Sickle cell disease and β-thalassemia continue to cause significant morbidity and mortality. Strategies to increase fetal hemoglobin (HbF) levels can ameliorate symptoms and improve the lives of patients with these diseases. While most previous studies have focused on induction of γ-globin gene expression as an approach to induce HbF, there is evidence that HbF may be post-transcriptionally regulated. For example, butyrate was shown to increase the translational efficiency of γ-globin mRNA and 5-azacytidine (5-Aza) induces HbF to a much greater degree than γ-globin mRNA steady state levels. These findings suggest that translational regulation may play an underappreciated yet important role in controlling HbF levels and that investigating the molecular mechanisms involved in this control may provide new therapeutic targets for HbF induction. We hypothesized that the Integrated Stress Response (ISR) pathway is involved in differentially regulating fetal and adult hemoglobin production. The ISR pathway has been shown to modulate globin protein synthesis in response to heme availability and other stresses. In the absence of heme, the heme-regulated inhibitor kinase phosphorylates eIF2α, downregulates general protein synthesis, but enables translation of a limited number of transcripts that are critical for coordinating the stress response. To test our hypothesis, we first evaluated the effects of salubrinal (Sal), a small molecule that activates ISR signaling by selectively inhibiting p-eIF2α dephosphorylation, in K562 cells. 3μM and 6μM Sal increased p-eIF2α and activated ISR signaling as evidenced by increased ATF4 and GADD34 protein levels and increased gene expression of ATF3 and CHOP, two transcriptional targets of ATF4. Once we verified that Sal increased p-eIF2α and ISR signaling, we extended testing to primary human erythroid cells to evaluate its effect on hemoglobin production. We first determined a dose range of Sal that increased p-eIF2α in primary cells without reducing cell viability. Both 3μM and 6μM Sal increased p-eIF2α and only reduced cell number by 15% when applied on days 15 and 18 of differentiation, the period of maximal hemoglobin synthesis. Next, we determined that 3μM and 6μM Sal slightly reduced γ-globin and β-globin steady state mRNA levels but did not change the γ/(γ+β) ratio relative to control. In contrast, Sal significantly induced HbF when evaluated by HPLC at the end of differentiation on day 20. Compared to untreated cells, 3μM Sal increased the percent HbF from 2.7% to 5.0% (1.8 fold) and 6μM Sal resulted in 12.9% HbF (4.7 fold) (n=4, p<0.05). The enhanced %HbF was due to increased HbF but also reduced HbA, providing evidence that HbF and HbA may be differentially or reciprocally regulated at the translational level. Importantly, Sal treatment did not significantly reduce the total hemoglobin content relative to the untreated control and did not alter cellular differentiation when assessed by flow cytometry for CD71 and CD235a. These results suggest that Sal increases HbF by a post-transcriptional mechanism potentially through ISR activation. Sal treatments earlier in the differentiation process (days 9 and 12) before considerable amounts of hemoglobin are synthesized failed to significantly increase HbF, further supporting this conclusion. We then evaluated whether Sal treatment could enhance HbF induction by known activators of γ-globin transcription, such as 5-Aza and hydroxyurea (HU). 200nM 5-Aza alone increased %HbF from 2.7% to 12.4%. When 200nM 5-Aza was combined with 3μM and 6μM Sal, the %HbF increased to 18.0% and 22.8%, respectively. Similarly, 10μM HU alone increased HbF from 2.9% to 4.9%, but co-treatment with 3μM and 6μM Sal increased HbF to 7.7% and 15.0%, respectively. For both HU and 5-Aza, combined treatment with Sal did not alter the γ/(γ+β) ratio from what was seen with HU or 5-Aza alone. Taken together, these results indicate that the novel method of HbF induction by Sal enhances the effect of transcriptional activators of γ-globin. In the future, utilization of transcriptional and translational mechanisms of HbF induction may provide an opportunity for combination therapy to achieve therapeutic HbF levels at reduced doses, thereby reducing toxicity.
No relevant conflicts of interest to declare.
Asterisk with author names denotes non-ASH members.