Abstract 3338: Novel caspase-3 activator, L14R8, induces apoptosis in chronic lymphocytic leukemia cells.

Abstract

Abstract Chronic lymphocytic leukemia (CLL) is a B-cell malignancy and leukemia cells accumulate in bone marrow, lymph node, and peripheral blood due to decreased apoptosis. High levels of Bcl-2 family anti-apoptotic and inhibitors of apoptosis proteins (IAPs) in leukemic lymphocytes are primarily responsible for defective apoptosis in CLL. Both intrinsic caspase-9-mediated and extrinsic caspase-8-dependent apoptosis pathways activate downstream procaspase-3 to active casapce-3. In normal (n=4) as well as CLL lymphocytes (n=9), there is consistent expression of procaspase-3. However, active caspase-3 was present in normal lymphocytes but not in CLL cells suggesting that CLL cells have defective endogenous apoptosis. Hence, we hypothesized that compounds that can directly activate the functional ability of procaspase-3, should overcome the checkpoints of cell death and trigger apoptosis in CLL lymphocytes. Procaspase activating compounds (PAC; Vanquish Oncology) convert executioner procaspases to their active forms by chelating labile zinc on these enzymes. We have tested L14R8, a potent analogue of PAC, in primary CLL cells. The IC50 of L14R8-induced apoptosis in CLL was 7.7μM (n=12, 24hrs). L14R8 (10 μM) induced apoptosis as early as 6 hrs along with mitochondrial outer membrane permeabilization (MOMP). The drug significantly induced apoptosis in CLL patient samples (n=4) cultured in suspension cultures (60%, p=0.023,) as well as CLL lymphocytes under microenvironmental protection of marrow stromal cells (40%, p=0.011) albeit at a lower rate. Mechanistically, L14R8-treatment reduced uncleaved procaspases-3 and -9 expression and increased cleaved active caspases-3 and -9 expression (n=3). Consistent with the mechanism of action of L14R18 on terminal caspases, WT as well as cells lacking BAX and BAK (mouse embryo fibroblasts) were equally sensitive to this agent. Consistent with L14R8 mechanism, addition of exogenous Zinc ion completely reversed L14R8-mediated apoptosis of CLL lymphocytes (p=0.0063, n=4), but did not affect staurosporin-induced apoptosis in these lymphocytes. Moreover, L14R8-induced 86% (range 58-90%, n=4) cell death in CLL lymphocytes, which was reduced by pan caspase-inhibitors, Z-VAD to 58% (range 32-79%) and Q-VD-OPh to 35% (range 20-55%) cell death, which indicated the mechanistic recovery of L14R8-mediated apoptosis. Smac (the inhibitor of IAPs) protein expression was increased in CLL cells after treatment with L14R8. With respect to the therapeutic index, at 24 hrs, L14R8 induced 79% apoptosis in CLL cells (range 46%-97%, n=14) and only 18% (16-28%, n=3) in lymphocytes from healthy donors. We conclude that L14R8 induced apoptosis through activation of procaspase-3 to active caspase-3 in CLL cells. L14R8 and other PAC provide therapeutic index and may be developed into targeted therapeutics for patients with CLL. *Funded by Vanquish Oncology Citation Format: Viralkumar M. Patel, Kumudha Balakrishnan, William Wierda, Varsha Gandhi. Novel caspase-3 activator, L14R8, induces apoptosis in chronic lymphocytic leukemia 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 3338. doi:10.1158/1538-7445.AM2013-3338