STAT1 Gain-of-Function Mutations Cause High Total STAT1 Levels With Normal Dephosphorylation

Abstract

Signal transducer and activator of transcription (STAT1)1 gain of function (GOF) pathogenic variants have been associated with increased levels of phosphorylated STAT1 and STAT1-dependent cellular responses. Delayed dephosphorylation was proposed as the underlying mechanism leading to the characteristically raised pSTAT1 levels. We examined the levels of STAT1 protein and message as well as rates of STAT1 phosphorylation, dephosphorylation, and degradation associated with STAT1 GOF pathogenic variants. Fresh peripheral blood mononuclear cells (PBMC) from 14 STAT1 GOF patients carrying 10 different pathogenic variants in the coiled-coil, DNA binding, and SH2 domains and healthy donors were used to study STAT1 levels and phosphorylation (pSTAT1) following IFNγ and IFNα stimulation. STAT1 protein levels were measured by flow cytometry and immunoblot. STAT1 mRNA levels were measured using quantitative reverse transcription PCR. STAT1 protein degradation was studied using cycloheximide. Patient IFNγ and IFNα induced peak pSTAT1 was higher than in healthy controls. The velocity of pSTAT1 dephosphorylation after treatment of IFNγ stimulated CD14 + monocytes with the Janus Kinase (JAK)-inhibitor ruxolitinib was significantly faster in patient cells. STAT1 protein levels in patient CD14 + monocytes and CD3 + T cells were higher than in healthy donors. There was a strong and positive correlation between CD14 + STAT1 protein levels and peak pSTAT1 levels. Patient fresh PBMC STAT1 mRNA levels were increased at rest and after 16 h of incubation. STAT1 protein degradation was similar in patient and healthy volunteer cells. Patient IFNγ receptors 1 and 2 and JAK2 levels were normal. One patient in our cohort was treated with the oral JAK inhibitor ruxolitinib. Treatment was associated with normalization of both STAT1 protein and peak pSTAT1 levels. After JAK inhibitor treatment was stopped the patient's CD14 + monocyte STAT1 protein and peak phosphorylation levels increased proportionally. These findings suggest that patients with STAT1 GOF mutations have higher levels of total STAT1 protein, leading to high levels of pSTAT1 after stimulation, despite rapid STAT1 dephosphorylation and normal degradation.

Live Cell Imaging Demonstrates Multiple Routes Toward a STAT1 Gain-of-Function Phenotype

“…We demonstrated that different mutations, associated with STAT1 GOF, result in a STAT1 GOF phenotype via distinct mechanisms using real-time imaging and tracking of GFPtagged proteins. Our results (summarized in Table 1) are in line with previous reports (50)(51)(52), indicating that different molecular mechanisms can result in a STAT1 GOF phenotype. It remains unknown whether these differences (altered nuclear mobility, increased DNA binding, or increased nuclear import) are biologically relevant in influencing the patients' phenotype.…”

“…On the other hand, Fujiki et al (50) studied the R274Q mutation, reporting an impaired dephosphorylation for this mutant. Finally, Zimmerman et al (52) included monocytes from 14 STAT1 GOF patients, of whom 1 bearing the R274W and 2 the R274Q mutant. They observed a similar dephosphorylation rate for the pooled GOF mutants compared with the healthy controls.…”

Heterozygous Cell Models of STAT1 Gain-of-Function Reveal a Broad Spectrum of Interferon-Signature Gene Transcriptional Responses

“…In this regard, some studies in patient samples found STAT1 itself to be elevated, suggesting that total STAT1, rather than pSTAT1, is the primary disease driver of STAT1 GOF. 58,59 In our current study, pSTAT1 was elevated in all GOF mutants following stimulation (as described in patients). In addition, increased total STAT1 was clearly seen in one GOF mutant, with a trend toward increased total STAT1 in two others.…”

Chronic Mucocutaneous Candidiasis in Early Life: Insights Into Immune Mechanisms and Novel Targeted Therapies

“…Delayed dephosphorylation of STAT1 in these patients impairs the function of IL-6 and IL-21, thus decreasing STAT3-dependent differentiation of naïve CD4 + T cells into Th17 cells (20). Of note, a recent report suggests that some STAT1 GOF mutations may cause STAT1 levels to be high, although phosphorylation is normal (21). Disease severity appears to vary according to the mutation.…”