Abstract 408: ATM function and mutation in CLL 11q deletion samples

Abstract

Abstract Chronic lymphocytic leukemia (CLL) is characterized by the accumulation of abnormal B-cell development in which cell death mechanisms have been altered. As the second most common type of leukemia in adults, it progresses more slowly than other types of leukemia and is considered incurable. Approximately 10% of previously untreated patients with CLL exhibit a substantial deletion in the q arm of chromosome 11(22-23), the site of the ATM gene, the product of which regulates homologous recombination repair of double strand DNA breaks (DSB). This lesion occurs at increased incidence, about 50% in patients relapsing on fludarabine-cyclophosphamide-rituximab (FCR) therapy. The residual ATM allele is often mutated, suggesting that the region containing ATM is important for response to chemoimmunotherapy. Although some mutations that cause non-synonymous substitution of amino acids associated with loss of ATM function are known, because the ATM protein contains more than 3,000 amino acids, it is unlikely that all most or all non-synonymous changes would cause loss of function. To address this issue, we developed an immunoblot assay to determine ATM function in del(11q22-23) samples from CLL patients. We confirmed that SMC1 and KAP1 are unique substrates of ATM. Their phosphorylation levels after irradiation (IR) were linearly correlated with ATM activity. This was demonstrated using cell lysates mixtures from cells derived from an individual with ataxia telangiectasia that are deficient in ATM, and those cells repleted with ATM. Consequently, the ratios of phospho to total proteins of SMC1 and KAP1 were employed as indicators of ATM function in response to IR treatment. Using a pool made up of lysates from FISH-negative CLL samples (n = 8) as a positive standard, we validated this assay in 46 del(11q22-23) CLL samples. The phosphorylation ratios of SMC1 and KAP1 from 46 CLL samples were analyzed simultaneously by the normal mixture model-based clustering method, and a formula was generated to determine ATM function. Using this formula, we identified 13 ATM function-deficient samples. To determine whether there is a correlation between ATM gene mutation and the function of the protein, we conducted targeted sequencing of the ATM gene in the 46 samples. Genomic DNA of T-cells isolated and expanded from each of these samples was extracted to serve as a germline reference for the CLL cells. Through a series of bioinformatics analyses, 12 different ATM somatic mutations were identified. Fifteen other ATM mutations were recognized as germ line mutations. No strong correlation was observed between ATM mutation and function. Therefore, alternative assays of ATM function, such as that we have described, are needed to identify ATM deficient tumors in order to develop selective therapies, e.g. agents that cause DSB or inhibit redundant repair pathways. Citation Format: Yingjun Jiang, Xiaojun Liu, Hsiang-Chun Chen, Kim-Anh Do, Xiaoping Su, William Wierda, Michael Keating, William Plunkett. ATM function and mutation in CLL 11q deletion samples. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 408.