Case Reports

. 2022 Jan 14;3(1):42-57.e5.

doi: 10.1016/j.medj.2021.12.003.

JAK inhibition in a patient with a STAT1 gain-of-function variant reveals STAT1 dysregulation as a common feature of aplastic anemia

Affiliations

¹ Department of Medicine, Massachusetts General Hospital, Boston, MA 02114, USA; Ragon Institute of MGH, MIT, and Harvard, Cambridge, MA, USA; Harvard Medical School, Boston, MA, USA; Harvard T.H. Chan School of Public Health, Boston, MA, USA. Electronic address: jrosenberg2@mgh.harvard.edu.
² Ragon Institute of MGH, MIT, and Harvard, Cambridge, MA, USA; Broad Institute of MIT and Harvard, Cambridge, MA, USA.
³ Ragon Institute of MGH, MIT, and Harvard, Cambridge, MA, USA; Harvard Medical School, Boston, MA, USA; Institute for Medical Engineering and Science, Department of Chemistry, and Koch Institute for Integrative Cancer Research, Massachusetts Institute of Technology, Cambridge, MA, USA.
⁴ Harvard Medical School, Boston, MA, USA; Broad Institute of MIT and Harvard, Cambridge, MA, USA.
⁵ Harvard Medical School, Boston, MA, USA; Department of Pathology, Massachusetts General Hospital, Boston, MA, USA.
⁶ Department of Medicine, Massachusetts General Hospital, Boston, MA 02114, USA; Center for Genomic Medicine, Massachusetts General Hospital, Boston, MA, USA; Laboratory for Molecular Medicine, Partners Personalized Medicine, Cambridge, MA, USA; Department of Pathology, Massachusetts General Hospital, Boston, MA, USA.
⁷ Department of Medicine, Massachusetts General Hospital, Boston, MA 02114, USA; Harvard Medical School, Boston, MA, USA; Broad Institute of MIT and Harvard, Cambridge, MA, USA; Center for Genomic Medicine, Massachusetts General Hospital, Boston, MA, USA; Department of Pathology, Massachusetts General Hospital, Boston, MA, USA.
⁸ Ragon Institute of MGH, MIT, and Harvard, Cambridge, MA, USA; Institute for Medical Engineering and Science, Department of Chemistry, and Koch Institute for Integrative Cancer Research, Massachusetts Institute of Technology, Cambridge, MA, USA.
⁹ Department of Medicine, Massachusetts General Hospital, Boston, MA 02114, USA; Massachusetts General Hospital Cancer Center, Boston, MA, USA.
¹⁰ Broad Institute of MIT and Harvard, Cambridge, MA, USA; Genentech, South San Francisco, CA, USA.
¹¹ Ragon Institute of MGH, MIT, and Harvard, Cambridge, MA, USA; Harvard Medical School, Boston, MA, USA; Institute for Medical Engineering and Science, Department of Chemistry, and Koch Institute for Integrative Cancer Research, Massachusetts Institute of Technology, Cambridge, MA, USA; Broad Institute of MIT and Harvard, Cambridge, MA, USA.
¹² Ragon Institute of MGH, MIT, and Harvard, Cambridge, MA, USA; Harvard Medical School, Boston, MA, USA; Harvard T.H. Chan School of Public Health, Boston, MA, USA.
¹³ Department of Medicine, Massachusetts General Hospital, Boston, MA 02114, USA; Center for Regenerative Medicine, Department of Medicine, Massachusetts General Hospital, Boston, MA, USA; Harvard Stem Cell Institute, Cambridge, MA, USA; Massachusetts General Hospital Cancer Center, Boston, MA, USA. Electronic address: dbsykes@mgh.harvard.edu.

PMID: 35590143
PMCID: PMC9123284
DOI: 10.1016/j.medj.2021.12.003

Case Reports

JAK inhibition in a patient with a STAT1 gain-of-function variant reveals STAT1 dysregulation as a common feature of aplastic anemia

Jacob M Rosenberg et al. Med. 2022.

. 2022 Jan 14;3(1):42-57.e5.

doi: 10.1016/j.medj.2021.12.003.

Authors

Affiliations

¹ Department of Medicine, Massachusetts General Hospital, Boston, MA 02114, USA; Ragon Institute of MGH, MIT, and Harvard, Cambridge, MA, USA; Harvard Medical School, Boston, MA, USA; Harvard T.H. Chan School of Public Health, Boston, MA, USA. Electronic address: jrosenberg2@mgh.harvard.edu.
² Ragon Institute of MGH, MIT, and Harvard, Cambridge, MA, USA; Broad Institute of MIT and Harvard, Cambridge, MA, USA.
³ Ragon Institute of MGH, MIT, and Harvard, Cambridge, MA, USA; Harvard Medical School, Boston, MA, USA; Institute for Medical Engineering and Science, Department of Chemistry, and Koch Institute for Integrative Cancer Research, Massachusetts Institute of Technology, Cambridge, MA, USA.
⁴ Harvard Medical School, Boston, MA, USA; Broad Institute of MIT and Harvard, Cambridge, MA, USA.
⁵ Harvard Medical School, Boston, MA, USA; Department of Pathology, Massachusetts General Hospital, Boston, MA, USA.
⁶ Department of Medicine, Massachusetts General Hospital, Boston, MA 02114, USA; Center for Genomic Medicine, Massachusetts General Hospital, Boston, MA, USA; Laboratory for Molecular Medicine, Partners Personalized Medicine, Cambridge, MA, USA; Department of Pathology, Massachusetts General Hospital, Boston, MA, USA.
⁷ Department of Medicine, Massachusetts General Hospital, Boston, MA 02114, USA; Harvard Medical School, Boston, MA, USA; Broad Institute of MIT and Harvard, Cambridge, MA, USA; Center for Genomic Medicine, Massachusetts General Hospital, Boston, MA, USA; Department of Pathology, Massachusetts General Hospital, Boston, MA, USA.
⁸ Ragon Institute of MGH, MIT, and Harvard, Cambridge, MA, USA; Institute for Medical Engineering and Science, Department of Chemistry, and Koch Institute for Integrative Cancer Research, Massachusetts Institute of Technology, Cambridge, MA, USA.
⁹ Department of Medicine, Massachusetts General Hospital, Boston, MA 02114, USA; Massachusetts General Hospital Cancer Center, Boston, MA, USA.
¹⁰ Broad Institute of MIT and Harvard, Cambridge, MA, USA; Genentech, South San Francisco, CA, USA.
¹¹ Ragon Institute of MGH, MIT, and Harvard, Cambridge, MA, USA; Harvard Medical School, Boston, MA, USA; Institute for Medical Engineering and Science, Department of Chemistry, and Koch Institute for Integrative Cancer Research, Massachusetts Institute of Technology, Cambridge, MA, USA; Broad Institute of MIT and Harvard, Cambridge, MA, USA.
¹² Ragon Institute of MGH, MIT, and Harvard, Cambridge, MA, USA; Harvard Medical School, Boston, MA, USA; Harvard T.H. Chan School of Public Health, Boston, MA, USA.
¹³ Department of Medicine, Massachusetts General Hospital, Boston, MA 02114, USA; Center for Regenerative Medicine, Department of Medicine, Massachusetts General Hospital, Boston, MA, USA; Harvard Stem Cell Institute, Cambridge, MA, USA; Massachusetts General Hospital Cancer Center, Boston, MA, USA. Electronic address: dbsykes@mgh.harvard.edu.

PMID: 35590143
PMCID: PMC9123284
DOI: 10.1016/j.medj.2021.12.003

Abstract

Background: Idiopathic aplastic anemia is a potentially lethal disease, characterized by T cell-mediated autoimmune attack of bone marrow hematopoietic stem cells. Standard of care therapies (stem cell transplantation or immunosuppression) are effective but associated with a risk of serious toxicities.

Methods: An 18-year-old man presented with aplastic anemia in the context of a germline gain-of-function variant in STAT1. Treatment with the JAK1 inhibitor itacitinib resulted in a rapid resolution of aplastic anemia and a sustained recovery of hematopoiesis. Peripheral blood and bone marrow samples were compared before and after JAK1 inhibitor therapy.

Findings: Following therapy, samples showed a decrease in the plasma concentration of interferon-γ, a decrease in PD1-positive exhausted CD8⁺ T cell population, and a decrease in an interferon responsive myeloid population. Single-cell analysis of chromatin accessibility showed decreased accessibility of STAT1 across CD4⁺ and CD8⁺ T cells, as well as CD14⁺ monocytes. To query whether other cases of aplastic anemia share a similar STAT1-mediated pathophysiology, we examined a cohort of 9 patients with idiopathic aplastic anemia. Bone marrow from six of nine patients also displayed abnormal STAT1 hyper-activation.

Conclusions: These findings raise the possibility that STAT1 hyperactivition defines a subset of idiopathic aplastic anemia patients for whom JAK inhibition may be an efficacious therapy.

Funding: Funding was provided by the Massachusetts General Hospital Department of Medicine Pathways Program and NIH T32 AI007387. A trial registration is at https://clinicaltrials.gov/ct2/show/NCT03906318.

Keywords: Aplastic anemia; JAK inhibition; JAK/STAT; STAT1 GOF; STAT1 gain-of-function; T-cell exhaustion; Translation to patients; autoimmune; interferon gamma; itacitinib.

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Conflict of interest statement

Declaration of interests Y.-B.C. reports consulting fees from Incyte. A.K.S. reports compensation for consulting and/or SAB membership from FL82, Merck, Honeycomb Biotechnologies, Cellarity, Repertoire Immune Medicines, Hovione, Third Rock Ventures, Ochre Bio, Relation Therapeutics, and Dahlia Biosciences. J.M.R. reports consulting fees from Third Rock Ventures. D.B.S. is a co-founder and holds equity in Clear Creek Bio, is a consultant and holds equity in SAFI Biosolutions, and is a consultant for Keros Therapeutics. A.R. is an advisory board member of Med, a founder and equity holder of Celsius Therapeutics, an equity holder in Immunitas Therapeutics and, until August 31, 2020, was an SAB member of Syros Pharmaceuticals, Neogene Therapeutics, Asimov, and Thermo Fisher Scientific. From August 1, 2020, A.R. is an employee of Genentech. All other authors declare no competing interests.

Figures

**Figure 1:. Clinical presentation and response to treatment with itacitinib.**
A. Family pedigree. The phenotype of severe oral ulcers is denoted by shaded symbols (males = squares, females = circles). The patient (red arrow) and his father (blue arrow) are both heterozygous for the A267V mutation. B. Schematic of STAT1 protein domains and its tyrosine phosphorylation site (pY). Black bars represent known STAT1 GOF mutations, the red bar represents the patient’s mutation, and confirmatory Sanger sequencing of the mutation is shown. C. Peripheral blood laboratory values including hematocrit, % reticulocytes, white blood cell count (WBC) and absolute neutrophil count (ANC) are plotted before and after treatment with itacitinib. The Green arrow at day 0 indicates the initiation of itacitinib. D. Bone marrow hematoxylin and eosin (H&E) and phosphorylated STAT1 (pSTAT1) immunohistochemical staining are shown for a healthy donor (left) and the patient (right), both before and after initiation of itacitinib.

**Figure 2:. Immunophenotyping.**
A. Phospho-CyTOF histograms of pSTAT1 after in vitro stimulation with interferon-γ. Shown are pSTAT1 levels in monocytes from a healthy control and from the patient before (2 time points) and after (2 time points) initiation of itacitinib treatment. B. Plasma cytokine levels measured in units of mean fluorescent intensity (MFI) from the patient at six time points (specified in c.) pre- and post-itacitinib. C. Intracellular IFN-γ following in vitro stimulation in CD4+ memory T cells (CD45RO+) shown both as histograms and as gated percent of cells. D. Intracellular IFN-γ following in vitro stimulation in CD8+ memory T cells (CD45RO+) shown both as histograms and as gated percent of cells. E. Programmed Death 1 (PD-1) expression levels on CD8+ T cells shown both as histograms and as the gated percent of cells. (c.– e.) N = 4 distinct healthy donors were tested. Patient samples were tested from n = 6 distinct time points, with the specified number of weeks before or after initiation of itacitinib. (c.– d.) PBMCs were stimulated *in vitro* with PMA/ionomycin. (b.- e.) Statistical analyses performed using unpaired t-test.

**Figure 3:. Single-cell transcriptional and epigenetic profiling.**
A. Unsupervised clustering of all cells by scRNA-Seq expression profiling in Uniform Manifold Approximation and Projection (UMAP) space. Cells are colored by sample (left) or cell type (middle). n=4 different healthy control samples, n=3 pre-itacitinib time points, and n=3 post-itacitinib time points. Stacked bar plot shows cell proportions by condition (right). “Myeloid” includes CD14+ monocytes, CD16+ monocytes, and type 2 conventional dendritic cells (cDC2). “Other” cells include plasma cells, platelet-like cells, progenitor-like cells, and proliferating cells. B. Sub-clustering of T cell-containing populations (top left). The CD8+ T cell population (olive green) was scored for exhaustion, cytotoxicity, and cytokine effector function. C. Sub-clustering of myeloid populations (top left). Each myeloid cell was scored (top right) by its Type I interferon score (x-axis) and Type II interferon score (y-axis). Histograms of these scores by condition are shown on the plot edge. Each myeloid cell was then displayed by condition and colored by its Type II interferon score (bottom figures). D. scATAC-seq accessibility peaks at the PD-1 locus in healthy control PBMC and the patient’s PBMC at n = 5 time points. Red box highlights peak differences between samples. E. scATAC-seq genome-wide accessibility at STAT1 binding motif sites. Each cell was scored by STAT1 accessibility across cell types and conditions. Patient PBMC were scored at the same n = 5 time points as listed in (d). b.-e. P values calculated using Wilcoxon rank-sum test using each cell as an individual observation.

**Figure 4:. Bone marrow STAT1 activation in historical aplastic anemia cases.**
a. Bone marrow hematoxylin and eosin (H&E) stains and phosphorylated STAT1 (pSTAT1) immunostains are shown for n = 4 distinct healthy controls and b. n = 9 distinct patients with idiopathic aplastic anemia (AA) at the time of diagnosis. Blinded hematopathologic interpretation of pSTAT1 (positive or negative) is denoted below each image. Note that HC 1 is also shown in Fig 1.

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JAK inhibition in a patient with a STAT1 gain-of-function variant reveals STAT1 dysregulation as a common feature of aplastic anemia

Affiliations

JAK inhibition in a patient with a STAT1 gain-of-function variant reveals STAT1 dysregulation as a common feature of aplastic anemia

Authors

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Abstract

Conflict of interest statement

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