Genetic and Epigenetic Profiling on 7q22 Identifies ATXN7L1 As Candidate Tumor Suppressor in Acute Myelogenous Leukemia

Abstract 1408

About 10 % of patients with acute myeloid leukemia (AML) present with either monosomy 7 or deletions on the long arm of chromosome 7. This chromosomal aberration is associated with poor prognosis and adverse therapeutic response. Following biallelic inactivation as proposed by Knudsen's hypothesis, a “second hit” of the remaining allele might be required for loss of gene function. So far, no consistent additional genetic hits could be identified in the minimally deleted regions. Thus, epigenetic silencing might display a local alternative mechanism ultimately causing silencing of a potential tumor suppressor. Based on cytogenetic data we defined a minimally deleted region on 7q22.2 of 2–3 Mb in size to narrow down the location of the putative tumor suppressor. It is flanked by the microsatellite markers D7S1503 and D7S1841. We utilized comprehensive DNA methylation profiling in the CpGrich areas of the minimal deleted regions by high resolution DNA methylation assessment by MassARRAY to identify aberrant epigenetic patterns in these regions. In this region we quantitatively analyzed CpG island methylation in a cohort of 64 AML patients with del(7q), 11 with monosomy 7, ten with normal karyotype and five CD34+ bone marrow cell DNA from healthy donors as controls. We identified four genes (PRES, LHFPL3, ATXN7L1, and CDH28) that are hypermethylated in their promoters in this region with significant mean methylation differences from 5 to 20 % comparing AML to healthy controls. Hypermethylation occurred both in patients with chromosome 7 aberrations and in patients with normal karyotypes. To narrow down aberrantly methylated genes to functional relevant candidates, we excluded those that were not expressed in healthy CD34+ cells. ATXN7L1, a gene located on 7q22.2 and coding for a potential subunit of the histone acetyl transferase (HAT) and deubiquitinase SAGA complex, was expressed in healthy CD34+ cells and granulocytes, but downregulated upon hypermethylation (>30%) in patients. To elucidate a potential functional role of ATXN7L1, we immunopurified SAGA from HeLa cells using antibodies against a SAGA subunit, GCN5,and analyzed the coimmunoprecipated proteins by subsequent mass spectrometry. Based on the identity of the co-precipitated proteins we demonstrate that ATXN7L1 is a bona fide component of the human SAGA complex. Thus our results suggest that ATXNL1 has a similar role as the closely related protein, ATXN7 that is known to be involved in transcription regulation via USP22 dependent deubiquitination of histones. Experiments to understand how ATXN7L1 silencing may affect the leukemic phenotype are in progress.