Empagliflozin restores neutropenia and neutrophil dysfunction in a young patient with severe congenital neutropenia type 4

Abstract

Clinical ImplicationsPatients with severe congenital neutropenia type 4 are prone to recurrent, often life-threatening bacterial infections. Empagliflozin, a potential therapeutic option, restores neutrophil counts and function, reduces the incidence of infection, and allows the termination of G-CSF treatment. Patients with severe congenital neutropenia type 4 are prone to recurrent, often life-threatening bacterial infections. Empagliflozin, a potential therapeutic option, restores neutrophil counts and function, reduces the incidence of infection, and allows the termination of G-CSF treatment. Severe congenital neutropenia type 4 (SCN-4) is a rare primary immunodeficiency caused by loss-of-function mutations in the gene G6PC3, encoding the ubiquitously expressed glucose-6-phosphatase-β enzyme, which hydrolyses glucose-6-phosphate to glucose in the endoplasmic reticulum (ER).1Boztug K. Appaswamy G. Ashikov A. Schaffer A.A. Salzer U. Diestelhorst J. et al.A syndrome with congenital neutropenia and mutations in G6PC3.N Engl J Med. 2009; 360: 32-43Crossref PubMed Scopus (288) Google Scholar Although it is described as a multisystem autosomal-recessive disorder with a diverse spectrum of nonhematological manifestations, the main life-threatening complication is associated with persistent severe neutropenia, neutrophil dysfunction, and subsequent recurrent bacterial infections in almost all patients.1Boztug K. Appaswamy G. Ashikov A. Schaffer A.A. Salzer U. Diestelhorst J. et al.A syndrome with congenital neutropenia and mutations in G6PC3.N Engl J Med. 2009; 360: 32-43Crossref PubMed Scopus (288) Google Scholar Although bone marrow exhibits premature arrest of myeloid maturation, functional defects such as impairments in the oxidative burst, neutrophil chemotaxis, and phagocytosis are also recognized in G6PC3-deficient patients.2Hayee B. Antonopoulos A. Murphy E.J. Rahman F.Z. Sewell G. Smith B.N. et al.G6PC3 mutations are associated with a major defect of glycosylation: a novel mechanism for neutrophil dysfunction.Glycobiology. 2011; 21: 914-924Crossref PubMed Scopus (75) Google Scholar In glycogen storage disease type Ib, caused by the defect of glucose-6-phosphate translocase (G6PT/SLC37A4), neutropenia and neutrophil dysfunction also contribute to characteristic features of the complex inborn disease. Although the introduction of subcutaneous injections of G-CSF in patients with SCN-4 and GSD-1b effectively reduced the rate of severe infections and improved quality of life by expanding absolute neutrophil counts, the main concern about long-term G-CSF treatment is derived from an increased risk of development of myelodysplastic syndrome, or acute myeloid leukemia, due to the shortening of telomere length.3Li A.M. Thyagu S. Maze D. Schreiber R. Sirrs S. Stockler-Ipsiroglu S. et al.Prolonged granulocyte colony stimulating factor use in glycogen storage disease type 1b associated with acute myeloid leukemia and with shortened telomere length.Pediatr Hematol Oncol. 2018; 35: 45-51Crossref PubMed Scopus (27) Google Scholar Addressing enzymatic, cell culture, and in vivo approaches, an extensive study described that G6PT and G6PC3 work in tandem to eliminate the toxic, noncanonical metabolite 1,5-anhydroglucitol-6-phosphate (1,5AG6P) in the ER.4Veiga-da-Cunha M. Chevalier N. Stephenne X. Defour J.P. Paczia N. Ferster A. et al.Failure to eliminate a phosphorylated glucose analog leads to neutropenia in patients with G6PT and G6PC3 deficiency.Proc Natl Acad Sci U S A. 2019; 116: 1241-1250Crossref PubMed Scopus (74) Google Scholar Till the G6PT transports 1,5AG6P into the lumen, G6PC3 negates its toxic effects by dephosphorylation. In the failure of hydrolysis, the cytosolic accumulation of 1,5AG6P inhibits glucose phosphorylation and therefore glycolysis, the sole energy source for mature neutrophils, thus leading to subsequent apoptosis. Expanding on this elegant thesis, they showed that the pretoxic molecule (1,5AG) can be abolished with the sodium-glucose transport protein 2 (SGLT2) inhibitor empagliflozin and so ameliorates neutropenia in G6PC3-deficient mice.4Veiga-da-Cunha M. Chevalier N. Stephenne X. Defour J.P. Paczia N. Ferster A. et al.Failure to eliminate a phosphorylated glucose analog leads to neutropenia in patients with G6PT and G6PC3 deficiency.Proc Natl Acad Sci U S A. 2019; 116: 1241-1250Crossref PubMed Scopus (74) Google Scholar After all, the first-in-human trial of using an SGLT2 inhibitor to treat neutropenia in patients with GSD-1b showed promising results in reversing neutrophil dysfunction and neutropenia in a small cohort.5Wortmann S.B. Van Hove J.L.K. Derks T.G.J. Chevalier N. Knight V. Koller A. et al.Treating neutropenia and neutrophil dysfunction in glycogen storage disease type Ib with an SGLT2 inhibitor.Blood. 2020; 136: 1033-1043Crossref PubMed Scopus (65) Google Scholar Because the potential clinical benefits of empagliflozin in pediatric patients with G6PC3 deficiency are still missing, we initiated a pilot trial to investigate the safety and efficacy of treatment in a young patient with a known mutation in the G6PC3 gene. Our male patient in this case report was born after an uneventful pregnancy and delivery. At the age of 5 months, he developed severe agranulocytosis with repeated skin infections, dystrophy, and septicemia. He had no hypoglycemia or lactic acidosis, as is seen in glycogen storage disorders. Bone marrow examination showed a maturation arrest at the promyelocyte/myelocyte stage. Accompanying symptoms were a dysmorphic face, prominent chest and abdominal veins, foramen ovale aperture, and mild pulmonary stenosis. At this point, direct sequencing of the G6PC3 gene disclosed a known nonsense mutation (c.141C>G; NM_138387.3), which eradicates the function of the protein.1Boztug K. Appaswamy G. Ashikov A. Schaffer A.A. Salzer U. Diestelhorst J. et al.A syndrome with congenital neutropenia and mutations in G6PC3.N Engl J Med. 2009; 360: 32-43Crossref PubMed Scopus (288) Google Scholar In parallel mutations, the Ela2 and HAX1 genes were excluded. During infancy, our patient received G-CSF treatment on request, depending on the severity of neutropenia and the need to control infections; however, because of the persistent neutropenia (absolute granulocyte count, 0.07-0.59 G/L) and large, painful mouth ulcers, regular subcutaneous G-CSF injections were instituted every other day at the age of 3 years. During long-term G-CSF therapy, serious infections were rarely reported, except for tooth loss due to chronic gingivitis. Thrombocytopenia first appeared in our patient at the age of 7 years and later persisted (between 60 and 120 G/L). Because of the possibility of myelodysplastic syndrome, bone marrow biopsies have been performed annually ever since, showing moderate dysplastic signs without blasts, initially on the megakaryocyte lineage and then on the granulocyte lineage as well. Because the first clinical trial of an SGLT2 inhibitor to treat neutropenia in patients with GSD1b showed safe results,5Wortmann S.B. Van Hove J.L.K. Derks T.G.J. Chevalier N. Knight V. Koller A. et al.Treating neutropenia and neutrophil dysfunction in glycogen storage disease type Ib with an SGLT2 inhibitor.Blood. 2020; 136: 1033-1043Crossref PubMed Scopus (65) Google Scholar at the age of 16 years we initiated an oral treatment with empagliflozin at a daily dose of 25 mg (0.6 mg/kg) for our patient. Through his treatment, the initial subcutaneous injections of 30 MU of G-CSF every other day were decreased by 1 day every 2 weeks and then discontinued completely after 2 months. During the transition period, the absolute neutrophil count remained in the normal range and remained so after stopping G-CSF, while platelet counts returned to normal after 2 weeks of empagliflozin treatment and have been normal ever since (Figure 1). To test the phagocytic activity of neutrophil granulocytes through empagliflozin treatment, we performed a dihydrorhodamine 123 assay. We experienced an improvement in oxidative burst for phorbol 12-myristate 13-acetate (PMA)-induced reactive oxygen species (ROS) production (stimulation index [SI] with PMA: 11.27 and 55.66, respectively; normal, >30); meanwhile, ROS production after Escherichia coli activation was not affected intensely by the treatment, and the SI remained in a normal percentile (SI with E coli 55.74 and 32.34, respectively; normal, > 30). Interpretation of the results could emphasize that the signaling pathway of PMA-induced ROS production could be affected in G6PC3 deficiency and seems to be mended by empagliflozin treatment. Although bone marrow biopsy after 6 months of treatment with empagliflozin continued to show hypercellular hematopoiesis with mild dysplastic signs in the megakaryocyte and myeloid lineage, our novel results from bone marrow assessment performed 1.5 years after the initial days of treatment present a normal range of bone marrow cellularity for selected age (40%-50%). Next, we investigated the differentiation status of peripheral neutrophils by detecting differentiation markers. Our results showed an increased expression of the β-integrin family of adhesion proteins, CD11b, and hexose-6-phosphate dehydrogenase during empagliflozin treatment (Figure 2). Besides the appearance of differentiated cell membrane receptor CD11b, with the marked increase in ER luminal resident, NADPH-producing hexose-6-phosphate dehydrogenase enzyme, the neutrophils may trigger themselves for respiratory oxidative burst.Figure 2Differentiation markers show increased protein expression in peripheral polymorphonuclear leukocytes. GAPDH, Glyceraldehyde-3-phosphate dehydrogenase; H6PD, hexose-6-phosphate dehydrogenase.View Large Image Figure ViewerDownload Hi-res image Download (PPT) One well-defined behavior of the circulating neutrophil granulocytes is that they rapidly undergo spontaneous apoptosis within a defined time, which is initiated by depolarization of the mitochondrial membrane, where caspase 3–mediated activation of protein kinase C-δ plays a crucial role. Therefore, we next determined the expression of procaspase-3 and the cleavage of its substrate poly(ADP-ribose) polymerase during the course of empagliflozin treatment (see Figure E1 in this article’s Online Repository at www.jaci-inpractice.org). In inflammatory processes, LPS and cytokines promote the activation of TNF receptors and FaS/CD95 through JNK and nuclear factor kappa B. In addition, ROS could influence the fate of neutrophils by inhibition of JNK-inactivating phosphatases, leading to JNK activation, and by the release of cytochrome c and the subsequent activation of caspase-3, which leads to poly(ADP-ribose) polymerase cleavage.6Davis R.J. Signal transduction by the JNK group of MAP kinases.Cell. 2000; 103: 239-252Abstract Full Text Full Text PDF PubMed Scopus (3688) Google Scholar However, empagliflozin could exhibit anti-inflammatory effects via MKK4/7-JNK pathways downregulation and could restore the antiapoptotic activity of X-linked inhibitor of apoptosis protein. In line with this, we confirmed a reduction in JNK phosphorylation during our treatment (Figure E1). Regarding G-CSF, it could delay neutrophil apoptosis and extend survival. The mechanism behind its action could be explained by the fact that G-CSF blocks Bid/Bax redistribution and subsequent cytochrome c release.7Kato T. Noma H. Kitagawa M. Takahashi T. Oshitani N. Kitagawa S. Distinct role of c-Jun N-terminal kinase isoforms in human neutrophil apoptosis regulated by tumor necrosis factor-alpha and granulocyte-macrophage colony-stimulating factor.J Interferon Cytokine Res. 2008; 28: 235-243Crossref PubMed Scopus (17) Google Scholar Thus, the above-mentioned increase in caspase-3 activity can also be explained by depletion and later termination of the G-CSF supplementation. To conclude, in repaired neutrophil life circle, key apoptotic signals compose the dominant effect and lead to spontaneous apoptosis, while discontinued G-CSF treatment just enhances it. In summary, the introduction of empagliflozin has allowed the termination of G-CSF therapy, thereby improving patients’ quality of life by removing painful injections and diminishing the long-term adverse effects associated with increased risk of malignancies and splenomegaly. In addition, our young patient remained infection-free, did not show symptoms of hypoglycemia, and during the first year and a half period of empagliflozin treatment, no side effects, such as metabolic enzyme changes and hypertension, were reported. A collective report of the clinical experience of 112 pediatric and adult individuals with GSD Ib also presented a favorable effect of empagliflozin on neutropenia and neutrophil dysfunction–related symptoms and safety profile.8Grunert S.C. Derks T.G.J. Adrian K. Al-Thihli K. Ballhausen D. Bidiuk J. et al.Efficacy and safety of empagliflozin in glycogen storage disease type Ib: data from an international questionnaire.Genet Med. 2022; 24: 1781-1788Abstract Full Text Full Text PDF PubMed Scopus (17) Google Scholar Our case report is one of the first instances of clinical evidence aiming to treat a patient with SCN-4,9Boulanger C. Stephenne X. Diederich J. Mounkoro P. Chevalier N. Ferster A. et al.Successful use of empagliflozin to treat neutropenia in two G6PC3-deficient children: impact of a mutation in SGLT5.J Inherit Metab Dis. 2022; 45: 759-768Crossref PubMed Scopus (12) Google Scholar and showing that the unpleasant, costly, and moderately effective G-CSF injections could be omitted and replaced with oral empagliflozin, resulting in a significant improvement in the quality of life and health. We thank the patient and his family for their participation in this study.

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