5-Azacytidine Induces Human Fetal Hemoglobin Production through Activation of the p38 MAPK and Integrated Stress Response Signaling Pathways
Authors: Mabaera Rodwell , Lowrey Christopher H. 
Published: April 2020

Abstract
Pharmacologic induction of fetal hemoglobin (HbF) has the potential to improve the health and quality of life of people with β-thalassemia and sickle cell disease. 5-Azacytidine (5-Aza) is a key lead compound as it, and the related drug decitabine, are active in most β-thal and SCD patients including those resistant to butyrate and because they have been shown to produce clinical benefits for selected patients. However, these drugs must be administered by injection, they depress blood counts and they are known to alter DNA structure and cause changes in genome-wide DNA methylation. Efforts to design improved inducing agents have been limited by an incomplete understanding of the mechanisms underlying pharmacologic induction of HbF. Current theories include proposals that 5-Aza and decitabine induce increased γ-globin gene expression by altering the kinetics of erythroid differentiation or by decreasing γ-globin promoter DNA methylation. We recently provided evidence that the primary action 5-Aza is not through either of these mechanisms. These results, and data from the literature, have led us to propose a new model of HbF induction based on activation of cell stress signaling pathways. To begin to evaluate this model, we used a human in vitro CD34 erythroid differentiation system to test the hypotheses that the p38 MAPK stress signaling pathway was involved in 5-Aza induction of HbF. Quantitative RT-PCR, Western blotting and HPLC were used to assess mRNA, protein and Hb levels. Our results showed that 500 nM 5-Aza causes p38 MAPK pathway activation as evidenced by phosphorylation of p38 MAPK, the downstream kinase MK2 and the downstream target Hsp27. The p38 MAPK inhibitor SB203580 (SB) prevented pathway activation and suppressed both baseline and 5-Aza-induced γ-globin mRNA production. On day +13 of differentiation, relative γ-globin mRNA levels were 1.00 (untreated), 0.40 (SB alone), 2.18 (5-Aza alone) and 0.46 (5-Aza + SB) All values were p&lt;0.05 vs. control. HbF levels at the end of differentiation were 2.1% (untreated), 2.2% (SB alone), 24.1% (5-Aza) and 10.7% (5-Aza + SB). These results indicate that the p38 MAPK pathway is activated by 5-Aza and that inhibition of this pathway suppresses 5-Aza-induced increases in γ-globin mRNA and HbF production. However, the fact that SB did not completely inhibit HbF production suggested other pathways might be involved. The Integrated Stress Response (ISR) pathway (also known as the Unfolded Protein Response pathway) responds to a variety of stresses with phosphorylation of translation factor eIF2α. While this initially inhibits translation of most proteins, production of the ATF4 transcription factor is increased and it mediates a secondary transcriptional response. This pathway is known to be activated in differentiating erythroid cells by inadequate heme and other stresses. 500 nM 5-Aza caused pathway activation as evidenced by phosphorylation of both heme-regulated eIF2α kinase (HRI) and eIF2α and by increased levels of ATF4. Expression of a dominant-negative ATF4 protein inhibited 5-Aza induction of γ-globin mRNA by more than 50%. Independent activation of the ISR pathway by L-azetidine-2-carboxylic acid (a proline analogue that causes protein miss-folding), induced γ-globin mRNA and HbF to levels equivalent to those seen with 5-Aza. 5-Aza also alters the polysome profiles of γ and β-globin mRNA in differentiating human cells (50% increase in polysome-associated γ-globin mRNA, p=0.02; 55% decrease in β-globin mRNA, p&lt;0.01). Taken together, these results suggest that 5-Aza induces HbF production through activation of the p38 MAPK and ISR cell signaling pathways and that this induction involves both transcriptional and translational effects. The identification of cell signaling pathways involved in HbF induction opens the possibility of future targeted drug development.