MSCV-IRES-EGFP

Pediatric myelodysplastic syndromes (MDS) are a heterogeneous disorder associated with impaired hematopoiesis, bone marrow cytopenia, and frequent deletions involving chromosome 7 (monosomy 7). Heterozygous germline mutations in SAMD9 and SAMD9L have recently been identified in children with monosomy 7 and MDS. Here, researchers used a lentiviral overexpression approach to evaluate 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 (HSPCs). By combining protein interactome analysis, transcriptional analysis, and functional validation, the researchers found that SAMD9 and SAMD9L are multifunctional proteins that induce profound changes in cell cycle, cell proliferation, and protein translation in HSPCs. Importantly, molecular and functional studies have also shown that expression of these genes and their mutations result in a cellular environment that promotes defects in DNA damage repair and ultimately promotes hematopoietic cell apoptosis.

Here, researchers developed lentiviral overexpression models of SAMD9 and SAMD9L in primary human or mouse hematopoietic stem and progenitor cells (HSPCs) to test the effects of these mutations in more relevant hematopoietic systems (Figure 1A). This method uses lentiviral MSCV-IRES-eGFP (MIG) vectors to overexpress wild-type SAMD9, SAMD9L, or mutations previously identified in children with MDS (SAMD9: E1136Q; SAMD9L: H880Q, W1180R, or R1281K). Overexpression of wild-type or mutant SAMD9 and SAMD9L in human CD34+ cells resulted in significant inhibition of colony formation (Figure 1B). Overexpression of human or mouse SAMD9L and its cognate W1180R/W1171R mutations resulted in a significant reduction of S-phase cells and accumulation of G2/M-phase cells, while the mouse Samd9l mutation exacerbated this effect (Figure 1C). Thus, the data suggest that overexpression of SAMD9 or SAMD9L impairs hematopoietic cell growth, whereas expression of their pathogenic mutations enhances this phenotype.

Figure 1. SAMD9 and SAMD9L alter hematopoietic cell proliferation, differentiation, and cell cycle. A The experimental model of lentivirus (MSCV-IRES-eGFP)-mediated overexpression of SAMD9, SAMD9L, and their mutations SAMD9-E1136Q, SAMD9L-H880Q, -W1180R, and -R1281K, in cord blood human CD34+ or Samd9l and Samd9l-W1171R in HSPCs cells from Samd9l−/− mice. B Colony forming units showing the total number of colonies from CD34+ (n = 4) (left) and Samd9l−/− HSPCs (n = 3) (right) transduced with GFP vector control (MSCV-IRES-eGFP), SAMD9, SAMD9L, or the indicated pathogenic mutations. C Flow cytometric analysis showing the percentage of EdU incorporation in Samd9l−/− HSPCs transduced with SAMD9L or Samd9l or their mutations. (Thomas III, Melvin E., et al., 2021)