Methylome-based cell-of-origin modeling (Methyl-COOM) identifies aberrant expression of immune regulatory molecules in CLL.

BACKGROUND: In cancer, normal epigenetic patterns are disturbed and contribute to gene expression changes, disease onset, and progression. The cancer epigenome is composed of the epigenetic patterns present in the tumor-initiating cell at the time of transformation, and the tumor-specific epigenetic alterations that are acquired during tumor initiation and progression. The precise dissection of these two components of the tumor epigenome will facilitate a better understanding of the biological mechanisms underlying malignant transformation. Chronic lymphocytic leukemia (CLL) originates from differentiating B cells, which undergo extensive epigenetic programming. This poses the challenge to precisely determine the epigenomic ground state of the cell-of-origin in order to identify CLL-specific epigenetic aberrations. METHODS: We developed a linear regression model, methylome-based cell-of-origin modeling (Methyl-COOM), to map the cell-of-origin for individual CLL patients based on the continuum of epigenomic changes during normal B cell differentiation. RESULTS: Methyl-COOM accurately maps the cell-of-origin of CLL and identifies CLL-specific aberrant DNA methylation events that are not confounded by physiologic epigenetic B cell programming. Furthermore, Methyl-COOM unmasks abnormal action of transcription factors, altered super-enhancer activities, and aberrant transcript expression in CLL. Among the aberrantly regulated transcripts were many genes that have previously been implicated in T cell biology. Flow cytometry analysis of these markers confirmed their aberrant expression on malignant B cells at the protein level. CONCLUSIONS: Methyl-COOM analysis of CLL identified disease-specific aberrant gene regulation. The aberrantly expressed genes identified in this study might play a role in immune-evasion in CLL and might serve as novel targets for immunotherapy approaches. In summary, we propose a novel framework for in silico modeling of reference DNA methylomes and for the identification of cancer-specific epigenetic changes, a concept that can be broadly applied to other human malignancies.

RAS-pathway mutation patterns define epigenetic subclasses in juvenile myelomonocytic leukemia.

Juvenile myelomonocytic leukemia (JMML) is an aggressive myeloproliferative disorder of early childhood characterized by mutations activating RAS signaling. Established clinical and genetic markers fail to fully recapitulate the clinical and biological heterogeneity of this disease. Here we report DNA methylome analysis and mutation profiling of 167 JMML samples. We identify three JMML subgroups with unique molecular and clinical characteristics. The high methylation group (HM) is characterized by somatic PTPN11 mutations and poor clinical outcome. The low methylation group is enriched for somatic NRAS and CBL mutations, as well as for Noonan patients, and has a good prognosis. The intermediate methylation group (IM) shows enrichment for monosomy 7 and somatic KRAS mutations. Hypermethylation is associated with repressed chromatin, genes regulated by RAS signaling, frequent co-occurrence of RAS pathway mutations and upregulation of DNMT1 and DNMT3B, suggesting a link between activation of the DNA methylation machinery and mutational patterns in JMML.

Epigenetic silencing of AKAP12 in juvenile myelomonocytic leukemia.

A-kinase anchor protein 12 (AKAP12) is a regulator of protein kinase A and protein kinase C signaling, acting downstream of RAS. Epigenetic silencing of AKAP12 has been demonstrated in different cancer entities and this has been linked to the process of tumorigenesis. Here, we used quantitative high-resolution DNA methylation measurement by MassARRAY to investigate epigenetic regulation of all three AKAP12 promoters (i.e., α, ß, and γ) within a large cohort of juvenile myelomonocytic leukemia (JMML) patient samples. The AKAP12α promoter shows DNA hypermethylation in JMML samples, which is associated with decreased AKAP12α expression. Promoter methylation of AKAP12α correlates with older age at diagnosis, elevated levels of fetal hemoglobin and poor prognosis. In silico screening for transcription factor binding motifs around the sites of most pronounced methylation changes in the AKAP12α promoter revealed highly significant scores for GATA-2/-1 sequence motifs. Both transcription factors are known to be involved in the haematopoietic differentiation process. Methylation of a reporter construct containing this region resulted in strong suppression of AKAP12 promoter activity, suggesting that DNA methylation might be involved in the aberrant silencing of the AKAP12 promoter in JMML. Exposure to DNMT- and HDAC-inhibitors reactivates AKAP12α expression in vitro, which could potentially be a mechanism underlying clinical treatment responses upon demethylating therapy. Together, these data provide evidence for epigenetic silencing of AKAP12α in JMML and further emphasize the importance of dysregulated RAS signaling in JMML pathogenesis.