Page 206 - 2021_12-Haematologica-web

Letters to the Editor
decreased by JQ1 and EPO, and the combination treat- ment showed an additive effect (Figure 2A). Since MYB is a target of miR-15A and miR-16-1, we measured the levels of these miR and found that both were upregulated by either JQ1 and EPO in an additive fashion (Figure 2B and C), showing an inverse correlation compared to MYB expression as expected. RNA-seq quantification of known miR-15A/16-1 targets indicated that most were downregulated by treatment with JQ1 and/or EPO (Figure 2D), further suggesting that these miR play a cen- tral role in mediating the effects of JQ1. Next, we exam- ined the genes encoding several well-established HBE1 and HBG1/2 inhibitors or HBB activators: IKZF1 (IKAROS), NR2F2 (COUP-TF2), BCL11A, and GATA1. Each of these genes was downregulated by JQ1 with or without EPO (Figure 2E to G). KLF1 promotes the expres- sion of BCL11A,9 and therefore as expected, we observed downregulation of BCL11A only when we also saw KLF1 repression in the setting of joint JQ1 and EPO treatment (Figure 2H and I). GATA1 western blots confirmed a sta- ble level of protein as expected from the qPCR data, whereas western blotting for BCL11A demonstrated almost no detectable protein after combination treatment (Figure 2J).
In order to examine how the spatial structure of the locus changes in response to JQ1 treatment, we per- formed chromatin conformation capture followed by qPCR (3C-qPCR) to quantify the LCR interactions with the b-globin genes under each treatment (Figure 3A). Interestingly, differentiation alone via EPO did not change the interaction frequency in this locus (Figure 3B). In contrast, JQ1 decreased the interaction at multiple loci near or in the fetal and adult g/b-globin genes (Figure 3B). Similarly, the LCR interaction in EPO+JQ1 double-treated cells is more similar to that of JQ1-treated cells than that of EPO-treated cells, again showing that JQ1, but not EPO, decreases interactions between the LCR and the g/b-globin genes (Figure 3C). Notably, JQ1 treatment did not decrease the interaction between the LCR and the HBE1 promoter (Figure 3B and C), suggesting that JQ1 treatment relaxes the looping between the LCR and the g/b-globin genes, which biases LCR contacts in favor of interactions with the promoter of embryonic HBE1.
Thus, the b-globin gene expression changes under BET inhibition are likely the result of both shifts in local chro- matin looping and expression changes of b-globin inhibitors and activators. The shifts in LCR interactions favor HBE1 expression over HBB or HBG1/2. Although the overall interaction between LCR and the b-globin genes decreases under JQ1 treatment, e/g-globin expres- sion still increased likely due to reduced inhibition and upregulation of genes involved in erythroid maturation. Together, these factors result in decreased HBB transcrip- tion and increased embryonic HBE1 transcription (Figure 1A).
Our data in TF-1 cells show that BETi induces partial erythroid maturation and reactivate the embryonic e-glo- bin HBE1 even without EPO-mediated erythroid matura- tion. An important clinical question is whether the HBE1 encoded e-globin could function as a reasonable substitute for the adult b-globin chain. Biochemical analysis of the embryonic hemoglobin Hb-Gower 2 (α2e2) shows that its P50 for oxygen, affinity to 2,3-BPG, Bohr coefficient, and Hill coefficient are comparable to those of adult hemoglo-
10
bin A (HbA). Hb-Gower 2 also has a comparable
tetramer-dimer dissociation constant to that of HbA.11 A study in transgenic α/b-thalassemia mice found that human embryonic hemoglobins consist of ζ-globin and e-globin rescue the lethal phenotype of
α/b-thalassemia.12 Similarly, a study in sickle cell mice found that the presence of human Hb-Gower 2 (α2e2) greatly alleviated sickle cell phenotypes, and Hb-Gower 2 inhibits sickle cell hemoglobin (HbS) polymerization.13
Our findings suggest that specific inhibition of bro- modomain-containing proteins could provide a reason- able alternative and/or adjuncts to the treatment of b-glo- binopathies like sickle cell anemia (SCA). SCA patients generally have elevated EPO levels,14 and thus treatment with JQ1 alone might be sufficient to induce e-globin in these patients. Currently, the most common treatment for SCA is administration of hydroxyurea, which elevates fetal hemoglobin production to alleviate symptoms.15 In recent years, multiple gene therapy strategies for SCA have emerged, including b-globin gene addition and nuclease-assisted b-globin gene modification/repair.16 Although these recent advances in gene therapy offer the potential for cure, they will not be accessible or afford- able for all patients, especially those from less affluent countries or health systems. We hope that our work motivates future studies that focus on the effect of BET inhibition on globin expression in other erythroid cell lines and primary cells and how BET inhibition could be harnessed for therapeutic purposes.
John Z. Cao,1 Kristina Bigelow,2 Amittha Wickrema1,2 and Lucy A. Godley1,2
1Committee on Cancer Biology, Biological Sciences Division, The University of Chicago and 2Section of Hematology/Oncology, Department of Medicine, The University of Chicago, Chicago IL, USA
Correspondence:
LUCY A. GODLEY - lgodley@medicine.bsd.uchicago.edu doi:10.3324/haematol.2021.278791
Received: March 16, 2021.
Accepted: August 20, 2021.
Pre-published: August 26, 2021.
Disclosures: LAG, AW and JZC are owners of the patent PCT/US20/52842 titled “Methods and compositions for treating sickle cell disease and thalassemia”, filed on September 25, 2020.
Contributions: JZC designed and performed the experiments, analyzed the data, and wrote the manuscript; KB performed additional experiments; LAG conceived of the study and provided insights in experimental design and data interpretation; AW provided additional input for experimental design and data interpretation.
Acknowledgments: we thank Dr. Alex Ruthenburg (University of Chicago) for providing the K562 cells used in this work and experimental advice; and Dr. Julie-Aurore Losman (Dana-Farber Cancer Institute) who provided the TF-1 cells.
Funding: this work was supported by a grant by the Edwards
P. Evans Foundation/EvansMDS to AW and LAG. Funding for JZC was supported by the University of Chicago Biological Sciences Division Dean’s Office, the University of Chicago Comprehensive Cancer Center Women’s Board, and the Goldblatt Scholarship.
References
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2. Wilber A, Nienhuis AW, Persons DA. Transcriptional regulation of fetal to adult hemoglobin switching: new therapeutic opportunities. Blood. 2011;117(15):3945-3953.
3. Aerbajinai W, Zhu J, Kumkhaek C, Chin K, Rodgers GP. SCF induces gamma-globin gene expression by regulating downstream transcription factor COUP-TFII. Blood. 2009;114(1):187-194.
4. Xu J, Bauer DE, Kerenyi MA, et al. Corepressor-dependent silencing of fetal hemoglobin expression by BCL11A. Proc Natl Acad Sci U S A. 2013;110(16):6518-6523.
5. Sankaran VG, Menne TF, Šćepanović D, et al. MicroRNA-15a and -16-1
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