Cooperating Effect of Rps14, Csnk1a1 and miRNA145/miRNA146a Haploinsufficiency in the Activation of the Innate Immune System in Del(5q) MDS

Heterozygous deletion of RPS14 occurs in del(5q) MDS and has been linked to impaired erythropoiesis, characteristic of this disease subtype. We previously generated a mouse model with conditional inactivation of Rps14 and demonstrated a p53-dependent erythroid differentiation defect with apoptosis at the transition from polychromatic to orthochromatic erythroblasts resulting in age-dependent progressive anemia, megakaryocyte dysplasia, and loss of hematopoietic stem cell (HSC) quiescence.

We now sought to determine the mechanistic basis for the anemia using unbiased, quantitative mass spectrometry in erythroid progenitor cells. We found powerful induction of proteins involved in innate immune signaling, particularly the danger associated molecular pattern (DAMP) heterodimeric S100A8/S100A9 proteins. We found significantly increased S100a8 in the erythroid progenitor populations affected by the differentiation block (RIII-RIV population) and in monocytes and macrophages of Rps14 haploinsufficient bone marrows, all representing cells of the erythroblastic niche. Recombinant S100A8 was sufficient to impair erythropoiesis in wild-type cells. We rescued the erythroid differentiation defect in Rps14 haploinsufficient HSCs by genetic inactivation of S100a8 expression using CRISPR/Cas-mediated gene inactivation in primary mouse Rps14 haploinsufficient HSPC.

We validated the association between induction of S100A8 and a severe erythroid phenotype in bone marrow samples of patients with del(5q) MDS. To examine whether ribosomal haploinsufficiency also leads to activation of S100A8 in patients with del(5q) MDS, we measured S100A8 expression using immunofluorescence in bone marrow biopsies from MDS patients with and without del(5q). In del(5q) MDS, the frequency of S100A8-positive cells was associated with disease severity, as reflected by transfusion burden.

RPS14, CSNK1A1 and miR-145 are universally co-deleted in the 5q- syndrome and each represent different clinical features of del(5q) MDS in murine models. Haploinsufficiency of miR-145 or -146a also induces inappropriate activation of innate immune signaling. To analyze the combinatorial effect of haploinsufficiency Rps14, Csnk1a1 and miRNA-145, we transduced hematopoietic stem and progenitor cells (HSPC) from compound haploinsufficient Rps14 and Csnk1a1 mice and stably knocked down both miR-145/miR-146a by retrovirus-mediated overexpression of respective target sequences. Compound haploinsufficiency of Rps14, Csnk1a1 and miR-145/146a led to a progressive anemia comparable to Rps14 haploinsufficiency with splenomegaly and an erythroid differentiation defect at the RIII/RIV population, indicating that the anemia is mainly driven by Rps14 haploinsufficiency. Bone marrow histology demonstrated the typical 5q-phenoytpe of megakaryocytes, in line with significant thrombocytosis. At 10 months of age, hematopoietic stem and progenitor cells were significantly increased (lineage^low ckit⁺ Sca1⁺; LSK), in particular multipotent progenitor cells (MPPs; lineage^low ckit⁺ Sca1⁺ CD48^- CD150⁺) to significantly higher extents than in solely Rps14 or Csnk1a1 haploinsufficient cells.

We next asked if compound haploinsufficiency of the three 5q-genes has combinatorial or synergistic effects on S100a8 expression. Compound haploinsufficiency of Csnk1a1, Rps14 and miR-145/146a induced the highest expression of S100a8 in monocytes, while haploinsufficiency of Rps14 alone induced the highest expression of S100a8 in the RIII erythroid population, suggesting that cell-type specific induction mediates the phenotype.

Our data indicate an unexpected link between haploinsufficiency for a ribosomal gene, Rps14, activation of S100A8, and inhibition of erythropoiesis. We demonstrate that compound haploinsufficiency for Csnk1a1 and miR145/146a with Rps14 haploinsuffciency increases the expression of S100a8, mainly in monocytes, and recapitulates the phenotype of del(5q) MDS by cooperating, cell-type specific effects.