Anemic Nan mice carry a mutation (E339D) in the second zinc finger of erythroid transcription factor KLF1. Nan-KLF1 fails to bind a subset of normal KLF1 targets and ectopically binds a large set of genes not normally engaged by KLF1, resulting in a corrupted fetal liver transcriptome. Here, we performed RNAseq using flow cytometric-sorted spleen erythroid precursors from adult Nan and WT littermates rendered anemic by phlebotomy to identify global transcriptome changes specific to the Nan Klf1 mutation as opposed to anemia generally. Mutant Nan-KLF1 leads to extensive and progressive transcriptome corruption in adult spleen erythroid precursors such that stress erythropoiesis is severely compromised. Terminal erythroid differentiation is defective in the bone marrow as well. Principle component analysis reveals two major patterns of differential gene expression predicting that defects in basic cellular processes including translation, cell cycle, and DNA repair could contribute to disordered erythropoiesis and anemia in Nan. Significant erythroid precursor stage specific changes were identified in some of these processes in Nan. Remarkably, however, despite expression changes in large numbers of associated genes, most basic cellular processes were intact in Nan indicating that developing red cells display significant physiological resiliency and establish new homeostatic set points in vivo.

RASA3 is a Ras GTPase activating protein that plays a critical role in blood formation. The autosomal recessive mouse model scat (severe combined anemia and thrombocytopenia) carries a missense mutation in Rasa3. Homozygotes present with a phenotype characteristic of bone marrow failure that is accompanied by alternating episodes of crisis and remission. The mechanism leading to impaired erythropoiesis and peripheral cell destruction as evidenced by membrane fragmentation in scat is unclear, although we previously reported that the mislocalization of RASA3 to the cytosol of reticulocytes and mature red cells plays a role in the disease. In this study, we further characterized the bone marrow failure in scat and found that RASA3 plays a central role in cell cycle progression and maintenance of reactive oxygen species (ROS) levels during terminal erythroid differentiation, without inducing apoptosis of the precursors. In scat mice undergoing crises, there is a consistent pattern of an increased proportion of cells in the G0/G1 phase at the basophilic and polychromatophilic stages of erythroid differentiation, suggesting that RASA3 is involved in the G1 checkpoint. However, this increase in G1 is transient, and either resolves or becomes indiscernible by the orthochromatic stage. In addition, while ROS levels are normal early in erythropoiesis, there is accumulation of superoxide levels at the reticulocyte stage (DHE increased 40% in scat; p = 0.02) even though mitochondria, a potential source for ROS, are eliminated normally. Surprisingly, apoptosis is significantly decreased in the scat bone marrow at the proerythroblastic (15.3%; p = 0.004), polychromatophilic (8.5%; p = 0.01), and orthochromatic (4.2%; p = 0.02) stages. Together, these data indicate that ROS accumulation at the reticulocyte stage, without apoptosis, contributes to the membrane fragmentation observed in scat. Finally, the cell cycle defect and increased levels of ROS suggest that scat is a model of bone marrow failure with characteristics of aplastic anemia.

Studies of the severely pancytopenic scat mouse model first demonstrated the crucial role of RASA3, a dual RAS and RAP GTPase activating protein (GAP), in hematopoiesis. RASA3 is required for survival in utero; germline deletion is lethal at E12.5–13.5 due to severe hemorrhage. Here, conditional deletion in hematopoietic stem and progenitor cells (HSPCs) using Vav-iCre recapitulates the null phenotype demonstrating that RASA3 is required at the stem and progenitor level to maintain blood vessel development and integrity and effective blood production. In adults, bone marrow blood cell production and spleen stress erythropoiesis are suppressed significantly upon induction of RASA3 deficiency, leading to pancytopenia and death within two weeks. Notably, RASA3 missense mutations in two mouse models, scat (G125V) and hlb381 (H794L), show dramatically different hematopoietic consequences specific to both genetic background and molecular variant. The mutation effect is mediated at least in part by differential effects on RAS and RAP activation. In addition, we show that the role of RASA3 is conserved during human terminal erythropoiesis, highlighting a potential function for the RASA3-RAS axis in disordered erythropoiesis in humans. Finally, global transcriptomic studies in scat suggest potential targets to ameliorate disease progression.

Ribosomopathies are a class of disorders caused by defects in the structure or function of the ribosome and characterized by tissue-specific abnormalities. Diamond Blackfan anemia (DBA) arises from different mutations, predominantly in genes encoding ribosomal proteins (RPs). Apart from the anemia, skeletal defects are among the most common anomalies observed in patients with DBA, but they are virtually restricted to radial ray and other upper limb defects. What leads to these site-specific skeletal defects in DBA remains a mystery. Using a novel mouse model for RP haploinsufficiency, we observed specific, differential defects of the limbs. Using complementary in vitro and in vivo approaches, we demonstrate that reduced WNT signaling and subsequent increased β-catenin degradation in concert with increased expression of p53 contribute to mesenchymal lineage failure. We observed differential defects in the proliferation and differentiation of mesenchymal stem cells (MSCs) from the forelimb versus the hind limbs of the RP haploinsufficient mice that persisted after birth and were partially rescued by allelic reduction of Trp53. These defects are associated with a global decrease in protein translation in RP haploinsufficient MSCs, with the effect more pronounced in cells isolated from the forelimbs. Together these results demonstrate translational differences inherent to the MSC, explaining the site-specific skeletal defects observed in DBA.

Inherited bone marrow failure syndromes (IBMFS) are a heterogenous group of disorders characterized by dysregulated hematopoiesis across various lineages, predisposition to malignancy, and diverse syndromic features. The scat (severe combined anemia and thrombocytopenia) mouse model has been characterized as a unique model of IBMFS. Scat carries an autosomal recessive missense mutation in the Rasa3 gene that results in RASA3 mislocalization and loss of function. RASA3 functions as a Ras-GTPase activating protein, and its loss of function in scat results in increased erythroid Ras activity, increased reactive oxygen species, and altered cell cycle progression, all culminating in delayed terminal erythroid differentiation. However, the precise mechanism of RASA3 regulation of erythroid differentiation remains undefined, and elucidation of this mechanism is crucial to identifying new therapeutic targets in inherited anemia. Considering the role of RASA3 as regulator of Ras signaling and cell cycle progression, and the importance of these processes to erythroid differentiation, we sought to first characterize the coordination of Ras signaling pathways and cell cycle progression in normal murine erythropoiesis, then observe how loss of RASA3 function alters this regulatory axis. We observed that wild type (WT) erythroblasts demonstrate population-specific influence of ERK and PI3K/AKT signaling in regulating cell cycle progression. Inhibition of both pathways with increasing doses of U0126 and LY294002, respectively, induced accumulation in G0/G1 from the proerythroblast stage until the late basophilic/polychromatic stage (U0126 vehicle vs. 1uM p= 0.0023; LY294002 vehicle vs 1uM p=0.0389). At these later stages, ERK and PI3K/AKT inhibition led to a decrease in G0/G1 percentages, suggesting a stage-specific switch in signaling mediated cell cycle regulation, with PI3K inhibition demonstrating more potent and consistent effects (U0126 vehicle vs 1uM p=0.0406, 0.0481; LY294002 vehicle vs 1uM p=0.0003, 0.0197, 0.0086, n=3). These patterns suggest that ERK and AKT may facilitate cell cycle progression past the G0/G1 checkpoint in early erythropoiesis while inducing cell cycle exit or accumulation in G0/G1 in late erythropoiesis. In scat, we previously characterized increased active Ras in erythroid cells and a delay in terminal erythroid differentiation with accumulation at the polychromatic stage. We therefore next sought to examine the potential mechanistic contribution of altered PI3K/AKT signaling and cell cycle progression to the differentiation delay seen in scat. Phospho-flow analyses demonstrate that scat bone marrow-derived basophilic and polychromatic erythroblasts have increased AKT activation compared to WT (p=0.0402, p=0.0559, respectively; n=4), with similar trends evident in scat spleen basophilic erythroblasts (p=0.064; n=4). These results are consistent with increased Ras activation in scat. Ex vivo EdU/PI analyses revealed that scat bone marrow-derived polychromatic erythroblasts demonstrate G0/G1 accumulation (p=0.0466) and decreased progression to S-phase (0.0414; n=6), also with similar trends in scat spleen basophilic erythroblasts (p=0.004; n=6). These results correlate with the observed differentiation delay in scat and indicate that RASA3 regulates stage-specific signaling and cell cycle progression during erythropoiesis. To study if cell cycle dysregulation in scat begins at an earlier stage of erythroid differentiation, we analyzed murine hematopoietic and erythroid progenitors as Ter119-, cKit+ cells expressing increasing levels of CD71 and found that both CD71lo and CD71med bone marrow-derived scat progenitors present with G0/G1 accumulation (p=0.0015, p=0.0073, respectively) and decreased progression to S-phase (p=0.0014, p=0.0241; n=7). This suggests a dynamic relationship between RASA3, Ras signaling, and cell cycle progression throughout early and late erythroid differentiation. Together, these findings support the role of RASA3 as a regulator of the signaling networks governing erythropoiesis and reveal a new targetable axis in a model of inherited bone marrow failure. Disclosures No relevant conflicts of interest to declare.