Patient-derived neomorphic SEPTIN6 mutations induce hematopoietic stem cell senescence underlying congenital neutropenia and myelodysplasia
Safa F. Mohamad, Meaghan McGuinness, Yu Zhou, Xin Jiang, Gabriele Casirati, Adele Mucci, Sara Canovas Nunes, Akiko Shimamura, Pietro Genovese, David A. Williams
Safa F. Mohamad, Meaghan McGuinness, Yu Zhou, Xin Jiang, Gabriele Casirati, Adele Mucci, Sara Canovas Nunes, Akiko Shimamura, Pietro Genovese, David A. Williams
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Research Article Hematology Oncology

Patient-derived neomorphic SEPTIN6 mutations induce hematopoietic stem cell senescence underlying congenital neutropenia and myelodysplasia

Abstract

Septins are RAS-like GTP-binding proteins involved in cytoskeletal organization and cellular division. We previously described 2 pediatric patients harboring SEPT6 germline mutations (GM1: SEPT6 c.1282T>C and GM2: SEPT6 c.1282T>A) presenting with congenital neutropenia associated with the development of myelodysplastic syndrome. In one patient, an additional somatic mutation (SM: SEPT6 c.43C>T) was detected at a low allelic fraction in the BM. Here, we describe the consequences of these mutations for hematopoiesis. Transgenic expression or gene editing of GM1 or GM2 in human CD34+ cells led to enlarged, multinucleated cells associated with accelerated exhaustion and premature senescence of hematopoietic stem and progenitor cells (HSPCs). Senescence defects were associated with the upregulation of mTOR phosphorylation, and pharmacologic inhibition of the mTOR pathway resulted in rescue of senescence. In parallel, HSPCs exhibited marked mitochondrial abnormalities, including increased expression of OPA-1, a key regulator of mitochondrial fusion, and enhanced oxygen consumption during mitochondrial respiration. Introduction of the SM in cis with GM1 mitigated all observed defects. Collectively, our findings demonstrate a gain-of-function mechanism underlying marrow failure in patients carrying germline SEPT6 mutations and suggest that acquisition of a secondary somatic SEPT6 variant may confer a compensatory effect within hematopoietic cells.

Authors

Safa F. Mohamad, Meaghan McGuinness, Yu Zhou, Xin Jiang, Gabriele Casirati, Adele Mucci, Sara Canovas Nunes, Akiko Shimamura, Pietro Genovese, David A. Williams

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Figure 2

GM1 causes a hematopoietic engraftment defect accompanied with a decrease in the number of LT-HSCs and ST-HSCs.

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GM1 causes a hematopoietic engraftment defect accompanied with a decreas...
(A) Schema of bidirectional LVV to simultaneously knock down WT SEPT6 and express mutated SEPT6 in CD34+ cells. SEPT6 cDNAs used as inserts are shown. The location of the shmiR containing shRNA targeting WT SEPT6 embedded in microRNA30 is shown. The same schema was used to make the lentivirus used in Supplemental Figures 1, 2, and 4. (B) Photomicrographs of cytospins of CD34+ cells 7 days after transduction. Multinucleated myeloid precursor is shown at the black arrow. Original magnification, ×100. n = 3 biological replicates with similar results. (C) Cells were stained with 7-AAD to calculate percentage of multinucleation, n = 3. One-way ANOVA followed by Tukey’s multiple comparisons test as a post hoc analysis. (D) Transduced GFP+ cells were sorted and plated 2 days after transduction in hematopoietic progenitor assays. Colonies were counted 14 days after plating. n = 3 independent experiments performed in triplicate. Two-way ANOVA followed by Tukey’s multiple comparisons test as a post hoc analysis. (E–G) Transduced CD34+ cells (1 × 106) 2 days after lentiviral transduction were injected retro-orbitally into NBSGW mice. (E) Percentage GFP+ donor chimerism was analyzed monthly in PB, and at the 4-month time point in BM. (F) Differences in donor engraftment in the myeloid and lymphoid compartment, and (G) immunophenotypically defined HSPC compartments analyzed in BM 4 months after xenotransplantation. (E–G) Graphs represent 1 of 2 xenotransplants, each containing an n of 5–10 mice and showing similar results. Two-way ANOVA followed by Tukey’s multiple comparisons test as a post hoc analysis. (H and I) Human CD34+ cells were transduced and flow sorted 2 days after lentiviral transduction into GFP+ cells. Transduced CD34+ cells were cultured for 10 days in serum-free culture and analyzed for immunophenotypically defined LT-HSCs (H) and ST-HSCs (I) at each time point. Both percentages (first panel) and absolute number of cells (second panel) were analyzed and calculated. n = 3. Two-way ANOVA followed by Tukey’s multiple comparisons test as a post hoc analysis. Data presented as mean ± SD (C–G) or mean ± SEM (H and I). *P < 0.05; **P < 0.005; ***P < 0.0005; ****P < 0.0001.