The combination of BH3-mimetic ABT-737 with the alkylating agent temozolomide induces strong synergistic killing of melanoma cells independent of p53

Abstract

Metastatic melanoma has poor prognosis and is refractory to most conventional chemotherapies. The alkylating agent temozolomide (TMZ) is commonly used in treating melanoma but has a disappointing response rate. Agents that can act cooperatively with TMZ and improve its efficacy are thus highly sought after. The BH3 mimetic ABT-737, which can induce apoptosis by targeting pro-survival Bcl-2 family members, has been found to enhance the efficacy of many conventional chemotherapeutic agents in multiple cancers. We found that combining TMZ and ABT-737 induced strong synergistic apoptosis in multiple human melanoma cell lines. When the drugs were used in combination in a mouse xenograft model, they drastically reduced tumor growth at concentrations where each individual drug had no significant effect. We found that TMZ treatment elevated p53 levels, and that the pro-apoptotic protein Noxa was elevated in TMZ/ABT-737 treated cells. Experiments with shRNA demonstrated that the synergistic effect of TMZ and ABT-737 was largely dependent on Noxa. Experiments with nutlin-3, a p53 inducer, demonstrated that p53 induction was sufficient for synergistic cell death with ABT-737 in a Noxa-dependent fashion. However, p53 was not necessary for TMZ/ABT-737 synergy as demonstrated by a p53-null line, indicating that TMZ and ABT-737 together induce Noxa in a p53-independent fashion. These results demonstrate that targeting anti-apoptotic Bcl-2 members is a promising method for treating metastatic melanoma, and that clinical trials with TMZ and Bcl-2 inhibitors are warranted.

Figure 1. TMZ and ABT-737 cause strong, synergistic cell death in melanoma cell lines.

(A) MTS assays of 1205Lu (top) and A375 (bottom) cell lines treated with varying doses of TMZ with or without 3.3 µM ABT-737 for 96 h. (B) The visual appearance of cells (100× magnification) treated with varying doses of TMZ with or without 1.1 µM ABT-737 for 72 h. TMZ treatment primarily reduced cell proliferation, while the combination treatment caused cell death. (C) Annexin V assays for various cell lines treated with TMZ (400 µM) and ABT-737 (3.3 µM) for 72 h.

Figure 2. p53 and Noxa are increased in TMZ/ABT-737 treated cells.

Immunoblots are of lysates from Annexin V experiments shown in Fig. 1C with 72 h drug exposure. PARP cleavage indicates high levels of apoptosis in combination treated cells. TMZ treatment alone increases levels of p53, but only the combination treatment increases Noxa. Other Bcl-2 family members tested remained unchanged. Relative ratios of Noxa and p53 to tubulin are shown below, with each value normalized to the control.

Figure 3. Synergy between TMZ and ABT-737 is Noxa-dependent.

Annexin V assays of 1205Lu (A) and A375 (B) shNoxa and shControl cells treated with or without 400 µM TMZ and 3.3 µM ABT-737 for 72 h. Immunoblots of cell lysates from Annexin V experiments for 1205Lu (C) and A375 (D) cells show Noxa knockdown and lack of PARP cleavage for shNoxa lines, while high levels of Noxa exist in combination-treated shControl lines.

Figure 4. p53 induction through nutlin-3 causes synergy with ABT-737 in a Noxa-dependent fashion.

(A) Annexin V assays of 1205Lu and A375 cells treated with or without 4 µM nutlin-3 and 3.3 µM ABT-737. (B) A375 shNoxa and shControl cells treated with or without 4 µM nutlin-3 and 3.3 µM ABT-737. (C) Immunoblots of lysates from the Annexin V experiments shown in B show Noxa knockdown and lack of PARP cleavage in the shNoxa line. Relative ratios of Noxa to tubulin are shown below, with each value normalized to the control.

Figure 5. p53 is not necessary for synergy between TMZ and ABT-737.

MTS assays of RPMI-7951 (a p53-null line) with varying doses of TMZ (A) or nutlin-3 (B) with or without 3.3 µM ABT-737. (C) Annexin V assays of RPMI-7951 cells treated with or without 400 µM TMZ and 3.3 µM ABT-737 (left) or 4 µM nutlin-3 and 3.3 µM ABT-737 (right). (D) Immunoblots of cell lysates from the Annexin V experiments shown in C show Noxa induction in TMZ/ABT-737-treated cells but not in nutlin-3/ABT-737-treated cells. Relative ratios of Noxa to tubulin are shown below, with each value normalized to the control. (E) Immunoblots of several melanoma cell lines show that p53 is undetectable in RPMI-7951 cells.

Figure 6. TMZ and ABT-737 reduce tumor growth in a mouse xenograft model.

(A) Tumor growth curves. (B) Kaplan-Meier curves for tumor doubling times. * p = 0.0005 vs. control.