Chronic lymphocytic leukemia (CLL) is characterized by the accumulation of abnormal B-cell development in which mechanisms for cell death have been altered. As the second most common type of leukemia in adults, it progresses at a very slow rate compared to other leukemias and is considered incurable. Approximately 10% of previously untreated patients with CLL exhibit a substantial deletion in the q arm of chromosome 11 (11q22.3), the site of the ATM gene (ataxia telangiectasia mutated). Deletion of this region is associated with rapid disease progression and shorter overall survival. This lesion occurs at increased incidence, 40-50%, in patients who relapse on fludarabine-cyclophosphamide-rituximab chemoimmunotherapy. The residual ATM allele is often mutated, suggesting that the region containing ATM is important for response to chemotherapy. Also, because ATM is critical for repairing double strand breaks that arise after exposure to PARP1 inhibitors and drugs that cause replication fork collapse, identification of tumors that lack ATM function may be a biomarker for specific therapies.

In order to distinguish CLL disease with ATM non-functional and functional among del(11q22.3) patients characterized by fluorescence in situ hybridization (FISH), we have established an assay of ATM activity in primary CLL cells. It is known that ATM is activated in response to ionizing radiation and will further phosphorylate its downstream targets. We have demonstrated that phosphorylation of KAP1 and Smc1 (both ATM downstream substrates) are primarily dependent on the activity of ATM using cell lines established from a patient with ataxia telangiectasia that are either deficient in ATM function or complemented with the full length gene. In this assay, CLL samples from eight patients with normal FISH karyotype were collected, mock treated or irradiated with a 10 Gy dose, and protein lysates were prepared. The lysates were then combined as a positive pool comparator for the del(11q22-23) CLL samples, and both total and phosphorylated KAP1 and Smc1 proteins were quantitated by immunoblotting. The averaged phosphorylation levels of the KAP1 and Smc1 proteins among the irradiated samples from patients with del(11q22.3) by FISH was calculated and compared with those of the ATM-positive pool. An averaged phosphorylation ratio ≤30% of the positive pool sample was taken as indicative of a deficiency in ATM function. Using this cutoff, we identified five ATM non-functional individuals among ten del(11q22.3) CLL samples isolated from archived stocks. The mutation status of DNA extracted from CLL cells is being analyzed by deep sequencing in reference to the subjects’ T-cell genomic DNA . This assay could be applied to CLL and other tumors at risk for loss of ATM activity, to identify those deficient in ATM function. These individuals may be considered for tumor-specific and personalized therapy targeting ATM deficiency.

Citation Format: Yingjun Jiang, Xiaojun Liu, Sijin Wen, Kim-Anh Do, Xiaoping Su, Michael J. Keating, William Wierda, William Plunkett. Quantitation of ATM function in primary CLL cells. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 3528. doi:10.1158/1538-7445.AM2013-3528