In vivo measurement of RBC survival in patients with sickle cell disease before or after hematopoietic stem cell transplantation.

ABSTRACT: Stable, mixed-donor-recipient chimerism after allogeneic hematopoietic stem cell transplantation (HSCT) for patients with sickle cell disease (SCD) is sufficient for phenotypic disease reversal, and results from differences in donor/recipient-red blood cell (RBC) survival. Understanding variability and predictors of RBC survival among patients with SCD before and after HSCT is critical for gene therapy research which seeks to generate sufficient corrected hemoglobin to reduce polymerization thereby overcoming the red cell pathology of SCD. This study used biotin labeling of RBCs to determine the lifespan of RBCs in patients with SCD compared with patients who have successfully undergone curative HSCT, participants with sickle cell trait (HbAS), and healthy (HbAA) donors. Twenty participants were included in the analysis (SCD pre-HSCT: N = 6, SCD post-HSCT: N = 5, HbAS: N = 6, and HbAA: N = 3). The average RBC lifespan was significantly shorter for participants with SCD pre-HSCT (64.1 days; range, 35-91) compared with those with SCD post-HSCT (113.4 days; range, 105-119), HbAS (126.0 days; range, 119-147), and HbAA (123.7 days; range, 91-147) (P<.001). RBC lifespan correlated with various hematologic parameters and strongly correlated with the average final fraction of sickled RBCs after deoxygenation (P<.001). No adverse events were attributable to the use of biotin and related procedures. Biotin labeling of RBCs is a safe and feasible methodology to evaluate RBC survival in patients with SCD before and after HSCT. Understanding differences in RBC survival may ultimately guide gene therapy protocols to determine hemoglobin composition required to reverse the SCD phenotype as it relates directly to RBC survival. This trial was registered at www.clinicaltrials.gov as #NCT04476277.

Calmodulin inhibitors improve erythropoiesis in Diamond-Blackfan anemia.

Diamond-Blackfan anemia (DBA) is a rare hematopoietic disease characterized by a block in red cell differentiation. Most DBA cases are caused by mutations in ribosomal proteins and characterized by higher than normal activity of the tumor suppressor p53. Higher p53 activity is thought to contribute to DBA phenotypes by inducing apoptosis during red blood cell differentiation. Currently, there are few therapies available for patients with DBA. We performed a chemical screen using zebrafish ribosomal small subunit protein 29 (rps29) mutant embryos that have a p53-dependent anemia and identified calmodulin inhibitors that rescued the phenotype. Our studies demonstrated that calmodulin inhibitors attenuated p53 protein amount and activity. Treatment with calmodulin inhibitors led to decreased p53 translation and accumulation but does not affect p53 stability. A U.S. Food and Drug Administration-approved calmodulin inhibitor, trifluoperazine, rescued hematopoietic phenotypes of DBA models in vivo in zebrafish and mouse models. In addition, trifluoperazine rescued these phenotypes in human CD34+ hematopoietic stem and progenitor cells. Erythroid differentiation was also improved in CD34+ cells isolated from a patient with DBA. This work uncovers a potential avenue of therapeutic development for patients with DBA.

Drug discovery for Diamond-Blackfan anemia using reprogrammed hematopoietic progenitors.

Diamond-Blackfan anemia (DBA) is a congenital disorder characterized by the failure of erythroid progenitor differentiation, severely curtailing red blood cell production. Because many DBA patients fail to respond to corticosteroid therapy, there is considerable need for therapeutics for this disorder. Identifying therapeutics for DBA requires circumventing the paucity of primary patient blood stem and progenitor cells. To this end, we adopted a reprogramming strategy to generate expandable hematopoietic progenitor cells from induced pluripotent stem cells (iPSCs) from DBA patients. Reprogrammed DBA progenitors recapitulate defects in erythroid differentiation, which were rescued by gene complementation. Unbiased chemical screens identified SMER28, a small-molecule inducer of autophagy, which enhanced erythropoiesis in a range of in vitro and in vivo models of DBA. SMER28 acted through autophagy factor ATG5 to stimulate erythropoiesis and up-regulate expression of globin genes. These findings present an unbiased drug screen for hematological disease using iPSCs and identify autophagy as a therapeutic pathway in DBA.

Whole-exome sequencing and functional studies identify RPS29 as a novel gene mutated in multicase Diamond-Blackfan anemia families.

Diamond-Blackfan anemia (DBA) is a cancer-prone inherited bone marrow failure syndrome. Approximately half of DBA patients have a germ-line mutation in a ribosomal protein gene. We used whole-exome sequencing to identify disease-causing genes in 2 large DBA families. After filtering, 1 nonsynonymous mutation (p.I31F) in the ribosomal protein S29 (RPS29[AUQ1]) gene was present in all 5 DBA-affected individuals and the obligate carrier, and absent from the unaffected noncarrier parent in 1 DBA family. A second DBA family was found to have a different nonsynonymous mutation (p.I50T) in RPS29. Both mutations are amino acid substitutions in exon 2 predicted to be deleterious and resulted in haploinsufficiency of RPS29 expression compared with wild-type RPS29 expression from an unaffected control. The DBA proband with the p.I31F RPS29 mutation had a pre-ribosomal RNA (rRNA) processing defect compared with the healthy control. We demonstrated that both RPS29 mutations failed to rescue the defective erythropoiesis in the rps29(-/-) mutant zebra fish DBA model. RPS29 is a component of the small 40S ribosomal subunit and essential for rRNA processing and ribosome biogenesis. We uncovered a novel DBA causative gene, RPS29, and showed that germ-line mutations in RPS29 can cause a defective erythropoiesis phenotype using a zebra fish model.

Simvastatin and t-butylhydroquinone suppress KLF1 and BCL11A gene expression and additively increase fetal hemoglobin in primary human erythroid cells.

UNLABELLED: Although increased fetal hemoglobin (HbF) levels have proven benefit for people with ß-hemoglobinopathies, all current HbF-inducing agents have limitations. We previously reported that drugs that activate the NRF2 antioxidant response signaling pathway increase HbF in primary human erythroid cells. In an attempt to increase HbF levels achieved with NRF2 activators, in the present study, we investigated potential complementary activity between these agents and HMG-CoA reductase inhibitors (statins) based on their ability to induce KLF2 protein levels. Experiments in K562 cells showed that simvastatin increased KLF2 mRNA and protein and KLF2 binding to HS2 of the ß-globin locus control region and enhanced -globin mRNA production by the NRF2 activator Tert-butylhydroquinone (tBHQ). When tested in differentiating primary human erythroid cells, simvastatin induced HbF alone and additively with tBHQ, but it did not increase KLF2 mRNA or locus control region binding above levels seen with normal differentiation. Investigating alternative mechanisms of action, we found that both simvastatin and tBHQ suppress ß-globin mRNA and KLF1 and BCL11A mRNA and protein, similar to what is seen in people with an HPFH phenotype because of KLF1 haploinsufficiency. These findings identify statins as a potential class of HbF-inducing agents and suggest a novel mechanism of action based on pharmacologic suppression of KLF1 and BCL11A gene expression. KEY POINTS: Simvastatin and tBHQ suppress KLF1 and BCL11 gene expression and additively increase fetal hemoglobin in primary human erythroid cells. Because both drugs are FDA-approved, these findings could lead to clinical trials in the relatively near future.

Induction of human fetal hemoglobin via the NRF2 antioxidant response signaling pathway.

Although hematopoietic stem cell transplantation and gene therapy have the potential to cure ß-thalassemia and sickle cell disease, they are not currently available to most people with these diseases. In the near term, pharmacologic induction of fetal hemoglobin (HbF) may offer the best possibility for safe, effective, and widely available therapy. In an effort to define new pathways for targeted drug development for HbF induction, we evaluated the nuclear factor erythroid 2-related factor 2 (NRF2) antioxidant response element signaling pathway. We found that 3 well-known activators of this pathway increased γ-globin mRNA at nontoxic doses in K562 cells. Tert-butylhydroquinone (tBHQ), the most active of these compounds, increased cellular levels and nuclear translocation of NRF2 and binding of NRF2 to the γ-globin promoter. siRNA knockdown of NRF2 inhibited γ-globin induction by tBHQ. When tested in human primary erythroid cells, tBHQ induced NRF2 binding to the γ-globin promoter, increased γ-globin mRNA and HbF, and suppressed ß-globin mRNA and HbA, resulting in a > 3-fold increase in the percentage of HbF. These results suggest that drugs that activate the NRF2/antioxidant response element signaling pathway have the potential to induce therapeutic levels of HbF in people with ß-hemoglobinopathies.

A cell stress signaling model of fetal hemoglobin induction: what doesn't kill red blood cells may make them stronger.

A major goal of hemoglobinopathy research is to develop treatments that correct the underlying molecular defects responsible for sickle cell disease and beta-thalassemia. One approach to achieving this goal is the pharmacologic induction of fetal hemoglobin (HbF). This strategy is capable of inhibiting the polymerization of sickle hemoglobin and correcting the globin chain imbalance of beta-thalassemia. Despite this promise, none of the currently available HbF-inducing agents exhibit the combination of efficacy, safety, and convenience of use that would make them applicable to most patients. The recent success of targeted drug therapies for malignant diseases suggests that this approach could be effective for developing optimal HbF-inducing agents. A first step in applying this approach is the identification of specific molecular targets. However, while >70 HbF-inducing agents have been described, neither molecular mechanisms nor target molecules have been definitively verified for any of these compounds. To help focus investigation in this area, we have reviewed known HbF-inducing agents and their proposed mechanisms of action. We find that in many cases, current models inadequately explain key experimental results. By integrating features of the erythropoietic stress model of HbF induction with data from recent intracellular signaling experiments, we have developed a new model that has the potential to explain several findings that are inconsistent with previous models and to unify most HbF-inducing agents under a common mechanism: cell stress signaling. If correct, this or related models could lead to new opportunities for development of targeted therapies for the beta-hemoglobinopathies.

ICP34.5-dependent and -independent activities of salubrinal in herpes simplex virus-1 infected cells.

The small molecule salubrinal has antiviral activity against herpes simplex virus-1 (HSV-1) and inhibits dephosphorylation of eIF2 alpha mediated by the HSV-1 protein ICP34.5. We investigated whether salubrinal's activities in infected cells depend on ICP34.5. An ICP34.5 deletion mutant was as sensitive as wild type HSV-1 to salubrinal inhibition of plaque formation in Vero cells. However, salubrinal induced formation of syncytia in infected Vero cells, which was enhanced by ICP34.5 mutations. Expression of HSV-1 US11 with immediate early kinetics, which is known to suppress the effects of ICP34.5 mutations, resulted in slight resistance to salubrinal in murine embryonic fibroblasts, and substantial resistance in those cells when ICP34.5 was additionally mutated. ICP34.5 mutations, but not immediate early expression of US11, prevented salubrinal's ability to increase phosphorylation of eIF2 alpha during HSV-1 infection of Vero cells. Taken together, our data indicate that salubrinal has both ICP34.5-dependent and -independent activities in HSV-1 infected cells.