Septin family proteins belong to the RAS-like GTPase superclass and are considered a structural element of the cytoskeleton. Septins are evolutionarily conserved and are important in cytokinesis and polarity. We previously described two non-syndromic patients with severe neutropenia which progressed to myelodysplastic syndrome (MDS) (Renella et al. AJH, 2022, Mohamad et al. ASH 2023). Next-generation sequencing revealed germline mutations (GM) at codon 428 of the SEPT6 gene (GM1: SEPT6 c.1282T>C; GM2: SEPT6 c.1282T>A). Both patient GMs were predicted to add 9 amino acids to the C-terminus creating a novel SEPT6 protein. One patient demonstrated an additional somatic stop-gain mutation of unknown significance in cis with GM1 (SM: SEPT6 c.43C>T) in ~14% of bone marrow (BM) cells. Here we use genetic models to define the biological relevance of these mutations in blood formation.

SEPT6 is located on the X-chromosome and neither male patient expressed wild-type protein. Single base-pair gene editing that introduced GM1 into normal male donor CD34+ cells resulted in multi-nucleation, dysplastic nuclei and enlargement of edited cells, phenocopying the patients' BM findings and defective hematopoiesis in both in vitro and in vivo assays. Bulk mRNA sequencing demonstrated changes in ribosome, DNA replication, homologous recombination and mismatch repair pathways implying DNA damage. Expression of the mutant SEPT6 via lentivirus vector (LVV) gene transfer in wild-type (WT) CD34+ cells also disrupted hematopoiesis suggesting acquisition of novel functions by the mutated protein.

We previously hypothesized that the somatic stop-gain mutation (SM) in the patient's BM cell genome may mitigate the deleterious effects of GM1 (Renella et al. AJH, 2022). To test this hypothesis and further explore similarities of each individual mutation, we created LVV which contained a shmiR targeting endogenous SEPT6 for knock-down of WT protein in combination with: (a) GM1 alone, or (b) SM in cis with GM1, or (c) GM2. A WT knock-down resistant SEPT6 cDNA was reintroduced as a control. Clonogenic assays demonstrated that transgenic expression of GM1 or GM2 in CD34+ cells led to a reduction in the total number of myeloid progenitor colonies (Control: 81.33+/-2.51 vs SEPT6 shmiR+GM1: 49.7+/-6.1, SEPT6 shmiR+GM2: 56.67+/-7.6; mean+/- SD, N=3, p<0.0001). Introduction of SM in cis with GM1 rescued the reduced clonogenic activity seen with expression of GM1 (SEPT6 shmiR+GM1+SM: 73+/-10.6). Transplantation of SEPT6 shmiR+GM1 hematopoietic stem cells (HSCs) and multilineage progenitors in NBSGW mice demonstrated an engraftment defect at 4 months both in PB and BM (%GFP+ cells, Control: PB: 75+/-8, BM: 82.9+/-9.2 vs SEPT6 shmiR+GM1: PB: 55.5+/-19.1, BM: 65.4+/-18.8; +/-SD, N=10-15 mice, p< 0.05). The reduced engraftment observed was completely mitigated by the introduction of SM (PB: 72.3+/-12.3; BM: 81.5+/-10.2;).

Mechanistically, these engraftment defects were accompanied by an accelerated exhaustion of phenotypically defined long-term and short-term HSC in vitro. Single cell mRNA sequencing in GM1 cells demonstrated a downregulation of macrophage migration inhibitory factor (MIF), upregulation of p21 and defects in mitochondrial and macromolecular pathways. Based on these data, we hypothesized that GM1 induces premature senescence. Introduction of GM1 in human CD34+ cells significantly increased the number of senescent cells at day 7 post transduction (Control: 31.45+/- 1.5% vs SEPT6 shmiR+GM1: 41.6+/-0.8%; mean+/-SD, N=3, p<0.05) as measured by β-galactosidase assays which persisted at day 15. This increase was accompanied by a 2.2-fold upregulation of mRNA expression of CyclinD1. Introduction of SM mitigated the senescence defect associated with expression of GM1 alone. SEPT6 shmiR+GM1 HSC also demonstrated a higher oxygen consumption rate during mitochondrial respiration which was not observed in the SEPT6 shmiR+GM1+SM group confirming the mitochondrial perturbations observed in the single cell data. In vitro data from GM2 demonstrated similar results. Overall, our data demonstrate the acquisition of de novo protein function due to patient-derived germline mutations in the SEPT6 gene associated with defective hematopoiesis that defines a novel form of congenital cytopenia developing into MDS due to premature senescence in HSCs.

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2025
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