Myelodysplastic Syndrome Mice Research Articles

Genetic iron overload aggravates, and pharmacological iron restriction improves, MDS pathophysiology in a preclinical study

AbstractAlthough iron overload is a common feature in myelodysplastic syndromes (MDSs), it remains unclear how iron excess is detrimental for disease pathophysiology. Taking advantage of complementary approaches, we analyzed the impact of iron overload and restriction achieved through genetic activation of ferroportin (FPN) via the C326S mutation (FPNC326S) and pharmacologic inhibition (vamifeport) of the iron exporter FPN, respectively, in a MDS mouse model. Although FPNC326S-induced iron overload did not significantly improve the late stages of erythroid maturation, vamifeport-mediated iron restriction ameliorated anemia and red blood cell maturation in MDS mice, through the reduction of oxidative stress and apoptosis in erythroid progenitors. Iron overload aggravated, and restriction alleviated, reactive oxygen species formation, DNA damage, and cell death in hematopoietic stem and progenitor cells, resulting in altered cell survival and quality. Finally, myeloid bias, indicated by expanded bone marrow myeloid progenitors and circulating immature myeloid blasts, was exacerbated by iron excess and attenuated by iron restriction. Overall, vamifeport treatment resulted in improved anemia and significant survival increment in MDS mice. Interestingly, the combined therapy with vamifeport and the erythroid maturation agent luspatercept has superior effect in improving anemia and myeloid bias as compared with single treatments and offers additive beneficial effects in MDS. Our results prove, to our knowledge, for the first time in a preclinical model, that iron plays a pathologic role in transfusion-independent MDS. This is likely aggravated by transfusional iron overload, as suggested by observations in the FPNC326SMDS model. Ultimately, the beneficial effects of pharmacologic FPN inhibition uncovers the therapeutic potential of early prevention of iron toxicity in transfusion-independent MDS.

GSH exhaustion via inhibition of xCT-GSH-GPX4 pathway synergistically enhanced DSF/Cu-induced cuproptosis in myelodysplastic syndromes

The clinical application of the therapeutic approach in myelodysplastic syndromes (MDS) remains an insurmountable challenge for the high propensity for progressing to acute myeloid leukemia and predominantly affecting elderly individuals. Thus, the discovery of molecular mechanisms underlying the regulatory network of different programmed cell death holds great promise for the identification of therapeutic targets and provides insights into new therapeutic avenues. Herein, we found that disulfiram/copper (DSF/Cu) significantly repressed the cell viability, increased reactive oxygen species (ROS) accumulation, destroyed mitochondrial morphology, and altered oxygen consumption rate. Further studies verified that DSF/Cu induces cuproptosis, as evidenced by the depletion of glutathione (GSH), aggregation of lipoylated DLAT, and induced loss of Fe–S cluster–containing proteins, which could be rescued by tetrathiomolybdate and knockdown of ferredoxin 1 (FDX1). Additionally, GSH contributed to the tolerance of DSF/Cu-mediated cuproptosis, while pharmacological chelation of GSH triggered ROS accumulation and sensitized cell death. The xCT-GSH-GPX4 axis is the ideal downstream component of ferroptosis that exerts a powerful protective mechanism. Notably, classical xCT inhibitors were capable of leading to the catastrophic accumulation of ROS and exerting synergistic cell death, while xCT overexpression restored these phenomena. Simvastatin, an inhibitor of HMG-CoA (3-hydroxy-3-methylglutaryl-coenzyme A) reductase, has beneficial effects in repurposing for inhibiting GPX4. Similarly, the combination treatment of DSF/Cu and simvastatin dramatically decreased the expression of GPX4 and Fe–S proteins, ultimately accelerating cell death. Moreover, we identified that the combination treatment of DSF/Cu and simvastatin also had a synergistic antitumor effect in the MDS mouse model, with the reduced GPX4, increased COX-2 and accumulated lipid peroxides. Overall, our study provided insight into developing a novel synergistic strategy to sensitize MDS therapy by targeting ferroptosis and cuproptosis.

HMGA2 promotes platelet-neutrophil complex formation and pulmonary tissue damage in myelodysplastic syndromes

AbstractHigh mobility group AT-hook 2 (HMGA2) is an architectural transcription factor that functions as an oncogene in various cancers. Although overexpression of HMGA2 has been reported in several myeloid malignancies, its role varies considerably in different disease contexts. Here, we identified a distinct role of HMGA2 as a mediator of noninfectious pneumonia in myelodysplastic syndrome (MDS). The expression level of HMGA2 in CD34+ hematopoietic stem cells and progenitors (HSC/Ps) was significantly associated with the incidence of noninfectious pneumonia, a common systemic complication in patients with MDS. Consistent with this clinical investigation, HMGA2 overexpression in a mouse model of an MDS-associated mutation led to the development of lethal noninfectious pneumonia. Mechanistically, HMGA2 overexpression conferred a megakaryocytic lineage bias to HSC/Ps and contributed to platelet activation in MDS mice. P-selectin–positive activated platelets interacted with MDS clone–derived neutrophils that exhibit increased susceptibility to cell death and formed platelet-neutrophil complexes (PNCs). Both the frequency of PNCs and neutrophil cell death within the lung microenvironment increased in MDS mice overexpressing HMGA2. Genetic inhibition of P-selectin attenuated pulmonary tissue damage in MDS mice. These findings indicate that PNCs could be a new therapeutic target for noninfectious pneumonia in patients with MDS and provide new insights into the mechanistic basis of the systemic complications of MDS.

Pathogenesis of myelodysplastic syndromes by excessive mitochondrial fragmentation

Myelodysplastic syndromes (MDS) are a group of heterogenous hematopoietic stem cell (HSC) malignancies characterized by ineffective hematopoiesis in which clonal progenitor expansion occurs alongside impaired myelopoiesis. Inflammatory signaling activation due to dysregulated innate immunity is also a hallmark of MDS pathogenesis. We recently established a useful preclinical tool that recapitulates bona fide MDS phenotypes and gene expression profiles based on previously unreported co-mutations discovered during our clinical surveillance of mutations in patients with MDS. Notably, we focused unbiased transcriptome analysis on determining the distinct underlying mediators of MDS etiology, and identified excessive mitochondrial fission-mediated fragmentation in mutant HSCs and progenitors (HSC/Ps). We confirmed excessive mitochondrial fragmentation in HSC/Ps obtained from patients with MDS regardless of the mutational profile. Importantly, in vivo pharmacological inhibition of mitochondrial fission significantly attenuated inflammatory signaling activation, dysplasia formation and ineffective hematopoiesis phenotype, and prolonged survival of MDS mice, suggesting that excessive mitochondrial fragmentation could be a fundamental trigger of MDS pathogenesis. These findings provide new insights into the mechanistic basis of ineffective hematopoiesis, and a clue for targeting bone marrow failure caused by ineffective hematopoiesis in MDS.

Iron chelation improves ineffective erythropoiesis and iron overload in myelodysplastic syndrome mice.

Myelodysplastic syndrome (MDS) is a heterogeneous group of bone marrow stem cell disorders characterized by ineffective hematopoiesis and cytopenias, most commonly anemia. Red cell transfusion therapy for anemia in MDS results in iron overload, correlating with reduced overall survival. Whether the treatment of iron overload benefits MDS patients remains controversial. We evaluate underlying iron-related pathophysiology and the effect of iron chelation using deferiprone on erythropoiesis in NUP98-HOXD13 transgenic mice, a highly penetrant well-established MDS mouse model. Our results characterize an iron overload phenotype with aberrant erythropoiesis in these mice which was reversed by deferiprone-treatment. Serum erythropoietin levels decreased while erythroblast erythropoietin receptor expression increased in deferiprone-treated MDS mice. We demonstrate, for the first time, normalized expression of the iron chaperones Pcbp1 and Ncoa4 and increased ferritin stores in late-stage erythroblasts from deferiprone-treated MDS mice, evidence of aberrant iron trafficking in MDS erythroblasts. Importantly, erythroblast ferritin is increased in response to deferiprone, correlating with decreased erythroblast ROS. Finally, we confirmed increased expression of genes involved in iron uptake, sensing, and trafficking in stem and progenitor cells from MDS patients. Taken together, our findings provide evidence that erythroblast-specific iron metabolism is a novel potential therapeutic target to reverse ineffective erythropoiesis in MDS.

Combination Therapy with Luspatercept and the Ferroportin Inhibitor Vamifeport Is Superior to Either Drug Alone in Improving Anemia and Reducing Myeloid Skewing in MDS

Background: Patients with myelodysplastic syndromes (MDS) are prone to develop iron overload as a consequence of ineffective erythropoiesis and chronic transfusion therapy. Although iron overload is a common feature in MDS, it has remained unclear whether and how iron excess is detrimental for MDS pathophysiology. Recently, we have shown that iron restriction by the oral ferroportin (FPN) inhibitor vamifeport improves anemia and reduces myeloid skewing, being of benefit for MDS as single treatment. Luspatercept is a recombinant fusion protein acting as TGF-β superfamily ligands trap which has recently gained FDA approval for the treatment of anemia in transfusion-dependent low-risk MDS patients. Luspatercept promotes erythropoietin (EPO)-independent maturation of late-stage erythroid cells, resulting in increased hemoglobin and red blood cells (RBCs) count. However, this drug - due to a lack of improvement in myeloid skewing and AML evolution - is considered a non-disease-modifying therapy. Aims: In this study we investigated the effect of a combined therapy using luspatercept and the FPN inhibitor vamifeport in a preclinical MDS mouse model, with the hypothesis that both drugs could have additive effects with further benefit for the disease. Methods: To this end, we administered the FPN inhibitor vamifeport in NUP98-HOXD13 MDS mice for 3 months (0.5 mg/ml into drinking water), combined with luspatercept for the last 2 months (5 mg/kg twice a week, subcutaneous injection), starting at 3 months of age. Mice receiving the combined treatment were compared to those untreated or receiving single treatments. Results: At steady-state MDS mice develop anemia, neutropenia and lymphopenia, together with an iron overload phenotype, hallmarked by inappropriately low hepcidin levels, elevated serum iron and transferrin saturation, non-transferrin-bound iron (NTBI) formation and tissue iron deposition. Vamifeport administration in MDS mice reduced serum iron and NTBI formation and prevented tissue iron loading, either as a single or combined treatment with luspatercept. Luspatercept and vamifeport, as single-agent treatment, ameliorated anemia and the maturation of bone marrow and splenic RBCs in MDS mice. The combined therapy further improved anemia compared to the treatments with either drugs alone, as suggested by the higher hemoglobin levels, hematocrit and RBC count, and the decreased number of early erythroid precursors, sign of more effective erythropoiesis. Importantly, vamifeport, but not luspatercept, significantly altered myeloid skewing in MDS, revealing a major role of the iron status in myeloid expansion. Myeloid bias, monitored as percentage of CD11b + Gr1 + myeloid cells in the bone marrow was attenuated by iron restriction in vamifeport-treated MDS mice compared to control and luspatercept-treated MDS animals. The combined treatment merkedly reduced myeloid skewing in MDS mice, similarly to the single vamifeport treatment. Furthermore, the number of immature cKit + myeloid blasts in the peripheral blood of MDS animals were decreased by vamifeport alone and in combination with luspatercept, but not by luspatercept alone. Finally, splenomegaly was reduced in MDS mice treated with vamifeport alone or in combination with luspatercept, confirming that iron restriction improves ineffective erythropoiesis and limits myeloid skewing. Overall, the combination therapy with vamifeport and luspatercept showed superior effects in improving anemia and myeloid bias as compared to single treatments. Conclusion: In conclusion, our results show that iron restriction by vamifeport enhances the erythroid maturation action of luspatercept, likely by improving iron utilization in erythroid cells and ameliorating their survival. Furthermore, vamifeport improves the myeloid skewing in the MDS model, suggesting disease-modifying activity as single agent as well as combined therapy with luspatercept. Together, these data prove that combo therapies aimed at restricting iron and boosting erythroid maturation may offer additive beneficial effects in MDS and provide pre-clinical evidence for combining iron restriction and TFG-β superfamily ligand-trap approaches as more effective therapeutic strategies for the treatment of MDS.

Myelodysplastic Syndromes Benefit from Iron-Restricted Bone Marrow Transplant in Preclinical Mouse Model

Background: Hematopoietic stem cell transplantation (HSCT) remains the only fully curative therapy for diseases such as b-thalassemia, sickle cell disease, MDS and AML and represents a relevant procedure for gene therapy .Tissue iron overload and non-transferrin bound iron (NTBI) have emerged as adverse prognostic factors for post-transplant survival. Recently, we showed that - in line with observations in transplanted individuals - preparatory chemotherapy conditioning with a combination of busulfan and fludarabine in wild-type mice resulted in elevated serum iron and transferrin (Tf) saturation and elicited NTBI formation. Tf saturation and NTBI levels increased proportionally with conditioning intensity. Interestingly, we found that pre-conditioning TMPRSS6 silencing through a GalNAc-conjugated siRNA - by increasing hepcidin levels - reduced the peri-transplant elevation of Tf saturation and limited the appearance of NTBI. These results proved that NTBI formation is induced by conditioning chemotherapy in the peri-trasplant period independently of the presence of pre-existing iron overload. Aims & Methods: Here we asked whether these observations hold true in conditions hallmarked by pre-existing iron overload and elevated NTBI and how iron restriction impacts on bone marrow transplant (BMT) outcome. To this aim, we applied the GalNAc TMPRSS6 siRNA as iron restriction strategy in a mouse model of myelodysplastic syndromes (MDS) and assessed its ability to reduce conditioning-elicited NTBI formation as well as its potential benefit on BMT. After TMPRSS6 siRNA administration, CD45.2 + MDS mice received conditioning chemotherapy and transplant of CD45.1 + wild-type bone marrow cells. Systemic iron parameters, engraftment and chimerism were then analyzed. Results: Importantly, conditioning exacerbated the elevated systemic iron levels in MDS mice, which was prevented by pre-conditioning TMPRSS6 silencing. Similar to our observation in wild-type mice, recipient CD45.2 + MDS mice showed a superior blood chimerism, resulting from increased bone marrow and spleen engraftment of CD45.1 + donor cells. Multilineage engraftment in bone marrow, spleen and peripheral blood was superior in iron-restricted recipient MDS mice compared to controls. Finally, donor chimerism of hematopoietic stem and progenitor cells (HSPCs) was higher in iron-restricted versus control MDS mice undergoing BMT. Conclusion: These findings demonstrate that pre-transplant conditioning profoundly alters iron homeostasis and restricting iron improves BMT outcome by increasing engraftment efficiency. Overall, these data suggest that conditioning-elicited NTBI is a critical mediator of peri-transplant toxicity and adversely impacts transplant outcome. Specifically, our observations prove that iron restriction improves donor HSPC engraftment in the recipient bone marrow, likely resulting in more efficient reconstitution of the HSPC pool and improved multilineage engraftment. Lastly, our results indicate that the peri-transplant application of novel iron restriction strategies aimed at reducing conditioning-induced NTBI - by positively affecting BMT outcome - is of potential benefit for patients undergoing BMT or gene therapy, presenting either with or without pre-transplant iron overload.

AG-946, an Activator of Pyruvate Kinase, Improves Ineffective Erythropoiesis in the Bone Marrow of Mouse Models of Myelodysplastic Syndromes

Background: Myelodysplastic syndromes (MDS) are a heterogeneous group of hematologic malignancies characterized by ineffective erythropoiesis. Anemia is the most common cytopenia and major clinical problem in MDS. Acquired pyruvate kinase (PK) deficiency has been observed in MDS, and a direct impact on the glycolytic pathway in the pathogenesis of anemia associated with MDS has been demonstrated. AG-946 is an investigational, small-molecule, allosteric activator of PK that has the potential to enhance red blood cell (RBC) functionality and survival by increasing glycolysis and adenosine triphosphate (ATP) production, and improve differentiation of erythroid cells in bone marrow, potentially improving anemia caused by ineffective erythropoiesis in MDS. Previous findings showed that RBCs from patients with MDS treated ex-vivo with AG-946, led to an increase in PK activity. Here, we used two MDS-related mouse models to evaluate the effects of the PK activator AG-946 in this mechanism of anemia. Aim: Evaluate the effect of AG-946 treatment on impaired erythropoiesis in the bone marrow of two mouse models of MDS. Methods:NUP98-HOXD13 (NHD13) is a fusion gene mutation observed in some patients with MDS. Mouse models with this mutation develop key characteristics of MDS such as impaired erythropoiesis and progressive anemia leading to malignant leukemia around 12 months of age. Male and female NHD13 and C57BL/6J mice were placed on 0.006% AG-946 (approximate dose 10 mg/kg/day) formulated diet starting at 10 months given ad libitum and continuing for 3 months. Numbers of animals for wild type (WT) Control, WT AG-946, NHD13 Control, NHD13 AG-946 were 10, 10, 13, and 11, respectively.Polg D257A (Polg) mouse model, while not a mutation observed in patients with MDS, does display many key features of MDS, such as impaired erythropoiesis and progressive anemia. Male and female Polg mice were placed on 0.006% AG-946 (approximate dose 10 mg/kg/day) formulated diet starting at 4 months given ad libitum and continuing for 8 months. Numbers of animals for WT Control, WT AG-946, Polg Control, Polg AG-946 were 10, 10, 19, and 18, respectively. Bone marrow from 1 femur per mouse was collected and analyzed by flow cytometry. Bone marrow cell pellets were washed and treated with ACK lysing buffer prior to staining. Samples with fewer than 1000 Ter119+ events were removed from analysis due to insufficient cell count. Bone marrow erythroblast populations were gated using Single Cells/Live/B220-/Ter119+/CD44 vs FSC, which were then gated into three populations: basophilic erythroblasts (BasoE), polychromatic erythroblasts (PolyE), and orthochromatic erythroblasts and reticulocytes (OrthoE + Retic). Results:No differences in any erythroblast populations were observed between untreated WT and NHD13 mice. No changes in the BasoE population were observed in WT or NHD13 mice. The PolyE population in NHD13 mice treated with AG-946 was significantly decreased compared with both the NHD13 and WT untreated mice (p=0.0021 and 0.0003, respectively). NHD13 mice treated with AG-946 showed significant increases in the OrthoE + Retic populations over the untreated NHD13 mice (p=0.0275).No differences in BasoE or OrthoE + Retic populations were observed between untreated WT and Polg mice while the PolyE population was significantly decreased in Polg mice compared with WT (p<0.0001). The BasoE population was significantly decreased in Polg mice treated with AG-946 compared with untreated Polg (p=0.0002). No difference in PolyE population was observed in Polg mice with AG-946. OrthoE + Retic population was significantly increased in Polg mice treated with AG-946 compared with WT and Polg untreated mice (p=0.0001 and 0.0304, respectively). Data graphed as average +/- SEM. Conclusions: Our data suggest AG-946 treatment improves erythroblast maturation in two MDS mouse models as demonstrated by a reduction in early-stage erythroblasts (BasoE, PolyE) coupled with an increase in late-stage erythroblast populations (OrthoE + Retic). These data are the first to suggest that PK activation by AG-946 could improve ineffective erythropoiesis in MDS.

Genetic Iron Overload Aggravates and Pharmacological Iron Restriction Improves MDS Pathophysiology in a Preclinical Mouse Model

Background: Patients with myelodysplastic syndromes (MDS) are prone to develop iron overload as a consequence of ineffective erythropoiesis and chronic transfusion therapy. Although iron overload is a common feature in MDS, it remains unclear whether and how iron excess is detrimental for MDS pathophysiology. Aims: This study aimed at characterizing altered iron homeostasis in a preclinical MDS mouse model, understanding the molecular mechanisms underlying iron toxicity and unraveling the potential therapeutic value of pharmacologic iron restriction in this disease condition. Methods: To this end, we took advantage of complementary approaches and analyzed the effect of iron overload obtained through genetic activation of the iron exporter ferroportin (FPNC326S), and iron restriction achieved through the administration of the FPN inhibitor vamifeport in NUP98-HOXD13 MDS mice, starting at 3 or 5 months of age. Results: At steady-state MDS mice develop an iron overload phenotype, hallmarked by low hepcidin levels, elevated serum iron and transferrin saturation, non-transferrin-bound iron (NTBI) formation and tissue iron deposition. This is associated with anemia and low WBCs in the peripheral blood. FPNC326S MDS mice show an aggravated iron phenotype, with further elevated NTBI and tissue iron accumulation and still inappropriately low hepcidin levels. Vamifeport administration in MDS mice reduced serum iron (194.8 vs 141.8 mg/dl; P<0.05) and NTBI formation (0.62 vs 0 μM; P<0.05) and prevented tissue iron loading. While iron excess in FPNC326S MDS mice did not improve erythropoiesis and hematologic parameters in the peripheral blood, iron restriction by vamifeport significantly ameliorated anemia (Hb 9.5 vs 11.4 g/dl; RBC 5.4 vs 6.9x106 cells/ml; P<0.05) and maturation of bone marrow as well as splenic RBCs in MDS mice. The improved ineffective erythropoiesis was associated with reduced oxidative stress and apoptosis in erythroid progenitors. Importantly, we observed a major role of the iron status in myeloid expansion in MDS. The number of immature myeloid blasts and myeloid progenitors in the bone marrow of MDS mice were aggravated by iron overload in FPNC326S MDS mice and attenuated by iron restriction in vamifeport-treated MDS mice compared to control MDS animals. Myeloid bias, monitored as percentage bone marrow myeloid cells, was exacerbated by iron overload (54.3 vs 43.1% bone marrow mononuclear cells - BMNCs) and alleviated by iron restriction (67.5 vs 74.8% BMNCs; P<0.05). This was associated with increased and reduced inflammatory activation of bone marrow macrophages in FPNC326S MDS and vamifeport-treated MDS mice, respectively. This observation demonstrates a role for NTBI in driving the reported pro-inflammatory activation of MDS monocytes/macrophages (Fuster JJ et al, Science 2017; Jaiswal S et al, NEJM 2017; Cull AH et al, Exp Hematol 2017) and suggests that this iron-driven mechanism contributes to myeloid expansion. Overall, these alterations translated into a faster and delayed transition to AML in iron-overloaded and iron-restricted MDS mice, respectively. Finally, iron overload aggravated and iron restriction alleviated ROS formation, DNA damage and pyroptotic cell death in HSPCs. By reducing the intracellular labile iron pool, vamifeport improved HSPC quality and survival, partially rescuing the stem cell pool size (0.16 vs 0.38% Lin-BMNCs; P<0.05) in MDS mice. The ameliorated HSPC pool, reduced myeloid bias and inflammation, and attenuated ineffective erythropoiesis resulted in improved hematologic parameters for more than 3 months, and a significant survival increment in iron-restricted compared to control MDS mice (>45 days; P=0.037). We further demonstrate that a subgroup of mice can benefit from late iron restriction by vamifeport administration at 5 months of age, resulting in improved anemia and prolonged survival. Conclusion: Our results show for the first time in a preclinical mouse model of MDS that iron excess driven by ineffective erythropoiesis has pathological implication in transfusion-independent MDS. These effects are likely aggravated by transfusions causing additional iron overload. Finally, iron restriction achieved through pharmacologic FPN inhibition by the oral FPN inhibitor vamifeport significantly improves MDS pathophysiology, uncovering the therapeutic potential of early prevention of NTBI formation in MDS.

Iron Chelation Improves Ineffective Erythropoiesis and Iron Overload in a Mouse Model of Myelodysplastic Syndrome

Myelodysplastic syndrome (MDS) is a heterogeneous group of bone marrow stem cell disorders characterized by ineffective hematopoiesis and cytopenias, most commonly anemia. Patients are treated with periodic red cell (RBC) transfusions, resulting in iron overload. Iron overload in MDS patients correlates with decreased survival, yet despite the availability of effective, safe, and orally administered iron chelation therapy, the treatment of iron overload in MDS remains controversial. The TELESTO trial, using deferasirox in low risk MDS patients, revealed an increase in event-free survival without impacting overall survival, hemoglobin, or RBC transfusion requirements [Angelucci Ann Int Med 2020]. However, iron chelators have different affinities for iron relative to transferrin; deferiprone has the lowest affinity of available iron chelators [Sohn Blood 2008], consistent with its ability to increase hepcidin expression in mice [Schmidt Am J Hematol 2015; Casu haematol 2016]. We hypothesize that deferiprone (DFP) impacts systemic and cellular iron distribution to enhance erythropoiesis in MDS. To test this hypothesis, we employed a highly penetrant well-established mouse model of MDS, NUP98:HOXD13 fusion transgenic mice. These MDS mice exhibit hypercellular marrow, increased apoptosis in the bone marrow compartment, and impaired survival relative to control; a proportion of mice progress to acute leukemic transformation after 7 months of age [Lin Blood 2005]. Using MDS mice, we further characterize parameters of iron trafficking in erythroblasts, evaluate the effects of DFP on erythropoiesis and iron distribution, and provide correlatory evidence in stem and progenitor cells from MDS patients. MDS mice were treated at 5 months of age with 1.25 mg/mL DFP in the drinking water for 4 weeks and compared with untreated MDS mice. Our results characterize an iron overload phenotype with aberrant erythropoiesis in MDS mice, reversed in DFP-treated MDS mice (Figure 1). Specifically, DFP leads to a redistribution of iron from liver and spleen to increase serum iron concentration and transferrin saturation, increased hemoglobin and RBC concentration in circulation, and normalized hepcidin responsiveness to iron. While MDS mice exhibit relatively decreased WBC count, no changes in WBC count are observed in DFP-treated MDS mice. Importantly, serum erythropoietin (Epo) concentration, splenomegaly, expanded erythropoiesis and erythroblast differentiation in the bone marrow are normalized in DFP-treated MDS mice. As expected when erythropoiesis is reversed, erythroblast expression of Fam132b (erythroferrone) is suppressed but no changes in STAT5 or AKT signaling, pathways downstream of Epo, are evident in DFP-treated MDS mice. These findings suggest that the expected changes in signaling downstream of Epo are unaffected by DFP administration, implicating possible changes in erythroblast Epor expression in DFP-treated MDS mice. We used sorted bone marrow to evaluate Epor expression in progressive stages of terminal erythropoiesis and demonstrate decreased Epor expression in later stage bone marrow erythroblasts from MDS mice, normalized in DFP-treated MDS mice. These findings provide evidence that increased Epor expression is a potential mechanism by which DFP leads to enhanced Epo-responsiveness and enhanced erythroblast differentiation despite decreased serum Epo concentration in MDS mice. In addition, we demonstrate for the first time increased ferritin concentration and normalized Tfr1, Pcbp1 and Ncoa4 expression in late stage erythroblasts from DFP-treated MDS mice, evidence of aberrant iron trafficking in MDS erythroblasts and their normalization with DFP treatment (Figure 2). Importantly, this effect is not observed in DFP-treated WT mice. Our data also provides evidence that reversal of ineffective erythropoiesis in DFP-treated MDS mice occurs independently of effects on erythroblast apoptosis. Finally, we confirm increased expression of genes involved in Epo-mediation as well as iron uptake and trafficking-Epor, Fam132b, Tfr1, Pcbp1 and Ncoa4-in stem and progenitor cells from MDS patients. Taken together, our findings provide evidence that erythroblast-specific iron metabolism is a novel potential therapeutic target to reverse ineffective erythropoiesis in MDS and provide a rationale for exploring the effect of DFP in patients with low risk MDS. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Mlkl Mediates Age-Related Attrition of Hematopoietic Stem Cells and Ineffective Hematopoiesis in Myelodysplastic Syndrome

Aging causes anemia, perturbed immunity, and increased propensity to age-related hematological diseases such as myelodysplastic syndrome (MDS). Age-dependent functional decline in hematopoietic stem cells (HSCs), which is accompanied by morphological and functional changes in their organelles as well as sterile inflammation in the bone marrow (BM), is one of the main reasons why the hematopoietic system ages. However, molecular mechanisms by which HSCs alter their function during aging remain unclear. Necroptosis is a form of programmed necrosis that underlies sterile inflammation and can take place upon exposure to inflammatory stimuli in apoptosis-resistant cells including HSCs. MLKL, the effector molecule of necroptosis that can form channel-like structure on lipid bilayers upon activation, not only induces cell death but also injures membrane-bound organelles such as mitochondria, though its significance in HSC aging remains elusive. Our quantitative RT-PCR revealed abundant expression of MLKL in murine BM immature hematopoietic cells including HSCs (Lin-/cKit+/Sca1+/Flk2-/CD48-/CD150+), which was validated by reanalysis of the published proteome datasets (Zaro et al., eLife 2020). Using FRET-based necroptosis reporter mice and Western blotting analysis, we found that while MLKL was inactivated at the steady state, it was readily activated in HSCs upon poly I:C injection. To study the effect of MLKL activation on HSCs, we assessed cell death of HSCs isolated from poly I:C-treated MLKL-deficient (Mlkl-/-) mice. Unexpectedly, Annexin V/DAPI staining revealed no changes in the frequencies of dying and dead cells between wild-type (WT) and Mlkl-/- HSCs. Nonetheless, PVA-based HSC culture revealed that the growth suppression observed in poly I:C-treated WT HSCs largely depended on MLKL (p=0.0003). Furthermore, HSC transplantation assay revealed that reduced engraftment and skewed myeloid differentiation were observed in poly I:C-treated WT HSCs but largely alleviated by MLKL deficiency (p=0.011). These results indicate that inflammation-induced MLKL activation does not cause HSC death but impairs their growth and lymphopoietic potential. To assess the relevance of MLKL activation in HSC aging, we treated WT and Mlkl-/- mice with serial rounds of 5-FU treatment, which were shown to induce age-related alterations in HSCs (Beerman et al., Cell Stem Cell 2013). Of note, we observed marked attenuation of age-related features such as myeloid-skewed hematopoiesis and expansion of phenotypic HSCs in 5-FU-treated Mlkl-/- mice (p=0.0051). HSC transplantation experiments further revealed that MLKL deficiency alleviated 5-FU-induced decrease in engraftment and lymphopoietic potential of HSCs (p=0.013). Indeed, analysis of 18-month-old WT and Mlkl-/- mice demonstrated that MLKL deficiency largely rescued myeloid-skewed hematopoiesis and loss of BM CLPs (Lin-/cKitlo/Sca1lo/Flk2+/IL-7Rα+) (p=0.0047), and serial HSC transplantation revealed better self-renewal and lymphopoietic potential of aged Mlkl-/- HSCs (p=0.0055). Importantly, multiplex ELISA assay using BM supernatants revealed no changes in concentrations of inflammatory cytokines, indicating that the observed effects in Mlkl-/- HSCs were likely caused by cell intrinsic changes. In line with this, electron microscopy analysis revealed that age-related changes in mitochondria, such as elongated and swollen structure with disorganized cristae, were significantly attenuated in aged Mlkl-/- HSCs (p<0.0001). JC-1 dye staining further revealed that age-related accumulation of damaged mitochondria was significantly attenuated in Mlkl-/- HSCs (p=0.049). Finally, to evaluate the effect of MLKL on development of age-related hematological disease, we established MDS mouse models by transplantation of RUNX1S291fs-transduced WT and Mlkl-/- HSCs. We found that WT MDS mice mainly died of severe anemia due to ineffective hematopoiesis, whereas Mlkl-/- MDS mice showed milder anemia and better overall survival (p=0.044), indicating that MLKL promotes ineffective hematopoiesis in MDS. Taken together, our results identify MLKL as a key mediator of age-related functional decline in HSCs and ineffective hematopoiesis in MDS. They also suggest that pharmacological inhibition of MLKL activity is a promising strategy to better control aging of the hematopoietic system and age-related hematological disease.

Rker-050, a Novel Activin Receptor Type II Ligand Trap, Rescued Anemia and Reduced Bone Loss in a Mouse Model of Myelodysplastic Syndromes

Myelodysplastic syndromes (MDS) are a group of hematopoietic stem cell diseases characterized by ineffective hematopoiesis, peripheral blood cytopenias and dysplasias resulting from molecular defects and dysregulated signaling that govern the regulation of hematopoietic stem and progenitor cells (HSPCs). Crosstalk between cells in the osteo-hematopoietic (O-H) niche are necessary for the maintenance and self-renewal of HSPCs. Additionally, MDS patients commonly present with osteopenia and osteoporosis further demonstrating the critical interplay between the O-H cells in the bone marrow (BM). Alterations in the interaction between bone precursors and HSPCs within the BM microenvironment have been linked to the pathophysiology of MDS. Transforming growth factor-beta (TGF-β) superfamily signaling is instrumental in regulating both normal hematopoiesis and bone homeostasis. KER-050 is a modified investigational activin receptor type II ligand trap designed to bind and inhibit select TGF-β superfamily ligands, including activin A, activin B, GDF8 and GDF11, to promote hematopoiesis and bone growth. Preclinical studies have demonstrated that treatment with a research form of KER-050 (RKER-050) increased bone mass and multi-lineage hematopoiesis in healthy mice. To further investigate the effect of RKER-050 on hematopoietic and bone systems within the MDS disease setting, the NUP98/HOX13 MDS mouse model was utilized. Five-month-old male MDS mice were treated with vehicle (VEH, n=10), or RKER-050 (050, 7.5mg/kg, n=9) once weekly for 6 weeks along with a wildtype vehicle (WT, n=7) control group. To assess changes in hematological parameters, CBCs were measured, bone marrow (BM) and splenocytes were characterized via flow cytometry. To investigate alterations of bone microarchitecture, femurs were assessed using microCT imaging. MDS-VEH mice showed a reduction in red blood cells (RBC; 30%), hemoglobin (HGB; 19%), hematocrit (HCT; 13%) and reticulocytes (Retic; 16%) compared to WT; all hallmarks of human MDS. Relative to MDS-VEH, MDS-050 showed increased RBC, HGB, HCT and Retic by 25%, 13%, 10% and 39%, respectively. The erythroid Ter119 positive (Ter119+) cell population was slightly reduced in BM and spleen of MDS-VEH mice compared to WT. The BM Ter119+ cells were unchanged in the MDS-050 cohort relative to MDS-VEH; however, the spleen Ter119+ cells were increased similar to the levels in WT mice. Specific erythroid precursor populations were increased in BM and spleen of MDS-VEH mice versus WT, consistent with the ineffective erythropoiesis in this model. Interestingly, erythroid precursor populations were similar between MDS-VEH and MDS-050, yet MDS-050 exhibited recovered RBC production, suggesting an increase in maturation of erythroid precursors with RKER-050 treatment. Given the importance of the O-H niche in regulating hematopoiesis along with osteoporosis and osteopenia being comorbidities of MDS, bone microarchitecture was also analyzed. In the distal femoral trabecular bone, relative to WT mice, MDS-VEH mice had significant increases in trabecular (Tb) separation (33%) and decreases in Tb number (28%), changes that are consistent with reduced bone strength. Bone volume (34%) and bone volume fraction (26%) decreased but were not statistically significant. MDS-050 treated mice had a significant increase in bone volume (71%), higher bone volume fraction (88%), increased Tb number (44%) and decreased Tb spacing (20%) compared to the MDS-VEH, supporting RKER-050's role in improving bone mass. Taken together, these results suggest that RKER-050 can mitigate anemia and bone loss in an MDS mouse model, potentially by rebalancing hematopoiesis and bone turnover. In particular, the ability of RKER-050 to improve bone microarchitecture within the trabecular region, where the O-H niche resides, could be an important step in reestablishing healthy blood cell production. Furthermore, exploratory PD data from an ongoing phase 2 clinical trial demonstrate improved blood parameters with KER-050 treatment, including platelet and RBC in MDS patients (Tan et al. 2022 EHA #P776), and changes in bone biomarkers are currently being evaluated in the study. KER-050, thus, represents a potentially promising approach for patients with MDS and other hematological diseases, including myelofibrosis, where ineffective hematopoiesis and defects in bone homeostasis occur.

The NLRC4 Inflammasome Drives Myelodysplastic Syndrome By Linking Epigenetic Reprogramming and Innate Immune Signaling

Introduction Myelodysplastic syndromes (MDS) are a heterogeneous group of hematopoietic stem cell (HSC) malignancies characterized by dysplastic and ineffective hematopoiesis. Despite emerging evidence that chronic inflammation contributes to the development of MDS, the mechanisms underlying the dysregulation of innate immune and inflammatory signaling pathways in hematopoietic stem and progenitor cells (HSPCs) remain poorly characterized. We have used multiple MDS mouse models to identify a tumor suppressive role for the EP300 epigenetic histone acetyltransferase enzyme in the progression of MDS to AML. Based on the gene expression changes we observed, we have used one specific mouse model, that lacks Tet2 expression, to define the role that chronic inflammation plays in promoting MDS development and the mechanisms involved. Methods We have conducted a series of in vitro, in vivo, and in silico experiments using Tet2-deficient mice, and chronic, low-dose LPS treatment to model chronic inflammation. Cytokine array immunoassays were performed to evaluate the inflammatory response of WT or Tet2-deficient mice to LPS treatment. Chimeric mice transplanted with bone marrow (BM) cells isolated from LPS or vehicle (PBS) treated WT or Tet2-deficient mice were monitored for the development of MDS and survival. Serial competitive bone marrow transplantation assays and flow cytometry profiling were used to determine the competitive advantage of LPS or PBS-treated BM cells, and their differentiation. Bioinformatic analyses of RNA sequencing (RNA-seq) results from our mouse model were performed to characterize the transcriptional profiling and identify novel innate immune pathways. Colony-forming unit (CFU) and qPCR assays were used to evaluate HSPC proliferative capacity, self-renewal, and gene expression changes. Results We find that mice lacking Tet2 develop a hyper-inflammatory status, marked by the expression of a number of pro-inflammatory cytokines, such as Il-1b and Il-6, which are further fueled by LPS-induced chronic inflammation. Chimeric mice transplanted with BM cells isolated from LPS-treated Tet2-deficient mice exhibit a significantly shorter life span and early onset of MDS phenotypes, including dysplastic hematopoiesis and thrombocytopenia, compared to age-matched chimeric mice transplanted with BM cells isolated from PBS-treated Tet2-deficient mice or LPS-treated WT mice. Chronic inflammation promotes the self-renewal and myeloid differentiation of Tet2-deficient HSPCs in the short term; however, it accelerates HSC exhaustion in the long run. Transcription profiling and subsequent bioinformatic analyses identify functional clusters in LPS-treated Tet2-deficient HSPCs, including genes involved in the regulation of epigenetics, immunity, metabolism, and cell cycle. Detailed inspection of the most highly differentially expressed genes reveals that many genes in the Nlrc4 inflammasome pathway are upregulated in LPS-treated Tet2-deficient HSPCs. We validate changes in the expression of these genes by qPCR, and then show that deleting Nlrc4 suppresses the colony formation of Tet2-deficient HSPCs in vitro. Ongoing work focuses on evaluating the effects of deleting Nlrc4 or blocking Nlrc4 function using a small molecule inhibitor on MDS development in vivo. Conclusions The combinatory role of intrinsic and extrinsic factors drives the development of MDS in this Tet2-deficient MDS mouse model. It is likely that different genetic mutations generate unique sensitivity to certain signaling pathways, and we demonstrate the epigenetic rewiring and activation of the Nlrc4 inflammasome pathway as a mechanistic basis for the inflammatory component of MDS [or clonal hematopoiesis of indeterminate potential (CHIP)] pathogenesis in Tet2-deficient cells. Our work has the potential to provide novel targeting opportunities in MDS management and confirm the importance of different components of inflammasome signaling in MDS.

Activation of SIRT1 Deacetylase As a Therapeutic Approach for Myelodysplastic Syndromes By Restoring TET2 Function

Myelodysplastic syndromes (MDS) which arise from a primitive CD34+ hematopoitic stem/progenitor cell (HSPC), are incurable by non-transplant therapy. Better understanding of MDS HSPC regulation is critical for developing targeted therapies against the fatal disease. Tet methylcytosine dioxygenase 2 (TET2), initiating DNA demethylation through converting 5-methylcytosine to 5-hydromethylcytosine (5hmC), is the most frequently mutated gene in MDS. TET2 alterations lead to DNA hypermethylation and dysregulated gene expression in HSPCs, resulting in enhanced self-renewal and skewed myeloid specific expansion. While TET2's biological function has been well studied, the knowledge of its regulation is limited. We had demonstrated the TET2 c-terminus acetylation was reversed by SIRT1 deacetylase which maintains normal HSPCs activity under stress. We also observed that SIRT1 protein in MDS CD34+CD38- was expressed below normal levels, and that knockdown (KD) of SIRT1 enhanced in vivo growth of MDS-L cells, a patient derived MDS cell line, is associated with TET2 acetylation (Blood. 2016;128:1053). These results indicate an important role for SIRT1 in MDS HSPCs maintenance.To further assess SIRT1 function in MDS, we ectopically expressed SIRT1 in primary MDS CD34+ cells enriched for HSPCs and MDS-L cells, respectively. Overexpression (OE) of SIRT1 resulted in inhibition of colony formation of MDS CD34+ cells (p<0.01, n=3) but not normal counterpart (p>0.05, n=3). SIRT1 OE also suppressed MDSL colony formation (p<0.01) and cell growth (p<0.01). SIRT1 OE significantly reduced MDS-L cell BM engraftment in NSGs mice relative to controls (human CD45+ cells: 5.7±0.8% of BM in SIRT1 OE group vs 31.4 ± 5.8% in control, p<0.01, n=7), resulting in delayed mortality of recipient mice (median survival: 52 days vs 35 days; p<0.05, n=5). Meanwhile, treatment of SIRT1 agonist SRT1720, reduced colony formation of primary MDS CD34+ cells in a dose dependent manner (p<0.01, n=9). Similar results were also achieved in SRT1720 treated MDS-L cells. Furthermore, TET2 was hyperacetylated in primary MDS cells (n=3) compared to normal counterpart (n=3). Next, we tested whether inhibition of MDS HSPCs following SIRT1 activation was related to TET2 activation via protein deacetylation. Indeed, enhanced TET2 activity marked by 5hmC was observed in MDS CD34+ cells upon SRT1720 treatment (P<0.05, n=3). TET2 KD diminished growth inhibition induced by SIRT1 OE or SRT1720, indicating a requirement of TET2 for SIRT1 function. Through mass spectrometry analysis, three conserved acetylated lysine residuals (K1472, K1473, K1478) were identified in TET2 catalytic domain (CD). Engineered lysines to glutamines (TET2 3KQ) mutant, mimicking acetylated TET2, produced lower 5hmC, and led to increased colony growth compared to MDS-L expressing TET2 WT (p<0.01). We further evaluated the effects of SRT1720 using NUP98-HOXD13 (NHD13)MDS mouse. This murine MDS is transferable and secondary recipients receiving primary NHD13 MDS cells succumb to MDS at 4 months, allowing generation of a cohort of MDS mice for preclinical studies of therapeutics against MDS. CD45.2+ BM cells from donor NHD13+ mice showed evidence of dysplasia were harvested, and transplanted into preconditioned congenic recipients expressing CD45.1. SRT1720 treatment (100mg/kg/day for 12 weeks, p.o.) resulted in partial reversal of MDS-like disease developed in recipients receiving MDS cells, including an increase in HGB, PLT, RBC and WBC (p<0.01,n=6). Importantly, significant reduction of NHD13+ CD45.2+ chimerism in LSK and other progenitors from BM were also observed (p<0.01, n=6). As expected, 5hmC level in lineage depleted cells from SRT1720 treated mice was increased (p<0.01). Secondary transplantation was performed to evaluate the effects of SRT1720 on MDS initiating cells. We observed that the mice receiving cells from SRT1720 treated donors showed reduced NHD13+ CD45.2+ cell long-term engraftment (p<0.01, n=5).In summary, we demonstrated that genetic overexpression or pharmacological activation of SIRT1 drastically inhibited MDS maintenance, and that TET2 function was required for the inhibitory effect of SIRT1. These results support the hypothesis that SIRT1 deficiency promotes MDS cell expansion through TET2 inactivation and support the rationale for exploring SIRT1 activation as a promising approach to selectively target MDS stem cells. DisclosuresNo relevant conflicts of interest to declare.

The High NRF2 Expression Confers Chemotherapy Resistance through Upregulated DUSP1 in Myelodysplastic Syndromes

Myelodysplastic syndromes (MDS) are a diverse group of clonal marrow stem cell disorders characterized by ineffective hematopoiesis and dysplasia of the cellular elements. Although low-dose cytarabine (Ara-c) has been considered as one of the frontline therapies for high-risk MDS, the overall response rate of single Ara-c treatment was only 30%~40%. Nuclear factor erythroid 2-related factor 2 (NRF2), a basic leucine zipper transcription factor, has been reported to play a pivotal role in chemical detoxification. It is not yet known whether NRF2 has utility as a prognostic biomarker, or whether an elevated NRF2 level is associated with Ara-c resistance in MDS patients.To investigate the expression and effects of NRF2 in MDS patients, gene set enrichment analysis (GSEA) and immunohistochemistry (IHC) were performed. GSEA in a published MDS cohort (n=183) revealed that expressions of NRF2 target genes was significantly enriched in high risk MDS (REAB-1/2) compared to low-risks (RARS and RA). We further detected NRF2 expression levels using IHC staining in the bone marrow (BM) biopsies from a cohort of MDS patients (n=111). Consistent with GSEA results, our IHC data indicated that the NRF2 expression levels of BM from high-risks exceeded low-risk MDS patients by Revised International Prognosis Scoring System (IPSS-R). Importantly, MDS patients with higher NRF2 levels (IHC scores, 4~6) displayed worse overall survival (OS) than patients with lower NRF2 levels (IHC scores, 0~3) (median, 15.7 vs. 22.7 months, P=0.007). Subgroup analysis showed that higher NRF2 levels correlated with poor OS only in chemotherapy group (P=0.04) but not in decitabine (P=0.38) or supportive care group (P=0.19).To recapitulate our findings regarding the function of NRF2 in chemo-resistance, we performed in-vitro and in-vivo experiments. All experiments were performed both in human MDS-derived cell line SKM-1 and MDS mouse model cell line RUNX1 mutant-transduced MllPTD/WT BM cells (MllPTD/WT/RUNX1-S291fs). The cytotoxicity of Ara-c was determined by calculating the half maximal inhibitory concentration (IC50). NRF2 downregulation in MDS cell lines, by inhibitor luteolin, increases IC50 of Ara-c. On the contrary, upregulation of NRF2, mediated by agonist sulforaphane, induces resistance of MDS cells to Ara-C. To better define how suppression of NRF2 sensitized MDS cells to Ara-c, we used lentivirus-mediated shRNA for knockdown of NRF2. Knockdown of NRF2 markedly enhanced early and late apoptosis in MDS cell lines treated with Ara-c. We also established xenograft mouse models through intravenously injecting NRF2 shRNA or scramble shRNA SKM-1 cells into NOD/SCID-IL2Rγnull-SGM3 (NSGS) mice. Knockdown of NRF2 significantly sensitized tumors to Ara-c in our SKM-1 transplanted mouse model. To further investigate the mechanisms involved in NRF2-mediated Ara-c resistance, we analyzed published gene-expression profiles of a large cohort of MDS patients (n=183) and Ara-C sensitive (IC50 <6 μM AraC) and resistant (IC50 >80 μM) AML patient samples (n=10). The data indicated that a group of NRF2 target genes might be responsible for Ara-C resistence. Interestedly, DUSP1 was one of the genes upregulated in both high-risk MDS patients and Ara-c resistant AML patients. Our chromatin immunoprecipitation (ChIP) and qPCR results validated that DUSP1 was a NRF2 direct target gene. Importantly, downregulation of DUSP1 by inhibitor or lentivirus shRNA could abrogate Ara-c resistance in NRF2 elevated MDS cell lines, indicating that NRF2 confers Ara-c resistance partly through its downstream target gene DUSP1 in MDS cells.In conclusion, our clinical and experimental results reveal that 1) NRF2 expression levels are elevated in high-risk MDS patients and serve as a statistically significant prognostic variable for OS in patients with MDS, especially for MDS patients who have received chemotherapy. 2) Pharmacological inhibition of NRF2 re-sensitizes MDS cells to Ara-c treatment while activation of NRF2 by agonist resulted in the reduced sensitivity to Ara-c. 3) NRF2 mediates Ara-c resistance through its direct target gene DUSP1 . Taken together, our findings suggest that silencing NRF2 sensitizes MDS cells to Ara-c treatment; targeting NRF2 in combination with conventional chemotherapy could pave the way for high risk MDS therapy. DisclosuresNo relevant conflicts of interest to declare.

Ker-050, a Modified Actriia Ligand Trap, Alleviates Cytopenia Arising from Multiple Etiologies

Hemopoietic system control the production of circulating blood cells that have important functions from transport of oxygen and carbon dioxide, blood clotting to fighting infections. Not surprisingly, the system is tightly regulated and failure to replenish RBC can result in anemia and inadequate platelets increases the risk of bleeding. Impaired hematopoiesis and the consequential cytopenias are associated with aging, diseases characterized by ineffective hematopoiesis including myelodysplastic syndrome (MDS) and myelofibrosis and diseases that lead to loss of growth factors. A therapeutic that could act more globally on the hematopoietic pathway would have the potential to overcome cytopenia in many diseases. Signaling of the TGFβ superfamily regulates several stages of RBC maturation, and recent studies demonstrate that inhibition of TGFβ signaling can induce RBC production and increase circulating RBCs, HGB, and hematocrit (HCT). KER-050, a modified ActRIIA ligand trap, has been shown to increase RBCs in rodents and non-human primates, and to increase both RBCs and PLTs in healthy human volunteers. In the current studies, we aimed to test the efficacy of KER-050 in alleviating cytopenias caused by multiple conditions and to further delineate the effect of KER-050 on platelets. First, we examined whether RKER-050 (a research form of KER-050) can reverse anemia associated with aging and frailty. After 6 weeks of twice weekly treatment, 2-year-old aged, vehicle-treated (AV) mice had significantly lower RBCs, HGB, and HCT (-14.0%, -13.5%, -10.9%, respectively) relative to 11-week-old young vehicle-treated mice (YV). However, aged mice treated with RKER-050 had higher RBCs, HGB, and HCT (+12.3%, +10.0%, +9.1%, respectively) compared to AV, with levels indistinguishable from those of YV. These data support that RKER-050 can improve anemia that arises from aging. Next, we evaluated the efficacy of RKER-050 in the treatment of anemia in an animal model of MDS. NUP98-HOXD13 mice, a murine model of MDS, aged to 6 months and confirmed as anemic prior to treatment, were dosed twice weekly with either vehicle or RKER-050 for 6 weeks. Over this treatment period, vehicle-treated MDS mice continued to have significantly reduced RBCs, HGB, and HCT compared to wildtype controls. In contrast, RKER-050-treated MDS mice had increases in RBCs, HGB, and HCT (+10.9%, +11.2%, + 9.8%, respectively), achieving values comparable to the wild type control animal of the same age. These data support that RKER-050 reverses anemia in a mouse model of MDS. Finally, we evaluated whether RKER-050 can improve anemia after acute blood loss. Anemia was induced by bleeding 20% of total blood volume in Sprague Dawley rats followed by treatment with RKER-050 twice weekly. After phlebotomy, the RKER-050-treated group had early, robust increases in both RBCs and HGB that exceeded baseline levels, whereas decreased RBCs and HGB in the vehicle-treated group persisted longer. Moreover, while vehicle PLTs were unchanged over the study, RKER-050 treatment resulted in an increase in PLT count at Day 3 post-phlebotomy which remained elevated at Day 6. These data suggest that in cases of acute bleeding, RKER-050 not only rapidly increases RBCs but also PLTs, potentially demonstrating a pancytopenic effect of RKER-050. Our data suggest that RKER-050 is a fast-acting modulator of RBC maturation that rapidly increases RBCs, HGB, and HCT. The increases were observed in aged mice showing signs of age-associated anemia, as well as in mice with chronic conditions such as MDS. Moreover, RKER-050 showed rapid recovery in a model of acute bleeding with an effect on both erythropoiesis and thrombopoiesis. These results suggest that KER-050 could be developed for the treatment of anemias and potentially other cytopenias, including thrombocytopenia, arising from a variety of causes. Disclosures Feigenson: Keros Therapeutics: Current Employment. Nathan:Keros Therapeutics: Current Employment. Babbs:Keros Therapeutics: Current Employment. Materna:Keros Therapeutics: Current Employment. Tseng:Mitobridge: Current equity holder in private company; Keros Therapeutics: Current Employment, Current equity holder in publicly-traded company, Patents &amp; Royalties. Fisher:Keros Therapeutics: Current Employment, Current equity holder in publicly-traded company. Seehra:Keros Therapeutics: Current Employment, Current equity holder in publicly-traded company. Lachey:Keros Therapeutics: Current Employment, Current equity holder in publicly-traded company.

R-2HG/KDM2B/RIPK1 Signaling Mediates Necroptosis in Myelodysplastic Syndromes

Introduction: Myelodysplastic syndromes (MDS) are a group of heterogeneous hematopoietic stem cell disorders and manifested as ineffective hematopoiesis, refractory cytopenia and a propensity to evolve into acute myeloid leukemia (AML). Isocitrate dehydrogenase 1/2 (IDH1/2) mutations are common in both MDS and AML. Mutated IDH produces R-2-hydroxyglutarate (R-2HG) which inhibits multiple α-ketoglutarate/α-KG-dependent dioxygenases by competing against α-KG binding. Recent studies have demonstrated that R-2-HG can abrogates leukemic growth and induce leukemia cell death. Several nonapoptotic cell death have been identified, including phosphoribosyl pyrophosphate (PPRP)-1 mediated necrosis, pyroptosis, ferroptosis and necroptosis. By now, the specific type of cell death by which R-2-HG exerts anti-tumor effects is still unknown. Results: (1) (2R)-Octyl-2-HG exhibited anti-tumor effect on SKM-1, THP-1, Molm-13, HL-60 cell lines and bone marrow mononuclear cells from MDS and AML patients in a dose- and time-dependent manner. The overexpression of IDH2 mutation induced by doxycycline significantly decreased the viability of AML cell lines, and the inhibition rate was related to the dose of doxycycline. (2R)-Octyl-2-HG promotes apoptosis and causes G0/G1 phase arrest. (2) R-2-HG leads to increased expression of RIPK1 in high-risk MDS cells. The results of gene enrichment analysis indicated that the apoptotic pathway was enriched in (2R)-Octyl-2-HG groups, and the expression of RIPK1 gene was increased in all three (SKM-1, NOMO-1 and MA9.3ITD) (2R)-Octyl-2-HG groups. After treatment with (2R)-Octyl-2-HG, the mRNA and protein expression levels of RIPK1 gene were increased. (3) R-2-HG triggers RIPK1-dependent necroptosis and occurs earlier than apoptosis. Within 10 hours after treatment with (2R)-Octyl-2-HG, cell membrane permeability was disrupted, interactions between RIPK1 and caspase 8 increased, as well as phosphorylated MLKL level. However, caspase activity did not increase significantly, suggesting that necroptosis occurs earlier than apoptosis. With RIPK1 inhibitor Necrostatin-1 and (2R)-Octyl-2-HG co-treating cells, proliferation inhibition was reduced, cell membrane permeability was more stable and RIPK1-caspase8 complex was difficult to form. The same phenomenon occurs in SKM-1 cells stably transfected with RIPK1 shRNA virus, suggesting that RIPK1-dependent necroptosis is involved in cell death caused by (2R)-Octyl-2-HG. (4) In vivo experiments demonstrated that necroptosis by R-2-HG is dependent on the expression of RIPK. In RIPK1 shRNA MDS mice, the tumor burden showed a decreasing trend, but did not show a significant change in the R-2-HG treatment group. Treatment of scramble shRNA MDS mice with R-2-HG resulted in significantly smaller tumors in the spleen and less engrafment of CD45+ cells in bone marrow. (5) Low RIPK1 expression predicts poor prognosis in MDS patients. Data from the TCGA and GEO public databases indicate that RIPK1 expression is reduced in MDS and AML patients compared to healthy controls. Survival analysis showed that patients with lower RIPK1 expression levels had significantly shorter overall survival (OS) than patients with higher RIPK1 expression levels. MDS patients with lower RIPK1 expression levels progress to leukemia more frequently. (6) Inhibition of KDM2B induces necroptosis independently. Western blot assay shows the knockdown of KDM2B, upregulation of RIPK1 and increased levels of p-MLKL. Analysis of cell numbers showed that proliferation ceased from 4 days after doxycycline treatment onwards in shKDM2B cells. Co-IP assay shows the formation of RIPK1-caspase8 complex. Conclusion: This study confirmed that R-2-HG inhibited the viability of MDS and AML cells. R-2-HG increased the expression of RIPK1 in MDS cells, inducing necroptosis. Necroptosis occurred earlier than apoptosis. Inhibition of RIPK1 can alleviate the inhibitory effect of R-2-HG on MDS and AML cells. Clinical studies have shown that low expression of the RIPK1 gene is associated with poor prognosis in patients with MDS and AML. MDS patients with low expression of RIPK1 was more likely to progress to leukemia. Inhibition of KDM2B can induce necroptosis independently. Disclosures No relevant conflicts of interest to declare.

GDF1 Regulation of Ceramide Metabolism Restores Effective Hematopoiesis in Myelodysplastic Syndrome

Myelodysplastic syndrome (MDS) is a clonal hematopoietic disorder characterized by ineffective hematopoiesis, cytopenias, and an increased risk of transformation to acute myeloid leukemia. New studies are urgently needed to understand the underlying processes that govern MDS and related bone marrow failure disorders and to develop better therapeutic modalities. Sphingolipids are an extensive classification of lipids which play prominent roles in cellular signaling in addition to being essential components of membranes. The most well studied sphingolipid is ceramide, which serves as a hypothetical center of sphingolipid metabolism. Ceramide is an important cellular signal that can lead to apoptosis. Overall ceramide biology is significant because novel metabolic routes that persist in MDS may be exploited for therapeutic development. Interestingly, the gene encoding ceramide synthase 1 is encoded from a bicistronic transcript that also encodes for GDF1. Little is known about the roles of GDF1 outside of cardiac development. However, GDF1 is in the TGF-beta superfamily of which many members have been attributed roles in stem cell biology. Therefore, a better understanding of the role of GDF1 and its regulation of ceramide metabolism and hematopoiesis may lead to better treatment approaches for MDS and other bone marrow failure syndromes. The current study tested an overarching hypothesis that GDF1 regulates SMAD and STAT signaling to promote ceramide generation and restore effective myelopoiesis in MDS. Initially, the expression of GDF1 was evaluated in hematopoietic cells isolated from transgenic murine models of MDS (Nup98-HoxD13; Srsf2P95H-mutant) as well as myeloproliferation (Flt3ITD). Interestingly, GDF1 expression was greatest in the bone marrow of MDS models. This was significant because we recently reported that nanoliposomal ceramide (Lip-C6), which delivers a bioactive ceramide analog, exerts unique therapeutic efficacy towards acute myeloid leukemia arising out of MDS (Barth et. al. Blood Advances 2019). Lip-C6 is a ceramide-based therapy that currently is in a clinical trial for solid tumor malignancies (ClinicalTrials.gov identifier: NCT02834611). Next, treatment of transgenic MDS mice with either recombinant GDF1 or Lip-C6 was shown to stimulate the expansion of erythroid progenitors. This was concomitant with a decrease in immature myeloid cells, which was revealed to be due to granulo-monocytopoietic differentiation. Mechanistic studies subsequently revealed that GDF1 increased SMAD2/3 phosphorylation while simultaneously down regulating STAT3 (Y705) phosphorylation, both in a TGF-beta receptor 1-dependent fashion. This dual effect was unique to GDF1, whereas TGF-beta or GDF3 only recapitulated individual the effects on SMAD2/3 and STAT3 signaling, respectively. Finally, STAT3 binding sites were identified in the promoter region of the ceramide detoxifying enzyme glucosylceramide synthase. This is important because glucosylceramide synthase expression was shown to be downregulated by recombinant GDF1 treatment. Therefore, GDF1-mediated downregulation of STAT3-dependent glucosylceramide synthase expression provides a mechanistic link where GDF1 can augment intracellular ceramide levels. Overall, this study enhances our understanding of a fundamental hematopoietic process where GDF1 regulates ceramide metabolism and myelopoiesis. This is impactful because the effects of GDF1, which are unique from other related factors including TGF-beta and GDF3, are due to a novel mechanism of action that both upregulates SMAD2/3 signaling while downregulating STAT3 signaling. More so, this study demonstrated that GDF1, and/or ceramide, can exert an anti-MDS therapeutic role by restoring normal aspects of myelopoiesis. This work was funded by NIH/NCI K22 CA190674 (B.M.B.) and University of New Hampshire COBRE Pilot Project Grant NIH/NIGMS P20 GM113131 (B.M.B.). The authors acknowledge US Provisional Patent 62/602,437, issued to B.M.B. and the University of New Hampshire. Disclosures Barth: University of New Hampshire: Patents &amp; Royalties: US Provisional Patent 62/602,437; NIH (NCI and NIGMS): Research Funding. Loughran:Bioniz: Membership on an entity's Board of Directors or advisory committees; Keystone Nano: Membership on an entity's Board of Directors or advisory committees.

Effects of rigosertib on the osteo-hematopoietic niche in myelodysplastic syndromes

Rigosertib is a novel multi-kinase inhibitor, which has clinical activity towards leukemic progenitor cells of patients with high-risk myelodysplastic syndromes (MDS) after failure or progression on hypomethylating agents. Since the bone marrow microenvironment plays an important role in MDS pathogenesis, we investigated the impact of rigosertib on cellular compartments within the osteo-hematopoietic niche. Healthy C57BL/6J mice treated with rigosertib for 3weeks showed a mild suppression of hematopoiesis (hemoglobin and red blood cells, both - 16%, p < 0.01; white blood cells, - 34%, p < 0.05; platelets, - 38%, p < 0.05), whereas there was no difference in the number of hematopoietic stem cells in the bone marrow. Trabecular bone mass of the spine was reduced by rigosertib (- 16%, p = 0.05). This was accompanied by a lower trabecular number and thickness (- 6% and - 10%, respectively, p < 0.05), partly explained by the increase in osteoclast number and surface (p < 0.01). Milder effects of rigosertib on bone mass were detected in an MDS mouse model system (NHD13). However, rigosertib did not further aggravate MDS-associated cytopenia in NHD13 mice. Finally, we tested the effects of rigosertib on human mesenchymal stromal cells (MSC) in vitro and demonstrated reduced cell viability at nanomolar concentrations. Deterioration of the hematopoietic supportive capacity of MDS-MSC after rigosertib pretreatment demonstrated by decreased number of colony-forming units, especially in the monocytic lineage, further supports the idea of disturbed crosstalk within the osteo-hematopoietic niche mediated by rigosertib. Thus, rigosertib exerts inhibitory effects on the stromal components of the osteo-hematopoietic niche which may explain the dissociation between anti-leukemic activity and the absence of hematological improvement.

Impairment of the Stromal SDF-1-Mediated Hematopoietic Support By GDF-11 in MDS Is Rescued By Luspatercept

Introduction:Myelodysplastic syndromes (MDS) are characterized by ineffective hematopoiesis due to genetic and functional abnormalities of hematopoietic stem and progenitor cells (HSPCs). Accumulating evidence now also points to the bone marrow microenvironment (BMME) as a key mediator of MDS pathophysiology.Increased levels of transforming growth factor beta (TGF-β) superfamily ligands, including growth differentiation factor 11 (GDF-11), in the bone marrow have been linked to ineffective erythropoiesis and activation of SMAD2/3 signalling in MDS. Luspatercept (ACE-536) is a novel recombinant fusion protein containing modified activin receptor type IIB linked to the fragment crystallisable (Fc) domain of human immunoglobulin G1 and is a first-in-class erythroid maturation agent. Luspatercept binds to selected TGF-β superfamily ligands, including GDF-11 and activin B, restoring late-stage erythropoiesis in MDS mice and patients. Whether luspatercept modulates the BMME is unknown.Aim:We investigated the potential impact of luspatercept on the biology of mesenchymal stromal cells (MSCs).Methods:MSCs from patients with either high-or low-risk MDS and age-matched healthy donors (HD) were treated with GDF-11 in the presence or absence of RAP-536, a homologue of luspatercept harboring the same activin receptor IIB domain. Subsequently, Smad signaling pathway activation was analyzed by Western blot. Moreover, MSC phenotype, stromal derived factor-1 (SDF-1/CXCL12) expression and secretion as well as the osteogenic differentiation potential were recorded. To study the impact on the hematopoietic support, MSCs were pre-treated for one week with GDF-11 ± RAP-536 before freshly isolated CD34+ HSPCs were seeded on the stromal layer. The colony formation (CAF-C) was analyzed weekly. After four weeks, the HSPCs were seeded in methylcellulose medium to perform a CFU-GEMM assay. Further, engraftment and migration potential of co-cultured HSPCs was analyzed in zebrafish embryos in vivo.Results:GDF-11/RAP-536 treatment did not impact viability, proliferation, and growth pattern of MSCs. The osteogenic differentiation was significantly improved by RAP-536 treatment which was shown by a 2.3-fold increase of ALP activity. GDF-11 induced Smad2/3 phosphorylation in MSCs which was inhibited by RAP-536 to about 50% of activity. The MSC phenotype characterized by the expression of CD73, CD90, CD105, CD146, CD29 and CD44 was not significantly influenced by RAP-536 treatment.Interestingly, the chemokine SDF-1 which plays an important role for the interaction and support of HSPCs was significantly up-regulated in MDS and HD MSCs by RAP-536 at both the mRNA (1.9-fold) and the protein level (2.1-fold; 313.0±35.0 vs. 649.7±76.8 pg/ml, *p< 0.05, n=3). This restored SDF-1 secretion of MSCs resulted in functional effects of HSPCs when co-cultured with MSCs. The number of CAF-C was significantly higher after two, three, and four weeks of HSPCs cultured on pre-treated MSC layers (12.0-fold / 3.4-fold / 1.75-fold). The phenotypical analysis of HSPCs demonstrated increased expression levels of CXCR4 in the adherent fraction. Addition of the CXCR4 antagonist AMD3100 blocked enhanced colony formation by RAP-536 confirming the previous observation on SDF-1 modulation. Clonal growth of CAF-C-derived HSPCs after four weeks of co-culture on un- or pre-treated MSC monolayers was analyzed in a secondary colony-forming cell assay (CFU) for an additional 14 days. The frequency of CFUs was increased in all lineages with significant differences in total colony numbers (15±5.8 vs. 35.8±14.7, *p< 0.05, n=6).These results were validated in vivo in a zebrafish model. HSPCs were co-cultured with GDF-11 and RAP-536 pre-treated MDS-MSCs for one week before injection into the zebrafish circulation. One and two days post-injection, a significantly higher cell proportion could be detected in the caudal hematopoietic (CHT) region as a result of RAP-536 pre-treatment (Fig.1).Conclusion:These data provide first evidence that RAP-536 has also the capacity to modulate MSCs which might contribute to the restoration of hematopoiesis in MDS. DisclosuresPlatzbecker:Celgene: Research Funding.