2.54 Targeting Refractory CLL Lacking ATM Function with Sapacitabine-Containing Therapy

Abstract

Cytogenetic abnormalities are common, and have prognostic implications, in chronic lymphocytic leukemia (CLL). In about 7080% CLL cases, chromosomal abnormalities can be identified by fluorescence in-situ hybridization (FISH) using a disease specific probe set. The most common genomic aberrations include deletion 13q, trisomy 12, and deletion in 11q, 17p and 6q. Approximately 10% of previously untreated patients with CLL exhibit a substantial deletion in the q arm of chromosome 11, the site of the ATM gene at (11q22.3). This lesion occurs at increased incidence of about 40% in patients relapsing on fludarabine-cyclophosphamide-rituximab (FCR) chemoimmunotherapy. The residual ATM allele is often mutated, suggesting that the region containing ATM may be important for response to chemoimmunotherapy. Sapacitabine is the orally bioavailable prodrug of the nucleoside analog CNDAC. It is undergoing clinical trials and has presented favorable clinical activity in advanced acute leukemias and myelodysplastic syndromes. CNDAC induces cell death by a unique mechanism of action. Incorporation of CNDAC triphosphate into DNA results in a single-strand break (SSB), which is converted to a one-ended double-strand break (DSB) upon collapse of the DNA replication fork. We have demonstrated that CNDAC-induced DSBs, which are lethal if not repaired, are repaired predominantly through homologous recombination (HR), relative to other repair pathways, such as transcription-coupled nucleotide excision (TC-NER) and non-homologous end-joining (NHEJ). CNDAC greatly sensitizes the viability of cells lacking HR components, such as ATM, Rad51D, Xrcc3 and Brca2. Based on this evidence, we hypothesize that CLL patients with 11q deletion that lack ATM function will be selectively sensitized to sapacitabinecontaining therapy. In order to distinguish ATM non-functional and functional individuals among del(11q22-23) patients characterized by FISH, we have established an assay of ATM activity. ATM is activated in response to irradiation and will further phosphorylate its downstream targets. We have shown that phosphorylation of KAP1 and Smc1 is dependent on the activity of ATM. In this assay, samples from eight patients with normal FISH karyotype were collected. These samples were mock treated or irradiated at dose of 10 Gy and protein lysates were harvested. The lysates were then combined as a positive pool comparator for the 11q deletion CLL samples, and both total and phosphorylated proteins were quantitated in immunoblots. The averaged phosphorylation level of the KAP1 and Smc1 proteins among the CLL patient samples was calculated and compared with those of the positive pool. The value of the samples to the positive pool of 30% was used as indicative of deficient ATM function. Using this cutoff, we identified five ATM non-functional individuals among ten del(11q22-23) CLL samples isolated from archived stocks. A phase II clinical trial of sapacitabine in combination with cyclophosphamide and rituximab (SCR) for previously treated patients with CLL having del(11q22-23) is open for patient recruitment. We will extend clinical investigations of sapacitabine to these CLL patients, focusing on the comparisons of responses of patients defined by the loss of ATM function. The results will test the hypothesis that loss of ATM function will sensitize the disease to sapacitabine therapy and prevent disease progression.