ALBERT

Description: Juvenile myelomonocytic leukemia (JMML) is an aggressive myeloproliferative disorder of early childhood with often fatal outcome. Despite many attempts to develop alternative treatment options allogeneic hematopoietic stem cell transplantation (HSCT) remains the only curative modality. In the past our group has linked the prognosis of JMML to differential DNA methylation patterns (Olk-Batz, Blood 2011;117:4871-80 and Poetsch, Epigenetics 2014;9:1252-60), suggesting a key role of epigenetic modifications in JMML pathophysiology. To overcome the lack of suitable preclinical JMML research models we have developed an ex vivo JMML xenotransplantation system using neonatal Rag2-/- gamma-c-/- mice. Transplantation of 1x106 primary JMML cells resulted in stable xenologous engraftment and reproduced a characteristic JMML phenotype including myelomonocytic expansion; infiltration of spleen, liver and, notably, lung; splenomegaly; and reduced survival (median 26 weeks). Persistent human engraftment and leukemic organ infiltration was confirmed by both flow cytometry and immunohistology. Ras pathway mutations present in xenotransplanted patient samples were invariably confirmed in engrafted tissues. In addition, the model sustained serial transplantations and can therefore be used to amplify scarce patient material. We first tested if DNA methylation patterns in JMML cells were stable even after xenologous engraftment because such stability would be a prerequisite if the model were to be used for preclinical investigation of DNA methyltransferase inhibitors. JMML cells before xenotransplantation and those retrieved from the bone marrow of engrafted mice were profiled for global CpG methylation using Illumina 450K arrays. DNA methylation patterns in JMML were patient-specific and surprisingly robust in functional regions over several months of engraftment time (on average, 0.29% of 30877 promoters and 0.25 % of 30725 intragenic regions were called as "differentially methylated" between source and xenograft; 0.2 β-value change cutoff). These findings confirm the suitability of the xenograft model to investigate JMML epigenetics and, more importantly, indicate that patient-specific epigenetic profiles originate in leukemia-initiating stem cells, reinforcing a fundamental role of these alterations in JMML biology. Our group recently published a retrospective case series demonstrating unprecedented clinical efficacy of the DNA methyltransferase inhibitor 5-azacytidine (5AC) to induce partial or complete remissions in JMML before allogeneic HSCT (Cseh, Blood 2015;125:2311-3). To further investigate the drug on the preclinical level we administered 5AC to Rag2-/- gamma-c-/- mice xenografted with primary JMML cells. After a leukemia establishment phase the mice were divided into treatment or mock groups and treated with 5AC (3mg/kg body weight i.p., N=6) or saline (N=6) for 2 cycles (1 dose daily for 5 days; 9 days of recovery). This regimen was tolerated well by the animals. We found that 5AC reduced JMML infiltration in all organs analyzed, with most pronounced effects in spleen (human CD45+ fraction of all CD45+ cells, 0.24% +/- 0.04% vs 39.78% +/- 10.72%; p

Description: 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) (P80%) 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.