Erythropoiesis defect observed in STAT3 GOF patients with severe anemia

Signal transducer and activator of transcription (STAT) 3 (STAT3) gain-of-function (GOF) germline mutations cause autoimmunity, lymphoproliferation, and short stature.1Milner J.D. Vogel T.P. Forbes L. Ma C.A. Stray-Pedersen A. Niemela J.E. et al.Early-onset lymphoproliferation and autoimmunity caused by germline STAT3 gain-of-function mutations.Blood. 2015; 125: 591-599Crossref PubMed Scopus (354) Google Scholar The disease phenotype is driven by increased STAT3 activity and reduced phosphorylation of STAT5 and STAT1. In turn, suppression of STAT5 and STAT1 activity is instigated by the promiscuous activity of suppressor of cytokine signaling 3 (negative regulator of STAT3).1Milner J.D. Vogel T.P. Forbes L. Ma C.A. Stray-Pedersen A. Niemela J.E. et al.Early-onset lymphoproliferation and autoimmunity caused by germline STAT3 gain-of-function mutations.Blood. 2015; 125: 591-599Crossref PubMed Scopus (354) Google Scholar,2Sediva H. Dusatkova P. Kanderova V. Obermannova B. Kayserova J. Sramkova L. et al.Short stature in a boy with multiple early-onset autoimmune conditions due to a STAT3 activating mutation: could intracellular growth hormone signalling be compromised?.Horm Res Paediatr. 2017; 88: 160-166Crossref PubMed Scopus (26) Google Scholar Patients are typically treated with various immunomodulatory drugs, although targeted therapies that interfere with upstream regulators of STAT3 activity (such as IL-6–receptor inhibitors and Janus kinase [JAK] inhibitors) are currently being assessed.3Forbes L.R. Vogel T.P. Cooper M.A. Castro-Wagner J. Schussler E. Weinacht K.G. et al.Jakinibs for the treatment of immune dysregulation in patients with gain-of-function signal transducer and activator of transcription 1 (STAT1) or STAT3 mutations.J Allergy Clin Immunol. 2018; 142: 1665-1669Abstract Full Text Full Text PDF PubMed Scopus (138) Google Scholar STAT signaling is central to many pathways, including the pathway downstream of erythropoietin (EPO, the key hormone in erythropoiesis) and exerts its effects downstream of the EPO receptor (EPOR). The EPO signaling pathway is mainly STAT5-dependent, although other activated STATs (including STAT3) can fine-tune the response.4Kim A.R. Ulirsch J.C. Wilmes S. Unal E. Moraga I. Karakukcu M. et al.Functional selectivity in cytokine signaling revealed through a pathogenic EPO mutation.Cell. 2017; 168: 1053-1064.e15Abstract Full Text Full Text PDF PubMed Scopus (78) Google Scholar The level of STAT5-phosphorylation downstream of EPOR determines the rate of erythropoiesis.5Porpiglia E. Hidalgo D. Koulnis M. Tzafriri A.R. Socolovsky M. Stat5 signaling specifies basal versus stress erythropoietic responses through distinct binary and graded dynamic modalities.PLoS Biol. 2012; 10e1001383Crossref PubMed Scopus (31) Google Scholar Low intracellular concentrations of phosphorylated STAT5 (pSTAT5) are needed for basal erythropoiesis, whereas an increase in pSTAT5 levels in response to elevated EPO levels is needed for “stress erythropoiesis.”6Socolovsky M. Nam H. Fleming M.D. Haase V.H. Brugnara C. Lodish H.F. Ineffective erythropoiesis in Stat5a(−/−)5b(−/−) mice due to decreased survival of early erythroblasts.Blood. 2001; 98: 3261-3273Crossref PubMed Scopus (578) Google Scholar The present study was prompted by our observations in a patient (P1) admitted to hospital for severe Coombs-positive autoimmune hemolytic anemia. Within a week, the hemolysis had disappeared and bone marrow (BM) biopsy revealed pure red cell aplasia (see Fig E1, A-C, in this article’s Online Repository at www.jacionline.org; for detailed clinical description, see Table E1 in this article’s Online Repository at www.jacionline.org). Treatment with several immunosuppressants failed to control the transfusion-dependent anemia (Fig E1, D). Whole-exome sequencing revealed a de novo heterozygous germline STAT3 c.2144C>T (P715L) GOF mutation (Fig E1, E-G).2Sediva H. Dusatkova P. Kanderova V. Obermannova B. Kayserova J. Sramkova L. et al.Short stature in a boy with multiple early-onset autoimmune conditions due to a STAT3 activating mutation: could intracellular growth hormone signalling be compromised?.Horm Res Paediatr. 2017; 88: 160-166Crossref PubMed Scopus (26) Google Scholar A transcriptomic analysis of P1’s PBMCs, performed at the age of 7 years and 4 months, highlighted elevated mRNA levels of STAT3 target genes such as IL6, IL8, IL1B, and suppressor of cytokine signaling 3 (Fig 1, A, and Fig E1, H). Consistently with the observation of anemia, HBB (coding for hemoglobin-β) was the most significantly downregulated gene in P1. Reporter activity assays revealed that the P715L mutation leads to enhanced transcriptional activity of STAT3, which could be modulated by the addition of IL-6 and STAT3 phosphorylation inhibitors (Fig 1, B). Furthermore, P715L-STAT3 was phosphorylated very rapidly in response to IL-6 (Fig 1, C), and overall increased maximal levels of pSTAT3 were found in nuclear and cytoplasmic extracts (see Fig E2, A and B, in this article’s Online Repository at www.jacionline.org). Along with these differences in transcriptional activity and phosphorylation dynamics, we found that P715L-STAT3 bound more readily to several known and hitherto unknown proinflammatory binding partners in both the presence and absence of IL-6 stimulation; however, these putative partners did not bind to wild-type STAT3 in an affinity purification/mass spectrometry analysis (Fig 1, D, and Fig E2, C and D). To determine whether P1’s hematopoietic defect was linked to the P715L mutation, we measured erythroid lineage commitment in a colony-forming unit (CFU) assay with bone-marrow–derived mononuclear cells (BMMCs). A significant reduction in erythroid burst-forming colonies was observed in patient-derived cells (Fig 1, E). These results could be reproduced by lentiviral transduction of healthy BMMCs with either wild-type-STAT3 or P715L-STAT3 (Fig 1, F, and Fig E2, E). We then analyzed later stages of erythropoiesis in cultured BMMCs (Fig E2, F) and PBMCs by measuring the expression profile of the markers CD71 (transferrin receptor) and CD235a (glycophorin A) (see Fig E3, A, in this article’s Online Repository at www.jacionline.org).7Merryweather-Clarke A.T. Atzberger A. Soneji S. Gray N. Clark K. Waugh C. et al.Global gene expression analysis of human erythroid progenitors.Blood. 2011; 117: e96-e108Crossref PubMed Scopus (90) Google Scholar Cultures of P1’s PBMCs showed a significantly lower number of erythroid precursor cells (Fig 1, G), with a comparable maturation profile (based on CD71 and CD235a expression) to healthy controls (Fig E3, B). Although most patients with STAT3 GOF mutations do not suffer from severe anemia,8Fabre A. Marchal S. Barlogis V. Mari B. Barbry P. Rohrlich P.S. et al.Clinical aspects of STAT3 gain-of-function germline mutations: a systematic review.J Allergy Clin Immunol Pract. 2019; 7: 1958-1969.e9Abstract Full Text Full Text PDF PubMed Scopus (100) Google Scholar these findings were reproduced in another 4 patients with STAT3 GOF mutations (n = 5; Fig 1, G; for clinical details, see Table E1). Although none of the other 4 patients showed obvious hematopoietic defects, all patients had elevated EPO levels (Fig 1, H), indicative of erythropoietic stress. A concomitant analysis of the STAT phosphorylation signature downstream of the EPOR revealed an overall increase in pSTAT3 levels at the CFU-E stage, whereas pSTAT5 levels were abnormally low in subsequent stages of erythropoiesis (Fig 1, I). Consistently, we were able to detect significantly higher pSTAT3/STAT3 ratios at all stages (Fig 1, J). The proportion of CD34+ PBMCs was normal or slightly elevated in the STAT3 GOF patients, which ruled out a lack of progenitors as the cause of reduced erythropoietic capacity in P1 (Fig E3, C). In all 5 patients, the erythropoietic potential was normal for the CD34+ PBMC fraction but abnormally low for the residual CD34− PBMC fraction (Fig 1, K). Aside from the peripheral autoimmunity observed in all patients, a more detailed analysis of P1’s BM revealed lymphoid infiltrates (Fig 2, A and B; see Fig E4, A and B, in this article’s Online Repository at www.jacionline.org) composed primarily of double-negative T cells (Fig 2, C). The polyclonal TCRγ chain distribution pattern of these CD8+ T cells (Fig E4, C) and oligoclonal IgH rearrangements (Fig E4, D) of the B cells isolated from P1 suggest oligoclonal lymphoproliferation. It is unclear whether these infiltrates are autoimmune in nature and how they contribute to an inflammatory microenvironment within the BM. Nevertheless, we were able to largely rule out autoimmune effects occurring during our in vitro experiments as being causative for the low erythropoietic capacity observed (Fig E4, E and F). Targeted treatment with a JAK inhibitor was initiated in P1, with the goal of normalizing the balance between STAT3 and STAT5 signaling (Fig 2, D). The treatment was associated with the first spontaneous increase in hemoglobin in over 5 years. However, the JAK inhibitor was discontinued after the appearance of severe thrombocytopenia (managed with intravenous immunoglobulins and high-dose corticosteroids). Treatment with tocilizumab and cyclosporine was then initiated; P1 has remained transfusion-free ever since, and erythropoiesis is now normal (Fig 2, E). Three months after initiation of treatment with cyclosporine, in combination with tocilizumab, we repeated a transcriptomic analysis of P1’s PBMCs (at the age of 10 years and 8 months). Before treatment (at the age of 7 years and 4 months), we had observed a shift toward a fetal hemoglobin gene expression signature; this is characteristic of an overall decrease in the effectiveness of erythropoiesis. Under treatment (at the age of 10 years and 8 months), hemoglobin gene expression levels were significantly upregulated, and the hemoglobin ratios (Fig 2, F) and STAT3 target gene expression had normalized (Fig 2, G). Our findings reveal a decreased stress erythropoietic response in patients with STAT3 GOF mutations. We suggest that prolonged, accentuated STAT3 activation in patients carrying the P715L-STAT3 mutation impairs the increase in pSTAT5 needed to respond to elevated EPO levels. The enhanced STAT3 activity seen in these patients is further accentuated by greater interaction between the mutant protein and proinflammatory binding partners. This suggests that inflammatory triggers could contribute to making inflammatory processes especially potent in patients with STAT3 GOF mutations. Given the malleability of P715L-STAT3’s activity, a clinically relevant increase in its activity might require a trigger. In P1, this may have been the autoimmune hemolytic anemia and/or the lymphoproliferative, potentially autoimmune infiltration of the BM. We and others have shown that in healthy controls, CD34− PBMCs contribute substantially to erythropoiesis in vitro.9van den Akker E. Satchwell T.J. Pellegrin S. Daniels G. Toye A.M. The majority of the in vitro erythroid expansion potential resides in CD34(−) cells, outweighing the contribution of CD34(+) cells and significantly increasing the erythroblast yield from peripheral blood samples.Haematologica. 2010; 95: 1594-1598Crossref PubMed Scopus (95) Google Scholar Irrespective of the composition of the CD34− population in PBMCs, our data clearly show that these cells’ proliferative capacity is low in patients with a STAT3 GOF mutation. In summary, we reveal that STAT3 activity in patients carrying the P715L-STAT3 mutation can be modulated by either IL-6 stimulation or targeted inhibition. Furthermore, based on our findings in cell lines, P715L-STAT3 is not constitutively active in patients carrying the mutation. In these patients, STAT3 interacts with proinflammatory binding partners. Finally, P715L-STAT3 GOF manifests with faster kinetic and prolonged STAT3 phosphorylation, causing deviated STAT5 activity in our in vitro model of hematopoiesis. This imbalance in STATs’ activity suppresses erythropoietic capacity as evidenced by low erythropoietic potential of peripheral CD34− precursors. The chronic state of severe anemia seen in P1 was accompanied by oligoclonal lymphoid infiltrates in the BM. This observation, together with successful treatment of pure red cell aplasia using a combination of high-dose methylprednisolone, IL-6R inhibitor, and cyclosporine A after the use of a JAK inhibitor, shows that this chronic state of severe anemia can be reversed. This indicates that erythropoiesis in patients with STAT3 GOF mutations is a system under strain, in which inflammatory triggers may unbalance erythroid differentiation and suppress proliferative capacity of red blood cell precursors, rendering patients less able to respond with stress erythropoiesis. We thank the Laboratory of Professor R. Aebersold for the support in the affinity purification and mass spectrometry work. Fig E2View Large Image Figure ViewerDownload Hi-res image Download (PPT)Fig E3View Large Image Figure ViewerDownload Hi-res image Download (PPT)Fig E4View Large Image Figure ViewerDownload Hi-res image Download (PPT) Download .docx (.02 MB) Help with docx files Online Repository Text Download .pdf (.07 MB) Help with pdf files Table E1