Fetal hemoglobin induction in azacytidine responders enlightens methylation patterns related to blast clearance in higher-risk MDS and CMML

Abstract

Background: As new treatment options for patients with higher-risk myelodysplastic syndromes are emerging, identification of prognostic markers for hypomethylating agent (HMA) treatment and understanding mechanisms of their delayed and short-term responses are essential. Early fetal hemoglobin (HbF) induction has been suggested as a prognostic indicator for decitabine-treated patients. Although epigenetic mechanisms are assumed, responding patients' epigenomes have not been thoroughly examined. We aimed to clarify HbF kinetics and prognostic value for azacytidine treated patients, as well as the epigenetic landscape that might influence HbF re-expression and its clinical relevance.

Results: Serial HbF measurements by high-performance liquid chromatography (n = 20) showed induction of HbF only among responders (p = 0.030). Moreover, HbF increase immediately after the first azacytidine cycle demonstrated prognostic value for progression-free survival (PFS) (p = 0.032, HR = 0.19, CI 0.24-1.63). Changes in methylation patterns were revealed with methylated DNA genome-wide sequencing analysis (n = 7) for FOG-1, RCOR-1, ZBTB7A and genes of the NuRD-complex components. Targeted pyrosequencing methodology (n = 28) revealed a strong inverse correlation between the degree of γ-globin gene (HBG2) promoter methylation and baseline HbF levels (p = 0.003, r_s = - 0.663). A potential epigenetic mechanism of HbF re-expression in azacytidine responders was enlightened by targeted methylation analysis, through hypomethylation of site -53 of HBG2 promoter (p = 0.039, r_s = - 0.504), which corresponds to MBD2-NuRD binding site, and to hypermethylation of the CpG326 island of ZBTB7A (p = 0.05, r_s = 0.482), a known HbF repressor. These changes were associated to blast cell clearance (p_HBG2 = 0.011, r_s = 0.480/p_ZBTB7A = 0.026, r_s = 0.427) and showed prognostic value for PFS (p_ZBTB7A = 0.037, HR = 1.14, CI 0.34-3.8).

Conclusions: Early HbF induction is featured as an accessible prognostic indicator for HMA treatment and the proposed potential epigenetic mechanism of HbF re-expression in azacytidine responders includes hypomethylation of the γ-globin gene promoter region and hypermethylation of the CpG326 island of ZBTB7A. The association of these methylation patterns with blast clearance and their prognostic value for PFS paves the way to discuss in-depth azacytidine epigenetic mechanism of action.

Keywords: Fetal hemoglobin; Methylation patterns; Myelodysplastic syndromes; Prognosis; ZBTB7A.

Fig. 1

Schematic representation of HBG2 and ZBTB7A/LRF gene loci and the LINE-1 region. The CG dinucleotides analyzed by pyrosequencing are presented. HBG2: hemoglobin subunit gamma 2, ZBTB7A: zinc finger and BTB domain containing 7A, LRF: leukemia/lymphoma-related factor, LINE-1: long interspersed element-1

Fig. 2

Early fetal hemoglobin increase is related to blast clearance and prognostic of progression-free survival. A Fetal hemoglobin (HbF) kinetics in marrow responders (mCR) (n = 11), and non-responding (NR) patients (n = 8). p < 0.05(*). B HbF kinetics in hematological responders (HI-R = mCR + HI) (n = 7) and non-responders (NHR) patients. C HbF after 1 cycle of azacytidine (HbF1) is related to blast clearance (blasts = 0.78–0.02*HbF1). D Increase of HbF levels between the 1st and 2nd treatment cycle is strongly associated with longer duration of hematological response in HI-R patients (p = 0.026, r_s = 0.665) [(HbF2-HbF1) =  − 0.72 + 0.05*(duration of HI)]

Fig. 3

Kaplan–Meier analyses of overall survival (A) and progression-free survival (B) according to whether HbF increased or not after one cycle of azacytidine. Patients in whom HbF was increased after the first cycle of AZA, OS and PFS, were longer, compared to those of patients, who exhibited decreased or stable HbF: median OS 19.7 vs 14.1 months and median PFS 19.5 vs 13 months (p_OS = 0.065, HR = 0.17, CI 0.22–1.4, p_PFS = 0.032, HR = 0.19, 95% CI 0.24–1.63)

Fig. 4

Gamma-globin promoter methylation status alteration in azacytidine responders. A Reduction in methylation of CG1 of the γ-globin gene (HBG2) promoter is greater in HI-R responders (n = 10) than NHR patients (p = 0.03). B The same trend was observed for mCR responders (n = 15) compared to non-responders (NR) (p = 0.08). mCR = marrow response (blasts ≤ 5% and decreased ≥ 50% over pretreatment and HI-R = marrow response and hematological improvement of at least one lineage according to revised IWG criteria 2018. p* < 0.05

Fig. 5

Gamma-globin promoter methylation status alteration correlates to fetal hemoglobin expression and blast clearance. A Mean methylation levels of HBG2 promoter post-AZA correlates to blast count (p = 0.011, r_s = 0.482). B Hypomethylation at CG1 relates to less blast count at evaluation of response (p₁ = 0.005, r_s1 = 0.527). C Mean methylation of the HBG2 promoter is reversely related to HbF levels [HbF = 5.29–0.06*(HG2 promoter methylation)] (p = 0.016, r_s =  − 0.398). D Hypomethylation at CG1 relates to higher HbF levels at evaluation of response (HbF6) (p = 0.039, r_s =  − 0.504)

Fig. 6

Changes in methylation patterns of the CpG326 of the ZBTB7A after azacytidine treatment. A Heat map of methylation patterns of CG sites of the CpG326 of ZBTB7A pre- and post-azacytidine treatment in marrow responders (mCR) (n = 15) and non-responders (NR) (n = 13). The heat map was created with TBtools. B Increase in methylation of CpG326 is related with HbF at evaluation of response to azacytidine (HbF6). C CG sites of CpG326 with hypermethylation in marrow responders. Significant increase in methylation of CG1.p < 0.05(*). D Kaplan–Meier analysis for progression-free survival according to having above or equal to median hypermethylation (≥ 7%) of CpG326 (p = 0.037 HR = 1.14, 95% CI 0.34–3.8)