Heme-Regulated Inhibitor (HRI) Loss and Pharmacologic Treatments Cooperate to Strongly Elevate Fetal Hemoglobin and Reduce Sickle Cell Formation

Abstract Increasing fetal hemoglobin (HbF) provides significant clinical benefit for patients with sickle cell disease (SCD) and is a critical goal that is being pursued with a variety of pharmacologic strategies. Hydroxyurea (HU) is currently the only FDA-approved drug aimed at raising HbF for SCD patients but is limited in its efficacy. Recent work in our laboratory utilizing a protein kinase-domain focused CRISPR-Cas9 based genetic screen identified heme-regulated inhibitor (HRI), an erythroid-specific protein kinase, as a novel HbF regulator (Grevet et al., Science 361:6399, 285-290). Depleting HRI in a human erythroid cell line and primary human erythroid cultures significantly raised HbF levels. Besides HU, several compounds have been recently identified as in vitro HbF inducers, including pomalidomide, a third-generation imide, and UNC0638, an EHMT 1/2 histone methyltransferase inhibitor. While all these approaches induce HbF in vitro, it remains to be seen whether these would achieve sufficient levels of HbF induction in patients when used as single agents. Although the specific mechanisms by which HRI, HU, pomalidomide and UNC0638 regulate HbF remain to be fully resolved, we hypothesized that they might work in distinct pathways, and that by combining them might improve effect size and diminish potential adverse effects on erythroid cell maturation and viability. We utilized a three-stage in vitro culture system that recapitulates normal erythropoiesis by terminal differentiation of adult CD34+ hematopoietic cells. Primary human CD34+ cells were treated with select pharmacologic inducers of HbF, including hydroxyurea, pomalidomide, or UNC0638, in combination with shRNA lentiviral knockdown of HRI. HbF levels were assessed by RT-qPCR, Western blot, flow cytometry, and cation-exchange HPLC. We find that knockdown of HRI leads to significant HbF induction as expected. We further observed an inverse correlation between remaining HRI levels and HbF induction with maximal effect size requiring 85%-90% HRI depletion. HU displayed comparatively little activity and failed to increase the effects of HRI knockdown. However, treatment with pharmacologic levels of pomalidomide or UNC0638 combined with HRI depletion each showed greater than additive effects in HbF with levels reaching 25-30% HbF for UNC0638-treated and 30-40% HbF for pomalidomide-treated combinations, suggesting underlying cooperativity of HbF induction. RT-qPCR and Western blot analyses suggest that the diminished expression of the HbF repressor BCL11A accounts in large part for HbF induction in HRI knockdown samples, particularly when combined with pomalidomide treatment in which Bcl11A depletion was greater than 90%, while other HbF repressors such as LRF were unchanged. To determine whether HbF induction results in reduced sickle cell formation, we performed combination HRI depletion and HbF pharmacologic induction in primary CD34+ cells derived from sickle cell patients. HRI depletion in primary SCD cells showed a marked increase in HbF from baseline levels; in addition, significant cooperativity with pomalidomide and UNC0638 was observed, achieving 45-50% HbF for UNC0638-treated and 50-60% HbF for pomalidomide-treated drug combinations with no apparent detrimental effects on erythroid differentiation or maturation. Importantly, combination of HRI knockdown and HbF pharmacologic treatments markedly reduced in vitro sickling as measured by low-oxygen sickling assays, suggesting significant amelioration of the sickle cell phenotype in vitro. Overall, we find that the combination of HbF pharmacologic induction and shRNA-mediated HRI inhibition results in significant cooperative upregulation of HbF levels in both normal and sickle cell-derived primary human cells without impairing red cell maturation. We are currently exploring additional potential synergies with HbF regulators to determine the optimal modalities to maximize HbF induction. While no effective and specific HRI inhibitors are currently available, our work suggests that future small molecule inhibition of HRI may be combined with other pharmacotherapies to achieve significant, clinically meaningful HbF induction for the treatment of SCD and other hemoglobinopathies. Disclosures No relevant conflicts of interest to declare.

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Heme-Regulated Inhibitor (HRI) Depletion Cooperates with Pharmacologic Inducers to Strongly Elevate Fetal Hemoglobin and Reduce Sickle Cell Formation

Blood ◽

10.1182/blood-2019-125664 ◽

2019 ◽

Vol 134 (Supplement_1) ◽

pp. 3557-3557

Author(s):

Scott A. Peslak ◽

Xianjiang Lan ◽

Eugene Khandros ◽

Peng Huang ◽

Jennifer A. Yano ◽

...

Keyword(s):

Sickle Cell ◽

Research Funding ◽

Fetal Hemoglobin ◽

Cell Maturation ◽

Erythroid Cell ◽

Dose Titration ◽

Cell Phenotype ◽

Erythroid Cells ◽

Hbf Induction

Increasing fetal hemoglobin (HbF) provides significant clinical benefit for patients with sickle cell disease (SCD) and is a critical goal that is being pursued with a variety of pharmacologic strategies. Hydroxyurea (HU) is currently the only FDA-approved drug aimed at raising HbF but is limited in its efficacy. Recent work in our laboratory identified heme-regulated inhibitor (HRI), an erythroid-specific protein kinase, as a novel HbF regulator (Grevet et al., Science 2018). Depleting HRI in primary human erythroid cells significantly raised HbF levels; however, maximal HbF induction by HRI knockdown required at least 80-85% HRI depletion. It is currently unknown whether this degree of HRI inhibition can be achieved pharmacologically. Besides HU, several compounds have been recently identified as in vitro HbF inducers, including pomalidomide, a third-generation imide, and UNC0638, an EHMT 1/2 histone methyltransferase inhibitor. We therefore set out to test whether combining HRI depletion with pharmacologic HbF inducers would cooperatively increase HbF levels and diminish potential adverse effects on erythroid cell maturation and viability. We performed three-stage in vitro culture of human CD34+ cells treated with select pharmacologic inducers of HbF, including HU, pomalidomide, or UNC0638, in combination with shRNA knockdown of HRI. HbF levels were assessed by RT-qPCR, Western blot, flow cytometry, and HPLC. We found that treatment with HU displayed comparatively little activity and failed to increase effects of HRI knockdown. However, treatment with UNC0638 combined with HRI depletion showed greater than additive effects on HbF with levels reaching 25-30% HbF, while HRI depletion combined with pomalidomide treatment showed the highest levels of cooperativity, reaching 30-40% HbF. Combination of HRI depletion and HbF pharmacologic inducers showed minimal effects on the erythroid transcriptome by RNA-Seq and did not significantly impair erythroid maturation. Intriguingly, dose-titration experiments indicated that HRI knockdown sensitizes erythroid cells to low doses of pomalidomide, maintaining HbF levels of greater than 40% in HRI-depleted samples despite a ten-fold decrease in pomalidomide concentration. Diminished expression of the HbF repressor BCL11A accounted in large part for HbF induction in HRI knockdown samples, particularly when combined with pomalidomide treatment in which BCL11A depletion exceeded 90%, while other HbF repressors such as LRF were unchanged. We will present RNASeq analyses aimed at elucidating mechanisms of HRI cooperativity. Finally, we found that HRI depletion in SCD patient-derived cells showed significant cooperativity with pomalidomide and UNC0638, achieving 45-50% HbF for UNC0638-treated and 50-60% HbF for pomalidomide-treated drug combinations. Importantly, combination of HRI knockdown and HbF pharmacologic treatments markedly reduced in vitro sickling as measured by low-oxygen sickling assays, suggesting significant amelioration of the sickle cell phenotype. In sum, we find that dual targeting of HbF induction via HRI inhibition and pharmacologic inducers results in successful cooperative upregulation of HbF levels in both normal and SCD primary human cells without impairing red cell maturation. Furthermore, our data suggest that dose titration of HbF inducers combined with HRI depletion could maximize HbF induction while potentially reducing off-target effects. Moreover, HRI is an attractive target for HbF induction as it is expressed in an erythroid-specific manner. While no specific HRI inhibitors are currently available, our work suggests that future small molecule inhibitors of HRI may be combined with other pharmacotherapies to achieve significant, clinically meaningful HbF induction for the treatment of SCD and other hemoglobinopathies. Disclosures Abdulmalik: The Children's Hospital of Philadelphia: Patents & Royalties: Provisional Patent. Blobel:Bioverativ: Research Funding; Pfizer: Research Funding.

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HRI depletion cooperates with pharmacologic inducers to elevate fetal hemoglobin and reduce sickle cell formation

Blood Advances ◽

10.1182/bloodadvances.2020002475 ◽

2020 ◽

Vol 4 (18) ◽

pp. 4560-4572

Author(s):

Scott A. Peslak ◽

Eugene Khandros ◽

Peng Huang ◽

Xianjiang Lan ◽

Carly L. Geronimo ◽

...

Keyword(s):

Sickle Cell ◽

High Performance ◽

Cell Formation ◽

Fetal Hemoglobin ◽

Specific Protein ◽

Erythroid Cell ◽

Cell Disease ◽

Hbf Induction ◽

Cell Functionality ◽

Specific Protein Kinase

Abstract Increasing fetal hemoglobin (HbF) provides clinical benefit in patients with sickle cell disease (SCD). We recently identified heme-regulated inhibitor (HRI, EIF2AK1), as a novel HbF regulator. Because HRI is an erythroid-specific protein kinase, it presents a potential target for pharmacologic intervention. We found that maximal HbF induction required >80% to 85% HRI depletion. Because it remains unclear whether this degree of HRI inhibition can be achieved pharmacologically, we explored whether HRI knockdown can be combined with pharmacologic HbF inducers to achieve greater HbF production and minimize potential adverse effects associated with treatments. Strongly cooperative HbF induction was observed when HRI depletion was combined with exposure to pomalidomide or the EHMT1/2 inhibitor UNC0638, but not to hydroxyurea. Mechanistically, reduction in the levels of the HbF repressor BCL11A reflected the cooperativity of HRI loss and pomalidomide treatment, whereas UNC0638 did not modulate BCL11A levels. In conjunction with HRI loss, pomalidomide maintained its HbF-inducing activity at 10-fold lower concentrations, in which condition there were minimal observed detrimental effects on erythroid cell maturation and viability, as well as fewer alterations in the erythroid transcriptome. When tested in cells from patients with SCD, combining HRI depletion with pomalidomide or UNC0638 achieved up to 50% to 60% HbF and 45% to 50% HbF, respectively, as measured by high-performance liquid chromatography, and markedly counteracted cell sickling. In summary, this study provides a foundation for the exploration of combining future small-molecule HRI inhibitors with additional pharmacologic HbF inducers to maximize HbF production and preserve erythroid cell functionality for the treatment of SCD and other hemoglobinopathies.

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Truncated Erythropoietin Receptors Confer an In Vivo Selective Advantage in Gene-Modified Erythroid Cells Expressing Fetal Hemoglobin Due to BCL11A Interference

Blood ◽

10.1182/blood-2019-122770 ◽

2019 ◽

Vol 134 (Supplement_1) ◽

pp. 2063-2063

Author(s):

Naoya Uchida ◽

Claire Drysdale ◽

Morgan Yapundich ◽

Jackson Gamer ◽

Tina Nassehi ◽

...

Keyword(s):

Rhesus Macaques ◽

Fetal Hemoglobin ◽

Selective Advantage ◽

Erythroid Cells ◽

Post Transplant ◽

Human Cd34 ◽

Cd34 Cells ◽

Hbf Induction

Hematopoietic stem cell gene therapy for hemoglobin disorders, such as sickle cell disease, requires high-level gene marking and robust therapeutic globin expression in erythroid cells (>20% of γ- or β-globin production) for widespread successful clinical application. We previously demonstrated that lentiviral transduction of a truncated human erythropoietin receptor (thEpoR) gene allows for erythropoietin-dependent selective proliferation of gene-modified human erythroid cells during in vitro differentiation (ASH 2017). In this study, we sought to evaluate whether thEpoR can enhance the phenotypic effect of a therapeutic vector in erythroid cells in xenograft mouse and autologous non-human primate transplantation models. To investigate this hypothesis, we designed lentiviral vectors encoding both thEpoR and BCL11A-targeting micro RNA-adapted short hairpin RNA (shmiBCL11A), driven off an erythroid specific ankyrin 1 (ANK1) promoter. Both selective proliferation and high-level fetal hemoglobin (HbF) induction were observed in in vitro erythroid differentiation cultures using transduced human CD34+ cells. Healthy donor CD34+ cells were transduced with shmiBCL11A vector, thEpoR-shmiBCL11A vector, and GFP vector (control). Transduced cells were transplanted into immunodeficient NBSGW mice. Five months post-transplant, xenograft bone marrow cells were evaluated for human cell engraftment (human CD45+) and vector copy number (VCN) in both human CD34+ progenitor cells and glycophorin A+ (GPA+) erythroid cells. HbF production was also measured in GPA+ erythroid cells by reverse phase HPLC. We observed efficient transduction in transduced CD34+ cells in vitro (VCN 2.1-5.1) and similar human cell engraftment among all groups (84-89%). The VCN with thEpoR-shmiBCL11A transduction was 3-fold higher in human erythroid cells when compared to CD34+ cells (p<0.01), but not with shmiBCL11A or GFP vectors. HbF levels were significantly elevated in thEpoR-shmiBCL11A vector (43±6%, p<0.01) when compared to no transduction control (1±0%), but not for either shmiBCL11A vector (3±1%) or GFP vector (1±0%). These data demonstrate selective proliferation of gene-modified erythroid cells, as well as enhanced HbF induction with thEpoR-shmiBCL11A transduction. We then performed autologous rhesus CD34+ cell transplantation using either shmiBCL11A vector (142562 and RA0706, n=2, compared to a GPA promoter-derived shmiBCL11A vector) or thEpoR-shmiBCL11A vector (ZL50 and ZM24, n=2, compared to a Venus-encoding vector). Transduced CD34+ cells were transplanted into autologous rhesus macaques following 2x5Gy total body irradiation. Efficient transduction was observed in CD34+ cells in vitro among all 4 macaques (VCN 3.8-8.7) using a high-density culture protocol (Uchida N, Mol Ther Methods Clin Dev. 2019). In shmiBCL11A transduction animals, engraftment of gene-modified cells (VCN 0.2-1.0) and robust HbF induction (14-16%) were observed 1 month post-transplant. However, VCN and HbF levels were reduced down to VCN ~0.1 and HbF ~0.4% in both animals 6 months post-transplant. In contrast, a thEpoR-shmiBCL11A transduction animal (ZL50) resulted in engraftment of gene-modified cells (VCN 0.8-1.0) and robust HbF induction (~18%) 1 month post-transplant, with both gene marking and HbF levels remaining high at VCN 0.6-0.7 and HbF ~15% 4 months post-transplant. These data suggest that shmiBCL11A transduction results in transient HbF induction in gene-modified erythroid cells, while thEpoR-based selective advantage allows for sustained HbF induction with shmiBCL11A. In summary, we developed erythroid-specific thEpoR-shmiBCL11A expressing vectors, enhancing HbF induction in gene-modified erythroid cells in xenograft mice and rhesus macaques. While further in vivo studies are desirable, the use of thEpoR appears to provide a selective advantage for gene-modified erythroid cells in gene therapy strategies for hemoglobin disorders. Disclosures No relevant conflicts of interest to declare.

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SGK1 Inhibition Induces Fetal Hemoglobin in Erythroid Cells

Blood ◽

10.1182/blood-2021-146071 ◽

2021 ◽

Vol 138 (Supplement 1) ◽

pp. 963-963

Author(s):

Yannis Hara ◽

Mark Stottlemyer ◽

Kim Alving ◽

Nis Halland ◽

Alexandra Hicks ◽

...

Keyword(s):

Western Blot ◽

Blood Donors ◽

Fetal Hemoglobin ◽

Erythroid Cell ◽

Erythroid Cells ◽

Target Engagement ◽

Western Blots ◽

Cd34 Cells ◽

Hbf Induction ◽

In Erythroid Cells

Abstract Introduction: Novel and safe therapeutic targets to increase expression of fetal hemoglobin (HbF) have potential to treat b-hemoglobinopathies (Platt, Brambilla et al. 1994, Steinberg 2020), including sickle cell disease (SCD) in which red blood cell (RBC) hemoglobin S resulting from a mutation in the hemoglobin β-globin subunit causes RBC sickling and hemolysis triggering vascular inflammation (Piel, Steinberg et al. 2017, Kato, Piel et al. 2018). Serum- and glucocorticoid-regulated kinase 1 (SGK1) is a serine/threonine kinase in the AGK kinase family that controls physiological processes such as cell growth, proliferation, migration, and apoptosis (Hayashi, Tapping et al. 2001, Sang, Kong et al. 2020). SGK1 is regulated by multiple ligands (insulin, cAMP, IGF-1, steroids, IL-2 and TGF-β) and phosphorylation by SGK1 modulates the activity of downstream effectors including ion channels (ENaC), Na-Cl cotransporters (NCC), membrane transporters, cellular enzymes (GSK3B) and transcription factors (FOXO3a, β-catenin, NF-κB and SP1) (Brunet, Park et al. 2001, Snyder, Olson et al. 2002, Loffing, Flores et al. 2006, Bruhn, Pearson et al. 2010, Boccitto and Kalb 2011, Wang, Hu et al. 2017). Previous studies show that SGK1 mediates survival signals in HEK cells by inhibiting FOXO3a through phosphorylation at Ser-315 (Brunet, Park et al. 2001). Recently, metformin was shown to induce HbF in erythroid cells through FOXO3a activation and metformin prevents RBC sickling in SCD (Zhang, Paikari et al. 2018). Thus, we hypothesized that inhibition of SGK1 and subsequent alleviation of SGK1-induced FOXO3a inhibition, may induce expression of erythroid cell HbF. Methods: We studied the ability of SGK1 to inhibit HbF induction in erythroid cells by culturing CD34+ hematopoietic progenitor stem cells from both healthy and SCD blood donors using a 21-day differentiation protocol. After confirming expression of SGK1 in CD34+ cells by Western blot, SGK1 activity was inhibited using the selective and potent SGK1 inhibitor RA04075215A (Halland, Schmidt et al. 2015). SGK1 is activated by phosphorylation at Thr256 and we confirmed target engagement through measurement of Thr256 phosphorylation on Western blots. To decipher the effect of SGK1 inhibition on the SGK1 downstream pathway, we assessed the inhibition of FOXO3a triggered by SGK1 through evaluation of FOXO3a phosphorylation Ser315. In parallel, we quantified HbF gene transcripts by qPCR, determined the level of HbF protein by Western blot, and quantified F-cells by flow cytometry. Finally, to evaluate the effect of SGK1 inhibition on RBC sickling, we performed a cell sickling assay upon completion of erythroid differentiation in culture. Fully differentiated CD34+ cells from SCD blood donors were incubated under in hypoxia (2% O 2) for 4 hours and then abnormal shaped cells were analyzed using the Amnis® ImageStream® flow cytometer. Results: By day 21 of differentiation, HbF protein expression in CD34+ cells increases significantly in RA04075215A-treated cells versus untreated controls. In addition, a combination of SGK1 inhibition and hydroxyurea treatment reveals a potential synergistic induction of HbF. Western blot analysis shows a decrease in phospho-SGK1 phosphorylated at Thr-256 with SGK1 inhibition, confirming target engagement and loss of SGK1 activity. Downstream of SGK1, phospho-FOXO3a phosphorylated at Ser-315 was also decreased significantly following SGK1 inhibition, demonstrating alleviation of FOXO3a inhibition. Finally, in the RBC sickling assay, RA04075215A-treated cells were significantly protected from sickling under hypoxia compared to controls. Conclusion: In summary, this study establishes SGK1 as a potential new therapeutic target in SCD. We demonstrate that SGK1 inhibition induces HbF in CD34+ cells through FOXO3a transcription factor activation and prevents CD34+ cells from sickling. In the future, in vivo studies are necessary to confirm the role of SGK1 in HbF induction and to assess the efficacy of SGK1 inhibition in improving markers of SCD. Disclosures No relevant conflicts of interest to declare.

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Pomalidomide Augments Erythropoiesis and Fetal Hemoglobin Production in a Humanized Mouse Model of Sickle Cell Disease

Blood ◽

10.1182/blood.v112.11.536.536 ◽

2008 ◽

Vol 112 (11) ◽

pp. 536-536 ◽

Cited By ~ 2

Author(s):

Steffen E. Meiler ◽

Marlene Wade ◽

Zhong Chen ◽

Preetha Ramalingam ◽

Laure A Moutouhde Parseval ◽

...

Keyword(s):

Sickle Cell Disease ◽

Mouse Model ◽

Sickle Cell ◽

Fetal Hemoglobin ◽

Spleen Weight ◽

Cell Disease ◽

Humanized Mouse ◽

Humanized Mouse Model ◽

Cd34 Cells

Abstract Introduction: Pomalidomide (PL) is an IMiD™ immunomodulatory compound. Thalidomide and lenalidomide have previously been shown to mitigate blood transfusion dependency in patients with thalassemia major and myelodysplastic syndromes, respectively.1,2 In a recent in vitro study, PL, which is currently being evaluated for the treatment of hematological cancers, stimulated erythropoiesis, F-cell production, total hemoglobin (Hb) and fetal hemoglobin (HbF) synthesis in human CD34+ cells.3 Furthermore, combined treatment of CD34+ cells with PL and Hydroxyurea (HU) synergistically upregulated HbF expression. These results suggest a promising role for PL in the treatment of β-hemoglobinopathies. To evaluate the in vivo efficacy of PL in sickle cell disease (SCD), we conducted an eight week study in a relevant knockout-transgenic (KT) mouse model. 4 Methods: Animals. Six week old KT homozygous sickle mice were treated daily (Mon–Fri; i.p. injections) for eight weeks with the following compounds: Grp 1. Vehicle (n=8); Grp 2. PL (10 mg/kg; n=9); Grp 3. HU (100 mg/kg; n=7); Grp 4. PL+ HU (n=8). Mice were maintained in an accredited pathogen-free animal facility according to NIH and institutional guidelines. Mice were anesthetized with Ketamine/Xylazine and blood collected by intracardiac puncture into 0.5 ml vacutainer EDTA tubes. Complete blood count (CBC) was analyzed with the CBC-Diff™ Veterinary Hematology System (Heska Inc., Loveland, CO). Reticulocyte counts were determined by supravital staining with methylene blue. HbF analysis of mouse hemolysates was done by HPLC using a weak cation-exchange column SynChropak CM-300 (Elchrom INC, Darien, IL). Organ analysis (liver, spleen, and femur marrow) included organ weights (%bw) and histology of H&E paraffin sections. Statistical analysis. One-Way ANOVA followed by Student-Newman-Kuels (Sigma Stat). Data are reported as the mean ± SE. A P-value of < 0.05 was considered significant. Results: Activity level and social behavior were unaffected by the treatment groups. There were no physical signs of drug toxicity and weight gains were comparable for all groups. PL significantly augmented HbF expression comparable to HU (HbF [peak%]: Veh: 6.24±0.35; PL: 9.51±0.83, P<0.01; HU: 10.54±0.77, P<0.01/total Hb [gm/dl]: Veh: 7.16±0.38; PL: 7.67±0.33; HU: 6.09±0.79). Surprisingly, PL-induced HbF production returned to control values after combinatory therapy with HU (HbF [peak%]: Veh: 6.24±0.35; PL+HU: 7.14±0.32). In contrast to HU, PL enhanced erythropoiesis as evidenced by increases in total RBCs, reticulocytes, and spleen weight (RBCs [×106/μl]: Veh: 4.84±0.14; PL: 5.49±0.19, P<0.05; HU: 4.47±0.51/Retics [%]: Veh: 36.81±5.78; PL: 40.47±4.73; HU: 16.41±3.94, P=0.01/Spleen weight [%bw]: Veh: 4.07±0.27; PL: 4.92±0.2, P=0.02; HU: 2.75±0.25, P<0.01). The total WBC count was largely unaffected by PL, but decreased significantly with HU (WBC [×103/μl]: Veh: 14.43±2.74; PL: 11.62±2.62; HU: 6.85±0.82, P<0.05). The PL group showed a trend toward increased marrow hyperplasia. A detailed analysis of cell density, erythroid and myeloid cells is underway. Liver histology revealed decreased tissue inflammation and focal necrosis in ~50% of PL-treated animals. Summary & Conclusions: 1. PL caused a robust induction of HbF in sickle mice. These results extend earlier in vitro findings of PL’s bioactivity in human erythroid progenitor cells. The HbF response to PL was similar to the established HbF-inducer HU, but surprisingly was lost in combination with HU. This inhibitory effect suggests that PL and HU stimulate HbF by distinct mechanisms. 2. Additionally, PL augmented erythropoiesis as demonstrated by increases in total RBCs, reticulocytes, spleen weight, and marrow hyperplasia. In contrast, HU showed the expected findings of generalized myelosuppression. 3. PL reduced liver injury and inflammation in ~50% of treated animals. Liver preservation tended to be associated with higher HbF values, though PL’s immunomodulatory properties may have contributed to this outcome. 4. In summary, PL exhibits a highly favorable hematological profile in a humanized mouse model of SCD. These results warrant further investigation in a Phase I trial of patients with SCD.

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Fetal Hemoglobin Rescues Ineffective Erythropoiesis in Sickle Cell Disease

Blood ◽

10.1182/blood-2020-137477 ◽

2020 ◽

Vol 136 (Supplement 1) ◽

pp. 14-15

Author(s):

Sara El Hoss ◽

Sylvie Cochet ◽

Auria Godard ◽

Hongxia Yan ◽

Michaël Dussiot ◽

...

Keyword(s):

Cell Death ◽

Fetal Hemoglobin ◽

Erythroid Differentiation ◽

Ineffective Erythropoiesis ◽

Hypoxic Conditions ◽

Cd34 Cells ◽

F Cells

Sickle cell disease (SCD) is an autosomal hereditary recessive disorder caused by a point mutation in the β globin gene resulting in a Glu-to-Val substitution at the 6th position of the β globin protein. The resulting abnormal hemoglobin (HbS) polymerizes under hypoxic conditions driving red blood cell (RBC) sickling (Pauling et al., 1949). While pathobiology of circulating RBCs has been extensively analyzed in SCD, erythropoiesis is surprisingly poorly documented. In β-thalassemia, ineffective erythropoiesis is characterized by high levels of apoptotic erythroblasts during the late stages of terminal differentiation, due to an accumulation of free β-globin chains (Arlet et al., 2016). Ineffective erythropoiesis is the major cause of anemia in β-thalassemia patients. In contrast, a marked decrease in life span of circulating red cells, a feature of sickle red cells, is considered to be the major determinant of chronic anemia in SCD. It is generally surmised that ineffective erythropoiesis contributes little to anemia. The bone marrow environment has been well documented to be hypoxic (0.1 to 6% O2) (Mantel et al., 2015). As hypoxia induces HbS polymerization, we hypothesized that cell death may occur in vivo because of HbS polymer formation in the late stages of differentiation characterized by high intracellular hemoglobin concentration. In the present study, using both in vitro and in vivo derived human erythroblasts we assessed the extent of ineffective erythropoiesis in SCD. We explored the mechanistic basis of the ineffective erythropoiesis in SCD using biochemical, cellular and imaging techniques. In vitro erythroid differentiation using CD34+ cells isolated from SCD patients and from healthy donors was performed. A 2-phase erythroid differentiation protocol was used and cultures were performed at two different oxygen conditions, i.e. normoxia and partial hypoxia (5% O2). We found that hypoxia induces cell death of sickle erythroblasts starting at the polychromatic stage, positively selecting cells with high levels of fetal hemoglobin (HbF). This inference was supported by flow cytometry data showing higher percentages of dead cells within the non-F-cell population as compared to the F-cell population for SCD cells. Moreover, SCD dead cells showed higher levels of chaperon protein HSP70 in the cytoplasm than live cells, while no difference was detected between both subpopulations for control cells, suggesting that cell death of SCD erythroblasts was probably due to HSP70 cytoplasmic sequestration. This was supported by western-blot experiments showing less HSP70 in the nucleus of SCD erythroblasts under hypoxia, associated with decreased levels of GATA-1. At the molecular level, HSP70 was co-immunoprecipitated with HbS under hypoxia indicating that both proteins were in the same complex and suggesting interaction between HSP70 and HbS polymers in the cyotplasm. Importantly, we confirm these results in vivo by showing that in bone marrow of SCD patients (n = 5) cell loss occurs during terminal erythroid differentiation, with a significant drop in the cell count between the polychromatic and the orthochromatic stages (Figure 1). In order to specifically address the role of HbF in cell survival, we used a CRISPR-Cas9 approach to mimic the effect of hereditary persistence of fetal hemoglobin (HPFH). CD34+ cells were transfected either with a gRNA targeting the LRF binding site (-197) or a gRNA targeting an unrelated locus (AAVS1) (Weber, Frati, et al. 2020). As expected, the disruption of the LRF binding site resulted in HbF induction as shown by higher %F-cells compared to AAVS1 control. These higher levels of F-cells resulted in decreased apoptosis, under both normoxic and hypoxic conditions, clearly demonstrating the positive and selective effect of HbF on SCD cell survival (Figure 2). In summary, our study shows that HbF has a dual beneficial effect in SCD by conferring a preferential survival of F-cells in the circulation and by decreasing ineffective erythropoiesis. These findings thus bring new insights into the role of HbF in modulating clinical severity of anemia in SCD by both regulating red cell production and red cell destruction. Disclosures No relevant conflicts of interest to declare.

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Reticulocyte Survival in Sickle Cell Anemia: Effect of Cyanate

Blood ◽

10.1182/blood.v40.5.733.733 ◽

1972 ◽

Vol 40 (5) ◽

pp. 733-739 ◽

Cited By ~ 5

Author(s):

Blanche P. Alter ◽

Yuet Wai Kan ◽

David G. Nathan

Keyword(s):

Cell Survival ◽

Sickle Cell ◽

Fetal Hemoglobin ◽

Red Cells ◽

Red Cell ◽

Hemoglobin Molecule ◽

Red Cell Survival ◽

And Control

Abstract Cyanate prevents sickling in vitro and apparently prolongs the survival of 51Cr-tagged sickle erythrocytes in vivo. Cautious interpretation is required because the effects of cyanate on 51Cr binding to sickle and fetal hemoglobin-containing red cells are unknown, and comparison of the effect of cyanate on sickle red cell survival to control red cell survival must be performed sequentially. We have studied the survival of sickle reticulocytes utilizing radioactive amino acids that are incorporated into hemoglobin. Two informed adult patients with sickle cell disease were studied. In each study, two 50-ml samples of blood were incubated separately with 14C- and 3H-leucine for 2 hr, after which 50 mM cyanate was added to one aliquot for 1 hr. The cells were then washed and reinfused. Frequent venous samples were obtained, and the specific activities of 14C and 3H in the hemoglobin were followed. The t ½ of the carbamylated cells was tripled, but remained below normal. This method provides a generally useful measurement of the influence of drugs bound to red cells on reticulocyte lifespan. The labels are incorporated into the hemoglobin molecule of the reticulocyte, and simultaneous comparison of the survivals of the same cohort of drug-treated and control cells is achieved.

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Altered Collection Interfaces in Hematopoietic Progenitor Collection of Sickle Cell Patients

American Journal of Clinical Pathology ◽

10.1093/ajcp/aqz112.000 ◽

2019 ◽

Vol 152 (Supplement_1) ◽

pp. S1-S1

Author(s):

Jonathan Tsai ◽

John Manis ◽

Kimberly Ching

Keyword(s):

Sickle Cell ◽

Globin Gene ◽

Fetal Hemoglobin ◽

Interface Layer ◽

Hematopoietic Stem ◽

Healthy Donors ◽

Cd34 Cells ◽

Stem And Progenitor Cells ◽

Cell Monitoring ◽

Collection Practices

Abstract Sickle cell disease (SCD) results from a point mutation in the beta globin gene leading to abnormal hemoglobin production and, correspondingly, abnormal sickling of erythrocytes that result in chronic anemia and vaso-occlusive crises. Patients with sickle cell are commonly treated with lifelong transfusions or hydroxyurea to increase fetal hemoglobin to ameliorate sickling. Evolving therapies are aimed at correcting known mutations with gene therapy on autologous hematopoietic stem and progenitor cells (HSCs), indicating a growing need for optimal stem cell collections from SCD patients. Recent studies have shown the safety and efficacy of plerixafor to increase peripheral CD34+ cells, enhancing collection. Here we show that standard apheresis collection procedures from sickle cell patients are inefficient when compared to healthy donors. Eleven patients with SCD were recruited to receive plerixafor and followed by peripheral CD34+ cell monitoring and apheresis collection. Overall, efficiency ranged from 2% to 55% with no correlation to total peripheral CD34+ cell count. To better understand where the CD34+ cells sedimented in the apheresis instrument, we collected different layers of the interface ranging from 3% to 10% estimated Hct and found that deeper layers with higher hematocrit (7.5%-10%) are enriched for CD34+ cells when compared to historical donors with healthy red cells. All patients had undergone red cell exchange prior to collection, yet this intervention did not prevent the altered sedimentation of CD34+ cells. These findings indicate that CD34+ cells from SCD patients sediment at a deeper, higher Hct interface layer during apheresis and support the altered collection practices for the efficient collection of HSCs for cellular therapies.

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Pomalidomide augments fetal hemoglobin production without the myelosuppressive effects of hydroxyurea in transgenic sickle cell mice

Blood ◽

10.1182/blood-2010-11-319137 ◽

2011 ◽

Vol 118 (4) ◽

pp. 1109-1112 ◽

Cited By ~ 48

Author(s):

Steffen E. Meiler ◽

Marlene Wade ◽

Ferdane Kutlar ◽

Shobha D. Yerigenahally ◽

Yongjun Xue ◽

...

Keyword(s):

Sickle Cell ◽

Ex Vivo ◽

Fetal Hemoglobin ◽

Structural Analog ◽

Hematologic Malignancies ◽

Similar Efficacy ◽

Novel Therapy ◽

New Class ◽

Transfusion Requirements ◽

Hbf Induction

Abstract Pharmacologic induction of fetal hemoglobin (HbF) expression is an effective treatment strategy for sickle cell disease (SCD) and β-thalassemia. Pomalidomide is a potent structural analog of thalidomide and member of a new class of immunomodulatory drugs. Recent reports demonstrated that pomalidomide reduced or eliminated transfusion requirements in certain hematologic malignancies and induced HbF ex vivo in CD34+ progenitor cells from healthy and SCD donors. We investigated the effects of pomalidomide on erythropoiesis and hemoglobin synthesis in a transgenic mouse model of SCD. We found that 8 weeks of treatment with pomalidomide induced modest increases of HbF with similar efficacy as hydroxyurea. However, in stark contrast to hydroxyurea's myelosuppressive effects, pomalidomide augmented erythropoiesis and preserved bone marrow function. Surprisingly, combinatory therapy with both drugs failed to mitigate hydroxyurea's myelotoxic effects and caused loss of HbF induction. These findings support further evaluation of pomalidomide as a novel therapy for SCD.

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Clinical Evaluation of Combined Epigenetic Therapies on the Induction of Fetal Hemoglobin in Patients with Hematologic Malignancies

Blood ◽

10.1182/blood.v126.23.960.960 ◽

2015 ◽

Vol 126 (23) ◽

pp. 960-960

Author(s):

Katharine Rose Press ◽

Jeffrey Keefer ◽

Steven D. Gore ◽

Hetty E. Carraway ◽

Sarah Sakoian ◽

...

Keyword(s):

Histone Deacetylase ◽

Hdac Inhibitors ◽

Fetal Hemoglobin ◽

Fold Increase ◽

Methyl Transferase ◽

Disease Response ◽

Deacetylase Inhibitor ◽

Hbf Induction ◽

Hbf Level

Fetal hemoglobin induction with hydroxyurea (HU) is a mainstay of therapy for β-hemoglobinopathies, especially sickle cell disease (SCD). A high level of fetal hemoglobin (HbF) has a direct relationship with acute clinical status in SCD patients including pain crises, acute chest syndrome, and death. However, not all patients benefit from HU, and more effective HbF induction strategies are needed. DNA methyl transferase (DNMT) inhibitors and histone deacetylase (HDAC) inhibitors have been shown in vitro to induce HbF production through epigenetic modification of the β-globin gene cluster. Azacitidine (AZA) is a DNMT already used in some SCD patients resistant to HbF modulation with HU. Entinostat (MS-275) is an orally available histone deacetylase inhibitor with a long half-life and established antitumor activity in preclinical models. Recent studies suggest that drugs, which act with different molecular and epigenetic mechanisms, have synergistic effects on induction of fetal hemoglobin (Fard et al. IJHOSCR 2013). In this study, we evaluated the effects of a combination of AZA and MS-275 on HbF levels. This was preformed as a correlative study of a phase I clinical trial (J0443 trial) of these drugs in patients with myelodysplastic syndrome (MDS) or acute myeloid leukemia (AML). We sequentially measured the level of HbF the peripheral blood in 33 patients receiving different doses of AZA (range: 30mg/m2 to 50mg/m2 per day for 10 doses) and MS-275 (range: 2 to 8 mg/m2 orally on days 3 and 10). Patients completed a minimum of four 28-day cycles of combined therapy. HbF levels were measured in peripheral blood at baseline, at day 15 or 16 and day 29 or 30 of cycle 1, and after cycles 2, 4, and if applicable 6. Azacitidine dose positively correlated with HbF fold increase (mean of 1.1, 2.3, and 2.1 for doses of 30, 40, and 50 mg respectively, p=0.07) while MS-275 dose had a slightly negative correlation with HbF level (mean of 3.0, 1.8, and 1.3 for doses of 2, 4, and 6mg respectively, p=0.13). There was no correlation between baseline HbF and HbF fold increase after exposure to treatment (p=NS) and no correlation between baseline HbF levels and clinical disease response (p=0.19). Interestingly, we demonstrated a correlation between HbF fold increase and clinical disease response: median fold increase of 3.5 for patients achieving hematologic normalization (complete response, partial response, or trilineage hematological improvement) versus 1.4 in non-responders (p=0.006). The positive correlation between AZA dose and HbF increase is consistent with prior work showing that this drug induces HbF production. The correlation between clinical response and HbF induction could reflect a greater susceptibility to AZA potentially related to differing methylomes. Alternatively, it may also represent a known increase in HbF in the setting of stress erythropoiesis. The slight inverse correlation between MS-275 and HbF level was surprising, as other HDAC inhibitors are known to induce HbF in vitro. However, these results are in line with the methylation data found in the more recent randomized phase 2 trial of AZA +/- MS-275 (E1905 trial) that showed a potential pharmacodynamic antagonism of the combination (Prebet et al. J Clin Oncol. 2014). Overall, this work supports the use of AZA as a clinical inducer of HbF. It also shows the importance of trialing various combinations of HbF inducers, as not all drugs work synergistically and some may even be antagonistic in combination. Disclosures Off Label Use: Azacitidine (AZA) is a DNA methyl transferase (DNMT) inhibitor. Entinostat (MS-275) is an orally available histone deacetylase inhibitor. Both drugs were used in a phase I clinical trial (J0443 trial) of these drugs in patients with myelodysplastic syndrome (MDS) or acute myeloid leukemia (AML). . Keefer:MAST therapeutics: Employment. Gore:Celgene: Consultancy, Honoraria, Research Funding. Prebet:CELGENE: Research Funding.

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