New Therapeutic Options For Cll Treatment: Src/C-Abl-Directed Molecular Re-Engineering Of Chlorambucil And Bendamustine

Proceedings: AACR Annual Meeting 2014; April 5-9, 2014; San Diego, CA B-cell chronic lymphocytic leukemia (CLL) is characterized by actively dividing B-lymphocytes in the lymph nodes and bone marrow, associated with the accumulation of quiescent lymphocytes in the peripheral blood of affected patients. Current treatments for this disease include chemotherapeutic (chlorambucil (CLB), cyclophosphamide, fludarabine) and immunotherapeutic agents (Rituximab, Alemtuzumab) or the combination of immunotherapy with chemotherapeutics drugs. In 2008, the FDA approved bendamustine, a bifunctional agent with a nitrogen mustard moiety and a purine analog, for the treatment of patients with CLL. Signals from the bone marrow and lymph node microenvironments protect CLL lymphocytes from drug-induced apoptosis. During treatment the enzyme-mediated repair of DNA damage can induce resistance to chemotherapeutic drugs. We have previously shown that small molecule inhibitors of c-abl (a key protein of the homologous recombinational repair pathway) such as imatinib, nilotinib or dasatinib, sensitize primary CLL lymphocytes to CLB. We also demonstrated that ZRF4, a combi-molecule designed to target c-abl (through an imatinib moiety) and to induce DNA damage (through a nitrogen mustard moiety), has a more potent cytotoxic effect than the combination of CLB plus imatinib in CLL lymphocytes. Here we report the anticancer effect of three different combi-molecules composed of a chlorambucil moiety and a dasatinib (a dual Src/c-abl inhibitor) moiety compare to the individual components in primary CLL lymphocytes in-vitro. Using MTT assays on CLL lymphocytes from 40 CLL patients, we found that the IC50 (concentration which kills 50% of the cells) of AL748, AL758 and AL816 are significantly lower than the IC50 of CLB when used alone and the combination of CLB with 0.1 μM dasatinib (median value = 1.7 μM, 0.6 μM, 0.9 μM, 11 μM and 3.1 μM respectively). Moreover, our biochemical results suggest that the mechanism of action of AL758, AL816, CLB and CLB plus dasatinib share common downstream targets including inhibition of Src kinase, chemotaxis, and cross-talk with the microenvironment along with induction of DNA damage (p53, p21, comet assay) and apoptosis (AnnexinV, cleaved caspase-3). We also determined, by the MTT assay, the cytotoxic effect of the combi-molecule AL887 composed of a bendamustine moiety and a dasatinib moiety, compare to the individual components in vitro in primary B-lymphocytes from 20 CLL patients. Our results demonstrated that this combi-molecule has a better anticancer activity than bendamustine in combination with 0.1 μM dasatinib (p<0.0005). Dose-limiting toxicity and drug pharmacokinetics are important and limiting factors to take into account for the development of chemotherapy and will be tested in a CLL mouse model. Our results suggest that the combi-molecules AL758, AL816, and AL887 may be useful alternatives for treatment of CLL patients. Citation Format: Lilian Amrein, Anne-Laure Larocque, David Davidson, Lisa Peyrard, Daniel Borrelli, Bertrand Jean-Claude, Lawrence Panasci. New therapeutic options for CLL treatment: Src/c-abl-directed molecular re-engineering of chlorambucil and bendamustine. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 1881. doi:10.1158/1538-7445.AM2014-1881