Erythroid Maturation Research Articles

AZD8055 Is More Effective Than Rapamycin in Inhibiting Proliferation and Promoting Mitochondrial Clearance in Erythroid Differentiation

Background: As an important downstream effector of various signaling pathways, mTOR plays critical roles in regulating many physiological processes including erythropoiesis. It is composed of two distinct complexes, mTORC1 and mTORC2, which differ in their components and downstream signaling effects. Our previous study revealed that the inhibition of mTORC1 by rapamycin significantly repressed the erythroid progenitor expansion in the early stage but promoted enucleation and mitochondria clearance in the late stage of erythroid differentiation. However, the particular roles and differences of mTORC1 and mTORC2 in the regulation of erythropoiesis still remain largely unknown. In the present study, we investigated the comparative effects of dual mTORC1/mTORC2 mTOR kinase inhibitor AZD8055 and mTORC1 inhibitor rapamycin on erythroid differentiation in K562 cells induced by hemin and erythropoiesis in β‐thalassemia mouse model.Materials and Methods: In vitro erythroid differentiation model of hemin‐induced K562 cells and β‐thalassemia mouse model were treated with AZD8055 and rapamycin. Cell Counting Kit‐8 was used to detect cell viability. The cell proliferation, cell cycle, erythroid surface marker expression, mitochondrial content, and membrane potential were determined and analyzed by flow cytometry and laser scanning confocal microscopy. Globin gene expression during erythroid differentiation was measured by RT‐qPCR. The mTORC2/mTORC1 and autophagy pathway was evaluated using western blotting.Results: Both AZD8055 and rapamycin treatments increased the expression levels of the erythroid differentiation‐specific markers, CD235a, α‐globin, γ‐globin, and ε‐globin. Notably, AZD8055 suppressed the cell proliferation and promoted the mitochondrial clearance of hemin‐induced K562 cells more effectively than rapamycin. In a mouse model of β‐thalassemia, both rapamycin and AZD8055 remarkably improve erythroid cell maturation and anemia. Moreover, AZD8055 and rapamycin treatment inhibited the mTORC1 pathway and enhanced autophagy, whereas AZD8055 enhanced autophagy more effectively than rapamycin. Indeed, AZD8055 treatment inhibited both mTORC2 and mTORC1 pathway in hemin‐induced K562 cells.Conclusion: AZD8055 is more effective than rapamycin in inhibiting proliferation and promoting mitochondrial clearance in erythroid differentiation, which might provide us one more therapeutic option other than rapamycin for ineffective erythropoiesis treatment in the future. These findings also provide some preliminary information indicating the roles of mTORC1 and mTORC2 in erythropoiesis, and further studies are necessary to dissect the underlying mechanisms.

Aggregates of nonmuscular myosin IIA in erythrocytes associate with GATA1- and GFI1B-related thrombocytopenia

BackgroundThe transcription factor GATA1 is an essential regulator of erythroid cell gene expression and maturation and is also relevant for platelet biogenesis. GATA1-related thrombocytopenia (GATA1-RT) is a rare X-linked inherited platelet disorder (IPD) characterized by macrothrombocytopenia and dyserythropoiesis. Enlarged platelet size, reduced platelet granularity, and noticeable red blood cell anisopoikilocytosis are characteristic but unspecific morphological findings in GATA1-RT. ObjectivesTo expand the investigation of platelet phenotype of patients with GATA1-RT by light- and immunofluorescence microscopy on a blood smear. MethodsWe assessed blood smears by light- and immunofluorescence microscopy after May-Grünwald Giemsa staining using a set of 13 primary antibodies against markers belonging to different platelet structures. Antibody binding was visualized by fluorescently labeled secondary antibodies. ResultsWe investigated 12 individuals with genetically confirmed GATA1-RT from 8 unrelated families. While confirming the already known characteristic of platelet morphology (platelet macrocytosis and reduced expression of markers for α-granules), we also found aggregates of nonmuscular myosin heavy chain II A (NMMIIA) in the erythrocytes in all individuals (1-3 aggregates/cell, 1-3 μm diameter). By systematically reanalyzing blood smears from a cohort of patients with 19 different forms of IPD, we found similar NMMIIA aggregates in the red blood cells only in subjects with GFI1B-related thrombocytopenia (GFI1B-RT), the other major IPD featured by dyserythropoiesis. ConclusionAggregates of NMMIIA in the erythrocytes associate with GATA1-RT and GFI1B-RT and can facilitate their diagnosis on blood smears. This previously unreported finding might represent a novel marker of dyserythropoiesis assessable in peripheral blood.

Mitapivat Treatment Increases β-Thalassemic Erythroblasts Energy Production and Responsiveness to Oxidative Stress

β thalassemia (β thal) is a worldwide red blood cell (RBC) genetic disorder with limited therapeutic tools. Standard of care includes chronic RBC transfusions and iron chelation. In β thal, pathophysiologic studies have shown that oxidation plays a key role in both ineffective erythropoiesis and reduced survival of mature red cells in the peripheral circulation (Matte et al ARS 28: 1-14, 2018; Matte A et al. ARS 23: 1284, 2015). Recently, in a mouse model of β thal, we showed that the investigational pyruvate kinase (PK) activator mitapivat significantly improved anemia by targeting both ineffective erythropoiesis and hemolysis (Matte et al. JCI 131: e144206, 2021). We also found that mitapivat ameliorated the erythroid cell maturation index for in vitro CD34+ derived erythroblasts from β thal (cod b039) patients (Matte et al. JCI 131: e144206, 2021). The results of phase 2 proof-of-concept study (NCT03692052) in non-transfusion-dependent thalassemic patients support the beneficial effects of mitapivat on β thal (Kuo KHM et al Lancet 400: 493-501, 2022). Here, we study CD34+ derived erythroblasts from either healthy controls or β thal (cod b039) patients (n=10). Mitapivat (2uM) was added to the cell cultures as previously described (Matte et al. JCI 131: e144206, 2021). Compared to healthy controls, we found up-regulation of PKM gene expression in the late phase of β thal erythropoiesis (14 days of culture). Whereas the expression of PKLR gene declined during erythroid maturation in both healthy and β-thal erythroblasts. We found higher total PK activity in β thal erythroblasts than in healthy cells. Using recombinant PKR and PKM2 isoforms (R&D systems), we found that mitapivat activates both PKR and PKM2 (EC50 13.6±3.3 and 30.1±1.5, respectively) (Kung C et al Blood 2017). Studying erythropoiesis in vitro we confirmed that mitapivat significantly increased β thal erythroid maturation as supported by increased late-erythroblasts at 11 days of cell culture (Matte et al. JCI 131: e144206, 2021). The metabolomic analysis of β-thal erythroblasts at 14 days of culture confirmed that mitapivat activated PK, as gleaned by PGLY/DPG levels and increased fluxes through glycolysis, as inferred from steady-state measurements of glycolytic metabolites, DPG, ATP, and GTP. In addition, we observed higher levels of methionine oxidation in both mitapivat-treated healthy and β-thal erythroblasts, suggestive of a regulatory PTM effect secondary to higher ATP availability and, possible increased longevity of the cells. The improvement in the intracellular milieu by mitapivat is also supported by the downregulation of peroxiredoxin-2 (Prx2) and heme-oxygenase-1 (Hmox1) gene expression, which are up-regulated in response to oxidative stress and free heme accumulation in vehicle treated β-thal erythroblasts (Matte et al. ARS 23: 1284, 2015). Noteworthy, we also observed a reduction in heat shock protein-70 (Arlet JB et al Nature 514: 242, 2014) in mitapivat treated β-thal erythroblasts, further supporting the protective effect of mitapivat during β-thal erythropoiesis. In conclusion, our data show that both pklr and pkm isoforms are expressed in the late phase of β-thal erythropoiesis. The ability of mitapivat to activate both PKLR and PKM2 might represent an added value to limit oxidation during b-thal erythropoiesis, ensuring an improved β-thal erythroblasts maturation and survival.

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.

Whole-Genome CRISPR-Cas9 Screen Identifies ZBTB7A As a Potential Therapeutic Target for Cda-II

Congenital dyserythropoietic anemia type II (CDA-II) is an autosomal recessive disease characterized by anemia, ineffective erythropoiesis, and increased bone marrow bi-nucleated erythroblasts. CDA-II is caused by loss-of-function mutations in SEC23B, which encodes a component of coat protein complex II (COP-II) vesicles/tubules that transport secretory proteins from the endoplasmic reticulum to the Golgi apparatus. Mammals express two SEC23 paralogs, SEC23A and SEC23B. We have previously shown that SEC23A is functionally interchangeable with SEC23B and that increased expression of SEC23A rescues the SEC23B-deficient erythroid differentiation defect observed in CDA-II. Here, we utilized our recently generated clonal SEC23B deficient HUDEP-2 cell line to identify novel therapeutic targets for CDAII. SEC23B deficient HUDEP2 cells survive and grow normally when cultured as pro-erythroblasts in ‘maintenance media’. However, upon culturing these cells in ‘differentiation media’, which results in semi-synchronous erythroid differentiation, differentiated SEC23B-null HUDEP2 cells exhibit reduced growth, impaired differentiation, increased bi-nucleated erythroid cells, and erythroid cell death, features of CDA-II. To identify novel genes that play important roles in the pathophysiology of CDAII, we developed a functional genome-scale CRISPR knockout screen to define genes that when deleted, rescue the SEC23B-null erythroid differentiation defect. SEC23B null HUDEP2 cells cultured in maintenance media were infected with the hGeCKO-v2 lentiviral library at a multiplicity of infection (MOI) of ~0.3, allowing most transduced cells to receive 1 sgRNA (and Cas9) to knockout 1 gene only. Transduced cells were puromycin selected and passaged in expansion media for 14 days. SEC23B-null HUDEP2 cells were then differentiated for 10 days. Cells were harvested prior to differentiation (Day 0, D0), and differentiated (orthochromatic) cells were sorted at D10 of differentiation. Integrated sgRNAs were quantified by deep sequencing. sgRNAs targeting ZBTB7A were amongst the most enriched in D10 compared to D0 erythroid cells. To validate these findings, we transduced SEC23B-null HUDEP2 cells with two independent and efficient ZBTB7A-targeting sgRNAs. We found that ZBTB7A deletion (using either of the 2 sgRNAs) rescued the lethality and differentiation defect of SEC23B null HUDEP2 cells undergoing differentiation. We next generated 7 clonal cell lines with combined deletion for ZBTB7A and SEC23B and confirmed that these cells exhibited normal growth and differentiation indistinguishable from wildtype HUDEP2 cells. The rescuing effect of targeting ZBTB7A was also validated in SEC23B mutated human CD34+ hematopoietic stem and progenitor cell culture. Since SEC23A can functionally replace SEC23B in erythroid cells, we quantified the SEC23A mRNA level in SEC23B-null HUDEP2 cells deleted for ZBTB7A, by qRT-PCR. In early preliminary results, deletion of ZBTB7A resulted in a profound (~10 fold) increase in SEC23A mRNA expression, to a level predicted to be sufficient to rescue the SEC23B null erythroid differentiation defect. ChIP-seq analysis demonstrated that ZBTB7A occupies the SEC23A promoter in HUDEP2 cells. Taken together, these data suggest that ZBTB7A represses SEC23A expression during erythroid maturation and that in the setting of ZBTB7A deletion, SEC23A expression is de-repressed, resulting in rescue of the CDA-II erythroid differentiation defect. Genome editing of the ZBTB7A binding sites in the SEC23A promoter is ongoing in SEC23B-null HUDEP-2 cells. We will determine the impact of the latter editing on SEC23A expression and erythroid differentiation.

The Condensin II Complex Subunit NCAPH2 Regulates Patterns of H4K20me1 Occupancy and Erythroid Gene Expression

During terminal maturation, erythroid precursors upregulate erythroid-specific genes while silencing non-erythroid genes in the setting of a rapid cell division and a nucleus that is dramatically condensing in preparation for enucleation. Setd8, the sole histone methyltransferase that can generate H4K20me1, plays an essential role in both chromatin condensation and the regulation of gene expression during erythropoeisis (Malik Cell Reports 2019, Myers Epigenetics and Chromatin, 2020). H4K20me1 regulates chromatin structure and gene expression through interaction with multiple partners, including the Condensin II Complex. The Condensin II complex is a ring-like structure composed of two conserved SMC components (SMC2 and SMC4), two HEAT subunits (NCAPG2 and NCAPD3), and a kleisin subunit NCAPH2. The Condensin II complex plays an important role in chromatin condensation during mitosis. It also regulates higher-order chromatin interactions and gene expression in interphase cells (Yuen Science Adv 2017; Iwasaki Nature Comm 2019). Similar to Setd8, many subunits of the Condensin II complex are highly expressed in erythroid cells (biogps.org; bloodspot.eu). We hypothesized that the Condensin II complex, Setd8, and H4K20me1 work in tandem to establish appropriate patterns of chromatin architecture and gene expression in maturing erythroblasts. To address this hypothesis, we first depleted NCAPH2 in erythroid cells by crossing mice with floxed alleles of NCAPH2 with mice expressing cre-recombinase under the direction of the Erythropoietin receptor promotor (EpoRCre). Homozygous disruption of NCAPH2 (NCAPH2 Δ/Δ) was embryonic lethal by E13.5. NCAPH2 Δ/Δ embryos were similar in appearance to littermate controls until E12.5 when they developed notable pallor and a dramatic decline in the number of benzidine positive cells. In contrast to cells from littermate controls, primitive erythroid NCAPH2 Δ/Δ cells at E11.5 were heterogenous in cell and nuclear size. NCAPH2 Δ/Δ embryos also had a dramatic failure of definitive erythropoiesis, as evidenced by significant pallor of the fetal liver at E13.5. To gain insights into the mechanisms underlying these findings, we used CUT&RUN to identify the genomic targets of NCAPH2 in E11.5 erythroblasts and to identify changes in H4K20me1 occuapncy in E11.5 NCAPH2 Δ/Δ erythroblasts compared to littermate control. In addition, the NCAPH2 and H4K20me1 CUT&RUN studies were compared to transcriptomic analyses of NCAPH2 Δ/Δ, NCAPH2 Δ/+, and NCAPH2 +/+ erythroblasts from E11.5 embryos. The majority of NCAPH2 peaks were located at transcripton start sites (TSS). Genes that were enriched for NCAPH2 at the TSS were also enriched for H4K20me1 across the gene body. At NCAPH2 target genes, enrichment for H420me1 occupancy increased significantly in NCAPH2 D/Derythroblasts compared to control. NCAPH2 was present at the TSS of differentially expressed genes that control cell cycle progression and erythroid maturation, and there were dynamic changes in H4K20me1 over the body of these genes following NCAPH2 deletion. Together, these results demonstrate that NCAPH2 is essential for erythropoiesis, and works in conjunction with Setd8 and H4K20me1 to establish appropriate patterns of gene expression in maturing erythroblasts.

Atg4a Promotes Mitochondrial Quality Control and Hemoglobin Production In Vivo

Mitochondrial quality control is essential for maintaining cellular homeostasis and contributes to the regenerative capacity of hematopoietic stem cells and the differentiation of lineage-committed progeny. During red blood cell (RBC) production, mitochondria contribute to the production of hemoglobin and are the site for heme synthesis. Mitochondrial dysfunction is found in patients with mtDNA mutations, sickle cell disease, and myelodysplastic syndromes; therefore, it is critical to delineate the molecular mediators of mitochondrial quality control during erythropoiesis. While it is established that autophagy contributes to mitochondrial clearance during reticulocyte maturation, less is known about the regulation of mitochondrial quality control in erythroid progenitors. Our previous study identified an erythroid-specific role for the autophagy-related protease ATG4A in human erythropoiesis (Stolla MC et al., Blood Advances, 2022). To better understand the contribution of Atg4a-dependent autophagy to erythropoiesis in vivo, we have developed a new genetically modified mouse model . To evaluate the role of Atg4a in vivo, we disrupted the expression of Atg4a by targeting exons 5/6 using CRISPR/Cas9 in C57/BL6 embryos. Analysis of the peripheral blood of adult Atg4a-/- (KO) and Atg4a+/+ (WT) mice revealed a normal hematocrit (47.5% WT vs. 47% KO), but elevated numbers of red blood cells (10.7 M/μL WT vs. 11.2 M/μL KO, p=0.012) and a compensatory 5.8 % decrease in RBC volume (MCV: 44.3 fl WT vs. 41.7 fl KO, p=0.0004). Consistent with a reduction in RBC size, we observed a modest reduction in total hemoglobin (16.8 g/dl WT vs. 16.0 g/dl KO, p=0.054) and a profound decrease in the mean corpuscular hemoglobin concentration (MCHC: 35.39 g/dl WT vs. 34.17 g/dl KO, p=0.038) in Atg4a -/- (KO) mice. To determine if Atg4a-/- mice are iron deficient, we evaluated iron parameters in peripheral blood. While Atg4a-/- mice had slightly lower values of serum iron (152 μg/dl WT vs. 128.7 μg/dl KO, p=0.21) and total-iron binding capacity (511 μg/dl WT vs. 456 μg/dl KO, p=0.04) they had comparable levels of transferrin saturation (29.9% WT vs. 28.1% KO, p=0.65). Furthermore, we found similar amounts of EPO in the serum of Atg4a-/- and Atg4a+/+ mice (314 pg/ml WT vs. 345 pg/ml KO, p=0.78). Thus, loss of Atg4a leads to a reduction in hemoglobin production and RBC size without overt iron deficiency. To determine if alterations in erythroid maturation contributed to the decrease in MCV and MCHC, we used flow cytometry to quantify bone marrow erythroid progenitors (I), basophilic erythroblasts (II), polychromatic and orthochromatic erythroblasts (III), and reticulocytes (IV) using CD44, Ter119, CD71, and lineage markers (CD11b, B220, Gr-1) in Atg4a-/- mice. Similar numbers of erythroid populations (I-IV) were observed in the bone marrow and spleen of Atg4a-/- and Atg4a+/+ mice suggesting that erythroid maturation was not significantly altered in vivo. Since Atg4a is known to facilitate mitochondrial clearance during human erythropoiesis, we next evaluated mitochondrial dynamics in murine erythroid populations I-IV using dyes for active (TMRM) and total mitochondria (Mitotracker Green, MTG). Compared to Atg4a+/+ mice, Atg4a-/- mice had increased MTG fluorescence in erythroid populations I and II and a 25% decrease in TMRM fluorescence. Therefore, loss of Atg4a significantly reduces the ratio of active-to-total mitochondria in erythroid progenitors and basophilic erythroblasts, indicating that elimination of inactive mitochondria promotes mitochondrial function early in erythropoiesis. Since mitochondria are the site for heme synthesis, we sorted erythroid populations from Atg4a-/- and Atg4a+/+ mice and measured total heme content. While in Atg4a +/+ mice heme content increased by 3-fold between erythroid populations III and IV, heme content did not increase between erythroid populations III and IV from Atg4a-/- mice resulting in significantly reduced heme content in reticulocytes. Our study highlights an important protective role for autophagy in erythropoiesis where it mediates the elimination of dysfunctional mitochondria. Loss of mitochondrial quality control restricts heme production reducing the hemoglobin concentration of circulating RBCs. Further investigation will determine if the absence of Atg4a compromises the response to stress erythropoiesis.

Identification of CNOT4 As a Positive Regulator of Erythroid Cell Growth Using a Genome Wide CRISPR Knock-out Screen

Red blood cells, also known as erythrocytes, are the principle means of oxygen delivery to the body, and inadequate or disordered erythrocyte production is a major cause of human disease. While the cell-extrinsic factors that control erythrocyte production have been well studied, our understanding of the cell-intrinsic regulation of this process is incomplete. To identify genes that regulate erythroid proliferation and/or maturation, we performed a genome-scale CRISPR knock-out screen in the immortalized human erythroid cell line, HUDEP-2. HUDEP-2 cells can be maintained at the pro-erythroblast stage and then sequentially differentiated into basophilic, polychromatic, and orthochromatic erythroblasts by changing culture conditions, which permits separate evaluation of the impact of genetic perturbations on pro-erythroblast viability and terminal erythroid differentiation capacity. To perform our screen, we used the commercially available GeCKO-v2 library, which includes 6 sgRNAs targeting virtually every protein-coding gene in the human genome. This library was packaged into lentiviral particles, each expressing Cas9, an sgRNA, and a puromycin resistance gene. HUDEP-2 cells were transduced with the pooled GeCKO-v2 library at low multiplicity of infection to ensure delivery of only one sgRNA per cell. Following puromycin selection, pro-erythroblasts were collected prior to differentiation and orthochromatic erythroblasts were sorted at day 12 of differentiation (selecting for cells that downregulated CD49d). By identifying sgRNAs that were under-represented in orthochromatic erythroblasts compared to the proerythroblasts, we identified several genes that when deleted lead to impaired erythroid differentiation. One of these genes was CNOT4, which encodes a RING E3 ligase member of the CCR4-NOT complex, a conserved transcriptional regulator. This gene has been previously reported to positively regulate JAK/STAT signaling, which is known to play a key role in erythroid growth and differentiation. Additionally, only 30 out of 1095 cell lines tested in the Depmap portal exhibit diminished growth following CNOT4 deletion, suggesting a rather specific impact on erythroid cells. To define the role of CNOT4 in erythropoiesis, we electroporated human CD34+ hematopoietic stem and progenitor cells (HSPCs) with a ribonucleoprotein (RNP) complex consisting of Cas9 protein and CNOT4-targeting sgRNA. Three unique CNOT4-targeting sgRNAs were tested. Following RNP electroporation, cells were differentiated using a four-phase erythroid differentiation culture protocol and assessed for growth, indel frequencies, and erythroid differentiation capacity. For all three sgRNAs tested, CNOT4 disruption resulted in 10-fold reduced proliferation over the course of differentiation compared to cells electroporated with non-targeting sgRNAs. Erythroid differentiation, assessed by flow cytometry using CD49d and CD233 expression, was identical in CNOT4 deleted and control cells. However, evaluation of cytospins revealed reduced cell size with diminished cytoplasm to nuclear ratio in CNOT4 deficient compared to control cells. Notably, the findings above were validated in erythroid cells differentiated from HSPCs harvested from two independent donors. Taken together, these findings suggest a novel role for CNOT4 and/or the CCR4-NOT complex in erythroid differentiation. Studies are ongoing to determine the mechanism by which CNOT4 disruption negatively impacts erythropoiesis and to examine if CNOT4 overexpression results in enhanced proliferation/differentiation of erythroid cells.

Inhibition of Toll-like Receptor 4 (TLR4) Promotes Erythroid and Myeloid Differentiation in Myelodysplastic Syndrome (MDS) and Acute Myeloid Leukemia (AML)

Introduction: The hallmark of Myelodysplastic Syndrome (MDS) is a block in differentiation of hematopoietic precursors resulting in single or multilineage cytopenias. Evaluation of regulatory pathways that are inappropriately overactive and lead to a blockade in erythroid and myeloid differentiation is an important process in determining future therapeutic targets for the treatment of MDS. The Toll-like receptor (TLR) pathways play an important role in the innate immune response and are also highly expressed on hematopoietic stem cells. Aberrant overactivation of these pathways can stimulate inflammation and cytokine production in the marrow microenvironment. Also, while the overactivation of interleukin-1 receptor-associated kinase 4 (IRAK4), tumor necrosis factor receptor associated factor 6 (TRAF6) and nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) pathways has been seen in Myelodysplastic Syndrome and Acute Myeloid Leukemia (AML), the upstream signals that drive their activation are not fully identified. Results: We investigated the expression of all TLRs in a large database of MDS CD34+ marrow samples and age-matched controls. Our findings revealed that TLR6 and TLR4 were the most significantly overexpressed receptors in MDS samples (FDR<0.001). Furthermore, we observed that the TLR4 cofactor lymphocyte antigen 96 (LY96), also known as myeloid differentiation factor 2 (MD-2), was also overexpressed in MDS samples when compared to controls. Furthermore, the S100A8 and S100A9 proteins are ligands for TLR4 and were found to be positively associated with severity of anemia in the cohort of MDS samples. These data provided the rationale for determining the functional role of the TLR4 pathway in MDS/AML models. To obtain a potent and specific antibody against TLR4, we inoculated rabbits with recombinant human TLR4 and obtained high affinity antibodies that neutralized TLR4 function at low nano-molar range of doses. The TLR4 polyclonal antibody was able to inhibit lipopolysaccharide (LPS)-induced NF-κb activation in leukemic THP-1 cells. The TLR4 antibody also led to a decrease in proliferative phosphorylated extracellular signal-regulated kinase (ERK) mitogen-activated protein (MAP) kinase in leukemic cells. We then treated samples from MDS/AML patients (n=14) with the TLR4 antibody (1 nM dosing) and assessed for erythroid and myeloid growth and differentiation. Fluorescence activated cell sorting (FACS) analysis for erythroid differentiation showed increased expression of either CD71, CD235a or both after treatment with the TLR4 antibody. FACS analysis for myeloid differentiation showed increased expression of either CD11b, CD14 or both after treatment with the TLR4 antibody. This effect was more pronounced in the intermediate/high risk group than in the low-risk group of patients. Conclusion: Inhibition of the TLR4 pathway with a potent TLR4 antibody promotes erythroid and myeloid maturation and relieves the differentiation block on these lineages in MDS patient samples in the in vitro setting. Our preclinical data supports further evaluation of TLR4 inhibition as a therapeutic target in patients with MDS, as anemia and neutropenia are hallmarks of the disease.

Targeting Complement Inhibition in Cold Hemolysis; A Multifaceted Approach Using Sutimlimab and Washed Red Cell Transfusions

Introduction: Cold agglutinin syndrome (CAS) is a transient disorder mediated by autoantibodies that cause red cell agglutination at cold temperatures. CAS is often associated with infections rather than malignant or autoimmune conditions. Sutimlimab, a monoclonal antibody directed against the complement component C1s, was previously shown to improve anemia and fatigue in cold agglutinin disease (CAD) in single-arm studies. However, its use in CAS has not yet been elucidated. Here, we describe a case where complement inhibition using the novel C1s inhibitor, as well as washed and warmed red cell transfusions, were used to treat CAS in the setting of a rare autoantibody directed toward the Pr antigen Case report: A healthy 46-year-old woman presented with severe acute hemolytic anemia: with nadir hemoglobin of 4.7, hematocrit of 13.4%, 0.2% of reticulocytes, total bilirubin of 9.8 mg/dL, and unconjugated bilirubin of 8.4 mg/dL. A peripheral blood smear review revealed severe anemia with agglutination of red blood cells. An extensive infectious workup was negative including COVID-19, Parvovirus B19, CMV, EBV, CMV, and HIV. Over a period of two weeks, she received three doses of IVIG, prednisone, and 17 transfusions units of warmed red blood cells in the setting of persistent hemolytic anemia. Despite the evidence of severe hemolysis, her reticulocyte count remained very low (<0.5%). A bone marrow biopsy showed trilineage hematopoiesis with erythroid hypoplasia and maturation arrest. A direct Coombs test was intermittently positive but probably confused by IgG infusions, with anti-I and anti-i titers of 1:64, and positive for C3. Thermal amplitude studies showed agglutination at 30°C, 37°C, and room temperature. Red blood cell antibodies were not identified in the eluate. The indirect antiglobulin test was negative for ficin-treated RBCs showing no agglutination. This led the reference laboratory to report that the cold agglutination was directed against the anti-pr antigen and that this patient had clinically significant anti-Pr antibodies. The patient was kept warm and started on Danazol and Sutimlimab. However, the patient continued to have hemolysis after transfusions. After receiving her second dose of Sutimlimab and in the context of persistent hemolytic anemia, she underwent another bone marrow biopsy on day 34 after presentation. The biopsy showed hypercellular bone marrow with erythroid hyperplasia and complete maturation. However, this was not reflected in reticulocytosis until day 44. The patient received her third dose of Sutimlimab, and red blood cell transfusions were washed in addition to being warmed, which resolved hemolysis after transfusion reactions. She received a total of 30 red blood cell transfusions since presentation. The patient's anemia improved, with a hemoglobin level of 8.8 mg/dL and a reticulocyte count of 13%, and she was discharged on Day 51 after presentation (figure 1). She received one more dose of Sutimlimab (total of four doses) with complete resolution of her anemia, a normal reticulocyte count, normal LDH, negative Coombs study, and negative cold agglutinin titers. Conclusion: Here, we describe a challenging case of CAS caused by a rare Pr autoantibody, with a failure of first-line treatments for CAS. This report is the first to describe the use of Sutimlimab as a treatment for CAS. After initiating Sutimlimab, the patient continued to experience significant hemolysis shortly after transfusions, exhibiting hemoglobinuria and an inadequate increase in hematocrit. Only when the red blood cell transfusions were washed to remove complement, the hemolytic reactions ceased and significant increases in hematocrit occurred. This finding may be a consideration in other cases of cold agglutinin anemia. As CAS is a transient disorder, Sutimlimab could ultimately be discontinued once the hemolysis resolved. This case illustrates the role of complement inhibition in CAS and the potential need to warm and wash red blood cells in severe cases of CAS.

Ubiquitin Ligase SCF-FBXO11 Controls a Network of RNA-Binding Proteins and Splicing in MDS

The F-Box protein FBXO11 is a substrate receptor of Skp-Cullin-F-Box (SCF) ubiquitin ligase complexes. Loss-of-function mutations in FBXO11 are associated with diffuse large B-cell lymphoma, and FBXO11 has been shown to regulate epigenetic drivers of erythroid cell maturation. In our prior study, we found that deletion of FBXO11 improved cell survival under cytokine deprivation in an MDS cell line. Furthermore, we had observed a striking decrease in FBXO11 protein levels in AML patient samples. However, how FBXO11 contributes to myeloid malignancies is unclear. Here, using RNA-sequencing, proteomics, and CRISPR-Cas9 screening, we reveal broad ubiquitylation of RNA-binding proteins (RBPs) and alternative splicing as a mechanism by which deregulation of FBXO11 enhances malignant cell function. In addition, we report the presence of mutations in the uncharacterized N-terminal extension of FBXO11 in both lymphoid and myeloid malignancies. We first observed that heterozygous deletion of FBXO11 increases colony-forming ability of MDS-AML cell lines in vitro whilea2-fold increase in expression results in decreased cell growth and apoptosis. In a transplant model with a retrovirally-driven Runx1 mutation, heterozygous deletion of Fbxo11 exacerbated neutropenia and shortened overall survival. However, depletion of FBXO11 in healthy human and murine HSPCs significantly impaired myeloid progenitor colony-formation, indicating a unique requirement for FBXO11 in myelopoiesis. Based on its tumor suppressive activity, we evaluated whole-exome sequencing data from patient samples of both lymphoid and myeloid malignancies for FBXO11 mutations. This analysis uncovered rare mutations in FBXO11 in myeloid malignancies and revealed a substantial cluster of previously unidentified mutations in the Q-rich, N-terminal extension of FBXO11. We found that this uncharacterized, extended protein isoform is predominantly expressed in both normal and malignant hematopoietic cells. In colony-forming assays, expression of the long isoform and several of the N-terminal mutations inhibited CD34+ progenitor function. On a cellular level, lack of this N-terminal extension in the short isoform resulted in aberrant subcellular localization of FBXO11 within the nucleus. To elucidate the mechanism of FBXO11 in myeloid malignancy, we performed immunoprecipitation of FBXO11 followed by proteomics to identify endogenous FBXO11 complexes in MDS-AML cells. We integrated these data with ubiquitin proteomics of WT and FBXO11-KO MDS cells to identify candidate substrates of SCF-FBXO11-mediated ubiquitylation. FBXO11 complexes are significantly enriched for RNA-binding proteins affecting mRNA splicing, a process commonly deregulated in MDS. FBXO11-KO MDS cells displayed broad depletion of ubiquitylation in a network of RBPs. As expected, targeting FBXO11 did not affect the level of transcripts of interacting proteins; however, there were variable changes in total protein levels indicative of post-transcriptional regulation. We then applied a focused CRISPR-Cas9-KO screen of candidate SCF-FBXO11 substrates in MDS-AML cells depleted of FBXO11. We observed significant bi-directional effects in colony-forming ability mediated by several of the FBXO11-regulated RBPs including TRIM28, HNRNPU, NPM1, and SYNCRIP. Though FBXO11 is significantly decreased in MDS compared to healthy controls, we saw no difference in FBXO11 expression between splicing factor WT and mutant samples, suggesting that the function of FBXO11 in regulating mRNA splicing in MDS is independent of these mutations. Compared to healthy controls, FBXO11-low MDS samples exhibited a greater number of alternative splicing events, predominantly in skipped or mutually-exclusive exons. To experimentally test whether FBXO11 indeed affects RNA splicing, we utilized a bichromatic splicing reporter assay in MDS-AML cells which showed that depletion of FBXO11 is sufficient to alter splicing of the reading frame in the cassette exon, read out as a shift from GFP to RFP. Finally, STRING protein network analysis revealed that the alternative splicing events occurring in FBXO11-low MDS patient samples affect key nodes in protein translation and metabolism. Collectively, our data support a model whereby the change in FBXO11-mediated ubiquitylation of RBPs drives alternative exon usage that exacerbates malignant cell function in MDS.

Pharmacologic Inhibition of XPO1 By Selinexor Improves Late-Stage Erythropoiesis in Severely Affected β 0-Thalassemia/Hemoglobin E

Ineffective erythropoiesis due to accumulation of excess α-globin chains profounds unwarranted consequences in β-thalassemia. In severe β-thalassemia, the excess α-globin chains trap HSP70-the guardian of GATA1-within the cytoplasm. Thus, in the nucleus, GATA1 is degraded and leads to late-stage erythropoiesis arrest. XPO1 is a nuclear exporter that exports HSP70 from the nucleus to the cytoplasm. Previous study showed that chemical downregulation of XPO1 in β-thalassemia major using an old generation of XPO1 inhibitor (KPT-251) could improve late-stage in vitro erythropoiesis. Moreover, XPO1 inhibitor treatments in clinical trials revealed improvement of transfusion burden and hemoglobin levels in some patients with myelofibrosis. Together suggested that inhibition of XPO1 might be considered as a possible option for β-thalassemia treatment. Here, we investigated the effects of pharmacological XPO1 inhibition using old and new generation of XPO1 inhibitors, and natural products in human CD34 + hematopoietic stem and progenitor cells (HSPCs) derived from β 0-thalassemia/HbE with severe phenotypes. Coinheritance of common α-thalassemia were ruled out in all patients and severity score was computed using Sripichai scoring system (Sripichai et al. Am J Hematol 2008). HSPCs were purified from peripheral blood and subjected to 3-phase erythroid culture system. Nine compounds with known XPO1 inhibitory effect including five synthetic (KPT-185, KPT-276, KPT-330 or selinexor, KPT-335 or verdinexor and KPT-8602 or eltanexor) and four natural active compounds (curcumin, oridonin, piperlongumine and plumbagin) were evaluated in patient-derived HSPCs (n=1) for their potency and efficacy. Of these, selinexor exhibited the maximal efficacy in which the accumulation of CD71 Low/GPA High cell population was predominate. To affirm this finding, HSPCs derived from six independent β 0-thalassemia/HbE patients were treated with selinexor (5 and 10 nM) at early-stage of the in vitro erythropoiesis and maintained throughout the culture system. After treatments (n=3), XPO1 was downregulated in both cytoplasmic (5 nM; 1.5±0.1-fold, p < 0.005 and 10 nM; 1.7±0.5-fold, p < 0.05) and nuclear compartments (5 nM; 1.1±0.3-fold and 10 nM; 1.3±0.2-fold, p < 0.05). No significant cytotoxic effects were observed. In addition, effects of selinexor on erythroid differentiation and maturation were assessed by flow cytometry using CD71/GPA immunostaining and Wright-Giemsa staining. The results revealed that selinexor treatments could augment late-stage in vitro erythropoiesis (orthochromatic+enucleated/basophilic+polychromatic erythroid cell) in dose-dependent manner (5 nM; 1.4±0.4-fold and 10 nM; 3.5±1.8-fold, p < 0.05). This finding was correlated with erythroid cell morphology under a light microscope. We further explored whether XPO1 inhibition by selinexor (n=3) affects HSP70 and GATA1 expression. Homogenous erythroid cell population (CD71 High/GPA High) were collected and subjected for western blot analysis. We found that HSP70 was upregulated in the cytoplasm (5 nM; 1.8±1.0-fold and 10 nM; 2.1±1.1-fold). However, HSP70 and GATA1 in the nucleus were insignificant changed. Our finding demonstrated that manipulations of XPO1-HSP70 axis in the cytoplasm by selinexor were associated with accelerated late-stage erythropoiesis in severely affected β 0-thalassemia/HbE HSPCs. We then hypothesized whether selinexor functioned through an accumulation of HbF. Therefore, Hb profiles were investigated using HPLC. However, non-significant changes of Hb profiles and HbF levels were observed. With this finding, we purposed that selinexor functioned independently of HbF regulatory pathways and precise mechanisms underlying the regulation of late-erythropoiesis by selinexor are required. According to our preliminary data, an FDA approval compounds for multiple myeloma-selinexor-is a potent compound for improving in vitro late-stage erythropoiesis in HSPCs derived from severe β 0-thalassemia/HbE. Therefore, repurposing of selinexor to improve ineffective erythropoiesis in β-thalassemia is compelling. Moreover, combination treatments of selinexor with HbF inducers and/or SMAD2/3 blockers could be further validated to possibly achieve greater therapeutic benefits for β-thalassemia treatments.

Single Cell Analysis and Functional Features of Circulating CD34 + Cells from Patients with Sickle Cell Anaemia Unveil a Defect in the Myeloid Lineage

Polymorphonuclear neutrophils (PMN) have long been suspected to be involved in the pathophysiology of Sickle cell anemia (SCA) since early studies have reported a higher neutrophil count in patients at steady-state and its positive correlation with SCA severity. This high leukocyte count has long be considered as a peripheral phenomenon, driven by RBC hemolysis causing systemic inflammation and neutrophils recruitment. Nevertheless, several lines of evidence suggest that hematopoietic stem/progenitor cell (HSPC) function might be perturbed in SCA. Given that recent report showed an increased apoptosis during terminal erythroid maturation of SCA cells and that erythroid and myeloid differentiation derive from the same common myeloid progenitor (CMP), we investigated a possible enhanced myelopoiesis in SCA and its causes. First, to better understand when and how hyperleukocytosis and chronic inflammation begin in these patients, we analyzed longitudinal biological data from 1250 infants with SCA aged 1-24 months. We observed that the increase in neutrophils count began as early as the first months of life, and was statistically correlated with the drop in HbF rate and with the increase in reticulocytes count, much more than with the increase of the LDH level. These results seem to contradict the hypothesis of chronic inflammation driven by intravascular hemolysis. To assess whether the abnormal hematopoiesis is associated with a defect on lineage commitment of Hematopoietic Stem/Progenitor Cells (HSPCs), we evaluated the phenotype of myeloid and erythroid progenitors isolated from peripheral blood (PB) of untreated patients with SCA. While the portion of the common myeloid progenitor (CMP) was similar among both groups, we observed a significant increase of granulocyte-monocyte progenitors (GMP) in SCA patients (n=6) compared to health donors (HDs) (n=9), as well as a significant decrease of erythroid progenitors (MEP). Interestingly, HSPCs from SCA PB produced more myeloid colonies in vitro than those in HD HSPCs. In addition, colony-forming assay of sorted SCA CMP displayed an increase of granulo-monocytes colonies CFU-GM and decrease of erythroid colonies BFU-E compared to HDs, indicating an increased myeloid commitment of SCA HSPCs. To comprehensively characterize HSPC composition, we performed 10´ scRNA-seq on CD34+ HSPCs isolated from HDs (n=3) and patients with SCA (n=4). We confirmed that SCA HSPCs have a high proportion of neutrophil progenitors. More interestingly, MPP/HSC populations from SCA patients highly express several genes involved in myeloid differentiation like SELL (L-Selectin) and CSF3R (G-CSF receptor) genes, indicating an enhanced myeloid commitment of early progenitor in SCA. To better characterize the myeloid features of these progenitors, we investigated ex- vivo granulopoiesis of PB HSPCs from SCA patients (n=10) and HDs (n=9) in presence of G-CSF as CSF3R is highly expressed in early SCA progenitors. While cell proliferation was similar among both groups, unexpectedly SCA myeloid cells presented a significant delayed expression of CD11b (p<0.01). Interestingly, we also observed an overexpression of monocytic markers HLA-DR and CD36 in a subset of differentiated cells, suggesting a potential commitment of part of the myeloid progenitors to a monocyte lineage , confirmed by MGG staining. To further understand this defect, we performed RNAseq analysis of the GMP isolated from PB of SCA patients (n=4) and HD (n=4). We identified a total of 420 differentially expressed genes (DEGs). Among these DEGs, significantly downregulated genes in SCA included granulocyte differentiation program such as GFI1, C/EBPa, and JAK3. In return, significantly upregulated genes included KLF4, supporting the monocytic bias observed in the early myeloid progenitors. In conclusion, these data reveal the myeloid-primed signature in PB HSPCs of patients with SCA, likely responsible for the elevated PMN and monocyte count in these patients. This initial breakthrough in the understanding of the development of hyperleukocytosis and chronic inflammation in these patients will open up new early therapeutic options. Moreover, since CD34+ cells of the peripheral blood are the main source of HSPCs for the development of gene therapy in SCA, deciphering their functional feature and myelo-monocytic skewing could be of interest to guide the gene editing strategy.

Real-World Impact of Luspatercept on Patients with Myelodysplastic Syndromes Requiring Red Blood Cell Transfusions and with Prior Exposure to Erythropoietin-Stimulating Agents: A United States Healthcare Claims Database Study

Background: Myelodysplastic syndromes (MDS) are among the most common hematologic cancers in older US adults. MDS are characterized by abnormal bone marrow function leading to impaired production of blood cells, persistent cytopenia, and variable risk of progression to acute myeloid leukemia (AML). Since anemia is a common complication of MDS, many patients (pts) become dependent on red blood cell transfusions (RBCTs) and erythropoiesis-stimulating agents (ESAs) are the standard of care for patients with lower-risk MDS. In a randomized, double-blind, placebo-controlled phase 3 MEDALIST clinical trial, luspatercept, a first-in-class erythroid maturation agent, was shown to reduce the severity of anemia among transfusion-dependent (TD) pts with lower-risk MDS with ring sideroblasts (RS) status and who were refractory to prior ESA treatment (Fenaux P, et al. N Engl J Med 2020; 382:140-151). However, real-world evidence regarding the effect of luspatercept on transfusion burden among pts with MDS requiring RBCTs and with prior ESA exposure is currently limited. Aim: To describe clinical outcomes and MDS-related treatment patterns before and after the initiation of luspatercept among real-world pts with lower-risk MDS in the USA who required RBCTs before luspatercept initiation and were previously exposed to ESAs. Methods:In this retrospective claims-based cohort study, data from Symphony Health, a nationally representative database, (Jan 1, 2010 to June 30, 2022) were used to identify adults with ≥1 claim with a diagnosis of MDS (initial diagnosis on or after Jan 1, 2015) before the first claim for luspatercept (index date). Pts were considered TD at luspatercept initiation if they had ≥1 claim for RBCT in the 8 weeks before index date, and ESA-exposed if they had ≥1 ESA claim at any point before or at luspatercept initiation. Pts with chemotherapy treatments, with a diagnosis for refractory anemia with excess blasts, or with AML during the study period were excluded as they were presumed to have higher-risk MDS. Pts were also excluded if they had prior use of hypomethylating agents (HMAs) or a diagnosis for β-thalassemia, myelofibrosis, aplastic anemia, or breast cancer in the 6 months prior to index date (baseline period). Pt demographics and clinical characteristics were assessed during the baseline period. Pts' RBCT status (transfusion independent [TI] or TD) was measured in the 6 months after index date. A pt was considered 8-week TI if they had 0 RBCT in ≥1 rolling 8-week period in the 6 months after index date. 12-week, 16-week, and 24-week TI pts were defined similarly, using increasingly longer periods without RBCT. Pts' use of MDS-related treatment classes (ie, chemotherapy, ESAs, granulocyte-colony stimulating factor [G-CSF], HMA, and immunomodulatory imide drug [IMiD] agent) was measured before and after index date until end of data availability or clinical activity (follow-up). Results:The population comprisedof240 pts with MDS who were TD and had ESA exposure before luspatercept initiation. The mean (standard deviation [SD]) age was 75.4 (5.4) years and 56.7% were male. About one fifth of pts received ≥2 treatment classes before luspatercept initiation (2: 17.5%; 3+: 1.7%). Most pts (64.2%) achieved 8-week TI in the 6 months after luspatercept initiation. Similarly, 53.8%, 43.3%, and 29.6% of pts achieved 12-, 16-, and 24-week TI in the 6 months after luspatercept initiation, respectively (Figure 1). The percentage of pts achieving 8-, 12-, 16-, and 24-week TI was numerically higher among pts with positive RS status than those with negative/unknown RS status (Figure 1). Most pts (64.2%) did not require any additional MDS-related treatments during the study follow-up period (mean (SD) 15.6 (7.3) months). The most common treatment classes used after luspatercept initiation were ESA (22.5%) and HMA (16.3%; Figure 2). Conclusions:The findings of this real-world study strongly corroborate clinical trial results, showing that luspatercept is highly effective in reducing transfusion dependence among ESA-exposed pts with lower-risk MDS who were TD before luspatercept initiation. In this study, over half of pts achieved 8-week and 12-week TI status during the follow-up period, and more than a quarter did not require any RBCT over a 24-week period. Further, most pts treated with luspatercept did not require any additional MDS-related treatments during the follow-up period.

The ELEMENT-MDS Trial: A Phase 3 Randomized Study Evaluating Luspatercept Versus Epoetin Alfa in Erythropoiesis-Stimulating Agent-Naive, Non-Transfusion-Dependent, Lower-Risk Myelodysplastic Syndromes

Background and Significance: Anemia is a common and debilitating symptom in patients with myelodysplastic syndromes (MDS), frequently leading to dependence on red blood cell (RBC) transfusions. Transfusion-dependent (TD) patients have worse overall survival and quality of life, and repeat transfusions are associated with complications, including iron overload. Erythropoiesis-stimulating agents (ESAs) are the standard treatment for anemia in patients with lower-risk (LR)-MDS, but many patients are ineligible for or refractory to ESAs, and responses are limited. Luspatercept, a late-stage erythroid maturation agent, is approved to treat TD patients with LR-MDS after ESA failure. In the phase 3 COMMANDS study, in ESA-naive patients with LR-MDS requiring transfusions, a significantly higher proportion of patients receiving luspatercept than epoetin alfa achieved RBC transfusion independence for ≥ 12 weeks with concurrent hemoglobin (Hb) increase ≥ 1.5 g/dL (weeks 1-24) (Platzbecker U, et al. Lancet 2023. doi: 10.1016/S0140-6736(23)00874-7). The potential of luspatercept to improve moderately severe chronic anemia and reduce RBC transfusion dependence has not yet been investigated in the majority non-transfusion-dependent (NTD) LR-MDS patient population. Preventing transfusion dependence is crucial for improving LR-MDS patient outcomes. The aim of this actively enrolling, phase 3, randomized, multicenter study is to compare the safety and efficacy of luspatercept versus epoetin alfa in reducing progression to RBC transfusion dependence in ESA-naive, NTD adult patients with anemia due to LR-MDS. Study design and methods: The ELEMENT-MDS study includes a 5-week screening period, a 96-week treatment period, an extension phase, and a post-treatment follow-up period (Figure). Target enrollment is 360 patients with anemia due to Revised International Prognostic Scoring System (IPSS-R) Very low-, Low-, or Intermediate-risk MDS. Eligible patients are ≥ 18 years of age with a confirmed MDS diagnosis, baseline serum erythropoietin (sEPO) levels ≤ 500 U/L, and symptomatic anemia defined as a score of moderate or worse on ≥ 1 Patient Global Impression of Severity (PGI-S) item (fatigue, shortness of breath, weakness, or dizziness). Participants must be RBC transfusion-independent per International Working Group 2018 criteria (no RBC transfusions within 16 weeks prior to randomization) and have baseline Hb ≤ 9.5 g/dL. Patients will be excluded if they have a del(5q) cytogenetic abnormality, unclassifiable MDS or myelodysplastic/myeloproliferative neoplasms, anemia due to reasons other than MDS, bleeding disorders, low absolute neutrophil or platelet counts, uncontrolled hypertension, previous acute myeloid leukemia diagnosis, or history of recent malignancies, among other reasons. Participants will be randomized 1:1 to receive luspatercept or epoetin alfa. Stratification factors include baseline sEPO levels, ring sideroblast status, and IPSS-R risk category. Patients will receive luspatercept once every 3 weeks at a starting dose of 1.0 mg/kg (escalation up to 1.75 mg/kg allowed) or epoetin alfa once a week at a starting dose of 450 IU/kg (escalation up to 1050 IU/kg allowed). The primary endpoint will compare the proportion of patients becoming TD (defined as requiring ≥ 3 RBC units/16 weeks) during any continuous 16-week interval in weeks 1-96. The key secondary endpoint is a Hb increase ≥ 1.5 g/dL sustained for ≥ 16 weeks during weeks 1-48. Additional secondary endpoints include time to RBC transfusion dependence, transfusion-free survival, time to achieve modified hematologic improvement-erythroid response, transfusion independence ≥ 24 weeks, and quality of life assessment. Safety endpoints include type and severity of adverse events and relationship to treatment, overall survival, and progression to high-risk MDS and acute myeloid leukemia. Summary: The results of this phase 3 study will determine the efficacy and safety of luspatercept as a therapy for ESA-naive NTD patients with LR-MDS, who have limited treatment options for anemia. The study is registered at ClinicalTrials.gov (NCT05949684) and EudraCT (2022-500430-29-00).

Srcap Plays an Essential Role in Normal Hematopoiesis

The incorporation of variant histones into chromatin has been revealed to be an important mechanism of epigenetic regulation. Deposition of H2A.Z, in particular, is known to mediate several essential cellular processes including chromatin organization, DNA damage response, and transcriptional regulation. Nonsense and frameshift mutations in the H2A.Z exchanger, SRCAP, have been recurrently identified in both clonal hematopoiesis and AML, suggesting the importance of this gene in both non-malignant hematopoiesis and leukemogenesis. However, the precise role of SRCAP and the impact of its dysregulation on hematopoiesis remain unknown. To investigate the role of Srcap in hematopoiesis, we generated a conditional Srcap loss of function murine model with loxP sites flanking exons 11-16. Cre-induced deletion of these exons results in a loss of the catalytic ATPase domain along with a frameshift within the remaining protein sequence. When Vav1-iCre +/-Srcap fl/+ and Srcap fl/fl mice are crossed, no Vav1-iCre +/-Srcap fl/fl pups are born, suggesting that hematopoietic-specific-loss of Srcap is embryonic lethal. At E14.5/15.5, Vav1-iCre +/-Srcap fl/fl embryos are paler than other genotypes ( Figure 1) with a reduced number of Ter119 + fetal liver cells. Interestingly, the decrease in Ter119 + cells upon Srcap loss occurs primarily in later stage erythroid progenitors, whereas the number of stage 1 erythroid progenitors is relatively well preserved, suggesting that loss of Srcap adversely impacts fetal erythroid maturation. Furthermore, Srcap loss impacts fetal liver myeloid maturation. While the number of granulocytes is unchanged in Srcap deficient embryos, they have a near complete absence of monocytes, monocyte-dendritic cell progenitors, and monocyte progenitors. In contrast to the changes observed within mature hematopoietic populations, phenotypic hematopoietic stem cells (HSCs) are maintained in Vav1-iCre +/-Srcap fl/fl fetal livers. However, their function is impaired as fetal liver HSCs fail to repopulate upon transplantation ( Figure 2). Importantly, adult HSCs isolated from Mx1-Cre +/-Srcap fl/flmice show a very similar phenotype in that they fail to maintain hematopoiesis when transplanted and treated with poly (I:C). In total, this data shows that Srcap is essential for the maintenance and proper function of both mature hematopoietic populations and hematopoietic progenitors with different lineages and developmental stages displaying varied sensitivities to Srcap loss. To investigate the cellular consequences of SRCAP loss, we knocked down SRCAP expression in hematopoietic cell lines using an inducible shRNA approach. In agreement with the results obtained from our murine model, SRCAP deficient MOLM13 cells show a baseline competitive growth disadvantage that becomes apparent within one week of culture. This is associated with increased γ-H2AX activity, suggesting enhanced DNA damage and consistent with the previously identified role of SRCAP in the cellular DNA damage response. Interestingly, murine fetal liver HSCs with loss of Srcap also exhibit increased γ-H2AX activity, and Srcap null fetal liver stage 1 erythroid progenitors display marked transcriptional upregulation of multiple p53 downstream genes. Our data demonstrate that, in hematopoietic populations, Srcap is an essential gene, with its complete loss resulting in enhanced cellular stress potentially due to increased DNA damage. In humans, nonsense and frameshift mutations in SRCAP appear to be heterozygous in nature, suggesting that the impact of SRCAP deficiency is dose dependent. Indeed, murine HSCs with heterozygous loss of Srcap maintain normal repopulating activity in both primary and secondary transplantation assays ( Figure 2). It remains to be determined how the intermediate deficiencies in SRCAP levels found in patients impact hematopoiesis and lead to clonal expansion and whether the impact of SRCAP loss on the cellular response to stress, including DNA damage, is mediated by its canonical function as a histone variant H2A.Z exchanger. These studies promise to reveal novel insights on the nature of H2A.Z-mediated epigenetic regulation in HSCs and on how its dysregulation may lead to leukemogenesis.

Protein Phosphatase 6 Complex: Novel Regulator of Fetal Hemoglobin and Potential Therapeutic Target in Sickle Cell Disease

Although increasing levels of fetal hemoglobin (HbF) in sickle cell disease (SCD) significantly reduces mortality, effective pharmacologic induction of HbF has remained elusive. To identify potentially druggable molecules involved in HbF control, we carried out a domain-focused CRISPR-Cas9-based genetic screen in HUDEP2 cells targeting all 218 serine/threonine protein phosphatases with 6 independent sgRNAs each. A single phosphatase, PPP6C, emerged as a potential HbF repressor. PPP6C is the catalytic subunit of protein phosphatase 6 (PP6), a serine/threonine phosphatase complex that has been implicated in cell cycle regulation, autophagy, and innate immunity mechanisms in other cell types, but its role in erythropoiesis and HbF regulation has not previously been described. CRISPR-Cas9-based depletion of PPP6C in primary human erythroid cells elevated mRNA levels of HBG (the fetal form of β-globin) in a dose-dependent manner up to 3-4 fold, increased HbF levels 3-fold as measured by HPLC (up to 15-20% of total hemoglobin), and doubled the number of HbF-expressing cells. PPP6C depletion caused relatively few changes in the erythroid transcriptome by RNA-seq analysis, and did not measurably impair erythroid maturation. In addition, PPP6C loss in primary SCD patient-derived cells was well-tolerated, led to robust levels of HbF induction, and reduced cell sickling in vitro by up to 60%. Mechanistically, loss of PPP6C reduced the levels of the HbF repressor BCL11A by nearly 50% but left unchanged the levels of other HbF regulators, such as HRI, LRF, EKLF, NFIA/X, ZNF410, or HIC2, suggesting that PPP6C-mediated HbF regulation proceeds at least in part via loss of BCL11A. Importantly, xenotransplantation data (NBSGW) showed ~4-fold induction of HBG at 16 weeks post-transplant, suggesting that PPP6C deficiency leads to effective, sustained HbF induction in vivo. We next set out to determine whether any PP6 subunits have erythroid specificity that could be exploited therapeutically. In contrast to PPP6C, which is highly expressed in all hematopoietic lineages, the PP6 facultative subunit PPP6R1 appears to be enriched in erythroid precursors, suggesting that depletion of PPP6R1 may function as an erythroid-selective target to increase HbF levels. CRISPR-Cas9-based depletion of PPP6R1 in primary human erythroid cells showed a 2-3-fold increase in HBG and near-doubling of HbF-expressing cells, recapitulating the majority of PPP6C-mediated HbF induction. Functional studies are currently in progress to specifically determine whether additional PP6 subunits play a key role in HbF regulation. Furthermore, ongoing work utilizing dTAG-based acute depletion of PPP6C and PPP6R1 paired with phospho-proteomic studies will narrow down direct targets of the PP6 complex and provide insights into erythropoietic and HbF regulatory pathways impacted by PP6. Taken together, our data indicate that PP6, including its catalytic subunit PPP6C and erythroid-enriched regulatory subunit PPP6R1, inhibit HbF production in part via modulating BCL11A levels, and may serve as a red-cell specific therapeutic target in the treatment of SCD.

Evaluation of the Impact of a Naturally Occurring Beta Hemoglobin Variant, Hb G-Makassar, on Mature Red Blood Cell Function and Pathology in a Sickle Cell Disease Mouse Model

Background: Gene based therapy may provide a cure for individuals with sickle cell disease (SCD); base editing is a novel strategy with the ability to directly edit the mutation responsible for disease. The sickle hemoglobin (HbS) point mutation cannot be converted to wild-type HBB by an adenine base editor, however it can be converted to a naturally occurring hemoglobin variant, HbG-Makassar (HbG). HbG is a non-pathogenic, non-sickling hemoglobin variant. In vitro crystal structure analysis of HbG shows that it is highly similar to that of wildtype hemoglobin (HbA). However, few HbGG individuals (with no overt red cell disease) and no HbSG individuals have been identified; thus, the red blood cells (RBCs) of HbGG and HbSG individuals have not been evaluated functionally. The Makassar base editing strategy produces greater than 80% homozygous Hb-G product and has been evaluated in vitro, however erythroid culture of edited hematopoietic stem cells do not produce fully mature, enucleated red cells. Therefore, mouse models of HbGG and HbSG could be used to evaluate the function of mature, enucleated HbGG or HbSG RBCs. Methods: A humanized beta globin locus mouse (Townes) with the Makassar allele was created at by knocking-in the Makassar point mutation to generated WT/Makassar heterozygotes (HbAG) which were backcrossed to generated HbGG homozygotes. HbSG genotypes were subsequently generated by crossing to HbSS Townes mice. We analyzed the red cell function of HbAA, HbAS, HbSS, HbGG, and HbSG mice. Whole blood was collected through terminal enucleation. Complete blood counts were measured on a hematology analyzer (Heska and, ADVIA). Red cell deformability (elongation index, EI) was measured using oxygen gradient ektacytometry (LoRRCA) under normoxic (EImax) and hypoxic (EImin) conditions. Viscosity measurements were obtained on a Beckman cone and plate viscometer. Percent sickle cells were quantified through chemically induced sickling with sodium metabisulfite and counted by an individual blinded to genotype using light microscopy. Kidney, liver, and spleen were fixed, preserved in paraffin, stained using hematoxylin and eosin stains and imaged using a Keyence microscope. Flow cytometry was performed on bone marrow and whole blood to determine the erythroid maturation and mitochondrial retention of RBCs. Analysis was performed with Stata 18.0 (College Station) across genotypes using Dunn Pairwise test with statistically significant values being selected at adjusted p < 0.05. Results Homozygous HbG mice (HbGG) had comparable white blood cell count (WBC) and absolute reticulocyte count (ARC) to that of mice with homozygous HbA (HbAA). HbGG mice had lower hemoglobin and mean corpuscular volume (MCV) in comparison to mice with HbAA. HbGG mice had a higher percentage of dense RBCs than HbAA mice. HbSG mice had comparable values to HbAS mice for MCV but had significantly lower hemoglobin and ARC. HbSG mice had significantly higher values for WBC, and % dense RBCs against HbAS mice. HbSS and HbSG mice had comparable values for hemoglobin, and ARC. Hematocrit to viscosity ratio (HVR) shows comparable oxygen transport effectiveness of HbGG, HbSG, HbAS and HbSS RBC. Further red cell function testing is shown in Figure 1 and Figure 2. Mitochondrial retention was highest in HbSS mice, followed by HbSG, while HbAS, HbAA, and HbGG had similar values. Microscopy analysis of renal sections of HbGG and HbSG mice sacrificed at 24-28 weeks did not show the characteristic morphologic changes seen in HbSS mice at 24-28 weeks. Conclusions- HbGG appears functionally similar to HbAA, with minimal sickling under hypoxia, no anemia, normal spleen size, and no organ damage at 24 weeks of age. HbGG does exhibit signs of dehydration, as evidenced by elevated %DRBC and reduced RBC deformability. RBC function, sickling under hypoxia, Hb, WBC, ARC, and mitochondrial retention measures places HbSG as intermediate in severity between HbAS and HbSS. However, organ pathology, spleen and liver weights for HbSG were more comparable to HbAS than HbSS. Base editing strategies to install Makassar therefore can potentially lead to >90% of RBCs that have therapeutic benefit due to rheological properties comparable to HbAA and can be seen as a promising treatment strategy for patients with sickle cell disease.

Efficacy and Tolerability of Luspatercept in Patients with Lower Risk Myelodysplastic Syndrome and Anemia: A Systematic Review and Meta-Analysis of Phase III Randomized Controlled Trials

Introduction: Erythropoietin Stimulating Agents (ESAs) are the standard-of-care treatment for most patients with lower risk myelodysplastic syndrome (MDS) and anaemia. However, there has been unmet need for those who failed ESAs other than regular blood transfusion. Luspatercept is a recombinant fusion protein which binds TGF beta ligands resulting in reduction of erythroid maturation through reduction of SMAD2 and SMAD3 signaling. In recent years, studies have shown that luspatercept has significantly improved efficacy in patients with lower risk MDS who failed ESAs with some notable adverse events. The purpose of our meta-analysis is to determine efficacy by observing the rate of transfusion independence for 12 weeks or longer and haematological improvement in erythroid (HIE), and tolerability by rate of treatment emergent serious adverse events (SAE), treatment discontinuation (TD) and treatment interruption (TI) or dose reduction and treatment related mortality in patients with lower risk MDS treated with luspatercept. Methods: We systematically conducted a comprehensive literature search using MEDLINE, EMBASE databases and meeting abstracts from inception through June 30 th, 2023. Phase III RCTs utilizing luspatercept in patients with lower risk MDS that mention efficacy i.e. transfusion independence 12 weeks or longer and HIE and tolerability i.e. rate of treatment discontinuation and interruption, dose reduction, treatment emergent serious adverse events, treatment related mortality were incorporated in the analysis. Mantel-Haenszel (MH) method was used to calculate the estimated pooled risk ratio (RR) with 95% confidence interval (CI). Heterogeneity was assessed with Cochran's Q- statistic. Fixed effects model was applied. Results: 2 phase III RCTs (MEDALIST and COMMANDS) including total 583 patients were eligible in our meta-analysis. MEDALIST trial compared luspatercept vs placebo and COMMANDS trial compared luspatercept vs erythropoietin stimulating agent. The randomization ratios were 2:1 in MEDALIST and 1:1 in COMMANDS trials. Transfusion independence for 12 weeks or longer was noted in 141 (47%) in study arm vs 77 (33.5%) in control arm at RR of 1.67 (95% CI: 1.35 -2.06; P <0.00001). 190 (63.3%) patients in study arm achieved HIE response compared to 88 (38.3%) in control arm at RR of 1.85 (95% CI: 1.53 -2.24; P<0.00001). TD due to TEAE was reported in 21 (6.3%) of luspatercept group vs 10 (4%) in control group with RR of 1.38 (95% CI: 0.67 -2.84; P = 0.39). The incidence for TI or dose reduction due to TEAE was similar between the two groups at 18% with RR of 1.23 (95% CI: 0.88 -1.72; P = 0.22). Treatment emergent SAEs were observed in 116 (35%) in the luspatercept group vs 83 (32.9%) in control group (RR, 1.09; 95% CI: 0.87-1.38; P=0.45). Treatment related mortality was noted in 13 (3.9%) of patients in the study group compared to 16 (6.3%) in the control group with RR of 0.65 (95% CI: 0.31 -1.33; P = 0.24). Conclusions: According to our meta-analysis, luspatercept significantly improved transfusion independent rate and HIE response in patients with lower risk MDS and anaemia compared to control arm. Furthermore, there was no statistically significant difference in the treatment emergent SAEs, treatment discontinuation, treatment interruption or dose reduction and treatment related mortality between the two groups.

A Pilot Multicentric Prospective Study on the Usefulness of Peripheral Blood Immunophenotyping As Erythroid Maturation Response Marker during Luspatercept Treatment in Patients Affected By ß-Thalassemia Major and SF3B1+ Myelodysplastic Syndrome

Introduction Erythropoiesis is a multi-step maturation process; luspatercept proved to be successful on ineffective erythropoiesis acting on the second phase of erythroid maturation reducing the hyper expression of transforming growth factor beta (TGF-beta) ligands via the Smad 2/3 signaling pathway, thus ameliorating the aberrant late stage erythroid maturation featured in the myelodysplastic syndrome (MDS) and thalassemia. MEDALIST and BELIVE studies showed efficacy and safety of Luspatercept in MDS and Thalassemic patients respectively, but, regarding erythroid maturation, a significant correlation has been revealed only between hematological response and the reticulocyte value or medullar erythroblasts counts. In order to avoid for patients extra scheduled bone marrow aspirate , in particular in thalassemic patients, in our pilot study we propose to explore, for the first time, the role of peripheral blood immunophenotyping (IF) as reproducible and more affordable erythroid maturation's marker respect to the medullar immunophenotyping described in the previous published papers. Our hypothesis is that peripheral IF could represent a new tool to detect the ameliorate of inefficacy erythropoiesis during Luspatercept therapy, acting as “minimal residual disease” in patients in response or that loss the response to Luspatercept. Patients, Material and Methods Approval by local Ethic Scientific Commission has be completed. Patients will be enrolling by Hematology Unit and by Hemoglobinophaties Unit from 2 different Institution in Italy (“A. Businco” Oncologic Hospital and “A. Cao” Hemoglobinopathies Hospital). Inclusion criteria: Patients must be affected by MDS with ringed sideroblasts (with or without SF3B1 mutation) very low, low and intermediate revised IPSS stage or affected by ß-thalassemia major; aged ≥ 18 years and able to understand and sign an informant consent; not renal or hepatic failure; EF > 50%. Duration of the study will be scheduled for 1 year. Prevision of 30 patient enrolled in the first 1 year of study. Primary endpoint is to confirm the role of peripheral blood immunophenotyping (IF) as reproducible and affordable erythroid maturation's marker in these setting of patients. Secondary endpoint is to evaluate the role of peripheral IF as “minimal residual disease” in particular during the loss of Lusplatercept response and also the assessments of iron homeostasis in responder's patients. Patients will be assessed for r-IPSS and NGS (only for MDS), endogenous erythropoietin, full blood count and reticulocyte, iron status, transfusion burden in the previous 24 weeks and peripheral IF (centralized in the laboratory of Hematology Unit). All patients will collect these samples at baseline timing and after 12,24,36 and 48 weeks. Peripheral IF is tested by 8-color EuroFlow antibody panels define progressive terminal stages of erythroblasts' maturation (10 health samples have been used as control). Hematological response and Luspatercept's dose escalation are considered as for MEDALIST study criteria for MDS and BELIVE study for thalassemia, as well adverse events. Results At the time of this report 26 patients have been enrolled in the two institution (10 MDS and 16 thalassemic patients). Baseline patients' characteristics are reported in TAB 1 and baseline peripheral blood erythroid maturation (based on centralized IF) are reported in TAB 2 Conclusion Our pilot study aims to explore, for the first time, the role of peripheral blood immunophenotyping (IF) as reproducible and affordable erythroid maturation's marker during Luspatercept therapy in Thalassemic and MDS patients.