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1041 Regulation of Fetal Hemoglobin Production in Adult Erythroid Cells By Protein Phosphatase 6C (PPP6C)

Program: Oral and Poster Abstracts
Session: 113. Hemoglobinopathies, Excluding Thalassemia: Basic and Translational: Poster I
Hematology Disease Topics & Pathways:
Research, Fundamental Science, Sickle Cell Disease, Translational Research, Genetic Disorders, Thalassemia, genomics, Hemoglobinopathies, hematopoiesis, Diseases, Therapies, Biological Processes, molecular biology, Technology and Procedures
Saturday, December 10, 2022, 5:30 PM-7:30 PM

Scott A. Peslak, MD, PhD1,2, Eugene Khandros, MD, PhD2, Peng Huang, PhD2*, Aoi Wakabayashi, BS2,3, Talla Abbas, BS1,2*, Osheiza Y. Abdulmalik, DVM2*, Cheryl A. Keller, PhD4,5*, Ross C. Hardison, PhD5*, Junwei Shi, PhD6* and Gerd A. Blobel, MD, PhD2

1Division of Hematology/Oncology, Department of Medicine, University of Pennsylvania Perelman School of Medicine, Philadelphia, PA
2Division of Hematology, The Children's Hospital of Philadelphia, Philadelphia, PA
3University of Pennsylvania, Philadelphia, PA
4Genomics Research Incubator, Pennsylvania State University, University Park, PA
5Department of Biochemistry and Molecular Biology, Pennsylvania State University, University Park, PA
6Department of Cancer Biology, University of Pennsylvania Perelman School of Medicine, Philadelphia, PA

Although increasing levels of fetal hemoglobin (HbF) in sickle cell disease (SCD) significantly reduces mortality, effective pharmacologic induction of HbF has remained elusive. To identify potentially druggable molecules involved in HbF control, we carried out a domain-focused CRISPR-Cas9-based genetic screen targeting all serine/threonine protein phosphatases (1308 independent sgRNAs representing 218 phosphatases). The phosphatase sgRNA library was cloned into a lentivirus scaffold and introduced into the adult-type erythroid cell line HUDEP2 stably expressing Cas9, the top and bottom 10% of HbF-expressing cells sorted, and the integrated sgRNAs sequenced. Of all protein phosphatases tested, this screen singly identified PPP6C as a novel HbF repressor.

PPP6C is the catalytic subunit of protein phosphatase 6, a serine/threonine protein phosphatase that has been implicated in numerous cellular functions, including cell cycle regulation, autophagy, and innate immunity, but its role in HbF regulation has not previously been described. Depletion of PPP6C by 5 independent sgRNAs in HUDEP2 cells resulted in significant HbF enrichment and did not adversely affect cell fitness. To validate these findings, we performed CRISPR-Cas9-based depletion of PPP6C in primary human erythroid cells. Quantitation of fetal-type globin genes (gamma-globin) was determined by RT-qPCR and Western blot, and HbF protein levels were determined by flow cytometry and HPLC. Depletion of PPP6C elevated gamma-globin mRNA levels in a dose-dependent manner to 3-4 times basal levels, increased HbF levels 3-4 fold as measured by HPLC (up to 15-20% of total hemoglobin), and doubled the number of HbF-expressing cells. PPP6C depletion caused relatively few changes in the erythroid transcriptome by RNA-seq analysis and did not ostensibly impair erythroid maturation. Importantly, xenotransplantation data (NBSGW) from two independent murine transplants for a total of 10 recipient mice showed ~4-fold induction of gamma-globin at 16 weeks post-transplant, suggesting that PPP6C deficiency is well-tolerated and leads to effective, sustained HbF induction in vivo.

To test whether interference with PPP6C raised HbF levels sufficiently to provide anti-sickling effects, we depleted PPP6C by CRISPR-Cas9 in SCD patient-derived cells. PPP6C depletion in SCD cells was well-tolerated, led to robust levels of HbF induction, and reduced cell sickling in vitro by greater than 60%. Mechanistically, loss of PPP6C reduced the levels of the HbF repressor BCL11A by nearly 50% but left unchanged the levels of other HbF regulators, such as HRI, LRF, EKLF, NFIA/X, ZNF410, or HIC2, suggesting that PPP6C-mediated HbF regulation may proceed at least in part via loss of BCL11A. Additional studies, including specific interrogation of potential downstream mediators of PPP6C in HbF regulation, are ongoing to further elucidate HbF regulatory pathways impacted by PPP6C.

Taken together, our data indicate that PPP6C functions in a dose-dependent manner to regulate HbF in primary erythroid cells and may serve as a therapeutic target in the treatment of SCD.

Disclosures: Peslak: Fulcrum Therapeutics: Consultancy. Blobel: Fulcrum Therapeutics: Research Funding; Pfizer: Consultancy.

*signifies non-member of ASH