Small-molecule Bcl-2 inhibitors sensitise tumour cells to immune-mediated destruction

Abstract

The cytotoxic effects of anticancer immune cells are mediated by perforin/granzyme-B, Fas ligand and tumour necrosis factor-related apoptosis-inducing ligand (TRAIL), and therefore depend on intact apoptotic responses in target tumour cells. As killing by all three of these mechanisms is blocked by the frequently overexpressed antiapoptotic oncoprotein Bcl-2, we hypothesised that coexposure to a Bcl-2 inhibitor might enhance anticancer immune responses. We evaluated this in U937 lymphoma cells, and A02 melanoma cells, which both show strong Bcl-2 expression. Va24 þ Vb11 þ natural killer T (NKT) cells expanded from peripheral blood of normal donors (n ¼ 3) were coincubated with PKH26-labelled U937 cells, and cytotoxicity was determined by flow cytometry after annexin-V-FITC and 7-AAD staining. In all cases, addition of the HA14-1 small-molecule Bcl-2 inhibitor to the cocultures significantly increased apoptosis in the target U937 cells. Using a similar assay, killing of A02 cells by the cytotoxic T-lymphocyte clone 1H3 was shown to be amplified by coexposure to the potent small-molecule Bcl-2 inhibitor ABT-737. Experiments with immune effectors preincubated with concanamycin-A suggested that sensitisation to perforin/granzyme-B may underlie enhanced target-cell killing observed in the presence of Bcl-2 inhibitors. We conclude that immune destruction of malignant cells can be amplified by molecular interventions that overcome Bcl-2-mediated resistance to apoptosis.

Dual Mechanisms of sHA 14-1 in Inducing Cell Death through Endoplasmic Reticulum and Mitochondria

“…HA 14-1 is an organic compound originally discovered by computer modeling that predicted binding to Bcl-2 (Wang et al, 2000). It has been reported to synergize with a variety of anticancer agents to promote apoptosis in hematopoietic and solid tumors (Milella et al, 2002(Milella et al, , 2004Nimmanapalli et al, 2003;Pei et al, 2003Pei et al, , 2004Sinicrope et al, 2004;Sinicrope and Penington, 2005;Manero et al, 2006;Niizuma et al, 2006;An et al, 2007;Lickliter et al, 2007). However, we have demonstrated previously that HA 14-1 is highly unstable when incubated in standard tissue culture conditions and has a half-life of ϳ15 min (Doshi et al, 2007).…”

“…As described under Materials and Methods, isHA 14-1 is an inactive variant of sHA 14-1. B, RS4;11, BLIN-1, and NALM-6 were incubated with various concentrations of sHA 14-1 for 18 h, and Annexin V al Lickliter et al, 2007), suggesting the potential utility of HA 14-1 in combination therapy for cancer treatment. However, HA 14-1 is highly unstable and rapidly decomposes to inactive species with a half-life of only 15 min in cell culture medium (Doshi et al, 2007).…”

Targeting the Apoptotic Pathway with BCL-2 Inhibitors Sensitizes Primary Chronic Lymphocytic Leukemia Cells to Vesicular Stomatitis Virus-Induced Oncolysis

“…2a), have been shown to sensitize apoptosisresistant cells to cytotoxic drugs by binding to BCL-2 and disrupting interactions with proapoptotic proteins, such as BAX and BAK. As such, investigations are under way to explore the use of small-molecule inhibitors as therapeutic agents for the treatment of malignancies that overexpress BCL-2 (33,34,59). To examine the effect of EM20-25 on VSV oncolysis in CLL cells, primary human CLL cells from seven different patients (CLL8 to CLL11 and CLL13 to CLL15) were pretreated with 10 M EM20-25 (IC 50 approximately 20 M [Fig.…”

“…Nontoxic doses of EM20-25 were used to restore the ability of CLL cells to induce the intrinsic mitochondrial pathway in response to a stress signal-in this case VSV-AV1. The BCL-2 inhibitor had a synergistic effect, only in combination with VSV-AV1 and only in leukemic cells, thus indicating a safer chemotherapeutic regimen (33,34,43,56). Remarkably, the combination selectively killed the CD5 ϩ CD19 ϩ CLL population, while sparing the normal PBMC population and did not render normal PBMCs susceptible to VSVinduced oncolysis.…”

Comparison of Clinical and Immunological Effects of Intravenous and Intradermal Administration of α-GalactosylCeramide (KRN7000)-Pulsed Dendritic Cells

“…Tumor cells resistant to CTL through downregulation of antigen presentation to CTL would not be predicted to be resistant to NKT cell killing (8,9). Human NKT cells activated by a-galactosylceramide (a-GalCer, KRN7000), presented by CD1d on antigen presenting cells, particularly dendritic cells (DC), have been shown to exert significant antitumor activity in vitro and in vivo, using murine models (10,11), against a variety of malignancies, including solid tumors (12)(13)(14)(15)(16) and myeloid leukemia (8).…”