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We describe the clinical course of a 65-year-old male patient who suffered from hydrocarbon-induced myelodysplasia and was successfully treated with the thrombopoietin receptor agonist (TPO-RA), romiplostim. Myelodysplastic syndromes (MDS) are characterized by ineffective hematopoiesis, cytopenias, and increased risk of leukemic transformation. Here, we present a clinical vignette of MDS-associated thrombocytopenia refractory to first-line drugs as well as the TPO-RA, eltrombopag. To date, romiplostim is an U.S. Food and Drug Administration (FDA)-approved drug for idiopathic thrombocytopenic purpura and thrombocytopenia secondary to liver disease. Of note, currently the FDA advises against its use in MDS based on previous long-term safety concerns. Since the therapeutic options for thrombocytopenia in MDS patients are sparse, repurposing and reassessing romiplostim in this setting have been the focus of recent studies. At the time of writing, no published double-blind randomized clinical trials have conducted a head-to-head comparison between romiplostim and eltrombopag in thrombocytopenic MDS patients. To the best of our knowledge, for a thrombocytopenic patient in the setting of MDS, this is the first documented report of refractory clinical response after a 2-year use of eltrombopag in which replacement of treatment with romiplostim resulted in sustained physiological counts of thrombocytes within four weeks.

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., Author summary Hematopoiesis is the process by which blood cells are formed. An individual must have a normal complement of red blood cells to prevent anemia, platelets to control bleeding, and white blood cells to maintain immune functions. All blood cells are derived from hematopoietic stem cells that differentiate into progenitor cells that then develop into mature circulating cells. We studied several mouse strains carrying different mutations in the gene encoding RASA3 and human CD34+ cells, which can be induced to produce blood cells in culture. We show that RASA3 is required at the earliest stages of blood formation, the stem and progenitor cells, and that the complement of genes other than RASA3, or the genetic background, profoundly alters the overall effect on blood formation. Further, the molecular nature of the mutation in RASA3 also has a profound and independent effect on overall blood formation. One mutant mouse strain, designated scat, suffers cyclic anemia characterized by severe anemic crisis episodes interspersed with remissions where the anemia significantly improves. Comparison of scat crisis and remission hematopoietic stem and progenitor cells reveals striking differences in gene expression. Analyses of these expression differences provide clues to processes that potentially drive improvement of anemia in scat and provide new avenues to pursue in future studies to identify novel therapeutics for anemia.

Inherited bone marrow failure syndromes (IBMFS) are heterogeneous disorders characterized by dysregulated hematopoiesis in various lineages, developmental anomalies, and predisposition to malignancy. The scat (severe combined anemia and thrombocytopenia) mouse model is a model of IBMFS with a phenotype of pancytopenia cycling through crises and remission. Scat carries an autosomal recessive missense mutation in Rasa3 that results in RASA3 mislocalization and loss of function. RASA3 functions as a Ras-GTPase activating protein (GAP), and its loss of function in scat results in increased erythroid RAS activity and reactive oxygen species (ROS) and altered erythroid cell cycle progression, culminating in delayed terminal erythroid differentiation. Here we sought to further resolve the erythroid cell cycle defect in scat through ex vivo flow cytometric analyses. These studies revealed a specific G0/G1 accumulation in scat bone marrow (BM) polychromatophilic erythroblasts and scat BM Ter119(−)/c-KIT(+)/CD71(lo/med) progenitors, with no changes evident in equivalent scat spleen populations. Systematic analyses of RNAseq data from megakaryocyte-erythroid progenitors (MEPs) in scat crisis vs. scat partial remission reveal altered expression of genes involved in the G1-S checkpoint. Together, these data indicate a precise, biphasic role for RASA3 in regulating the cell cycle during erythropoiesis with relevance to hematopoietic disease progression.

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.

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.

The Ras GTPase-activating protein, RASA3, plays a critical non-redundant role in vertebrate erythropoiesis. The autosomal recessive scat mouse model carries a missense (G125V) mutation in Rasa3 that results in severe anemia, thrombocytopenia and leukopenia with markedly reduced survival. Terminal erythroid differentiation is dysregulated in scat bone marrow (BM) and spleen with delayed cell cycle progression, decreased apoptosis, and increased reactive oxygen species. To further study the role of RASA3 in hematopoiesis, we created a conditional Rasa3 allele. Germline null mice die at E12.5-13.5 of severe hemorrhage, pallor and reduced fetal liver size. Specific ablation of Rasa3 in hematopoietic stem and progenitor cells (HSPCs) and endothelial cells (ECs) driven by Vav-Cre and Tek-Cre recapitulates the null phenotype, highlighting the specific requirement of RASA3 in hematopoietic and vascular compartments. To study RASA3 deficiency specifically in terminal erythropoiesis, we utilized EpoR-Cre. Surprisingly, EpoR-Cre driven deletion does not produce an abnormal erythroid phenotype, and normal levels of RASA3 protein are seen on western blots of Ter119+ spleen and BM cells despite efficient deletion of the floxed Rasa3 allele, suggesting that RASA3 produced early in HSPCs is sufficient to maintain normal terminal erythroid differentiation. Indeed, deletion of Rasa3 in the entire hematopoietic system at 8 weeks of age via inducible Mx1-Cre results in death within 2 weeks of induction with profound anemia, thrombocytopenia, and leukopenia, recapitulating the scat phenotype. While HSC numbers are normal in the RASA3 depleted BM of this model, the frequency and absolute numbers of all progenitors are significantly decreased, and spleen stress erythropoiesis is significantly blunted. Altered Ras activity is associated with delayed erythropoiesis. ERK signaling, a key downstream Ras effector, is hyperactivated 3-fold by western blotting in scat BM CD45-depleted cells vs. wild type (WT). To extend RASA3 studies to human cells, we utilized cord blood CD34+ cells in a three-phase erythroid differentiation culture system. Similar to the mouse models, shRNA knockdown of RASA3 leads to delayed erythroid differentiation at culture day 11 based on glycophorin A, Band 3 and a4-integrin expression levels. Dysregulated cell cycle progression with accumulation in the S-phase is also evident at day 12 of culture. Unlike the mouse models, however, ERK activation was decreased (67-72%) in serum-starved sorted proerythroblasts and later day 11 populations of RASA3 knockdown cells treated with erythropoietin (Epo), stem cell factor (SCF), or both. To elucidate additional molecular mechanisms downstream of RASA3, we performed RNAseq on total spleen and BM, as well as sorted stem and myeloid progenitors (SMPs) of scat mice and WT littermate controls. Principle component analysis confirms that genotype is the primary contributing factor to differential gene expression. In preliminary analyses, MAPK signaling, including negative regulators of ERK, phosphatidyl-inositol signaling, cell cycle regulation, and transcriptional regulation emerge from pathways and GO term analyses of differentially expressed genes. In conclusion, we show that (1) absence of RASA3 results in embryonic lethality with evidence of severe HSPC/EC defects; (2) RASA3 is required for hematopoiesis starting at the HPC stage or earlier; (3) missense mutations (scat) or loss of RASA3 in adults cause pancytopenia and premature death; (4) mechanistically, aberrant ERK signaling and cell cycle defects contribute to the RASA3 deficient erythroid phenotype, while RNAseq studies provide further potential mechanistic targets. Disclosures No relevant conflicts of interest to declare.

RASA3, a Ras GTPase activating protein, is critical to vertebrate erythropoiesis and megakaryopoiesis. The autosomal recessive mouse model scat (severe combined anemia and thrombocytopenia) carries a G125V mutation in Rasa3 that leads to profound bone marrow failure with characteristics of aplastic anemia. The phenotype is cyclic, and mice alternate between periods of crisis and remission. Our previous studies demonstrated that this mutation in Rasa3 causes defects in several aspects of erythropoiesis, including a significant delay of erythroid differentiation at the polychromatophilic stage, decreased hemoglobinization, defects in cell cycle progression past the G1 checkpoint, and increased reactive oxygen species (ROS) during terminal erythroid differentiation as well as in scat peripheral blood reticulocytes and red blood cells. We previously reported that the mislocalization of mutated RASA3 to the cytosol of reticulocytes and mature red cells plays a role in the erythropoietic defect in scat, and the observed cell cycle arrest and increased ROS likely also contribute to this unique disease phenotype. Our current efforts are focused on further elucidation of the mechanism and specific disruptions in Ras signaling that lead to anemia, membrane fragmentation, and the cyclic phenotype in scat. Interestingly, we report here that apoptosis is not increased during scat crisis, and that mitochondria, a potential source of ROS, are normally eliminated at the reticulocyte stage. The dramatic nature of remission, with complete normalization of all hematologic parameters, led us to hypothesize that a secreted factor may be mediating the cyclic phenotype of scat. Differences in the cytokine profile of the serum of scat mice compared to wild type suggest that, indeed, one or several secreted factor(s) may be influencing the occurrence of bone marrow failure. Levels of galectin-1, a known mediator of cell-cell interactions and intracellular signaling in the hematopoietic niche, are consistently decreased in scat serum according to a multispot anti-cytokine antibody array (23,326.5 ± 21,439.7 integrated density in scat vs. 31,019.6± 20,110.7 in controls, p Strengthening the notion that RASA3 has a critical conserved role in vertebrate terminal erythropoiesis, the characteristics of bone marrow failure seen in scat have been reproduced in human CD34+ cells using siRNA and shRNA knockdowns of Rasa3 . Similar to the changes seen in scat, cells with decreased RASA3 demonstrated delayed terminal erythroid differentiation and defective hemoglobinization. Finally, analysis of Ras expression and functional pull-down studies in human CD34+ cells revealed that, while K-Ras is the major active isoform expressed during terminal erythroid differentiation, H-Ras is also active during human erythropoiesis. Future studies with CD34+ Rasa3 knockdown cells will explore the influence of RASA3 on human K- and H-Ras signaling in erythropoiesis. Taken together, our studies further characterize the vital role of RASA3 in hemoglobinization, cell cycle progression, and cell survival during terminal erythroid differentiation, as well as identify novel targets for investigation of unknown mechanisms (e.g., dysregulated cytokine secretion) of bone marrow failure syndromes. Disclosures No relevant conflicts of interest to declare.