Sickle cell disease (SCD) causes erythrocyte sickling and downstream sequelae, including hemolysis, iron mishandling, and subclinical renal dysfunction, thereby reducing erythropoietic drive. These effects result in chronic anemia that drives SCD-related complications. Treatment of severe anemia in SCD with frequent subcutaneous injections of erythropoiesis-stimulating agents is often poorly tolerated by patients, underscoring the need for oral treatments. Here, we provide key insights into how daprodustat, an oral hypoxia-inducible factor prolyl hydroxylase inhibitor (HIF-PHI) that stimulates red cell production, may serve as a targeted treatment for anemia in SCD. In a mouse model of SCD, daprodustat increases red blood cell production, reduces sickling, and improves iron mobilization by suppressing hepatic hepcidin expression, thereby enhancing iron availability for erythropoiesis and ameliorating chronic anemia. In addition to these systemic, non-cell-autonomous effects on iron homeostasis, daprodustat directly induces γ-globin expression and increases the production of fetal hemoglobin-containing erythroid cells (F-cells) derived from CD34+ hematopoietic stem and progenitor cells of patients with SCD, conferring cell-autonomous therapeutic benefits. These findings demonstrate that HIF-PHIs act on multiple therapeutic targets in SCD and represent a promising oral treatment option for chronic anemia in this disease.

Cytochrome b5 reductase 3 (CYB5R3) or met-hemoglobin reductase is an oxidoreductase that maintains hemoprotein and cellular redox balance, yet its contribution to erythropoiesis under stress conditions remains unclear. Motivated by prior observations that the hypomorphic CYB5R3 T117S blunts hydroxyurea-induced fetal hemoglobin responses in patients with sickle cell disease, we tested whether CYB5R3 contributes to the regulation of erythropoiesis. Hematopoietic lineage-specific CYB5R3 knockout mice exhibited markedly impaired erythropoietic induction in response to chronic hypoxia compared to controls, with males showing a more pronounced deficit, and splenectomy further exacerbating this impairment. Genetic deletion of CYB5R3 in human CD34⁺ progenitors reduced globin expression and disrupted terminal erythroid differentiation. Meanwhile, CYB5R3 knockdown in K562 cells produced a heme-deficient state whereby only exogenous heme but not hydroxyurea, iron, or upstream precursors restored globin synthesis. Transcriptomic profiling revealed coordinated downregulation of erythroid transcription factors and multiple enzymes in the heme biosynthetic pathway, which was reversed with heme treatment. Together, these results reveal an unexpected function for CYB5R3 beyond met-hemoglobin reduction, positioning it as a central metabolic regulator of sex-specific stress erythropoiesis and unveiling a heme-restricted vulnerability that may augment disease severity in anemia, hemoglobinopathies, and individuals carrying CYB5R3 loss-of-function variants. ### Competing Interest Statement Dr. Straub received research funds from Bayer Pharmaceuticals and holds stock options in Creegh Pharmaceuticals. Dr. Schopfer has a financial interest in Creegh Pharmaceuticals Inc. and Furanica Inc. NIH, R35 HL161177 NIH, R01 GM 125944

Sickle cell disease (SCD) is a monogenic disorder that results in chronic anemia and painful vaso-occlusive episodes. Chronic anemia increases the risk for development of chronic kidney disease, pulmonary hypertension, and stroke in SCD. The current standard of care therapy, hydroxyurea (HU), inhibits polymerization of abnormal hemoglobin S (HbS) by inducing fetal hemoglobin (HbF) expression, preventing vaso-occlusive complications. However, HU only modestly improves anemia, which can persist due to inadequate erythropoietic (EPO) production. Hypoxia-inducible factor prolyl hydroxylase inhibitors (HIF-PHIs) are a class of orally bioavailable drug that stimulate erythropoiesis by inhibiting degradation of HIF1/2α, increasing EPO expression. In addition, the HIF-PHI roxadustat (roxa) has been shown to induce HbF expression in CD34+ hematopoietic stem and progenitor cells (HPSCs) in vitro, suggesting multiple therapeutic benefits in SCD. Daprodustat (dapro) is the first FDA-approved HIF-PHI for dialysis-dependent chronic kidney disease. In this study, we sought to evaluate the HbF-inducing and erythropoietic potential of daprodustat in both a CD34+ HSPC in vitro model and an in vivo mouse model of SCD. CD34+ HSPCs were isolated from 3 patients with SCD, differentiated in vitro to erythroblasts, and treated with vehicle, 30μM dapro, or 50μM of HU. Flow cytometric analysis on Day 15 (D15) showed that dapro treatment increased % F-cells by 2-fold (p<0.05) compared to vehicle, similar to the effect of roxa on HbF induction (Feng et al., Nature 2022; Sharma et al., ASH abstract 2022). HU treatment increased % F-cells by 3-fold (p<0.001) compared to vehicle. We then evaluated the effect of dapro in an in vivo SCD model using male 10 wk old C57BL/6J mice transplanted with bone marrow from transgenic SCD Townes mice (Jackson Laboratory). Engrafted mice were treated by oral gavage daily for 3 weeks with either vehicle (10%DMSO;80%PEG;10%saline) (n=4) or 30 mg/kg dapro (n=5). All mice underwent retro-orbital bleeding at baseline (24 hours pretreatment), D10 and D21 for hematological evaluation. Reticulocyte% was also determined by flow cytometry at D21. At D10, the increase in Hb concentration compared to baseline was higher in the dapro vs. vehicle treated mice (44% vs. 2%, p<0.05). This difference persisted at D21, with an increase in Hb of 50% in the dapro group and a decrease of 30% in vehicle group (p<0.01). Similar trends were observed in Hct and RBC count. Interestingly, at D21, reticulocyte% was lower in the dapro group vs. vehicle group (39% vs. 55%, p<0.01). RBC indices (mean corpuscular volume (MCV), mean corpuscular hemoglobin (MCH), and MCH concentration (MCHC)) decreased earlier in the treatment course and to a greater extent in the dapro vs. vehicle treated mice, but these differences were not significant. Additionally, by D21, there was a significantly greater increase in platelet count in the dapro treated group vs. the vehicle treated group (75% vs. -20%, p<0.001). White blood cell (WBC) count decreased in both groups; the difference was not significant. Our study demonstrates that dapro has the potential to both induce HbF and erythropoietic drive in preclinical models of SCD. Despite the significant increase in Hb, Hct and RBC count, reticulocyte% was lower in the dapro vs. vehicle treated group at D21, suggesting that dapro may influence Hb through multiple mechanisms, such as decreasing hemolysis through HbF induction or decreasing stress erythropoiesis by correcting anemia. Notably, there was a decrease in Hb, Hct, MCV, MCH, and MCHC in the vehicle group at D21, concerning for development of iron deficiency anemia. There was a similar decrease in MCV, MCH, and MCHC at D21 in the dapro group, in line with the hypothesized vehicle-induced iron deficiency, but dapro treatment may have protected against the development of anemia from iron deficiency. Finally, we saw a decrease in all three cell lines (WBC, RBC, and platelets) in the vehicle group at D21, concerning for bone marrow toxicity from the vehicle, suggesting that the erythropoietic effect of dapro treatment may have been even greater without this confounding effect. In summary, daprodustat proves to be a promising candidate for treatment of anemia in SCD, and the long-term effects of daprodustat on erythropoiesis, HbF induction, and iron metabolism in SCD should be further evaluated.

Background: Cytochrome b5 reductase 3 (CYB5R3) is critical for sustaining vital physiological reactions connected to oxidative equilibrium, heme reduction, cholesterol biosynthesis, lipid desaturation and drug metabolism. Hemoglobin, Coenzyme Q, soluble guanylate cyclase (sGC), Vitamin E, Vitamin C, etc., are important substrates of CYB5R3 that get activated through reduction. With numerous genetic variants spotted till date, CYB5R3 T117S stands out with ~50% loss of function and a high allele frequency in persons of African origin, an ethnic group that mostly bears the burden of sickle cell disease (SCD). In an oxidative systemic phenomenon like SCD where stress erythropoiesis is induced to constantly replace hemolyzed erythrocytes, CYB5R3 may be critical. Hydroxyurea (HU) is the FDA-approved first line therapy for SCD. We have recently discovered that CYB5R3 T117S is a modifier of HU efficacy for treating anemia through fetal hemoglobin (HbF) synthesis and erythrocyte formation in SCD patients. Since NO-cGMP signaling is known to induce erythropoiesis and HbF under stress via CYB5R3-regulated sGC, this also makes the reductase a logical actor in this pathway. Hence, we hypothesized that CYB5R3 is crucial for erythropoiesis. Methodology and result: CRISPR was utilized to create a non-targeting (NT) control and CYB5R3 knockout (KO) CD34+ hematopoietic stem cells (HSCs). Cells were collected on day 11 to check the hemoglobin transcripts using qPCR and on day 15 to check protein level using western blot. In comparison to the NT group, the expression of beta globin (HBB) and gamma globin (HBG) were significantly lower in the KO cells (n=8-11, p<0.05). This was followed by a fewer percentages of CD44lowFSClow cells in the KO cells, representing fewer mature erythrocytes and enucleated reticulocytes on day 18 (n=4, p<0.05), determined using flow cytometry. Knocking down (KD) CYB5R3 in K562 cells using lentiviral transduction depleted the transcript (40-60%) and protein levels (60-80%) of HBB and HBG significantly (n=4, p<0.05). Treatment with HU did not induce globin accumulation as expected in the KD cells neither at transcript nor protein level, although there was a 1.5-fold induction in the NT group (n=4, p<0.05). Since hemoglobin is made up of heme and globin and heme induces globin formation, the level of heme and heme intermediate protoporphyrinogen were then measured in the cells using High Performance Liquid Chromatography-Mass-spectrometry (HPLC-MS). Both the protoporphyrinogen and heme levels were reduced by approximately 40% in the KD cells (n=8, p<0.05), demonstrating heme deficiency in absence of CYB5R3. Addition of hemin, an iron (III) chloride heme, although did not rescue the transcripts, significantly rescued the globin proteins in the KD cells in comparison to the baseline (n=4, p<0.05). However, treatment with iron alone did not have any effect on globins. Supplementing the cells with 5-amino-levulinic acid (5-ALA), the key substrate of the heme biosynthesis pathway, did boost the globin proteins by 2-3 fold in comparison to the untreated KD cells, but that was not enough to rescue the proteins back to the baseline as in NT (n=3). Both RNA-seq analysis and qPCR revealed suppressed levels of key erythroid transcription factors, including GATA-binding factor 1 (GATA1) and enzymes in heme biosynthesis pathway, including 5-amino-levulinate synthase 2 (ALAS2), Porphobilinogen Deaminase (PBGD), Coproporphyrinogen Oxidase (CPOX), along with other important erythroid specific genes (n=3, p<0.05) in the KD cells, thus leading to heme deficiency which was reversed to some extent by hemin supplementation (n=3, p<0.05). Conclusion: In conclusion, it can be said that the absence of CYB5R3 causes downregulation of erythroid specific transcription factors and heme biosynthesis enzymes, hindering heme formation. Lack of heme affects globin formation and stability, thereby affecting erythrocyte maturation in the long run. Thus, these new insights about the role of CYB5R3 in erythropoietic pathway may enable better understanding of the complex mechanisms regulating erythropoiesis and lead to novel drug development strategies for erythropoietic disorders.

Sickle cell disease (SCD) is a hereditary hematological disease with high morbidity and mortality rates worldwide. Despite being monogenic, SCD patients display a plethora of disease-associated complications including anemia, oxidative stress, sterile inflammation, vaso-occlusive crisis-related pain, and vasculopathy, all of which contribute to multiorgan dysfunction and failure. Over the past decade, numerous small molecule drugs, biologics, and gene-based interventions have been evaluated; however, only four disease-modifying drug therapies are presently FDA approved. Barriers regarding effectiveness, accessibility, affordability, tolerance, and compliance of the current polypharmacy-based disease-management approaches are challenging. As such, there is an unmet pharmacological need for safer, more efficacious, and logistically accessible treatment options for SCD patients. Herein, we evaluate the potential of small molecule nitroalkenes such as nitro-fatty acid (NO2-FA) as a therapy for SCD. These agents are electrophilic and exert anti-inflammatory and tissue repair effects through an ability to transiently post-translationally bind to and modify transcription factors, pro-inflammatory enzymes and cell signaling mediators. Preclinical and clinical studies affirm safety of the drug class and a murine model of SCD reveals protection against inflammation, fibrosis, and vascular dysfunction. Despite protective cardiac, renal, pulmonary, and central nervous system effects of nitroalkenes, they have not previously been considered as therapy for SCD. We highlight the pathways targeted by this drug class, which can potentially prevent the end-organ damage associated with SCD and contrast their prospective therapeutic benefits for SCD as opposed to current polypharmacy approaches.

The fetal-to-adult switch in hemoglobin production is a model of developmental gene control with relevance to the treatment of hemoglobinopathies. The expression of transcription factor BCL11A, which represses fetal β-type globin (HBG) genes in adult erythroid cells, is predominantly controlled at the transcriptional level but the underlying mechanism is unclear. We identify HIC2 as a repressor of BCL11A transcription. HIC2 and BCL11A are reciprocally expressed during development. Forced expression of HIC2 in adult erythroid cells inhibits BCL11A transcription and induces HBG expression. HIC2 binds to erythroid BCL11A enhancers to reduce chromatin accessibility and binding of transcription factor GATA1, diminishing enhancer activity and enhancer-promoter contacts. DNA-binding and crystallography studies reveal direct steric hindrance as one mechanism by which HIC2 inhibits GATA1 binding at a critical BCL11A enhancer. Conversely, loss of HIC2 in fetal erythroblasts increases enhancer accessibility, GATA1 binding and BCL11A transcription. HIC2 emerges as an evolutionarily conserved regulator of hemoglobin switching via developmental control of BCL11A.

Sickle cell disease (SCD) is a devastating monogenic disorder caused by a mutation in the β-globin gene, leading to production of mutant hemoglobin S (HbS). Abnormal polymerization of deoxygenated HbS causes sickling of red blood cells (RBCs), resulting in painful vaso-occlusive crises and chronic hemolytic anemia. Etiologies of chronic anemia in SCD are multifactorial, including intravascular and extravascular hemolysis, inadequate erythropoietin (EPO) production, and abnormal iron homeostasis (Xu and Thein, Blood 2022). Hydroxyurea (HU) is the standard-of-care drug therapy for SCD and primarily reduces HbS polymerization through induction of fetal Hb (HbF) expression. However, it only modestly improves anemia and does not address other causes of anemia in SCD. Additionally, lower Hb levels in SCD are associated with chronic kidney disease (CKD), stroke, and pulmonary hypertension, arguing for the need for additional therapies targeting anemia in SCD. Hypoxia-inducible factor prolyl hydroxylase inhibitors (HIF-PHIs) are a class of drugs developed for treatment of anemia in CKD and end-stage renal disease. HIF-PHIs stimulate erythropoiesis by inhibiting prolyl hydroxylase and von Hippel-Lindau (VHL)-mediated degradation of HIF-α, leading to increased hypoxic signaling and promotion of HIF-induced EPO expression in the kidneys and liver. Stimulation of erythropoiesis also increases production of erythroferrone, the main regulator of hepcidin, and suppression of hepcidin levels by HIF-PHIs may improve iron mobilization. Furthermore, HIF-PHIs can induce HbF expression in erythroblasts derived from CD34+ hematopoietic stem and progenitor cells (HSPCs) (Hsieh et al.,Blood 2007, Feng et al., ASH abstract 2021), suggesting multiple potential therapeutic benefits in SCD. Therefore, we characterized the effect of the HIF-PHI FG-4592 (roxadustat), on HbF induction in CD34+ HSPCs and on hematologic markers, EPO and hepcidin expression in a mouse model of SCD. CD34+ HSPCs from 3 donors were differentiated in vitro to erythroblasts with vehicle, 20 μM of FG-4592, or 50 μM of HU. Flow cytometric analysis on Day 9 showed that FG-4592 treatment increased % F-cells by 1.47-fold (±0.45), compared to a 2.21-fold (±0.89) increase with HU (Fig. 1). We then transplanted lethally irradiated (1000 rads) male 6-8-week-old C57BL/6J mice with bone marrow from transgenic sickle cell mice (Hb SS; Townes model, Jackson Laboratory). Twelve weeks after transplantation, mice with >80% engraftment of the donor HbSS phenotype, determined by Hb electrophoresis, were used for subsequent experiments. Vehicle, 25 mg/kg FG-4592, or 50 mg/kg FG-4592 was administered three times weekly through intraperitoneal injection for 8 weeks. All mice underwent retro-orbital bleeding at baseline (48 h pretreatment), 2, 4, 6, and 8 weeks of treatment for hematological evaluation, including Hb, hematocrit, and reticulocyte % (Fig. 1). At the end of 8 weeks, livers, kidneys, and plasma were collected. Hb and hematocrit levels increased in the 50 mg/kg FG-4592 group compared to the other groups at 4 weeks but decreased by 8 weeks for all groups. Reticulocyte % also decreased in all groups at the 4- and 8-week timepoints. Red cell distribution width (RDW) and platelet count up-trended while mean corpuscular volume (MCV) and mean corpuscular Hb (MCH) down-trended over the 8 weeks, suggestive of an evolving iron deficiency contributing to worsening anemia. Quantitative PCR detected a 182% increase in liver EPO transcript and a 68% increase in kidney EPO transcript in the 50 mg/kg FG-4592 treated group, compared to vehicle. No changes in liver hepcidin transcription were observed in any group. ELISA analysis detected increased plasma EPO and decreased plasma hepcidin levels with increasing doses of FG-4592. None of these trends, except increases in RDW, were statistically significant. Our study is the first to characterize the effect of HIF-PHI drugs in a mouse model of SCD. Preliminary data confirm an effect of FG-4592 on HbF induction and suggest that FG-4592 may increase EPO expression and decrease hepcidin levels in SCD mice. These results highlight the potential of prolyl hydroxylase inhibition as a novel therapeutic approach for chronic anemia in SCD. Future directions include testing higher doses of FG-4592 to induce a stronger erythropoietic effect and monitoring additional markers of iron homeostasis in our mouse model of SCD. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Background: Sickle cell anemia (SCA) is a hereditary disorder caused by the formation of hemoglobin S (HbS). HbS undergoes polymerization and generates sickled red blood cells (RBCs). Along with having reduced oxygen carrying capacity, sickled RBCs are also prone to frequent hemolysis releasing the pro-oxidant heme in the circulation. Oxidative stress created by the free heme leads to sterile inflammation, vaso-occlusion and cardiovascular complications. The prevalence of SCA is greatest in the poorer regions of Sub-Saharan Africa and there are around 100,000 new incidents of SCA every year in the USA alone. Although a number of therapeutic options are currently FDA-approved for the treatment of SCA patients, there is considerable interpatient variability in terms of their efficacy and safety, along with high morbidity and mortality rates. We have developed a novel quinone-nitroalkene hybrid molecule called CP50 (figure 1), which has been shown to activate two important proteins: 1) nuclear factor erythroid 2-related factor 2 (Nrf2), a transcriptional regulator of cellular resistance to oxidants and 2) cytochrome b5 reductase 3 (CYB5R3), an anti-stress enzyme in the cardiovascular system. Methodology and results: Primary human aortic endothelial cells (HAECS) were incubated with 5 µM CP50 for 6 hours to perform a genomic screening. The mRNA expression profile revealed that CP50 differentially regulated pathways associated with oxidative balance, inflammation, and erythropoiesis. CP50 was associated with upregulation of genes responsible for counteracting stress, including heat shock protein family, HSPA6, HSPA1A, HSPA1B and heme-oxygenase 1 (HMOX-1) (n=5, p<0.05). CP50 downregulated genes that inhibit effective erythropoiesis, such as ZFP36 Ring Finger Protein Like 2 (ZFP36L2) and Growth/differentiation factor 15 (GDF15), thus imparting a positive effect on the process (n=5, p<0.05). Immunoblotting for heme-oxygenase 1 (HO-1) using wild type and Nrf2-knockdown HAECs demonstrated that HO-1 was induced by 5 µM CP50 by 5 fold (±1.36) after 24 hours of exposure, in a Nrf2 dependent manner (n=3, p<0.0001). CYB5R3 activity measured by its impact on myoglobin reduction rate, was also found to increase dose-dependently by CP50 (n=3, p<0.001). Afterwards, CD34+ hematopoietic stem cells (HSCs, n=3) were cultured with 5 µM CP50 and 50 µM hydroxyurea (HU) to observe the effect on erythropoiesis, using CD71, CD235a and fetal hemoglobin (HbF) as markers for flow cytometry. CP50 augmented the percentage of HbF-expressing F cells by 1.7 fold (±0.54) in comparison to 2.21 fold (±0.89) by HU on day 9. F cells are known to impart resilience against sickling and hemolysis in sickle cell anemia. We then observed the effect of CP50 administered by osmotic pump implantation on hematopoiesis in Townes HbSS mice (vehicle, 5mg/kg/day, 15mg/kg/day, n=3/4 in each group). After a period of 4 weeks, we collected whole bone marrow from the mice and using flow cytometry checked for HSCs. CP50 (15 mg/kg/day) significantly increased (p<0.001) the HSC pool by 59% in comparison to vehicle. Additionally, it also enhanced the differentiation of megakaryocyte-erythroid progenitors (MEP) in bone marrow cells by 11% (p<0.05). In a separate experiment, peripheral blood samples from Townes HbSS mice were collected and incubated with 5 µM CP50 before chemically inducing oxidative hemolysis. CP50 reduced hemolysis by 73.68% in comparison to vehicle. Conclusion: Together, our studies indicate that CP-50 has the potential to correct oxidative imbalance by activating antioxidant genes, reverse anemia by inducing hematopoiesis, and inhibit hemolysis by increasing F cell percentage. Thus, we can conclude that CP50 is a prospective drug candidate with the promise to satisfy the unmet need for additional safe and effective therapeutic options for patients with sickle cell anemia. Legend (figure 1): The quinone head group in CP50 serves as an antioxidant, whereas the nitro group in the fatty acid side chain acts as an electrophile. The electrophile modifies the cysteine maintaining the Keap1-Nrf2 interaction and releases the Nrf2. Nrf2 enters the nucleus and initiates the expression of genes that counteract oxidative stress. Keap1: Kelch-like ECH-associated protein 1 Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

The fetal-to-adult hemoglobin transition is clinically relevant because reactivation of fetal hemoglobin (HbF) significantly reduces morbidity and mortality associated with sickle cell disease (SCD) and β-thalassemia. Most studies on the developmental regulation of the globin genes, including genome-wide genetics screens, have focused on DNA binding proteins, including BCL11A and ZBTB7A/LRF and their cofactors. Our understanding of RNA binding proteins (RBPs) in this process is much more limited. Two RBPs, LIN28B and IGF2BP1, are known posttranscriptional regulators of HbF production, but a global view of RBPs is still lacking. Here, we carried out a CRISPR/Cas9-based screen targeting RBPs harboring RNA methyltransferase and/or RNA recognition motif (RRM) domains and identified RNA binding motif 12 (RBM12) as a novel HbF suppressor. Depletion of RBM12 induced HbF expression and attenuated cell sickling in erythroid cells derived from patients with SCD with minimal detrimental effects on cell maturation. Transcriptome and proteome profiling revealed that RBM12 functions independently of major known HbF regulators. Enhanced cross-linking and immunoprecipitation followed by high-throughput sequencing revealed strong preferential binding of RBM12 to 5' untranslated regions of transcripts, narrowing down the mechanism of RBM12 action. Notably, we pinpointed the first of 5 RRM domains as essential, and, in conjunction with a linker domain, sufficient for RBM12-mediated HbF regulation. Our characterization of RBM12 as a negative regulator of HbF points to an additional regulatory layer of the fetal-to-adult hemoglobin switch and broadens the pool of potential therapeutic targets for SCD and β-thalassemia.

Reversal of the developmental switch from fetal (HbF, α 2γ 2) to adult (HbA,α 2β 2) hemoglobin is an important therapeutic approach for sickle cell disease (SCD) and β-thalassemia. It has been noted since the 1950s that a small number of circulating red blood cells, called F-cells, produce elevated levels of HbF; these cells are resistant to sickling and are present in increased numbers in patients with SCD and those treated with pharmacological HbF inducers such as hydroxyurea. Because successful therapy for SCD requires increasing the number of F-cells, it is imperative to understand how these cells arise. This can potentially occur through a shift towards a global fetal-like program, selective variation in levels of known HbF silencers such as BCL11A or LRF, or through discrete epigenetic changes at the β-globin locus. We previously began to address this clinically important question using a novel experimental approach of sorting cultured primary human erythroblasts into HbF-high (F-cell) and HbF-low (A-cell) populations (Khandros et al, Blood 2020). We showed that surprisingly, F-cells from healthy donor primary erythroid cultures have minimal transcriptional differences with A-cells. Unexpectedly, this was also the case when comparing responders (F-cells) and non-responders (A-cells) to treatment with the HbF inducers pomalidomide and hydroxyurea, and there were no differences in the expression of known HbF regulators. We therefore hypothesize that HbF synthesis in F-cells is determined by epigenetic variation confined to the β-globin locus (and not by global changes in the cell fate or nuclear milieu).

Elevated levels of fetal hemoglobin (HbF) significantly ameliorate clinical outcomes for patients with beta-hemoglobinopathies, such as sickle cell disease (SCD). The only FDA-approved drug for treating SCD through inducing HbF is hydroxyurea, however the mechanism of action is unknown with variable effectiveness among patients. Thus, there remains a strong interest to identify more robust means of upregulating HbF, such as specific inhibition of HbF repressors. BCL11A and LRF are well-characterized transcription factors that independently repress the fetal type b-globin like genes HBG1 and HBG2 but their therapeutic potential is limited by challenging druggability and critical developmental function. However, upstream regulation of these factors, such as post-transcriptional mechanisms, are not well studied and may house novel therapeutic targets. To this end, we employed a CRISPR/Cas9 based screening approach to interrogate a library of RNA binding proteins (RBP) in the context of HbF regulation. Using HUDEP2 cells, a human adult-type erythroid progenitor cell line, we screened 341 human RBPs and identified four candidate RBPs, none of which have previously been implicated in HbF regulation. Of these candidates, RNA Binding Motif 12 (RBM12) showed the greatest level of HbF induction following in vitro depletion.

Glaucoma is clinically characterized by elevated intraocular pressure (IOP) that leads to retinal ganglion cell (RGC) and optic nerve damage, and eventually blindness if left untreated. Even in normal pressure glaucoma patients, a reduction of IOP is currently the only effective way to prevent blindness, by either increasing aqueous humor outflow or decreasing aqueous humor production. The trabecular meshwork (TM) and the adjacent Schlemm’s canal inner wall play a key role in regulating IOP by providing resistance when aqueous humor drains through the tissue. TM dysfunction seen in glaucoma, through reduced cellularity, abnormal extracellular matrix accumulation, and increased stiffness, contributes to elevated IOP, but current therapies do not target the TM tissue. Stem cell transplantation for regeneration and re-functionalization of damaged TM has shown promise in providing a more direct and effective therapy for glaucoma. In this review, we describe the use of different types of stem cells for TM regeneration in glaucoma models, the mechanisms of regeneration, and the potential for glaucoma treatment using autologous stem cell transplantation.

Animal chromosomes are partitioned into contact domains. Pathogenic domain disruptions can result from chromosomal rearrangements or perturbation of architectural factors. However, such broad-scale alterations are insufficient to define the minimal requirements for domain formation. Moreover, to what extent domains can be engineered is just beginning to be explored. In an attempt to create contact domains, we inserted a 2-kb DNA sequence underlying a tissue-invariant domain boundary—containing a CTCF-binding site (CBS) and a transcription start site (TSS)—into 16 ectopic loci across 11 chromosomes, and characterized its architectural impact. Depending on local constraints, this fragment variably formed new domains, partitioned existing ones, altered compartmentalization and initiated contacts reflecting chromatin loop extrusion. Deletions of the CBS or the TSS individually or in combination within inserts revealed its distinct contributions to genome folding. Altogether, short DNA insertions can suffice to shape the spatial genome in a manner influenced by chromatin context.

Upregulation of fetal hemoglobin (HbF, α2γ2) by reversing the developmental switch to adult HbA (α2β2) is a key approach for both pharmacologic and gene targeting therapies in the treatment of sickle cell disease (SCD) and β-thalassemia. HbF expression in healthy individuals, patients with SCD, and those treated with hydroxyurea is restricted to a subset of red blood cells known as F-cells; effective SCD therapy requires increasing the proportion of F-cells expressing sufficient HbF to block sickling. Although these cells have been observed since the 1950s, there have not been previous direct comparisons of F-cells to matched HbF-low A-cells from the same individual. Fetal erythroblasts have distinct global transcriptional programs and distinct long-range chromatin looping at the β-globin locus when compared to adult erythroblasts. An important question is therefore whether F-cells are formed through reversion to a fetal-like state at the transcriptional and epigenetic level. To address this clinically important question, we previously reported development of new techniques for the purification of stage-matched F- and A-erythroblasts from primary human CD34+ cell erythroid cultures and their downstream analysis (Khandros et al, Blood 2020). We demonstrated that F-cells in primary erythroid cultures have minimal transcriptional differences with A-cells and that the few differentially expressed transcripts do not overlap with fetal-specific transcripts. Furthermore, treatment with hydroxyurea or pomalidomide did not enhance transcriptional differences between F- and A-cells. Surprisingly, we did not find differences in the expression of any known HbF regulators such as BCL11A, LRF, or NuRD complex members that would account for differential HbF expression. Based on these findings, we hypothesized that F-cells are distinguished by epigenetic variation specifically at the β-globin locus. Given that fetal erythroblasts differ from adult erythroblasts in the chromatin architecture of the β-globin locus (e.g. Huang et al, Genes and Development 2017), we compared the higher order chromatin organization of the β-globin locus between F- and A-cells by Capture-C, a next-generation sequencing-adapted form of chromatin conformation capture. We found that in F-cells, contacts between the distal enhancer and the promoters of the fetal globin genes HBG1 and HBG2 were increased, while those between the enhancer and adult globin genes (HBB and HBD) were reduced. Other architectural changes associated with fetal globin gene expression, including fetal specific contacts of an intergenic non-coding gene with chromatin domain boundaries at the β-globin locus were also partially enriched in F-cells. We also did not find any differences in promoter-enhancer contacts between F- and A-cells for other developmentally regulated genes BCL11A, LIN28B, and THRB. Together these results are consistent with the concept that epigenetic changes associated with nuclear architecture that occur specifically at the β-globin locus underlie the difference in globin gene expression profiles between F- and A-cells. In sum our data demonstrate that in adult erythropoiesis, F-cells do not arise through either a wholesale reversion to a fetal-like genetic program or through variation in any known HbF regulators. Instead, modulation of chromatin architecture intrinsic to the β-globin locus, perhaps in a stochastic manner, accounts for elevated fetal globin expression in F-cells. We are currently performing mechanistic studies to elucidate the basis for the epigenetic regulation of the β-globin locus in F-cells. These studies will further our understanding of fetal hemoglobin regulation in adult cells and might inform new therapeutic approaches for SCD and β-thalassemia. Disclosures Blobel: Fulcrum Therapeutics: Consultancy; Pfizer: Research Funding.

Elevated levels of fetal hemoglobin (HbF) can alleviate symptoms of hemoglobinopathies, such as sickle cell disease (SCD). Hydroxyurea is the only FDA approved drug that works through this mechanism of HbF induction; however, its efficacy is variable among patients and its mechanism of action is not well understood. Therefore, significant clinical benefit would arise from a more reliable treatment to upregulate HbF, such as developing inhibitors that target HbF repressors. The transcription factors, BCL11A and LRF, are two major independent repressors of HbF however, they have been challenging to control via pharmacologic means. While these transcription factors and their co-factors have been extensively studied, upstream regulation of these transcription factors, such as potential post-transcriptional regulators, are not as well studied. Exploration of these upstream regulators might yield new insights into basic mechanisms of transcriptional and post-transcriptional regulation of HbF, which has the potential to uncover novel therapeutic targets. For example, we have previously used a novel screening approach to successfully identify the protein kinase HRI as a regulator of HbF through BCL11A expression (Grevet and Lan et al., Science, 2018). Novel targets such as HRI may be more amenable to pharmacologic regulation. To uncover novel upstream regulators of HbF, we employed a CRISPR/Cas9 based screening approach to target a spectrum of RNA binding proteins (RBPs) potentially involved in post transcriptional regulation of HbF expression. Using a human erythroid progenitor cell line, termed HUDEP2, we interrogated 342 human RBPs using an sgRNA library that targets RBPs harboring RNA methyltransferase and RNA recognition motifs. This screen yielded four candidate RBPs, in which their disruption or depletion in human primary erythroid cultures and HUDEP2 cells raised HbF levels. Three of these are members of the heterogeneous nuclear ribonucleoprotein (hnRNP) family, which have not previously been implicated in HbF regulation. Of these candidates, polypyrimidine tract binding protein 1 (PTBP1) showed the greatest level of HbF induction following in vitro depletion. Significant depletion of PTBP1 protein (>60%) in HUDEP2 cells and human CD34+ derived erythroid progenitors via CRISPR/Cas9 editing raised HbF production 2-4 fold as assessed by measuring % HbF positive cells, γ-globin (HBG, fetal β-like globin) mRNA, and HBG protein levels. Cell viability of PTBP1 perturbed samples are largely unaffected, however there is a delay in terminal differentiation as assessed by cell surface markers CD71 and CD235a (2-3 fold decrease in CD71-/CD235a+ cells at day 15 of differentiation). Unexpectedly, depletion of PTBP1 had minimal effect on BCL11A and LRF mRNA or protein levels. This suggests PTBP1 might impact the expression or activities of co-factors or upstream regulators of these transcription factors. Ongoing work is aimed at defining the mechanism of PTBP1 action by identifying its molecular targets. In sum, the identification of PTBP1 as a regulator of HbF production represents a previously undescribed layer of hemoglobin gene regulation. In pursuing this path, we hope to gain a deeper understanding of this process which might in turn lead to the identification of potential therapeutic targets for the treatment of SCD and other hemoglobinopathies. Disclosures Blobel: Bioverativ: Research Funding; Pfizer: Research Funding.

Reactivation of fetal hemoglobin in adult red blood cells benefits patients with sickle cell disease and β-thalassemia. BCL11A is one of the predominant repressors of fetal γ-globin transcription and stands as an appealing target for therapeutic genome manipulation. However, pharmacologic perturbation of BCL11A function or its co-regulators remains an unmet challenge. Previously, we reported the discovery of the erythroid-enriched protein kinase HRI as a novel regulator of γ-globin transcription and found that HRI functions in large part via controlling the levels of BCL11A transcription (Grevet et al., Science, 2018). However, the specific mechanisms underlying HRI-mediated modulation of BCL11A levels remain unknown. To identify potential HRI-controlled transcription factors that regulate BCL11A, we performed a domain-focused CRISPR screen that targeted the DNA binding domains of 1,447 genes in the human erythroid cell line HUDEP2. Activating transcription factor 4 (ATF4) emerged as a novel γ-globin repressor. Prior studies reported that ATF4 production is under positive influence of HRI. Specifically, HRI phosphorylates translation factor EIF2α which in turn augments translation of ATF4 mRNA. As expected, HRI deficiency reduced ATF4 protein amounts in HUDEP2 and primary erythroid cells. We further found that the degree of γ-globin reactivation was similar in ATF4 and HRI-depleted cells. ATF4 ChIP-seq in both HUDEP2 and primary erythroblast identified 4,547 and 3,614 high confidence binding sites, respectively. Notably, we did not observe significant enrichment of ATF4 binding or even the presence of an ATF4 consensus motif at the γ-globin promoters, suggesting that ATF4 regulates the γ-globin genes indirectly. However, ATF4 specifically bound to one of the three major BCL11A erythroid enhancers (+55) in both cell types. This was the sole binding site within the ~0.5Mb topologically associating domain that contains the BCL11A gene. Eliminating this ATF4 motif via CRISPR guided genome editing lowered BCL11A mRNA levels and increased γ-globin transcription. Capture-C showed that ATF4 knock-out or removal of the ATF4 site at the BCL11A (+55) enhancer decreased chromatin contacts with the BCL11A promoter. Forced expression of BCL11A largely restored γ-globin silencing in cells deficient for ATF4 or lacking the ATF4 motif in the BCL11A (+55) enhancer. An unexplained observation from our prior study was that HRI loss did not significantly lower Bcl11a levels in murine erythroid cells. Therefore, we mutated the analogous ATF4 motif in the Bcl11a enhancer in the murine erythroid cell line G1E. Unlike in human cells, Bcl11a mRNA synthesis was decreased only very modestly, and there was no effect on the murine embryonic globin genes whose silencing requires Bcl11a. This suggests that the species specific regulation of BCL11A by HRI results from divergent functional roles of ATF4 binding at the BCL11A (+55) enhancer. In sum, our studies uncover a major pathway that extends linearly from HRI to ATF4 to BCL11A to γ-globin. Moreover, these results further support HRI as a pharmacologic target for the selective regulation of BCL11A and γ-globin. Disclosures Blobel: Pfizer: Research Funding; Bioverativ: Research Funding.

Reversing the developmental switch from fetal (HbF, α2γ2) to adult (HbA, α2β2) hemoglobin is an important therapeutic approach in sickle cell disease (SCD) and β-thalassemia. Elevated HbF levels due to genetic variation or through therapeutic induction by hydroxyurea (HU) attenuate the severity of both disorders. HbF in healthy individuals, SCD patients, and patients treated with HU is present in a heterocellular fashion in a subset of red blood cells known as F-cells. Despite over 50 years of observations of F-cells, it is not known why only some cells in a genetically identical population are able to express HbF or respond to pharmacological inducers. Adult F-cells can potentially represent a reversion to a fetal-like epigenetic and transcriptional program, or alternatively isolated transcriptional or posttranscriptional events at the γ-globin genes. Here we set out to understand the heterogeneity of HbF activation and gain insights into whether the mechanisms underlying the heterocellular response are similar or distinct in response to different HbF inducers. To this end we developed techniques to purify differentiation stage-matched late erythroblast F-cells and non-F cells (A-cells) from the human HUDEP2 erythroid cell line and primary CD34 cell erythroid cultures using a reversible fixation protocol enabling extraction of high-quality RNA and protein. Purified F-cells from both sources were enriched for γ-globin transcripts by 200-500 fold by RT-PCR, validating the purification scheme. We profiled these cells by RNA-seq using a modified method that depletes globin mRNAs and ribosomal RNAs and is capable of detecting low abundance transcripts, as well as by mass spectrometry using size fractionation to increase the number of detected proteins. In differentiated clonal HUDEP2 cells, differences between F-cells and A-cells were remarkably small, with only 62 differentially expressed transcripts and 20 differentially expressed proteins. Top differentially expressed transcripts were γ-globin and the non-coding β-globin locus transcripts BGLT3 and HBBP1. Interestingly, there were no significant changes in known HbF regulators BCL11A, LRF, and HRI at the RNA or protein level. Gene set enrichment analysis (GSEA) using a previously generated set of differentially expressed transcripts from adult and fetal-derived CD34 erythroid cultures showed enrichment of fetal transcripts in F-cells and adult transcripts in A-cells. We also carried out transcriptome analysis of sorted matched late erythroblast F-cells and A-cells from human CD34+ cell erythroid cultures at different time points. Similar to HUDEP2 cells, only small numbers of transcripts were differentially expressed (33 at 8 days, 17 at 11 days, and 261 at 14 days). BCL11A, LRF, and HRI were not differentially expressed at the earlier timepoints, and BCL11A and HRI were at most decreased by about 20% at the 14-day mark. GSEA analysis did not show fetal transcript enrichment in day 8. At days 11 and 14, there was some enrichment of fetal transcripts in F-cells but not to the degree of HUDEP2 cells. Finally, we analyzed sorted F- and A-cells from day 11 CD34+ erythroid cultures treated with hydroxyurea and pomalidomide. Again, differences between F- and A-cells were small with hydroxyurea treatment (53 transcripts) and more significant with pomalidomide treatment (400 transcripts). We have successfully established an approach to analyze stage-matched γ-globin containing cells from a genetically identical starting population, with high degree of enrichment. Our preliminary data indicate that these cells are overall highly similar to non-γ-containing cells, but do show some enrichment of fetal-specific transcripts, more so in HUDEP2 cells. The differences between F- and A- cells are overall smaller than those observed by us and others in profiling of fetal and adult-derived erythroblasts. This suggests that F-cells are not formed by reversion to a fetal-like state but rather through specific changes at the β-globin locus. Importantly, we do not find differential levels of any known γ-globin regulators, suggesting an alternative mechanism for the heterocellular expression pattern. Studies are currently ongoing to carry out epigenetic profiling of F-cells. Disclosures Blobel: Bioverativ: Research Funding; Pfizer: Research Funding.