Background <p>Fetal hemoglobin (HbF; α₂γ₂) induction is a well-established approach for β-hemoglobinopathies, including sickle cell disease (SCD) and β-thalassemia. Decitabine, a DNA methyltransferase 1 (DNMT1) inhibitor, has been shown to effectively induce HbF production with a favorable safety profile. However, more potent therapeutic strategies are needed, particularly for β-thalassemia/HbE patients.</p> Methods <p>We evaluated the HbF-inducing efficacy of ten DNMT1 inhibitors in erythroid progenitor cells derived from β-thalassemia/HbE patients. To further enhance HbF induction, we investigated a combination treatment with decitabine and RN-1, a lysine-specific demethylase 1 (LSD1) inhibitor. HbF expression, cell viability, erythroid differentiation, and proliferation were assessed. Additionally, we investigated the association between treatment response and well-characterized single-nucleotide polymorphisms (SNPs) previously linked to HbF expression.</p> Results <p>Of the ten DNMT1 inhibitors tested, SGI-110, a dinucleotide analog of decitabine, exhibited similar HbF-inducing efficacy and toxicity profiles as decitabine at equivalent molar dose. The combination treatment with decitabine and RN-1 resulted in a robust additive increase in HbF expression in β-thalassemia/HbE erythroid progenitor cells, albeit with a slight reduction in cell viability. Additionally, the combination treatment improved the delayed differentiation phenotype in β-thalassemia/HbE erythroid cells, accompanied by a reduction in cell proliferation. Interestingly, individual variability in response to RN-1 and the combination treatments was observed, with major responders exhibiting significantly greater increases in HbF compared to minor responders. We identified two SNPs in the <i>BCL11A</i> gene (rs766432 and rs1427407) that were potentially associated with a higher likelihood of major response to treatments.</p> Conclusions <p>Our findings highlight the potential of targeting two distinct epigenetic corepressors within the γ-globin repressor complex to achieve robust HbF induction. The combination of decitabine and RN-1 represents a promising therapeutic strategy for β-thalassemia, warranting further investigation into the molecular mechanisms underlying individual response variability.</p> Graphical Abstract <p>-&#xa0;Decitabine and its analog SGI-110 have similar effects in inducing HbF.</p> <p>-&#xa0;Combined decitabine and RN-1 treatment resulted in an additive increase in HbF.</p> <p>-&#xa0;Responses to the RN-1 and combined treatments were divided into major and minor responders.</p> <p>-&#xa0;Specific SNPs in the BCL11A gene may contribute to the observed response variability.</p> <p></p>