Septin-6 Regulates Murine and Human Hematopoiesis, and Its Dysregulation Is Associated with Pediatric Myelodysplasia

Septin family proteins are key regulators of cytoskeletal organization and cytokinesis although little is known about their function in normal hematopoiesis. Septins are implicated in T and B cell lymphomas; and acute myeloid leukemias, where they frequently act as fusion partners with the Mixed-Lineage Leukemia (MLL) gene. Previously, we identified a non-syndromic newborn with severe neutropenia which progressed to myelodysplastic syndrome (MDS) (Renella et al. AJH, 2022). Next-generation sequencing revealed a novel X-linked germline mutation in the SEPTIN-6 gene associated with dysmyelopoiesis (SEPTIN6 c.1282T>C); and a second stop-gain somatic mutation hypothesized to rescue hematopoietic function, but the mechanism of MDS was not defined. Based on our initial publication, a second patient with neutropenia/MDS has since been identified in Seattle with a mutation in the same codon (SEPTIN6 c.1282T>A) suggesting an important new molecular mechanism for pediatric MDS. Both identified patients had a high degree of myeloid tetraploidy in the marrow and were refractory to G-CSF. To assess the pathobiology of the published patient’s mutation, we replicated the germline T>C mutation using single base-pair gene editing in normal donor primary CD34+ cells. Editing rates were ~50-70%. Morphological studies demonstrated that SEPTIN-6 edited human CD34+ cells demonstrated enlarged, multi-nucleated and dysplastic nuclei. These findings were similar to the appearance of the patient’s bone marrow myeloid precursor cells. These edited cells also demonstrated a decline in clonogenic activity notably with a selective reduction in granulocyte-macrophage (GM) colonies (Table 1). Both myeloid and erythroid colonies showed a reduction in colony size. These data directly confirm an important role for this mutated SEPTIN-6 in abnormal hematopoietic function.

The septin-6 family of proteins consist of septin-6, 8, 10, 11 and 14 which may have overlapping functions in cells. Murine single cell mRNA expression data demonstrate that hematopoietic stem and progenitor cells (HSPC) express significant levels of Septin-6, Septin-11 and 8, with little to no expression of Septin-10 and 14. To better understand the functional role of Septin-6, we created lentivirus vectors tagged with a GFP reporter that contained shRNAs against Septin-6. Culture of Septin-6 knockdown (Septin-6 KD) murine HSPC in IMDM (10ng/ml IL-3, 25ng/ml of SCF, TPO, Flt-3 and IL-6) resulted in an average of 18-fold decrease in cell counts compared to controls (p=0.02). This was accompanied by an increase in cell cycle: S-phase: Septin-6 KD 55.7%; controls 42.5%, p=0.05 and a cumulative decrease in the G0/G1 and G2/M phase suggesting that Septin-6 is important in regulating cell cycle in HSPC. Controls were a non-targeting shRNA and an empty vector for all experiments described. Furthermore, knockdown of Septin-6 caused a decrease in total clonogenic activity with defects seen in both myeloid (GM) and erythroid (BFU-E) colonies (Table 1). Competitive repopulation assays, wherein transduced BoyJ HSPC (GFP+ Septin KD and BFP+ control were mixed 1:1) were injected into lethally irradiated C57Bl/6 mice, demonstrated a reduction in multilineage chimerism in the peripheral blood, bone marrow, spleen and thymus in primary and secondary transplant recipients after knockdown of Septin-6 (Table 2). To better define function in specific hematopoietic populations of HSPC, we performed knockdown in FACS-sorted purified cells. Engraftment studies demonstrated a significant reduction in bone marrow chimerism in primary transplant recipients after knockdown of Septin-6 most clearly in ST-HSC (Table 2) with a trend towards a decrease in LT-HSC and MPP. These data support a key role of Septin-6 in murine and human hematopoiesis via alterations in cell cycle and validate the pathogenic nature of a specific mutation in human SEPTIN-6.