Small Molecules Targeting Rad51 Recombinase Synergize with Imatinib and Overcome Imatinib-Resistance in CML.

Rad51 recombinase is a key downstream effector of BCR-ABL, essential for DNA repair, cell proliferation and survival. In BCR-ABL positive leukemia cells, Rad51, itself a direct substrate of BCR-ABL, is transcriptionally activated through the BCR/ABL-Stat5 pathway. We have identified a novel compound IBR2, which targets Rad51 and inhibits its functions both in vitro and in vivo. We investigated whether targeted inhibition of Rad51 by IBR2 could overcome imatinib-resistance in CML. IBR2 potently inhibited the proliferation of Ba/F3 cells expressing wild-type BCR-ABL (P210), T315I or E255K mutant with the IC₅₀ values (12–20 μM), but had much lesser effect on the Ba/F3 parental cells. By annexin-V staining and FACS analysis, treatment with 20 μM IBR2 for 48 hours significantly killed Ba/F3 cells expressing P210, E255K or T315I with 33.6, 35.8 and 66.5% of apoptotic cells, respectively. The effects of IBR2 on protein expression and phosphorylation of BCR-ABL and Stat5 and Rad51 were examined by Western blotting after 48 hours of exposure to IBR2 (10–20 μM). In Ba/F3 cells expressing BCR-ABL, dose-dependent decreases of Rad51, BCR-ABL, phosphorylated BCR-ABL, and phosphorylated Stat5 were revealed, while c-Abl, p-c-Abl and total Stat5 protein were unchanged. In contrast, there was little effect on Ba/F3 parental cells. In a murine imatinib-resistant chronic myelogenous leukemia (CML) model bearing T315I mutant BCR-ABL, IBR2 at the dose of 100mg/kg daily for 20 days (i.p.) significantly prolonged animal survival. By colony-forming cell (CFC) assays, IBR2 did not significantly inhibit the growth of cord blood progenitor cells (less than 10% up to 40 μM). However, IBR2 at concentrations of 25, 30 and 40 μM could effectively inhibit the growth of CD34⁺ progenitor cells from CML patients with demonstrated clinical resistance to imatinib and cross-resistance to Nilotinib by 38.9, 54.8 and 95.5%, respectively. In contrast, these cells were inhibited with 5 μM imatinib by 35.2%; with 5 μM nilotinib by less than 9%; and with 0.5 μM dasatinib by up to 41.3%. Synergistic effects were observed upon co-treatment of IBR2 with imatinib in Ba/F3 cells expressing wild-type BCR-ABL. Treatment with 15 μM IBR2 alone resulted in <10% killing of these Ba/F3 cells expressing wild-type BCR-ABL, whereas treatment with 0.1, 0.2, 0.4 and 0.6 μM imatinib induced 6.8, 7.6, 23.1 and 39.3%, apoptosis respectively. IBR2 in combination with imatinib resulted in substantial increases of apoptosis to 28.4, 62.4, 73.6 and 86.4%, respectively. The effects of co-treatment on Rad51, BCR-ABL, and Stat5 protein expression levels were also examined. Treatment with 0.1–0.6 μM imatinib did not influence the Stat5 protein level, while Rad51, BCR-ABL, p-BCR-ABL and p-Stat5 levels were modestly decreased in a dose-dependent manner. In contrast, co-treatment of IBR2 (15 μM) with imatinib, resulted in a remarkable decrease in Rad51, BCR-ABL, p-BCR-ABL and p-Stat5 levels in a dose-dependent manner, but not Stat5 protein level. The significant inhibitory effects elicited by IBR2 or IBR2-imatinib co-treatment support potential clinical applications, which may be especially beneficial for late stage or imatinib-resistant CML patients. Corresponding author: whlee@uci.edu (WHL) and plchen@uci.edu (PLC).