Mark G. Frattini, MD, PhD
2010-12-06
One of the challenges facing researchers who identify novel mutations in cancer is to discover the function of the protein that is mutated and ultimately describe how the identified mutation is involved in the pathogenesis of the disease.
The first abstract presented during yesterday’s Plenary Scientific Session, by Anna Jankowska, MSc, from the Cleveland Clinic Taussig Cancer Institute, reported the identification of the function of one of these mutated proteins. Her talk, “Impaired Hydroxylation of 5-Methylcytosine in TET2 Mutated Patients with Myeloid Malignancies” represented a collaborative study with investigators in the United Kingdom, at Harvard Medical School and the Dana-Farber Cancer Institute in Boston, and the National Institutes of Health.
Ms. Jankowska was introduced by Dr. Radek Skoda from the University Hospital of Basel in Basel, Switzerland, and began her talk by detailing the frequency of TET2mutations in the various myeloid malignancies including myelodysplastic syndromes (MDSs), myeloproliferative neoplasms (MPNs), and both primary and secondary acute myeloid leukemias (AMLs and sAMLs, respectively). Results from their study showed that normally TET2 converts 5-methylcytosine (5-mC) to 5-hydroxymethylcytosine (5-hmC) therefore predicting that bone marrow cells of patients with mutated TET2 would have lower levels of 5-hmC incorporated into their DNA.
In order to identify the level of 5-hmC in DNA, investigators first developed an immunoblot assay to detect 5-hmC in genomic DNA and then went on to develop a more sensitive test indirectly measuring 5-hmC by measuring the accumulation of 5-methylenesulfonate (CMS) after treating DNA with bisulfite. Using the later technique, they were able to show in more than 100 patient samples of the various myeloid malignancies that mutations found in the TET2 coding region resulted in enzymatic inactivation and resultant lower levels of 5-hmC incorporated into the genomic DNA of mutant patient samples compared to wild-type controls. Interestingly, 18 percent of all wild-type TET2 patients also showed low levels of 5-hmC indicating that there may be as yet undiscovered TET2 mutations or mutations in other currently unidentified proteins (possibly IDH1 or DNMT3) that may be involved in the regulation of TET2 activity.
Ms. Jankowska and colleagues then returned to the lab bench and confirmed that the specific TET2 mutations seen in the patient samples did indeed result in decreased 5-hmC incorporation in murine cells in vitro. A retrovirally mediated depletion of TET2 in mouse hematopoietic precursors described a role for TET2 in normal hematopoiesis, as an association with an increase in the monocyte/macrophage lineage was seen in cells depleted of TET2. They next studied the consequences of overexpression of the enzyme in vitro in cell culture and as predicted found promoter DNA hypermethylation. Given these results, the investigators returned to the primary patient samples and using a methylation array assay showed a distinct increase in hypomethylation in patients who were found to have low levels of genomic 5-hmC.
Ms. Jankowska and colleagues described a unique function of the TET2 protein being involved in the conversion of 5-mC to 5-hmC in genomic DNA and proposed a role for mutated TET2 in the epigenetic dysregulation seen in myeloid neoplasia.
Dr. Frattini indicated no relevant conflicts of interest.
