Contributions
Abstract: EP320
Type: E-Poster Presentation
Session title: Acute lymphoblastic leukemia - Biology & Translational Research
Background
The JAK2 kinase plays an essential role in mediating growth-promoting activities of various cytokines. Constitutive activation of this kinase, via chromosomal translocations, has been previously described in acute lymphoblastic leukemia (ALL), with PAX5, BCR and ETV6 being the most common partner genes. ALLs with JAK2 fusions usually have the BCR-ABL1-like phenotype associated with unfavorable outcome. The metabolic consequences potentially contributing to leukemogenesis in cells harboring JAK2 fusion genes have not been investigated so far.
Aims
We identified and studied in detail a novel JAK2 involving fusion gene, NPAT-JAK2, in a patient with high-risk B-cell precursor ALL.
Methods
Seahorse Metabolic Analysis
Results
The NPAT gene is located on chromosome 11 and encodes a nuclear protein acting as a co-activator of histone gene transcription. No functional domains in the N-terminal part of the NPAT protein involved in the NPAT/JAK2 chimera, have been described, but, importantly, the chimera involved the complete JAK2 kinase domain. We cloned the full-length coding sequence of NPAT/JAK2 and studied its biological properties in model cell lines. We found that the NPAT/JAK2 protein is localized both in the cytoplasm and in the nucleus of transfected cells and that it is autophosphorylated on Y1007/Y1008 in the activation loop of JAK2 whose phosphorylation is essential for JAK2 kinase function. Next, we tested the oncogenic potential of NPAT/JAK2 in the Ba/F3 cells dependent on interleukin 3 (IL3), in which the fusion induced cytokine independent growth. This effect was abolished by a mutation of the NPAT/JAK2 ATP-binding site, demonstrating its dependence on the JAK2 kinase activity. Western blot analysis of the transformed BAF3 cells showed increased phosphorylation of STAT1 and STAT5. Importantly, the phosphorylation and pro-proliferative effect was blocked by the JAK1/2 inhibitor Ruxolitinib.
The cell energy phenotype test revealed an overall enhanced metabolic state of the NPAT/JAK2 transformed BA/F3 cells grown without IL3, with elevations of both the mitochondrial and the glycolytic function. Differences were visible already under steady-state cultivation conditions, however the major divergence between the NPAT/JAK2 transformed cells and controls occurred after stress induction. Concomitantly, the ATP production was increased in NPAT/JAK2 transformed cells, together with the NAD+/NADH ratio when compared to the empty vector and NPAT/JAK2-positive BAF3 cells dependent on IL3 (in pre-transformation phase), as assessed by a luciferase assay. To validate our result, we used 2-deoxy-D-glucose (2DG), a competitor of glucose which is a main substrate utilized in bioenergetics pathways of cancer cells. The proliferation of NPAT/JAK2 transformed cells treated with 2DG was dramatically inhibited, compared to controls. Interestingly, metabolic differences were not observed in the case of the recurrent JAK2 kinase fusion PAX5-JAK2. Therefore we presume that the here described metabolic alterations most likely do not represent a general mechanism for all of the fusion genes involving the JAK2 kinase domain.
Conclusion
To conclude, we identified and characterized in detail a novel druggable JAK2 fusion gene, NPAT/JAK2, with a constitutively active chimeric tyrosine kinase. The oncogenic potential of cells harboring this fusion is likely driven by signaling pathway alterations and intensive metabolic changes with dependencies on sufficient energy substrate supply, a mechanism that could potentially be exploited in future therapeutical strategies.
Support: AZV NV18–07-00129
Keyword(s): ALL, Fusion, Kinase, Pediatric
Abstract: EP320
Type: E-Poster Presentation
Session title: Acute lymphoblastic leukemia - Biology & Translational Research
Background
The JAK2 kinase plays an essential role in mediating growth-promoting activities of various cytokines. Constitutive activation of this kinase, via chromosomal translocations, has been previously described in acute lymphoblastic leukemia (ALL), with PAX5, BCR and ETV6 being the most common partner genes. ALLs with JAK2 fusions usually have the BCR-ABL1-like phenotype associated with unfavorable outcome. The metabolic consequences potentially contributing to leukemogenesis in cells harboring JAK2 fusion genes have not been investigated so far.
Aims
We identified and studied in detail a novel JAK2 involving fusion gene, NPAT-JAK2, in a patient with high-risk B-cell precursor ALL.
Methods
Seahorse Metabolic Analysis
Results
The NPAT gene is located on chromosome 11 and encodes a nuclear protein acting as a co-activator of histone gene transcription. No functional domains in the N-terminal part of the NPAT protein involved in the NPAT/JAK2 chimera, have been described, but, importantly, the chimera involved the complete JAK2 kinase domain. We cloned the full-length coding sequence of NPAT/JAK2 and studied its biological properties in model cell lines. We found that the NPAT/JAK2 protein is localized both in the cytoplasm and in the nucleus of transfected cells and that it is autophosphorylated on Y1007/Y1008 in the activation loop of JAK2 whose phosphorylation is essential for JAK2 kinase function. Next, we tested the oncogenic potential of NPAT/JAK2 in the Ba/F3 cells dependent on interleukin 3 (IL3), in which the fusion induced cytokine independent growth. This effect was abolished by a mutation of the NPAT/JAK2 ATP-binding site, demonstrating its dependence on the JAK2 kinase activity. Western blot analysis of the transformed BAF3 cells showed increased phosphorylation of STAT1 and STAT5. Importantly, the phosphorylation and pro-proliferative effect was blocked by the JAK1/2 inhibitor Ruxolitinib.
The cell energy phenotype test revealed an overall enhanced metabolic state of the NPAT/JAK2 transformed BA/F3 cells grown without IL3, with elevations of both the mitochondrial and the glycolytic function. Differences were visible already under steady-state cultivation conditions, however the major divergence between the NPAT/JAK2 transformed cells and controls occurred after stress induction. Concomitantly, the ATP production was increased in NPAT/JAK2 transformed cells, together with the NAD+/NADH ratio when compared to the empty vector and NPAT/JAK2-positive BAF3 cells dependent on IL3 (in pre-transformation phase), as assessed by a luciferase assay. To validate our result, we used 2-deoxy-D-glucose (2DG), a competitor of glucose which is a main substrate utilized in bioenergetics pathways of cancer cells. The proliferation of NPAT/JAK2 transformed cells treated with 2DG was dramatically inhibited, compared to controls. Interestingly, metabolic differences were not observed in the case of the recurrent JAK2 kinase fusion PAX5-JAK2. Therefore we presume that the here described metabolic alterations most likely do not represent a general mechanism for all of the fusion genes involving the JAK2 kinase domain.
Conclusion
To conclude, we identified and characterized in detail a novel druggable JAK2 fusion gene, NPAT/JAK2, with a constitutively active chimeric tyrosine kinase. The oncogenic potential of cells harboring this fusion is likely driven by signaling pathway alterations and intensive metabolic changes with dependencies on sufficient energy substrate supply, a mechanism that could potentially be exploited in future therapeutical strategies.
Support: AZV NV18–07-00129
Keyword(s): ALL, Fusion, Kinase, Pediatric