112-OR: Critical Role of Tyrosine Kinase 2 (TYK2) in Human Pancreatic Differentiation

Type 1 diabetes (T1D) is caused by the autoimmune destruction of pancreatic beta cells. Genome wide association and fine mapping studies to identify causal variants predicted several loci in the human genome that are associated with T1D risk. One such candidate gene is a member of the JAK (Janus Kinase) family of tyrosine kinases, TYK2, which plays a critical role in intracellular signaling stimulated by cytokines through STATs. Loss-of-function variants of TYK2 are associated with protection against T1D. In line with this, silencing of TYK2 in beta cells attenuated inflammatory signaling pathways and apoptosis. To study the role of TYK2 in human beta-cell development, we now generated TYK2- knockout (KO) human iPSC lines using CRISPR-Cas9 genome editing and induced them to differentiate to pancreatic endocrine lineage. Our results suggest that absence of TYK2 does not compromise the pluripotency or the early pancreatic endoderm differentiation potential of iPSC. However, in the later stages of differentiation, the mRNA levels of key pancreatic TFs (PDX1(55±17%, p=0.03), NGN3(65±13%, p=0.008)), RFX6(58±14%, p=0.014), NKX6.1(39±19%, p=0.114) and NKX2.2(68±7%, p=0.0008)) were significantly reduced in the TYK2-KO cells. In the final stages of controlled induction to endocrine lineage TYK2-KO lines expressed lower levels of INS(53±9%, p=0.004), GCG(70±10.5%, p=0.002) and SST(62±8.6%, p=0.002) compared to control lines (mean ± SEM of 3 independent experiments). Furthermore immunostaining for INS, GCG and SST confirmed a lower number of endocrine hormone positive cells in TYK2-KO lines. In contrast to the wild type controls, the TYK2-KO iPSC-derived pancreatic cells displayed no activation of STAT1 and STAT2 and showed impaired STAT3 activation in response to IFNαor stimulation by polyinosinic-polycitidilic acid treatment, a viral infection mimetic. The present study suggests that TYK2 plays an important early role in pancreatic endocrine differentiation.