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Genetic mutational drivers of Philadelphia chromosome-negative myeloproliferative neoplasms (MPN) are mutations in the genes of Janus kinase (JAK)2, calreticulin (CALR), and the thrombopoietin receptor myeloproliferative leukemia (MPL). JAK2V617F-mutant cells are shown to be more sensitive to interferon alpha (IFNα) treatment compared with CALR-mutant (CALRmut) cells due to greater phosphorylation of JAK1 and signal transducer and activator of transcription (STAT)1 in JAK2V617F‑mutant cells.
Stimulation of IFNα elicits phosphorylation of tyrosine kinase 2 (TYK2) and JAK1, which are known to be critical regulators of signal transduction pathways. TYK2 and JAK1 are associated with IFNα receptor-1 and -2, respectively, and activate the downstream signaling pathways, which in turn recruits specific STAT proteins that translocate to the nucleus and regulate gene expression. Inhibition of TYK2 has shown promising clinical efficacy and favorable safety profiles in auto-inflammatory diseases. However, there is limited evidence on the role of TYK2 in the survival and IFNα response to JAK2 in MPN cells versus CALRmut MPN cells.
During the European Hematology Association (EHA)2021 Virtual Congress, Rebecca Lemanzyk1 presented the findings from their study on the role of TYK2 in MPN cells. The MPN Hub is pleased to present the key findings here.
The aims and methods of the study included:
p-TYK2 was increased in the 32DMPL CALRdel52-mutant cells compared with 32DMPL JAK2V617F-mutant cells and empty vector cells (p < 0.001). Whereas, phosphorylated‑JAK2 (p-JAK2) was increased in 32DMPL JAK2V617F cells compared with 32DMPL CALRdel52-mutant cells.
Genotyping of initial clones demonstrated an altered TYK locus either homozygously or heterozygously. Base pair at 400 represented TYK2 KO cells and a raised base pair suggested wildtype (WT) cells. However, p‑TYK2 was retained in the initial clones as observed with western blotting. Regenotyping with new clones from the same cell lines demonstrated only WT remained at Day 21.
Using western blotting, 32DMPL JAK2V617F clones demonstrated the expression of TYK2. However, clones 19 and 43 were p‑TYK2-deficient, while clones 2 and 6 only showed a faint line for the p‑TYK2 band, suggesting failure to generate TYK2 KO cells in CALRdel52-mutant cells, and the importance of TYK2 in CALR-mutant cells. Clones 19 and 43 both showed the same heterozygous alterations of the TYK2 locus, with one allele harboring an in-frame deletion and the other harboring a frameshift mutation leading to a premature stop codon.
Clones treated with IFNα demonstrated a dose-dependent decrease in metabolic activity in the parental bulk and in the WT clones 37 and 42, compared with an increased metabolic activity in p‑TYK2-deficient clones 19 and 43. Significant induction of apoptosis was observed on treatment with IFNα in all cell clones. However, there were no significant differences between p‑TYK2-deficient and TYK2 WT 32DMPL JAK2V617F cells.
IFNα-treated parental bulk and WT cells showed a significant expression of Mx1 and Stat1 genes. However, no significant gene expression was observed for the p‑TYK2-deficient clone 43.
p-TYK2-deficient clones, 19 and 43, treated with IFNα, showed a reduction of p‑STAT3, while STAT1 expression was increased. Although there was similarity in the p‑STAT1 band in all clones, overexpression of STAT1 lead to reduction in p‑STAT1 in p‑TYK2-deficient clones
The TYK2 inhibitor deucravacitinib demonstrated a significant reduction in metabolic activity in CALRdel52-mutant cells even at low concentrations of 0.5 µM, but this was not observed in JAK2V617F-mutant cells. p-TYK2 was present only in CALRdel52-mutant cells but was ablated with deucravacitinib. Deucravacitinib did not show any effects on p-JAK2; however, it reduced p‑STAT3 and p‑STAT5 in CALRdel52-mutant cells.
The TYK2 inhibitor deucravacitinib had less of an effect on PBMCs from patients with polycythemia vera compared with PBMCs from patients with ET or PMF. No significant differences were observed between CALRdel52-mutant cells and JAK2V617F-mutant cells from patients with ET or PMF. CALRdel52 positive patients showed a reduced allele burden with TYK2 inhibition.
The findings demonstrated that TYK2 was required to deliver an adequate response to IFNα. TYK2 played an important role in CALRdel52-mutant cells but not in JAK2V617F-mutant cells, and it demonstrated the potential to be a novel treatment approach in CALRmut MPN. Future studies should investigate the interaction of TYK2 and MPL, establish successful generation of KO TYK2 in cell lines, and validate the findings in vivo studies.
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