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2526 Investigating Neutropenia in FNIP1 Deficiency: A CRISPR/Cas9 Model in HL-60 Cells

Program: Oral and Poster Abstracts
Session: 201. Granulocytes, Monocytes, and Macrophages: Poster II
Hematology Disease Topics & Pathways:
Research, Translational Research, Biological Processes, Pathogenesis
Sunday, December 8, 2024, 6:00 PM-8:00 PM

Francesco Saettini, MD1*, Fabiola Guerra2,3*, Benedetta Elena Di Majo2*, Mario Mauri, PhD4*, Federica Malighetti, MS4*, Angela Maria Savino5*, Stefano Rebellato2*, Arnalda Lanfranchi6*, Cristina Bugarin, MSc7*, Cecilia Poli8,9*, Elma Nievas10*, Tim Niehues11*, Peter McNaughton12*, Selami Ulaş13*, Sven Kracker14*, Rocco Piazza, MD, PhD15,16*, Grazia Fazio, PhD7,17* and Andrea Biondi17,18,19

1Centro Tettamanti, IRCCS San Gerardo dei Tintori, Monza, Italy
2Dipartimento di Medicina e Chirurgia, Università degli Studi Milano-Bicocca, Monza, Italy
3Pediatria, Fondazione IRCCS San Gerardo dei Tintori, Monza, Italy
4Department of Medicine and Surgery, University of Milano-Bicocca, Monza, ITA
5Università Degli Studi di Milano-Bicocca, Department of Medicine, Milano, Italy
6Laboratorio Cellule Staminali, Laboratorio Centrale di Chimica Clinica, Spedali Civili, Brescia, Italy
7Tettamanti Center, Fondazione IRCCS San Gerardo dei Tintori, Monza, Italy
8Facultad de Medicina, Universidad del Desarrollo-Clínica Alemana, Santiago, Chile
9Unidad de Inmunología y Reumatología, Hospital Roberto del Río, Santiago, Chile
10Alexander Fleming Hospital, Mendoza, Argentina
11Department of Pediatrics, Helios Klinik Krefeld, Krefeld, Germany
12Queensland Children's Hospital, Queensland, Australia
13Department of Pediatric Allergy and Immunology, Basaksehir Cam and Sakura City Hospital, Istanbul, Turkey
14Rene Descartes University of Paris and Necker Children's Hospital, Paris, FRA
15Department of Medicine and Surgery, University of Milano-Bicocca, Monza, Italy
16Hematology Division and Bone Marrow Unit, Fondazione IRCCS San Gerardo dei Tintori, Monza, Italy
17School of Medicine and Surgery, University of Milano-Bicocca, Monza, Italy
18Pediatrics, Fondazione IRCCS San Gerardo dei Tintori, Monza, Italy
19Centro Tettamanti, Fondazione IRCCS San Gerardo dei Tintori,, Monza, Italy

Background

FNIP1 (Folliculin Interacting Protein 1) deficiency is a rare inborn error of immunity characterized by heart defects and B-cell deficiency. Neutropenia is frequently reported in FNIP1-deficient patients, but its underlying mechanism has not been elucidated, with no evidence of neutropenia in the Fnip1 mouse model.

Aims

To test the hypothesis that FNIP1 deficiency could be directly associated with neutropenia we collected updated clinical and laboratory data of FNIP1-deficient patients. To unveil the role of FNIP1 in neutrophil development and function, we employed CRISPR/Cas9 genome editing to create a FNIP1-deficient model in HL-60 cells.

Methods

Ten (8 published and 2 unpublished) patients were enrolled. CRISPR/Cas9 was used to knock out (KO) FNIP1 in HL-60 cells. Whole RNA-sequencing, expression of pAKT, pS6, p4EBP1, mitochondrial abundance and membrane potential (MMP), oxygen consumption rate (OCR), ATP production and extracellular acidification rate (ECAR) of FNIP1-deficient and wild-type HL-60 cells were performed to assess effects of FNIP1 deficiency.

Results

All the patients (male:female = 5:5, age range <1-40 years, median 5.5 years) showed hypogammaglobulinemia/agammaglobulinemia with B-cell deficiency, heart defects, severe/recurrent infections and were receiving immunoglobulin replacement treatment. Crohn disease and seronegative arthritis were diagnosed in 1 individual each. Eight out of 10 patients showed decreased absolute neutrophil count (ANC; four chronic and four intermittent neutropenia). All the patients with chronic neutropenia had ANC <500/µl (severe neutropenia). Neutrophil oxidative burst was normal in 2 patients while chemotaxis resulted defective in 1 case. Bone marrow (BM) analysis showed left-shifted granulopoiesis with no promyelocyte/myelocyte arrest in 3/3 cases. Monocytosis and monocytopenia were present in 4 and 1 out of 10 patients, respectively. G-CSF was started in 3 patients, who responded to <5 mcg/kg/day. To date, no individual has developed a clonal hematopoietic disorder. Six patients were alive at the last follow-up.

Whole RNA-seq in FNIP1 KO HL-60 cells showed aberrant transcriptional control of differentiation from HSC to myeloid cells (increased C/EBPα and decreased MEIS1 and HOXA9). Enrichment in gene sets related to cell differentiation (Notch Transcriptional and post-Translation regulation), motility/cytoskeletal remodeling (Rap1, Rac1, Rho, RhoA and Cdc42 pathways), mitochondrial (oxidative stress induced senescence) and neutrophil (degranulation, chemotaxis and extracellular trap formation) function was observed in FNIP1 KO HL-60 cells.

FNIP1 KO HL-60 cells did not show differences in total mitochondria but MMP was significantly reduced (p<0.0001). Although transcripts involved in mitochondrial electron transport chain (COX16, COX19, COX6C, COX7A2 and COX7B) and OXPHOS (ATP5F1) were dysregulated, FNIP1 KO HL-60 cells showed no differences in ATP production and OCR, a measure of OXPHOS. Decrease of PPARγ and increase of key glycolytic enzymes (HK2 and LDHA) suggested altered glucose metabolism. ECAR, a measure of glycolysis, was increased in FNIP1 KO HL-60 cells (p<0.0001). We examined the PI3K/AKT pathway and did not observe differences in pAKT473, p4EBP1 and pS6 between WT and FNIP1 KO HL-60 cells.

Conclusions

Neutropenia is a frequent finding and part of the phenotypic spectrum of FNIP1 deficiency. FNIP1-deficient patients do not lack mature neutrophils in the BM and respond to G-CSF treatment. FNIP1 deficiency profoundly impacts the mechanisms underlying critical neutrophil activities, ultimately impairing neutrophil differentiation and function. Similarly to FNIP1-deficienct B cells, FNIP1 acts in neutrophil’s progenitors regulating glycolysis. Our work shows that FNIP1 deficiency alters previously unrecognized pathways (cytoskeletal dynamics/cell migration). We have previously described increased MMP and phosphorylation of pAKT473 and p4EBP in FNIP1-deficient B cells. As neutropenia is not associated with promyelocyte/myelocyte arrest, FNIP1 may have a prominent role in later stages of granulocyte’s differentiation, thus explaining the difference between B cells and HL-60 observed in MMP and PI3K pathway. Future work will focus on targeted interventions to restore neutrophil function in FNIP1 deficiency.

Disclosures: Saettini: Pharming Technologies B.V.: Research Funding. Kracker: UCB Pharma: Consultancy; Pharming: Consultancy; NA: Patents & Royalties: designated inventor on published patent application WO2017/198590. Biondi: CoImmune, Galapagos, Amgen, Novartis, BMS: Consultancy, Research Funding, Speakers Bureau.

*signifies non-member of ASH