Base editing platform to investigate pathogenic <i>SAMD9</i> mutations in paediatric myelodysplastic syndromes

Paediatric myelodysplastic syndromes (pMDS) are a group of haematological disorders characterized by impaired haematopoiesis and potential clonal evolution.1 Among these syndromes, those associated with mutations in the Sterile α motif domain-containing protein 9 (SAMD9) and SAMD9-like (SAMD9L) genes have drawn attention due to their involvement in bone marrow (BM) failure and other haematopoietic defects.2 These mutations can lead to a spectrum of clinical manifestations, including cytopenia, transfusion dependence and severe bone marrow hypoplasia.3, 4 Allogeneic haematopoietic stem cell transplantation (HSCT) remains the primary curative option for these patients, but the procedure carries significant risks, including graft-versus-host disease and transplant-related complications.1, 5 In this study, we describe a base editing approach aimed at evaluating the pathogenetic role of SAMD9 mutations in pMDS. In particular, we narrowed our attention to an SAMD9 mutation which was detected in one of our patients with pMDS. The 3-year-old boy referred to our hospital due to severe hepatitis and trilinear cytopenia. After performing a bone marrow biopsy, the diagnosis of Refractory Cytopenia of Childhood (RCC) was made. RCC is a peculiar paediatric disorder characterized by bone marrow hypocellularity and dysplastic features, potentially evolving to BM failure or frank myelodysplasia.6 The clinical exome sequencing uncovered the germline mutation in the SAMD9 gene (c.4496A>G), classified as variant of uncertain significance (VUS). Sahoo et al. reported that the majority of SAMD9 and SAMD9L mutations found in patients with pMDS are categorized as VUS, hence unveiling a complex and poorly understood genotype–phenotype correlation.7, 8 To assess the pathogenetic role of this specific SAMD9 mutation, we used adenine base editors (ABEs) to reproduce and explore its effects in vitro. ABEs enable the conversion of adenine to guanine at precise target sites in the genome, without inducing a DNA double-strand break.9 We introduced the mutation into the K562 cell line, a well-established platform for studying haematological diseases. Hence, the effects of this mutation on SAMD9 expression, apoptosis and cell proliferation were investigated upon interferon α (IFNα)-induced inflammatory stress, which is involved in the pathogenesis, thereby mimicking the disease condition.10 In detail, K562 cells were transfected with the ABE system using a Lonza Nucleofector device, a highly efficient electroporation method for transfecting plasmid DNA into haematopoietic cells. The ABE system consists of a Cas9 nickase fused to an adenine deaminase, guided to the target DNA sequence by a single guide RNA (sgRNA).9, 11 To determine the most effective editing system for targeted modification of the SAMD9 gene, different combinations of ABEs and sgRNAs were tested (Figure S1A). Following transfection of the selected sgRNA and ABE into K562 cells, genomic DNA was extracted, and the efficiency of base editing was assessed by polymerase chain reaction (PCR) amplification of the target region followed by Sanger sequencing. Quantitative analysis using the EditR web tool revealed a base editing efficiency of 18% for sgRNA#3, with minimal bystander editing detected at adjacent sites, indicating the best specificity and efficacy among the designed strategies (Figure S1B). To better characterize the effects of the c.4496A>G SAMD9 mutation, the edited K562 cells with sgRNA#3 have been subcloned to establish isogenic cell lines. Thus, we obtained: (i) unedited wild-type (WT) cells; (ii) cells carrying the mutation on one allele (30%); (iii) cells with the mutation on two alleles (60%), consistent with the pseudo-triploid nature of K562 cells (Figure S1C).12 Clones with bystander effect were excluded. We decided to use the cells with 60% editing for studying the impact of the mutation on SAMD9 expression and cell growth. Under basal conditions, SAMD9 expression was undetectable by real-time quantitative PCR, indicating that the gene is not constitutively active in K562 cells (Figure S2). Hence, cells were cultured with increasing doses of IFNα, a cytokine known to stimulate SAMD9 and SAMD9L gene expression under inflammatory conditions. Upon stimulation with IFNα, cells with 60% editing exhibited higher levels of SAMD9 expression compared to WT cells, suggesting that the mutation enhances the gene's responsiveness to inflammatory signals (Figure 1). This observation supports the hypothesis that SAMD9 mutations contribute to the increased susceptibility to apoptosis and haematopoietic failure observed in patients with pMDS. Apoptosis was assessed by Annexin V staining and flow cytometry. We documented an increased rate of apoptosis in the mutant cells, particularly after IFNα treatment (Figure S3). This finding is consistent with the known pathogenic role of SAMD9 mutations, which are thought to promote excessive cell death in response to stress, contributing to bone marrow failure in affected patients. The cell proliferation assay revealed a reduction in the growth rate of mutated K562 cells as compared to WT cells upon IFNα stimulation (10 μg/mL), indicating that the mutation may have an anti-proliferative effect under inflammatory conditions (Figure 2). This study demonstrates the successful use of adenine base editing to introduce the SAMD9 c.4496A>G mutation into K562 cells, representing a suitable model to explore the mutation's impact on pMDS. Cells with the c.4496A>G mutation exhibited elevated SAMD9 expression after IFNα stimulation, suggesting that the mutation enhances responsiveness to inflammatory stimuli, a hallmark feature of pMDS pathophysiology. This heightened sensitivity may contribute to increased apoptosis and impaired proliferation in patients with SAMD9 mutations, as we observed in our in vitro model. The anti-proliferative effects of the SAMD9 mutation align with its known role as a tumour suppressor. The mutant cells showed a marked decrease in growth and an increased apoptosis as compared to wild-type cells, particularly under IFNα stimulation, supporting the mutation's role in driving bone marrow failure through excessive cell death. Our findings underscore the promise of base editing as a tool for modelling pMDS-causing mutations. While this study primarily focused on modelling the mutation, the success of base editing raises the possibility of therapeutic applications, particularly for correcting deleterious mutations in haematopoietic stem cells.13 However, optimizing editing specificity and assessing long-term stability in patient-derived haematopoietic stem cells will be critical steps towards clinical use. Base editing could offer a safer, less invasive alternative to allogeneic HSCT for treating pMDS. This work was also supported by the Ministero della Salute, Current Research. G.CA. and M.L. collected clinical data. G.CA., M.L., G.F., V.D. and M.G. performed the experiments and data analysis. G.CE. conceptualized and supervised the experiments. G.CA. and V.D. drafted the manuscript. F.D.B., G.P., P.P., F.L. and G.CE. edited and revised the final version of the paper. The authors declare no conflicts of interest. The study was approved by the Ethics Committee of Bambino Gesù Children's Hospital. All data are available from the corresponding author upon reasonable request. Data S1. Please note: The publisher is not responsible for the content or functionality of any supporting information supplied by the authors. Any queries (other than missing content) should be directed to the corresponding author for the article.