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.
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