G-CSF resistance of ELANE-mutant neutropenia depends on SERF1-containing truncated-neutrophil elastase aggregates

Abstract

Severe congenital neutropenia (SCN) is frequently associated with dominant point mutations in ELANE, the gene encoding neutrophil elastase (NE). Chronic administration of granulocyte colony-stimulating factor (G-CSF) is a first-line treatment of ELANE-mutant (ELANEmut) SCN. However, some ELANEmut patients, including patients with ELANE start codon mutations, do not respond to G-CSF. Here, through directed granulopoiesis of gene-edited isogenic normal and patient-derived iPSCs, we demonstrate that ELANE start codon mutations suffice to induce G-CSF-resistant granulocytic precursor cell death and refractory SCN. ELANE start codon-mutated neutrophil precursors express predominantly nuclear N-terminally truncated alternate NE. Unlike G-CSF-sensitive ELANE mutations that induce endoplasmic reticulum and unfolded protein response stress, we found that the mutation of the ELANE translation initiation codon resulted in NE aggregates and activated proapoptotic aggrephagy, as determined by downregulated BAG1 expression, decreased BAG1/BAG3 ratio, NE colocalization with BAG3, and localized expression of autophagic LC3B. We found that SERF1, an RNA-chaperone protein, known to localize in misfolded protein aggregates in neurodegenerative diseases, was highly upregulated and interacted with cytoplasmic NE of mutant neutrophil precursors. Silencing of SERF1 enhanced survival and differentiation of iPSC-derived neutrophil precursors, restoring their responsiveness to G-CSF. These observations provide a mechanistic insight into G-CSF-resistant ELANEmut SCN, revealing targets for therapeutic intervention.

Keywords: Hematology; Neutrophils.

Figure 1. Neutrophil precursors with mutation at the translation initiation codon (c.1A>G) of ELANE that is associated with cell death, differentiation arrest, and G-CSF resistance.

(A) PB ANCs and AMCs of a patient with ELANE translation initiation codon mutation (c.1A>G, NEp.M1V) from diagnosis at 3 months after birth up to 16 months. The patient was found to have leukopenia, severe neutropenia (ANC = 0), and monocytosis (AMC = 2720/mm³). G-CSF (20 μg/kg/day) administration showed minimal effect on ANCs. Donor granulocyte transfusion did not increase neutrophil counts. This patient underwent allogeneic bone marrow transplantation, resulting in ANC recovery. (B) Experimental schema of modeling of SCN with ELANE translation initiation codon mutation employing directed hematopoietic and granulopoietic differentiation of normal (healthy donor iPSCs), isogenic gene-edited GTG-KI (GTG knockin in place of ATG in one allele of ELANE gene of normal iPSCs), GTG-P1 (ELANE c.1A>G SCN patient–derived iPSCs), and GTG-C (correction of GTG to ATG in SCN patient GTG-P1 iPSCs). (C) Cell growth of normal, GTG-KI, GTG-P1, and GTG-C iPSC–derived hematopoietic progenitors (CD34⁺CD45⁺). (D) Quantification of the apoptosis of normal, GTG-KI, GTG-P1, and GTG-C neutrophil precursors (CD45⁺CD34^–CD14^–CD11b^–CD15^+/dim). (E) Flow cytometry analyses of the granulopoiesis (mature neutrophils: CD45⁺CD14^–CD11b⁺CD16⁺CD66b⁺) of normal, GTG-KI, GTG-P1, and GTG-C iPSC–derived hematopoietic progenitors in the presence of 50 ng/mL G-CSF. (F) Quantification of the apoptosis of normal, GTG-KI, GTG-P1, and GTG-C neutrophil precursors (CD45⁺CD34^–CD14^–CD11b^–CD15⁺) in the presence of 50 ng/mL and 1000 ng/mL G-CSF. (G and H) Quantification (G) and representative morphological microphotographs (H) of output cells of G-CSF–induced (50 ng/mL) differentiation of normal, GTG-KI, GTG-P1, and GTG-C iPSC–derived hematopoietic progenitors (Wright-Giemsa staining; original magnification, ×40). Data are presented as individual data and mean ± standard deviation of a minimum of 3 replicates per experiment and a minimum of 2 independent experiments. Differences between groups were evaluated using 1-way ANOVA. *P < 0.05; **P < 0.01; ***P < 0.001.

Figure 2. Neutrophil precursors with mutation at translation initiation codon of ELANE express low molecular weight alternate NE (NE^ALT), generate NE aggregates (NE^AGR), and induce aggrephagy.

(A) Representative immunoblot analyses of full-length NE (NE^Normal) expression using α-NE Ab against N-terminal region (aa 13–39) of NE. (B) Representative immunoblot of NE expression in neutrophil precursors derived from normal, GTG-KI, GTG-P1, and GTG-C iPSC lines using an α-NE Ab against the C-terminal (aa 225–238) region of the protein. GTG-KI and GTG-P1 iPSC–derived neutrophil precursors express alternate NE (NE^ALT) and possibly aggregates (NE^AGR) of alternate NE peptides, and mutation correction rescues full-length NE expression. Some of the NE^ALT and NE^AGR expression in the GTG-C line could be due to contamination of mutant iPSCs during clonal isolation. (C) Representative immunoblots f NE in the pellet and soluble fractions of granulocyte precursor lysates after ultracentrifugation. (D) Quantification of NE and BAG3 in C in pellets and soluble fractions and presented as a normalized ratio over BAG3 (from panel B). (E) Representative confocal microscopic images of NE in association with ProteoStat fluorescent molecular rotor dye. (F) MFI of ProteoStat-stained aggresomes. (G) Representative immunoblots of BAG1 isoforms (BAG1L, BAG1M, and BAG1S) in normal, GTG-KI, GTG-P1, and GTG-C neutrophil precursors. (H and I) Representative confocal microscopic images (H) and MFI (I) of proximity ligation assay (PLA) signal between NE and BAG3 in normal, GTG-KI, GTG-P1, and GTG-C neutrophil precursors. (J) Schematic depiction of BAG3 and BAG1 protein ratio and association with NE^ALT aggregation. Scale bars: 10 μm. The MFIs of more than 10 cells from 2 independent experiments were quantified. Data are presented as individual data and mean ± standard deviation of 2 or 3 replicates per experiment and a minimum of 2 independent experiments. Differences between groups were evaluated using 1-way ANOVA. ***P < 0.001.

Figure 3. Expression of SERF1, an RNA binding chaperone, is upregulated and interacts with NE aggregates in ELANE translation initiation codon–mutant neutrophil precursors.

(A) Representative immunoblots of SERF1A and SERF1B in normal, GTG-KI, GTG-P1, and GTG-C neutrophil precursors. SERF1A, but not SERF1B, expression is upregulated in ELANE translation initiation–mutant neutrophil precursors. (B) Representative Western blots of SERF1A and SERF1B in healthy donor (normal) and ELANE^EX-3 mutant (I118N, Q97P) iPSC–derived neutrophil precursors. SERF1A expression is marginally reduced in ELANE^EX-3 mutant SCN. (C and D) Representative confocal microscopic images of NE and SERF1 (C) and MFIs (D) of SERF1A in normal, GTG-KI, GTG-P1, and GTG-C neutrophil precursors. (E) Quantification of SERF1 cytoplasmic expression in normal, GTG-KI, GTG-P1, and GTG-C neutrophil precursors. Increased SERF1 expression is associated with translocation to cytoplasm. (F and G) Representative confocal images (F) and MFIs (G) of PLA signal between SERF1 and NE normal, GTG-KI, GTG-P1, and GTG-C neutrophil precursors. Scale bars: 10 μm. The MFIs of more than 10 cells from 2 independent experiments were quantified. Data are presented as individual data and mean ± standard deviation of 2 or 3 replicates per experiment and a minimum of 2 independent experiments. Differences between groups were evaluated using 1-way ANOVA. **P < 0.01; ***P < 0.001.

Figure 4. SERF1 downregulation restores survival and granulocytic differentiation of ELANE translation initiation codon–mutant neutrophil precursors.

(A) Apoptosis of non-targeting (Ntg) shRNA– and SERF1 shRNA–transduced GTG-KI and GTG-P1 neutrophil precursors. SERF1 downregulation led to increased survival of GTG-KI and GTG-P1 neutrophil precursors. (B) Granulopoietic differentiation of Ntg shRNA– and SERF1 shRNA–transduced GTG-KI and GTG-P1 iPSC–derived hematopoietic progenitors at 50 ng/mL G-CSF. (C) Granulopoietic differentiation of Ntg shRNA– and SERF1 shRNA–transduced GTG-KI and GTG-P1 iPSC–derived hematopoietic progenitors at 1000 ng/mL G-CSF. (D and E) Representative confocal microscopic images (D) and MFIs (E) of ProteoStat fluorescent molecular rotor dye in Ntg shRNA– and SERF1 shRNA–transduced GTG-KI and GTG-P1 neutrophil precursors. (F and G) Representative confocal microscopic images (F) and MFIs (G) of PLA signals between NE and BAG3 in Ntg shRNA– and SERF1 shRNA–transduced GTG-KI and GTG-P1 neutrophil precursors. (H and I) Representative confocal microscopic images and MFIs of BAG1 expression in Ntg shRNA– and SERF1 shRNA–transduced GTG-KI and GTG-P1 neutrophil precursors. (J and K) Representative confocal microscopic images and MFIs of PLA between NE and BAG1. SERF1 downregulation enhanced NE interaction with BAG1 in GTG-P1 and GTG-KI neutrophil precursors. Scale bars: 10 μm. The MFIs of more than 10 cells from 2 independent experiments were quantified. Data are presented as individual data and mean ± standard deviation of 2 or 3 replicates per experiment and a minimum of 2 independent experiments. Differences between groups were evaluated using 1-way ANOVA. *P < 0.05; **P < 0.01; ***P < 0.001.