SEPTIN6 mutations in pediatric myelodysplastic syndrome disrupt nuclear actin organization Free

Septins are a family of evolutionarily conserved GTP-binding proteins that play critical roles in cytoskeletal organization, cytokinesis, and intracellular trafficking forming cytoplasmic filaments interacting with each other, Rho GTPases and other cytoskeleton proteins. We previously identified germline mutations of SEPTIN6 in two unrelated infants with congenital neutropenia that evolved to myelodysplastic syndrome (MDS). Each mutation resulted in the addition of nine amino acids to the C-terminus and led to aberrant localization and expression of SEPTIN6 in myeloid precursors and megakaryocytes, causing multinuclearity and cell cycle abnormalities (Renella et al. 2022, Mohamad et al. 2022). While the cytoplasmic role of Septin6 and its interaction with actin has been characterized (Kinoshita et al., 2002), its nuclear functions and potential involvement in MDS are unexplored. Nuclear actin is increasingly recognized as a key regulator of chromatin organization, DNA repair, and transcription (Hurst et al., 2019). Notably, hematopoietic Rho GTPases and specifically CDC42, which we and others have extensively studied, have been shown to be involved in Septin-mediated cell polarity (Althoff et al., 2020; Woods et al., 2021).

We hypothesized that the reported SEPTIN6 mutations would disrupt nuclear actin organization and Rho GTPase functions, resulting in chromatin disorganization, and aberrant gene expression associated with hematopoietic failure. To investigate the role of MDS-associated mutations on nuclear actin dynamics, we used lentiviral vector (LVV) constructs encoding a shmiR to knock-down endogenous SEPTIN6, coupled with the transgenic expression of either siRNA-resistant wild-type (WT) SEPTIN6 cDNA, two previously identified germline mutations (GM1: c.1282T>C; GM2: c.1282T>A), and an acquired somatic mutation (SM: c.43C>T) found in cis with GM1 at a low VAF in the bone marrow (BM) of one patient. We previously hypothesized that the somatic variant could mitigate the deleterious effects of the germline mutation.

Human CD34⁺ hematopoietic stem cells (HSC) and progenitors (HSC/P) were transduced with each LVV construct and analyzed on day 6 post-transduction using super-resolution microscopy. In WT cells, SEPTIN6 co-localized with nuclear F-actin in distinct puncta. Knockdown of

endogenous SEPTIN6 reduced nuclear actin by 45% compared with control (N=6 different donors, p < 0.05) while re-expression of WT SEPTIN6 cDNA restored nuclear actin to WT levels. In contrast, expression of both MDS-associated germline mutants significantly decreased nuclear F-actin intensity compared with control (GM1:46% less, N=6; GM2: 31% less, N=4, p < 0.05). The deleterious effects of GM1 were mitigated by co-expression of the somatic mutation (c.43C>T), which restored nuclear actin to levels comparable with WT (N=6).

To elucidate the downstream effects of mutant SEPTIN6-dependent nuclear actin dysregulation in pediatric MDS, we performed single-cell RNA sequencing on GM1-expressing HSC. This analysis revealed widespread transcriptional changes affecting nuclear actin dynamics, chromatin remodeling and epigenetic regulation. Notably, ACTB (encoding β-Actin) was downregulated, PFN1 (Profilin-1), which regulates nuclear actin export and HSC metabolism and retention, was upregulated; WAS (encoding WASp), a CDC42 effector, whose deficiency causes Wiskott-Aldrich Syndrome, was downregulated, and the GTPase activating protein ARHGAP15 (RhoGAP15), a negative regulator of Rho GTPase RAC which is closely related to CDC42, was strongly upregulated. Key chromatin regulators were also affected, with upregulation of the histone acetyltransferase KAT6A and downregulation of SMARCE1, a core subunit of the SWI/SNF complex critical for maintaining HSC identity. These transcriptomic changes were validated by qRT-PCR of transduced human CD34⁺ which showed a 91% reduction in ACTB expression, a 57% reduction in WAS expression, >40-fold increase in ARHGAP15, >3-fold increase in PFN1, a 1.65-fold increase in KAT6A expression and a 65% reduction in SMARCE1 expression (p < 0.05 for each gene).

These findings support a model in which SEPTIN6 maintains nuclear actin architecture through interactions with Rho GTPases, affecting chromatin structure and epigenetic regulators. Germline mutations associated with SEPTIN6 identified in pediatric MDS impair nuclear actin organization and downstream gene regulation contributing to hematopoietic dysfunction.