SLGT2 Inhibitor Rescues Myelopoiesis in G6PC3 Deficiency

The energy metabolism of myeloid cells depends primarily on glycolysis. 1,5-Anhydroglucitol (1,5AG), a natural monosaccharide, is erroneously phosphorylated by glucose-phosphorylating enzymes to produce 1,5-anhydroglucitol-6-phosphate (1,5AG6P), a powerful inhibitor of hexokinases. The endoplasmic reticulum transporter (SLC37A4/G6PT) and the phosphatase G6PC3 cooperate to dephosphorylate 1,5AG6P. Failure to eliminate 1,5AG6P is the mechanism of neutrophil dysfunction and death in G6PC3-deficient mice. Sodium glucose cotransporter 2 (SLGT2) inhibitor reduces 1,5AG level in the blood and restores the neutrophil count in G6PC3-deficient mice. In the investigator-initiated study, a 30-year-old G6PC3-deficient woman with recurrent infections, distressing gastrointestinal symptoms, and multi-lineage cytopenia was treated with an SLGT2-inhibitor. A significant increase in all the hematopoietic cell lineages and substantial improvement in the quality of life was observed.


Introduction
The enzyme glucose-6-phosphatase (G6PC1) hydrolyzes glucose-6-phosphate in the endoplasmic reticulum of liver, kidney, and intestinal cells, a final step of glycogenolysis and neoglucogenesis. Glucose-6-phosphatase catalytic subunit 3 (G6PC3) is a ubiquitously expressed glucose-6-phosphatase homologue which has a role in differentiation and survival of myeloid cells. [1,2] G6PC3-deficient mice have neutropenia with defective respiratory burst, chemotaxis, increased susceptibility to bacterial infection, elevated plasma glucagon, and low serum cholesterol. [3] Humans with G6PC3 deficiency have variable hematological and non-hematological manifestations. Non-hematological features include prominent superficial venous pattern, congenital cardiac anomalies, renal system malformations, and genital anomalies. [2] Severe neutropenia is a consistent hematological feature of all patients with G6PC3 deficiency. [4] Two-thirds of patients present with intermittent thrombocytopenia [4].
Neutropenia causes significant morbidity. [4] Patients with G6PC3 deficiency commonly present with recurrent bacterial infections in the first few months of life. [5] Common infections are sino-pulmonary, otitis media, urinary tract infections, skin abscesses, and sepsis. Oral ulcers, gingivitis, periodontitis, stomatitis, inflammatory bowel disease, and fungal infections have also been reported. Although the neutropenia of G6PC3 deficiency is GCSFresponsive, inflammatory colitis secondary to G6PC3 deficiency does not respond to GCSF [6,7].
Neutropenia was recently explained by the accumulation of toxic amounts of 1,5-anhydroglucitol-6-phosphate (1,5AG6P) in neutrophils in a G6PC3-deficient mouse model. [8] Veiga-da-Cunha et al. have also shown that the administration of a sodium glucose cotransporter 2 (SLGT2) inhibitor, lowered plasma 1,5AG, reduced the intracellular accumulation of 1,5AG6P, and restored normal neutrophil counts in G6PC3-deficient mice [8] as well as in patients with neutropenia-associated glycogen storage disease type 1b (GSD1b), deficient in SLC37A4/G6PT, the transporter that collaborates with G6PC3 to dephosphorylate 1,5AG6P in neutrophil's endoplasmic reticulum. [9,10] Now, we provide the first evidence for the role of empagliflozin, an SLGT2 inhibitor, in rescuing neutropenia and neutrophil dysfunction in a patient with G6PC3 deficiency.

Case Description
A 30-year-old female patient, born to non-consanguineous parents, developed recurrent infections since birth. There was a history of a male sibling death due to sepsis in infancy (Fig. 1A).
The patient presented to us at the age of 27 years. Her height was 152 cm and weight was 58 kg. She had recurrent infections since birth. Infection profile is shown in Fig. 1B, and the frequency of the visits to a physician for infections and/or abdominal symptoms are shown in the Supplemental Fig. 1. She had multiple episodes of upper and lower respiratory tract infection, skin abscesses, otitis media, and urinary tract infections (Supplemental Table 1).
Recurrent tongue ulcers were common since she was 12 years old. These often lasted for 10 to 20 days (Fig. 1C). Bone marrow test on four occasions confirmed left shifted myelopoiesis, indicating an increasing number of promyelocytes and myelocytes (Fig. 1D).
Episodes of abdominal pain and diarrhea developed since the age of 17 years. She had one to two episodes every 3 months and each episode lasted for 15 to 20 days. She had 11 severe episodes of abdominal pain and diarrhea requiring visits to a physician but was never investigated for it. She also had mild thrombocytopenia and a small patent foramen ovale with left to right flow as often seen in G6PC3-deficient patients [5].
Clinical exome sequencing was performed to identify the cause of congenital neutropenia-a novel homozygous missense variant in the gene coding for G6PC3 (LRG -182t1:c482G > C; p.Arg161Pro) was identified. The parents were heterozygous carriers for the same variant. A variant affecting the same amino acid (p.Arg161Gln) has been described as part of compound heterozygous mutation in a patient with severe congenital neutropenia. [11] An in vitro study showed that Arg161Gln variant abolishes the catalytic activity of G6PC3. [12] Therefore, Arg161Pro variant was reported as pathogenic mutation.

Methods
Informed consent was taken for treatment with empagliflozin. The data was collected prospectively. Whole blood was obtained in EDTA and lithium heparin tubes under institutional research board-approved protocols in accordance with the Declaration of Helsinki. Blood samples were obtained weekly for 7 weeks and then at every outpatient visit. Blood counts were monitored at every clinic visit. 1,5AG was monitored every week for 7 weeks and then on further two clinic visits. Flow cytometry for CD14, CD15, CD66B, CD18, and dihydrorohodamine-123 (DHR) assay was monitored for 4 weeks. The dose of empagliflozin was titrated to the neutrophil count.

Quantification of 1,5-Anhydroglucitol in Human Plasma by LC-MS
Plasma was isolated after centrifugation (5 min at 500 × g; 22 °C) of 0.4 ml of freshly collected EDTA blood and heated for 5 min at 90 °C and immediately frozen at − 80 °C. The samples were shipped from Mumbai to Brussels at room temperature. Plasma samples were centrifuged and the supernatant was used for 1,5AG measurement by LCMS as previously described. [8] Plasma was collected before starting empagliflozin and weekly during treatment for 7 weeks and for two subsequent clinic visits on day 52 and day 58. Plasma samples were also collected at a later time-point on day 585, day 586, day 609, and day 610.

Dihydrorhodamine (DHR) Assay
Neutrophil function test was performed on whole blood using dihydrorohodamine-123 (DHR) and phorbol myristate acetate (PMA) (a protein-kinase C activator) in the laboratory (Sigma-Aldrich) (NIIH, Mumbai) as previously described. [13] Flow cytometry-based DHR assays can detect, upon PMA stimulation, reduced production of ROS; especially hydrogen peroxide and abnormal levels indicate defective NADPH complex activity. A stimulation index (SI) was calculated for gated neutrophils by estimating the ratio of median fluorescent intensity (MFI) of PMA-stimulated and PMA-unstimulated cells in the DHR assay. SI values for patient and healthy control sample were evaluated simultaneously.

Assays for Granulocyte Activation
Granulocytes were examined for abundance of CD14, CD15, CD18, and CD66B neutrophil antigens by flow cytometry analysis. Whole blood samples collected in EDTA were processed within 3 h of collection. Fluorescence staining of granulocytes was carried out with CD14-PE, CD66B-FITC, CD18-PerCP, and CD15-APC labelled antibodies (Becton Dickinson) directly on whole blood followed by lysis using Optilyse C (Beckman Coulter). A total of 20,000 events were acquired on Navioe EX (Beckman Coulter) and data analysis was done using Kaluza® software. Quantification of neutrophil antigens in the patient before and during treatment with empagliflozin was compared with normal untreated controls.

QoL-PROMIS Scoring System
The patient was requested to complete a quality-of-life questionnaire for two time points: (1) before starting empagliflozin and (2) 1 year on treatment with empagliflozin.
Three domains of quality of life were assessed: (1) cognitive function; (2) emotional distress such as anger, depression, and anxiety; (3) clinical symptoms such as pain interference, belly pain, and diarrhea. T score of 50 is the average for the general population in the PROMIS scoring system [14 15].

Results and Discussion
A significant increase in the absolute neutrophil counts (ANC) was observed during treatment with empagliflozin ( Fig. 2). Increase in ANC was associated with a drop in blood 1,5AG levels ( Fig. 2A). When blood samples from before (n = 17) and during empagliflozin treatment (n = 20) were compared, a significant increase in the monocyte counts, platelet counts, and hemoglobin were also observed during empagliflozin treatment ( Fig. 2B; p < 0.0001 unpaired t test). To examine changes in neutrophil function, we used a DHR-based assay that showed an improvement of the neutrophil SI from 9.46 (before treatment) to a median of 51.9 (during treatment on days 7, 14, and 21), i.e., similar to untreated normal controls ( Fig. 3A and B).
Neutrophils in patients with G6PC3 deficiency have been extensively studied by Goenka et al. [7] They reported an altered inflammatory activation status of neutrophils in patients with G6PC3 deficiency, possibly due to dependence on glycolysis as their energy source. [16] Therefore, we measured surface expression of neutrophil markers [17][18][19] (before and during empagliflozin treatment) to determine neutrophil activation status in our G6PC3-deficient patient. Before treatment, we found higher expression of CD14, CD15, CD18, and CD66B in neutrophils of the G6PC3deficient patient, similar to the previous report. [7] Interestingly, we observed prompt normalization of the mean fluorescence intensity during treatment with empagliflozin-an observation similar to G6PC3-deficient patients that underwent hematopoietic cell transplantation (HCT) (Fig. 3B). [7] Thus, there was correction of neutrophil activation during treatment with empagliflozin. Taken together, these results indicate a possible role of empagliflozin in normalizing the neutrophil phenotype by activating glycolysis due to the release of hexokinase inhibition.
The improvement in ANC and neutrophil function was associated with resolution of clinical symptoms. The patient has not developed new infections nor tongue ulcers since starting the treatment with empagliflozin. There has been no episode of abdominal pain or diarrhea during 521 days, since treatment was started. Moreover, our patient reported a significant improvement in the quality of life assessed by PROMIS questionnaire. Cognitive function, emotional distress, and clinical symptom scores were below average before treatment. There was significant improvement (T score of > or < 10 i.e. > 1 standard deviation) in all 3 domains during empagliflozin treatment (Supplemental Table 2). It is noteworthy that the clinical symptom scores improved to above average for the general population.
There was no evidence of hypoglycemic episodes or any other adverse event during the treatment. Empagliflozin is a frequently used drug in type 2 diabetes with good safety profile. [20] The usual starting dose of empagliflozin in an adult with type 2 diabetes is 10 mg daily. To determine the dose of empagliflozin that would be sufficient to treat neutropenia and neutrophil function in G6PC3 deficiency, we started with the daily dose of 5 mg (0.09 mg/kg/day). The dose of empagliflozin was increased to 10 mg/day (0.2 mg/kg/ day) on day 42 of treatment, to achieve a normal neutrophil count as shown in Fig. 2A and Supplemental Table 3. After achieving a normal neutrophil count, on day 52, we reduced the dose to 10 mg every second day (back to 0.09 mg/kg/ day) to consistently maintain ANC above 500 × 10 9 /L. Since day 182, our patient has been kept on a maintenance dose of 5 mg every 3rd day (0.03 mg/kg/day). Her neutrophil count has remained consistently above 700 × 10 9 /L on the maintenance therapy for 345 days (since day 182 of treatment) (see Fig. 2A). Patient is asymptomatic without any evidence of infection, tongue ulcers, nor gastrointestinal symptoms. It is important to note that the empagliflozin maintenance dosage (0.03 mg/kg/day) that is used in this patient is more than tenfold lower in comparison to the previously reported dose (about 0.4 mg/kg/day) used to treat neutropenia in GSD1b patients. [9] As yet, we do not have an explanation for this, but it is possible that for G6PC3deficient patients who do not take cornstarch, which is likely an important source of 1,5-anhydroglucitol, [21] once they have taken enough empagliflozin to lower the concentration of 1,5-anhydroglucitol in blood (as seen during the initial days of treatment, Fig. 2A), a lower maintenance dose can be used to keep 1,5-anhydroglucitol in blood sufficiently low for neutrophils to remain functional and infections to be prevented, as is seen in this particular patient. Clinical trials enrolling more G6PC3-deficient patients treated with empagliflozin are necessary before recommending an empagliflozin dosage for the treatment of neutropenia in G6PC3 deficiency. Currently, HCT is considered the only curative option for G6PC3 deficiency. Few cases of HCT have been successfully applied in patients with G6PC3 deficiency. [6,7,22,23] Seven patients have been reported to have received HCT for G6PC3 deficiency with very good outcomes. The curative potential of HCT for patients with congenital neutropenia is encouraging; however, there is morbidity and mortality associated with HCT. In 136 recipients of HCT for congenital neutropenia, the cumulative incidence of acute graft-versus-host disease was 21%, with 17% transplant-related mortality. [22] Typically, HCT is performed in children and adults with G6PC3 deficiency and inflammatory colitis in this GCSF-responsive neutropenia. [6,7,22] Although the neutrophils are GCSF-responsive, they exhibit an activated phenotype and accelerated apoptosis. [7] Despite some response to GCSF, patients with G6PC3 deficiency often develop inflammatory colitis. This is not surprising since as shown by Veiga-da-Cunha et al. despite GCSF treatment, neutrophils continue to accumulate toxic amounts of 1,5AG6P. [8,9] The inflammatory colitis is attributed to abnormal myeloid biology due to G6PC3 deficiency. [7] Our patient also had gastrointestinal symptoms, abnormal neutrophil function and counts, and neutrophils with an activated phenotype, before starting empagliflozin treatment. The neutrophil count, function of the neutrophils, and their phenotype were normalized, and the gastrointestinal symptoms resolved on empagliflozin. However, it is important to note that there is no objective measure of gastrointestinal pathology either existed or was resolved besides the patient report of symptoms. Patient report of improvement in debilitating symptoms is an encouraging finding and suggests that reduction in 1,5AG with SLGT2-inhibitor improves the quality of myeloid cells, and hence could have a therapeutic role in inflammatory colitis of G6PC3 deficiency. Accordingly, a very recent report, describing the role of empagliflozin in the treatment of neutropenia associated with GSD1b also demonstrated a clear improvement of inflammatory colitis [9]. This is the first-in-human report of an SLGT2 inhibitor in G6PC3 deficiency advancing the proof-of-concept demonstrated by G6PC3-deficient mouse models. Complex molecular and genetic techniques are being employed to develop therapeutic options for G6PC3 deficiency and other enzyme deficiencies. [24,25] However, the approach of eliminating the toxic metabolite is simple and costeffective. Similar approaches were tried in the past in a few patients with adenosine deaminase deficiency but the results were not encouraging. [26,27] The success of empagliflozin in rescuing the myelopoiesis in G6PC3deficiency indicates that such simple approaches should be investigated in the laboratory. More trials are now underway to test the definitive role of SLGT2 inhibitor in neutropenia of G6PC3 deficiency and GSD1b. However, our report suggests that patients with G6PC3 deficiency and poor quality of life due to recurrent infections or endorgan involvement should be offered the treatment with an SLGT2 inhibitor before proceeding to high-risk procedure of HCT.