Contributions
Abstract: S197
Type: Oral Presentation
Session title: New pathways and molecular mechanisms in MPN
Background
The classical Philadelphia-chromosome-negative (Ph-) myeloproliferative neoplasms (MPNs) consist of three entities: polycythemia vera (PV), essential thrombocythemia (ET) and primary myelofibrosis (PMF), carrying three mutually exclusive driver mutations in the genes of janus kinase 2 (JAK2), calreticulin (CALR) and the thrombopoietin (TPO) receptor (MPL). JAK2V617F-mutant cells were shown to be more sensitive to interferon alpha (IFNa) treatment compared to CALR-mutant cells and this IFNa “priming” involved increased phosphorylation of JAK1 and signal transducer and activator of transcribtion (STAT) 1 in JAK2V617F- vs. CALR-mutant cells. IFNa stimulation leads to phosphorylation of tyrosine kinase (TYK) 2 and JAK1, which are constitutively associated with the IFNa receptor-1 or -2 (IFNAR1/2), respectively, and activate downstream signaling pathways. However, the role of TYK2 in the survival and IFNa response of JAK2- vs. CALR-mutant MPN cells is still incompletely understood.
Aims
Analysis of the role of TYK2 in MPN cells to further investigate the differential response of JAK2V617F- and CALR-mutant cells to IFNa treatment and evaluate TYK2 as a potential target in MPN.
Methods
We used CRISPR/Cas9 technology to generate TYK2-deficient 32DMPL JAK2V617F or CALRdel52 cells and subsequently analyzed the IFNa response on cell viability (MTT assays), downstream JAK-STAT signaling and gene expression. Furthermore, we evaluated the effects of the TYK2-specific inhibitor deucravacitinib in 32DMPL mutant cells and in primary MPN patient material using colony formation unit assays.
Results
Basal phosphorylation of TYK2 (pTYK2) was increased in 32DMPL CALRdel52-mutant cells compared to JAK2V617F-mutant as well as empty vector cells (p<0.001). Generation of a TYK2 knockout in 32DMPL CALRdel52 cells was not successful, while pTYK2-deficient JAK2V617F-mutant cells were readily produced, underlining an important role of TYK2 in CALR-mutant cells. In pTYK2-deficient 32DMPL JAK2V617F cells, IFNa effects were significantly reduced, including loss of viability, activation of pSTAT1 and pSTAT3 and IFNa target genes. However, induction of apoptosis after IFNa treatment was not altered. We analyzed the effect of deucravacitinib and found that cell viability of 32DMPL CALRdel52- but not JAK2V617F-mutant cells was significantly decreased even at low concentrations [0.5 µM] of the inhibitor. Western Blotting confirmed complete loss of TYK2 phosphorylation after deucravacitinib treatment in both cell lines as well as reduced phosphorylation of downstream signaling molecules. In primary peripheral blood-derived mononuclear cells of patients with MPN, the TYK2 inhibitor decreased CALR- but not JAK2V617F-mutant allele burden in vitro, and clonogenic growth of ET- and PMF- but not PV-derived cells was inhibited.
Conclusion
Our data demonstrate an essential role for TYK2 in CALR-mutant but not JAK2V617F-mutant MPN cells and suggest pharmacologic TYK2 inhibition as a novel promising treatment approach in CALR-mutant MPNs.
Keyword(s): Interferon-alpha, Myeloproliferative disorder
Abstract: S197
Type: Oral Presentation
Session title: New pathways and molecular mechanisms in MPN
Background
The classical Philadelphia-chromosome-negative (Ph-) myeloproliferative neoplasms (MPNs) consist of three entities: polycythemia vera (PV), essential thrombocythemia (ET) and primary myelofibrosis (PMF), carrying three mutually exclusive driver mutations in the genes of janus kinase 2 (JAK2), calreticulin (CALR) and the thrombopoietin (TPO) receptor (MPL). JAK2V617F-mutant cells were shown to be more sensitive to interferon alpha (IFNa) treatment compared to CALR-mutant cells and this IFNa “priming” involved increased phosphorylation of JAK1 and signal transducer and activator of transcribtion (STAT) 1 in JAK2V617F- vs. CALR-mutant cells. IFNa stimulation leads to phosphorylation of tyrosine kinase (TYK) 2 and JAK1, which are constitutively associated with the IFNa receptor-1 or -2 (IFNAR1/2), respectively, and activate downstream signaling pathways. However, the role of TYK2 in the survival and IFNa response of JAK2- vs. CALR-mutant MPN cells is still incompletely understood.
Aims
Analysis of the role of TYK2 in MPN cells to further investigate the differential response of JAK2V617F- and CALR-mutant cells to IFNa treatment and evaluate TYK2 as a potential target in MPN.
Methods
We used CRISPR/Cas9 technology to generate TYK2-deficient 32DMPL JAK2V617F or CALRdel52 cells and subsequently analyzed the IFNa response on cell viability (MTT assays), downstream JAK-STAT signaling and gene expression. Furthermore, we evaluated the effects of the TYK2-specific inhibitor deucravacitinib in 32DMPL mutant cells and in primary MPN patient material using colony formation unit assays.
Results
Basal phosphorylation of TYK2 (pTYK2) was increased in 32DMPL CALRdel52-mutant cells compared to JAK2V617F-mutant as well as empty vector cells (p<0.001). Generation of a TYK2 knockout in 32DMPL CALRdel52 cells was not successful, while pTYK2-deficient JAK2V617F-mutant cells were readily produced, underlining an important role of TYK2 in CALR-mutant cells. In pTYK2-deficient 32DMPL JAK2V617F cells, IFNa effects were significantly reduced, including loss of viability, activation of pSTAT1 and pSTAT3 and IFNa target genes. However, induction of apoptosis after IFNa treatment was not altered. We analyzed the effect of deucravacitinib and found that cell viability of 32DMPL CALRdel52- but not JAK2V617F-mutant cells was significantly decreased even at low concentrations [0.5 µM] of the inhibitor. Western Blotting confirmed complete loss of TYK2 phosphorylation after deucravacitinib treatment in both cell lines as well as reduced phosphorylation of downstream signaling molecules. In primary peripheral blood-derived mononuclear cells of patients with MPN, the TYK2 inhibitor decreased CALR- but not JAK2V617F-mutant allele burden in vitro, and clonogenic growth of ET- and PMF- but not PV-derived cells was inhibited.
Conclusion
Our data demonstrate an essential role for TYK2 in CALR-mutant but not JAK2V617F-mutant MPN cells and suggest pharmacologic TYK2 inhibition as a novel promising treatment approach in CALR-mutant MPNs.
Keyword(s): Interferon-alpha, Myeloproliferative disorder