The CDK-Inhibitor p27 Kip1 Regulates Fetal Hemoglobin Expression

Abstract

γ-globin upregulation has been shown to be beneficial for individuals with Sickle Cell Disease, leading to improvement in morbidity and mortality rates. Intriguingly, small molecules that modulate the cell cycle have been observed to impact γ-globin expression in erythroid cells, though a causal relationship has not been definitively established. Understanding the connection between cell cycle dynamics/regulation and γ-globin production may shed light into a novel molecular mechanism by which γ-globin expression is regulated, with potential therapeutic implications for Sickle Cell Disease. To evaluate if cell cycle speed correlates with γ-globin expression, we labeled HUDEP-2 cells (which express beta-globin) with carboxyfluorescein diacetate succinimidyl ester (CFSE) at different days of differentiation. With each cell division, CFSE dye intensity is expected to be reduced by half; therefore, faster cycling cells retain less CFSE than slower cycling cells. Forty-eight hours post-CFSE labeling, HUDEP-2 cells were analyzed for γ-globin expression by intracellular flow cytometry at days 2, 4, 6, and 8 of differentiation. Starting at Day 6 of differentiation, the slowest cycling (Top 5% CFSE) HUDEP-2 cells exhibited >2-fold increase in γ-globin expressing cells (F-cells) compared to the fastest cycling (bottom 5% CFSE) cells. Notably, these results were confirmed in erythroid cells differentiated from primary human CD34+ hematopoietic stem and progenitor cells (HSPCs), therefore ruling out the possibility that the above findings were an artifact of the immortalized HUDEP-2 cell line. The correlation between slow cell cycling speed and increased γ-globin expression prompted us to genetically perturb the cell cycle machinery and define the impact of these genetic perturbations on γ-globin expression. Using CRISPR activation (CRISPRa), we increased the expression of 8 Cyclin Dependent Kinase Inhibitors (CDKN2A, CDKN2B, CDKN2C, CDKN2D, CDKN1A, CDKN1B, CDKN1C and CDKN3) in HUDEP-2 cells. To do so, we first generated a HUDEP-2-MPH cell line that stably expresses the transcriptional activator complex MPH (MS2-P65-HSF1). We then transduced this cell line with a virus that expresses one of 3 sgRNAs targeting each of the 8 CDKIs, a VP64 activation domain fused to catalytically dead Cas9 (dCas9-VP64), and a blasticidin resistance cassette. We found that activation of CDKN1b (P27 Kip1) in particular, resulted in increased F-cell percentage. Indeed, compared to cells transduced with control sgRNAs, HUDEP-2-MPH cells transduced with a CDKN1B-targeting sgRNA (resulting in a 2-fold higher CDKN1B mRNA level; p= 0.006) exhibited an increased proportion of F-cells, from a baseline of ~6% up to ~20% (p= 0.024). Increased CDKN1B expression resulted in a ~14-fold increase in γ-globin mRNA levels (p= 0.026), associated with a mild reduction in beta-globin mRNA level and a reduction in BCL11A mRNA level to ~65% of normal. These results suggest that the increased γ-globin expression resulting from CDKN1B overexpression may result, at least in part, from reduced BCL11A expression (which we are currently validating). To rule out an off-target effect of the CDKN1B targeting sgRNA, we overexpressed CDKN1B in HUDEP-2 cells, using a cDNA expression construct, and found comparable results as observed with CDKN1B CRISPRa. We next overexpressed CDKN1B cDNA in erythroid cells differentiated from CD34+ HSPCs. In early preliminary results, increased CDKN1B expression in the latter cells resulted in increased γ-globin expression, both at the mRNA and protein level, validating the HUDEP-2 data. Additional studies are ongoing to define the role of CDKN1B in the regulation of γ-globin expression, and to define the impact of CDKN1B overexpression on erythroid differentiation. In summary, our preliminary studies have uncovered a link between cell cycle regulation and γ-globin production. These findings may lay the foundation for the development of new therapeutic strategies for Sickle Cell Disease.