Sharon A. Singh

Congenital dyserythropoietic anemia type I (CDA-I) is an autosomal recessive disease characterized by anemia due to ineffective erythropoiesis and is caused primarily by mutations in CDAN1, which encodes codanin-1. Research efforts to understand CDA-I pathogenesis have been impeded by the embryonic lethality of germ line Cdan1-deleted mice, as well as mice with Cdan1 deleted in the erythroid compartment using the constitutively active EpoR-Cre allele. To study the function of codanin-1 in adult erythropoiesis, we generated mice with inducible erythroid-specific, biallelic Cdan1 deletion using the Gata1-CreERT2 allele. Following tamoxifen administration to adult mice, Cdan1 is excised, resulting in features of CDA-I, including anemia, impaired erythroid differentiation, disturbances in erythroblast cell cycle progression, and the finding of “spongy” heterochromatin in bone marrow erythroblasts. These findings confirm a critical role for codanin-1 in effective adult erythropoiesis and demonstrate the successful generation of an inducible CDA-I mouse model, which serves as a valuable platform for testing novel therapies for this orphan disease.

Analysis of Diamond Blackfan anemia syndrome (DBAS) cohorts and animal models has not revealed a potential mechanism for the variable anemia phenotype, a key feature of this disease. Here, we used an established Rpl5Skax23-Jus/+ murine DBAS model to study this dynamic erythropoiesis deficiency. These haploinsufficient mice exhibit variably penetrant craniofacial and cardiac defects mimicking the phenotypes of patients with DBAS bearing RPL5 mutations. We additionally discovered that this specific heterozygous splicing mutation is pathogenic and leads to partial intron retention. By examining the transcriptome of fetal liver erythroid progenitors at embryonic day 12.5 (E12.5), we demonstrate downregulation of erythroid differentiation pathways consistent with the DBAS phenotype. We also identified dysregulated transcription of lipid metabolism genes with significant reduction in Scd1 expression in the subset of E12.5 mutant embryos at risk for erythroid failure. Stearoyl–coenzyme A desaturase 1, a key enzyme that converts saturated to monounsaturated fatty acids, has not been previously linked to erythropoiesis or DBAS. When anemia was induced in adult Rpl5Skax23-Jus/+ mice, mutant mice exhibited delayed erythroid recovery, whereas pretreatment with an stearoyl–coenzyme A desaturase 1 inhibitor resulted in improved erythropoiesis in both wild-type and mutant mice. This analysis suggests a potential role of lipid metabolism in the variable anemia penetrance in DBAS and highlights a previously unappreciated pathway that requires further study as a potential target for drug development.

Sickle cell disease (SCD) and β-thalassemia are devastating genetic disorders resulting from defects in the β-globin subunit of adult hemoglobin. Both disorders are ameliorated by the induction of γ-globin, a component of fetal hemoglobin (HbF). Therefore, the development of safe, effective, and widely available inducers of HbF is needed. Here, we discovered that slow cycling erythroid cells exhibit increased γ-globin expression. To understand the molecular basis of this, we screened all cyclin-dependent kinase inhibitors (CDKIs) for their ability to induce HbF using CRISPR activation. We found that overexpression of CDKN1B, which encodes p27Kip1 (but not overexpression of other CDKIs), induces γ-globin expression at the transcriptional level. CDKN1B mutants expressing proteins unable to bind/inhibit CDKs and/or cyclins revealed that γ-globin induction by p27Kip1 depends largely on the domains involved in its cell cycle function. Pharmacological inhibition and genetic reduction of CDK4/6 also result in increased HbF. In genetic rescue experiments, we show that p27Kip1 induces HbF by inhibiting CDK4/6, through a mechanism that is likely BCL11A and ZBTB7A independent. Furthermore, palbociclib, an oral CDK4/6 inhibitor, significantly increases HbF in a murine SCD model at doses that are well tolerated. Moreover, we show that HbF induction by hydroxyurea, a drug currently in use to treat SCD, may be mediated in part by CDK4/6 inhibition. Overall, our findings establish a causal relationship between CDK4/6 activity and γ-globin production and suggest that single or dual CDK4/6 inhibitors might be therapeutically beneficial for SCD and β-thalassemia.

Analysis of neither Diamond Blackfan anemia syndrome (DBAS) cohorts nor animal models has revealed a potential mechanism for the variable anemia phenotype, a key feature of this disease. Here, we utilized an established Rpl5 ^Skax23-Jus/+ murine DBAS model in order to study this dynamic erythropoiesis deficiency. These haploinsufficient mice exhibit variably penetrant craniofacial and cardiac defects mimicking the phenotypes of DBAS patients bearing RPL5 mutations. We additionally discovered that this specific heterozygous splicing mutation is pathogenic and leads to partial intron retention. By examining the transcriptome of fetal liver erythroid progenitors at E12.5, we demonstrate that the downregulation of erythroid differentiation pathways is consistent with the DBAS phenotype. We also identified dysregulated transcription of lipid metabolism genes with significant reduction in the abundance of Scd1 in a subset of E12.5 mutant embryos at risk for erythroid failure. SCD1, a key enzyme that converts saturated to monounsaturated fatty acids, has not been previously linked to erythropoiesis or DBAS. When anemia was induced in adult mice, pretreatment with an SCD1 inhibitor resulted in improved erythropoiesis. This analysis suggests a key role of lipid metabolism in the variable anemia penetrance in DBAS and highlights a previously unappreciated pathway that may serve as a potential target for drug development. Key Points: The variable anemia in Rpl5 ^Skax23-Jus/+ mice is triggered by intrinsic/extrinsic stress Rpl5 haploinsufficient murine and human erythroid progenitors exhibit a lipid metabolism signature with downregulation of Scd1 / SCD .

Small molecules that inhibit LSD1 (lysine-specific demethylase 1, KDM1A) have been shown to induce abundant fetal hemoglobin (HbF) levels in red blood cells both in vitro and in vivo, therefore potentially serving as potent and cost-effective therapeutics to treat the β-globinopathies, sickle cell disease (SCD) and β-thalassaemia major (TM). However, most LSD1 inhibitors (LSD1i) that induce HbF in vivo are covalent and irreversible, which leads to adverse effects. In this study, we utilized structure-aided drug design to develop potent new reversible LSD1i's leading to robust γ-globin expression in vitro. Moreover, in a mouse model of SCD, oral administration of these novel inhibitors lead to significant HbF elevation and alleviation of multiple features of disease pathology that are the usual consequences of SCD. In addition, we discovered that combined treatment of an LSD1i with a BRD4 degrader (BD-9136) represses the induction of RUNX1 and PU.1, thereby rescuing the erythroid to myeloid lineage conversion that accompanies LSD1i in hematopoiesis. The data indicate that this new generation of LSD1i can effectively induce HbF levels, reduce SCD pathologies, and are well-tolerated by oral administration in SCD mice. We anticipate that the combination of these or related binary compounds offer exciting new therapeutic possibilities for treating SCD and TM.

Nuclear receptor TR4 (NR2C2) was previously shown to bind to the –117 position of the γ-globin gene promoters in vitro, which overlaps the more recently described BCL11 transcription factor A (BCL11A) binding site. The role of TR4 in human γ-globin gene repression has not been extensively characterized in vivo, whereas any relationship between TR4 and BCL11A regulation through the γ-globin promoters is unclear at present. We show here that TR4 and BCL11A competitively bind in vitro to distinct, overlapping sequences, including positions overlapping –117 of the γ-globin promoter. We found that TR4 represses γ-globin transcription and fetal hemoglobin accumulation in vivo in a BCL11A-independent manner. Finally, examination of the chromatin occupancy of TR4 within the β-globin locus, compared with BCL11A, shows that both bind avidly to the locus control region and other sites, but only BCL11A binds to the γ-globin promoters at statistically significant frequency. These data resolve an important discrepancy in the literature and, thus, clarify possible approaches to the treatment of sickle cell disease and β-thalassaemia.

Human γ-globin is predominantly expressed in fetal liver erythroid cells during gestation from 2 nearly identical genes, HBG1 and HBG2, that are both perinatally silenced. Reactivation of these fetal genes in adult red blood cells can ameliorate many symptoms associated with the inherited β-globinopathies, sickle cell disease, and Cooley anemia. Although promising genetic strategies to reactivate the γ-globin genes to treat these diseases have been explored, there are significant barriers to their effective implementation worldwide; alternatively, pharmacological induction of γ-globin synthesis could readily reach the majority of affected individuals. In this study, we generated a CRISPR knockout library that targeted all erythroid genes for which prospective or actual therapeutic compounds already exist. By probing this library for genes that repress fetal hemoglobin (HbF), we identified several novel, potentially druggable, γ-globin repressors, including VHL and PTEN. We demonstrate that deletion of VHL induces HbF through activation of the HIF1α pathway and that deletion of PTEN induces HbF through AKT pathway stimulation. Finally, we show that small-molecule inhibitors of PTEN and EZH induce HbF in both healthy and β-thalassemic human primary erythroid cells.

Histone H3 lysine 4 methylation (H3K4Me) is most often associated with chromatin activation, and removing H3K4 methyl groups has been shown to be coincident with gene repression. H3K4Me demethylase KDM1a/LSD1 is a therapeutic target for multiple diseases, including for the potential treatment of β-globinopathies (sickle cell disease and β-thalassemia), because it is a component of γ-globin repressor complexes, and LSD1 inactivation leads to robust induction of the fetal globin genes. The effects of LSD1 inhibition in definitive erythropoiesis are not well characterized, so we examined the consequences of conditional inactivation of Lsd1 in adult red blood cells using a new Gata1creERT2 bacterial artificial chromosome transgene. Erythroid-specific loss of Lsd1 activity in mice led to a block in erythroid progenitor differentiation and to the expansion of granulocyte-monocyte progenitor–like cells, converting hematopoietic differentiation potential from an erythroid fate to a myeloid fate. The analogous phenotype was also observed in human hematopoietic stem and progenitor cells, coincident with the induction of myeloid transcription factors (eg, PU.1 and CEBPα). Finally, blocking the activity of the transcription factor PU.1 or RUNX1 at the same time as LSD1 inhibition rescued myeloid lineage conversion to an erythroid phenotype. These data show that LSD1 promotes erythropoiesis by repressing myeloid cell fate in adult erythroid progenitors and that inhibition of the myeloid-differentiation pathway reverses the lineage switch induced by LSD1 inactivation.

Ribosome dysfunction is implicated in multiple abnormal developmental and disease states in humans. Heterozygous germline mutations in genes encoding ribosomal proteins are found in most individuals with Diamond-Blackfan anemia (DBA), whereas somatic mutations have been implicated in a variety of cancers and other disorders. Ribosomal protein-deficient animal models show variable phenotypes and penetrance, similar to human patients with DBA. In this study, we characterized a novel ENU mouse mutant (Skax23m1Jus) with growth and skeletal defects, cardiac malformations, and increased mortality. After genetic mapping and whole-exome sequencing, we identified an intronic Rpl5 mutation, which segregated with all affected mice. This mutation was associated with decreased ribosome generation, consistent with Rpl5 haploinsufficiency. Rpl5Skax23-Jus/+ animals had a profound delay in erythroid maturation and increased mortality at embryonic day (E) 12.5, which improved by E14.5. Surviving mutant animals had macrocytic anemia at birth, as well as evidence of ventricular septal defect (VSD). Surviving adult and aged mice exhibited no hematopoietic defect or VSD. We propose that this novel Rpl5Skax23-Jus/+ mutant mouse will be useful in studying the factors influencing the variable penetrance that is observed in DBA.