Abstract
Elevated levels of fetal hemoglobin (HbF) are beneficial to patients with sickle cell disease (SCD) and b-thalassemia. The identification of pharmacologically controllable pathways that regulate HbF levels remains an important goal in the treatment of these disorders. BCL11A and LRF (ZBTB7A) are the two major transcriptional repressors of g-globin (fetal beta-type globin). However, since transcription factors are challenging to inhibit with small molecules, we aimed to identify novel HbF regulators that might be amenable to control by pharmacologic means.
Using an improved protein-domain based CRISPR-Cas9 screen recently developed in our laboratory (Grevet et al., Science 361:6399, 285-290), we screened for new HbF regulators using a gRNAs library targeting chromatin associated proteins including BTB domain containing factors. We identified SPOP, a substrate adaptor of the CUL3 ubiquitin ligase complex, as a novel inhibitor of g-globin expression. Depletion of SPOP strongly elevated the mRNA and protein levels of g-globin in both HUDEP-2 and primary human CD34+ derived erythroid cells. The effects of SPOP loss were remarkably selective as evidenced by RNA-seq and whole cell mass spectrometry. Importantly, SPOP depletion did not appear to impair cell growth or differentiation. Mechanistically, we demonstrated that the repressive role of SPOP on g-globin levels is dependent on the CUL3 ubiquitin ligase complex. Additionally, forced expression of a dominant negative form of SPOP (Y87N) unable to bind to its substrates strongly induced HbF production. We also found that SPOP depletion did not measurably alter LRF levels and modestly reduced BCL11A levels. Overexpression of BCL11A did not restore g-globin silencing in SPOP depleted cells, indicating that SPOP works largely independently of these two factors. Finally, we tested whether the effects of SPOP depletion can be augmented through the use of experimental pharmacologic HbF inducers, such as pomalidomide. In proof of principle experiments we found that SPOP depletion and pomalidomide treatment induced HbF production to levels higher than either treatment alone.
In sum, our work uncovered SPOP as a new regulator of HbF and suggests targeting SPOP alone or in combination with other HbF inducers may provide a novel therapeutic strategy for the treatment of hemoglobinopathies such as sickle cell disease and b-thalassemia.
Disclosures
No relevant conflicts of interest to declare.
Phyllopod Acts as an Adaptor Protein To Link the Sina Ubiquitin Ligase to the Substrate Protein Tramtrack
