Epigenetic Changes Accompany Disordered Hemoglobin Regulation in Juvenile Myelomonocytic Leukemia
Authors: Fluhr Silvia , Krombholz Christopher Felix , Meier Angelina , Plass Christoph , Niemeyer Charlotte , Flotho Christian 
Published: October 2019

Abstract
Juvenile myelomonocytic leukemia (JMML) is an aggressive myeloproliferative disorder of early childhood characterized by massive proliferation of not only the monocytic and granulocytic lineage but also of erythropoietic precursors. Elevated levels of fetal hemoglobin (HbF) are found at time of diagnosis in more than half of JMML cases and are associated with poor outcome. We and others previously found that a key molecular feature of many JMML cases is DNA hypermethylation of distinct target genes, which also has adverse prognostic relevance. It is long known that epigenetic processes are involved in hemoglobin regulation. We therefore hypothesized that epigenetic dysregulation would also be involved in aberrant hemoglobin expression in JMML erythroblasts.
ɣ-globin silencing, which is completed one year after birth under physiological conditions, is associated with dense CpG methylation of the ɣ-globin promoter. Consistent with re-expression of ɣ-globin in JMML cases with elevated HbF, CpG methylation at the ɣ-globin promoter was decreased in purified erythroblasts from JMML patients with elevated HbF compared to JMML with normal HbF (average methylation 40.0% ±4.2%, N=6 vs. 72.0% ±3.5%, N=4) (P<0.001). CpG methylation at the ɣ-globin promoter inversely correlated with the relative amount of ɣ-globin expression (ɣ/(β+ɣ)) as determined by RT-qPCR in JMML erythroblasts. Correspondingly, the β-globin promoter is normally unmethylated in erythroblasts from healthy adults, and was also found to be unmethylated in erythroblasts from a JMML patient with normal HbF, whereas it was hypermethylated in JMML with elevated HbF (average methylation 67.0% ±26.0%, N=2). We also assessed CpG methylation at the KLF1 gene which encodes an erythroid-specific transcription factor and strong β-globin activator. We found that the KLF1 enhancer (-593 to -312 relative to the transcription start site of KLF1) and promoter (-182 to +203) regions were unmethylated in healthy erythroblasts irrespective of neonatal or adult origin (N=11; enhancer, 4.5% ±0.9%; promoter, 2.2% ±0.6%), whereas both regions were densely methylated (>80%) in healthy non-erythroid cell populations. By contrast, the KLF1 enhancer and promoter were aberrantly methylated in JMML erythroblasts (N=11; enhancer, 21.2% ±4.7%, P=0.002; promoter, 11.5% ±2.9%, P=0.004). This suggested a regulatory role of KLF1 methylation in aberrant globin expression in JMML. To explore this functionally, we used a dual luciferase reporter assay, ligating the KLF1 enhancer into a CpG-free reporter vector and comparing the reporter activity of unmethylated vector with that of in vitro methylated vector after transfection into K562 cells. CpG methylation at the KLF1 enhancer abolished its activity, supporting methylation-associated silencing of KLF1 in JMML erythroblasts. Compatible with this, KLF1 transcript levels were reduced 2.1-fold in JMML erythroblasts (N=12) compared to healthy erythroblasts (N=9) as assessed by RT-qPCR (P=0.018). We additionally assessed the protein expression of the direct KLF1 target CD44 on the surface of JMML erythroblasts by FACS analysis. CD44 expression was 2.4-fold lower in JMML erythroblasts with high KLF1 enhancer methylation compared to JMML erythroblasts with normal KLF1 enhancer methylation.
In summary, we show that multiple layers of hemoglobin regulation are affected by epigenetic changes in JMML erythroblasts. The globin genes themselves are targets of aberrant DNA methylation in JMML erythroblasts with elevated HbF. Moreover, the β-globin activator KLF1 is aberrantly methylated and repressed in JMML erythroblasts. These findings provide for the first time a mechanistic explanation for the strong correlation and uniform prognostic relevance of elevated HbF and gene hypermethylation in JMML.

Disclosures
No relevant conflicts of interest to declare.