic: E-poster
Keyword(s): Tyrosine kinase, Tyrosine kinase inhibitor
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