Mutant Samd9l expression impairs hematopoiesis and induces bone marrow failure in mice
Sherif Abdelhamed, Melvin E. Thomas III, Tamara Westover, Masayuki Umeda, Emily Xiong, Chandra Rolle, Michael P. Walsh, Huiyun Wu, Jason R. Schwartz, Virginia Valentine, Marcus Valentine, Stanley Pounds, Jing Ma, Laura J. Janke, Jeffery M. Klco
Sherif Abdelhamed, Melvin E. Thomas III, Tamara Westover, Masayuki Umeda, Emily Xiong, Chandra Rolle, Michael P. Walsh, Huiyun Wu, Jason R. Schwartz, Virginia Valentine, Marcus Valentine, Stanley Pounds, Jing Ma, Laura J. Janke, Jeffery M. Klco
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Research Article Hematology

Mutant Samd9l expression impairs hematopoiesis and induces bone marrow failure in mice

Abstract

SAMD9 and SAMD9L germline mutations have recently emerged as a new class of predispositions to pediatric myeloid neoplasms. Patients commonly have impaired hematopoiesis, hypocellular marrows, and a greater risk of developing clonal chromosome 7 deletions leading to MDS and AML. We recently demonstrated that expressing SAMD9 or SAMD9L mutations in hematopoietic cells suppresses their proliferation and induces cell death. Here, we generated a mouse model that conditionally expresses mutant Samd9l to assess the in vivo impact on hematopoiesis. Using a range of in vivo and ex vivo assays, we showed that cells with heterozygous Samd9l mutations have impaired stemness relative to wild-type counterparts, which was exacerbated by inflammatory stimuli, and ultimately led to bone marrow hypocellularity. Genomic and phenotypic analyses recapitulated many of the hematopoietic cellular phenotypes observed in patients with SAMD9 or SAMD9L mutations, including lymphopenia, and pinpointed TGF-β as a potential targetable pathway. Further, we observed nonrandom genetic deletion of the mutant Samd9l locus on mouse chromosome 6, mimicking chromosome 7 deletions observed in patients. Collectively, our study has enhanced our understanding of mutant Samd9l hematopoietic phenotypes, emphasized the synergistic role of inflammation in exaggerating the associated hematopoietic defects, and provided insights into potential therapeutic options for patients.

Authors

Sherif Abdelhamed, Melvin E. Thomas III, Tamara Westover, Masayuki Umeda, Emily Xiong, Chandra Rolle, Michael P. Walsh, Huiyun Wu, Jason R. Schwartz, Virginia Valentine, Marcus Valentine, Stanley Pounds, Jing Ma, Laura J. Janke, Jeffery M. Klco

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Figure 5

The lack of fitness in Samd9l-mutant cells is partly via TGF-β activation.

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The lack of fitness in Samd9l-mutant cells is partly via TGF-β activatio...
(A) Model of pI:pC treatment regimen followed by sorting the Lin–cKit+ (LK) population from Samd9l-WT and Samd9l-Mut mice to perform RNA-seq. (B) Heatmap showing the up- and downregulated pathways in Samd9l-WT and Samd9l-Mut treated with vehicle or pI:pC. (C) A plot of pathway enrichments of DEGs downregulated in pI:pC-treated Samd9l-Mut mice relative to vehicle-treated. The size of the circles represents gene counts, and the significance was determined by FDR. The color is dependent on the fold of enrichment. (D) Gene set enrichment analysis (GSEA) showing TGF-β pathway activation in pI:pC-treated Samd9l-Mut mice relative to vehicle-treated Samd9l-Mut mice. Normalized enrichment score (NES), FDR, and P value are indicated. (E) Phospho-SMAD2/3 expression in Samd9l-KO, Samd9l-WT, and Samd9l-Mut BM cells treated with IFN-α or vehicle (n = 4 per group). (F) Representative histograms of phospho-SMAD2/3 expression in B cells of Samd9l-WT and Samd9l-Mut cells after IFN-α or vehicle. (G and H) Serial CFU-C replating of Samd9l-WT and Samd9l-Mut cells (G) or human cells from a patient with SAMD9L-S626L mutation or control (H) with or without TGF-β inhibitor (SD-208). Data show at least 3 independent experiments. For panel E, Kruskal-Wallis test was used to perform an initial comparison across all groups and followed by pairwise comparisons with Wilcoxon’s rank-sum test. For panel G, for each genotype/time point, we used Wilcoxon’s rank-sum test to compare across the 2 treatments because data were not normally distributed. For panel H, a longitudinal mixed-effects regression model was used for statistical analysis followed by pairwise Tukey-adjusted tests evaluating the equality of means across each pair of groups at each time point. *P < 0.05, **P < 0.01, ***P < 0.001 compared with vehicle-treated groups. #P < 0.05 compared with pI:pC-treated groups. Error bars indicate the SEM for biological replicates. Blue, Samd9l-KO; black, Samd9l-WT; red, Samd9l-Mut (red); stripes, IFN-α or pI:pC; solid, vehicle. Color indicates the comparison group.