Pediatric MDS and bone marrow failure-associated germline mutations in SAMD9 and SAMD9L impair multiple pathways in primary hematopoietic cells

Abstract

Pediatric myelodysplastic syndromes (MDS) are a heterogeneous disease group associated with impaired hematopoiesis, bone marrow hypocellularity, and frequently have deletions involving chromosome 7 (monosomy 7). We and others recently identified heterozygous germline mutations in SAMD9 and SAMD9L in children with monosomy 7 and MDS. We previously demonstrated an antiproliferative effect of these gene products in non-hematopoietic cells, which was exacerbated by their patient-associated mutations. Here, we used a lentiviral overexpression approach to assess the functional impact and underlying cellular processes of wild-type and mutant SAMD9 or SAMD9L in primary mouse or human hematopoietic stem and progenitor cells (HSPC). Using a combination of protein interactome analyses, transcriptional profiling, and functional validation, we show that SAMD9 and SAMD9L are multifunctional proteins that cause profound alterations in cell cycle, cell proliferation, and protein translation in HSPCs. Importantly, our molecular and functional studies also demonstrated that expression of these genes and their mutations leads to a cellular environment that promotes DNA damage repair defects and ultimately apoptosis in hematopoietic cells. This study provides novel functional insights into SAMD9 and SAMD9L and how their mutations can potentially alter hematopoietic function and lead to bone marrow hypocellularity, a hallmark of pediatric MDS.

Structure and function of an effector domain in antiviral factors and tumor suppressors SAMD9 and SAMD9L

“…We thus have focused on translational control as the potential underlying mechanism and found that GoF SAMD9/9L variants profoundly inhibit global protein synthesis. Several papers recently reported a similar inhibitory effect of the GoF variants on cellular protein synthesis while our manuscript was being prepared ( 6 , 7 , 27 ). We showed further that the same DBD residues that are essential for the antiviral and antiproliferative activities are also critical for SAMD9/9L to inhibit protein synthesis, suggesting that translational control is an important underlying mechanism for SAMD9/9L functions.…”

“…The binding affinity of DBD with a model DNA substrate is relatively weak (K D = 16 μM). Considering their cytoplasmic site of action and the recently reported interactions with ribosomal components ( 27 ), SAMD9/9L may bind to double-stranded regions of cytoplasmic RNA such as ribosomal RNA. Since the antiproliferative effect depends on the NA binding, DBD binding of NA in full-length SAMD9/9L is likely subject to tight regulation by other domains or cellular partners.…”

“…Affinities were obtained by nonlinear regression curve fitting of the fluorescence polarization data. Plasmids for transient expression of mCherry and SAMD9/9L fusions, pcDNA6.2/mCherry-SAMD9/9L, were a generous gift of Jeffery M. Klco, St. Jude Research Hospital, Memphis, TN ( 27 ). Specific mutations of SAMD9/9L were introduced into the plasmids through recombinant PCR-based site-directed mutagenesis as described previously ( 30 ).…”

Genetic barcoding systematically compares genes in del(5q) MDS and reveals a central role for CSNK1A1 in clonal expansion

“…These HSC-extrinsic effects are particularly interesting as MDS co-occurs or is preceded by chronic inflammatory conditions such as autoimmune disease 35,[40][41][42][43] or involves mutation of inflammatory-inducible genes such as SAMD9L [44][45][46] . The germline mutations in SAMD9/9L in monosomy 7 and MDS encode gain-of-function proteins that appear over-activated in response to inflammatory stress.…”

Convergence of specified RUNX2, SALL1, and SAMD9 variants permits normal global immune and endocrine function despite bone marrow hypocellularity and reduced ovarian reserve

“…Computer modeling has envisioned a protein-protein interaction network of differentially expressed genes, and human SAMD9 was among the 'hub genes' having special relevance after SARS-CoV-2 infection [20]. Thomas et al [21] assessed the functional impact of wild-type and mutant SAMD9 in primary mouse or human hematopoietic stem and progenitor cells. Using protein interactome analyses, transcriptional pro ling, and functional validation, it was concluded that SAMD9 mutations tend to favor interference with DNA damage repair, and ultimately apoptosis, in hematopoietic cells [21].…”