DNA Methylation of CDKN2A-B suppressor genes: mechanism and consequences

Tumor suppressor genes, p15INK4B and p16INK4A p14ARF, are the most frequently silenced genes in human cancers (Kim and Sharpless, 2006) because they represent a biological barrier to transformation (Skalska et al., 2013). Frequently, silencing is the result of DNA methylation of promoter regions, and to date, the precise mechanism is not known. Here, we present evidence that DNA methylation is associated with DNA damage caused by collisions between DNA and RNA polymerases near the transcription start sites. Inhibition of replication or transcription greatly reduces DNA damage and ultimately, local CpG methylation. This mechanism generates in vivo many variants (alleles) of p15INK4B p16INK4A and p14ARF genes, which differ only by the occurrence of methylated CpG within the sequence (epialleles). If some epialleles are positively selected, we expect to find them amplified in a complex population of proliferating cells. We have tested this notion by systematically investigating: 1. the configuration of methylated CpGs in each p16INK4A sequenced molecule (epiallele) and; 2. the frequency of families of epialleles during the progression of acute myeloid leukemia (AML) in cells from the bone marrow or the blood of three patients. The relevant results can be summarized as follows: 1. The epialleles, defined by unique methylation profiles, were stable and patient-specific. 2. The epialleles displayed gene specific methylation signatures and were somatically inherited; 3. Some epialleles were eliminated by the demethylating therapy (5-AzadC, Vidaza®), others were resistant or amplified by the demethylating drug and their frequency increased before and during the disease relapse; 4. The presence of high levels of families of epialleles after the therapy predicted the chemoresistance; 5. The epialleles of another suppressor, p21/WAF, in the same patients, did not evolve and remained stable throughout the progression of the disease; 6. Specific p16INK4A gene CpGs function as “seeds” for further methylation. Collectively, these data indicate: 1. An epigenetic mechanism driving the evolution of AML by dynamic methylation of alleles of p16INK4A suppressor gene; 2. The methylation changes during AML progression may be facilitated by DNA damage; 3. The distribution and the frequency of families of p16INK4A epialleles overtime accurately describe the evolution of myeloid acute leukemia.