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Non-small-cell lung cancer (NSCLC) is the most deadly type of cancer in the United States and worldwide. Although new therapy is available, the survival rate of NSCLC patients remains low. One hallmark of cancer cells is defects in the apoptotic cell death program. In this study, we investigate the role of B-cell lymphoma 2 (Bcl-2) family members Bcl-2, Bcl-x(L) and Mcl-1, known to regulate cell survival and death, in a panel of fourteen NSCLC cell lines. NSCLC cell lines express high levels of Mcl-1 and Bcl-x(L), but not Bcl-2. Silencing the expression of Mcl-1 with small interfering RNA (siRNA) oligonucleotides potently killed a subgroup of NSCLC cell lines. In contrast, Bcl-x(L) siRNA had no effect in these lines unless Mcl-1 siRNA was also introduced. Interestingly, high MCL1 to BCL-xl messenger RNA determines whether the cells depend on Mcl-1 for survival. We further investigated the role of Mcl-1 in NSCLC cells using a Mcl-1-dependent cell line, H23. The expression of a complementary DNA containing only the coding region of MCL1 rescued H23 cells from the toxicity of a 3' untranslated region (UTR) targeting Mcl-1 siRNA but not a siRNA targeting the coding region of MCL1. Furthermore, we show that Mcl-1 sequesters the BH3-only protein Noxa and Bim and the apoptotic effector Bak. Not surprisingly, Noxa, Bim, or Bak knockdown partially rescued H23 cells from toxicity mediated by Mcl-1 siRNA to different degrees. Collectively, our results indicate that targeting Mcl-1 may improve therapy for a subset of NSCLC patients.

ABT-263 inhibits the antiapoptotic proteins Bcl-2, Bcl-x(L), and Bcl-w and has single-agent efficacy in numerous small cell lung carcinoma (SCLC) and leukemia/lymphoma cell lines in vitro and in vivo. It is currently in clinical trials for treating patients with SCLC and various leukemia/lymphomas. Identification of predictive markers for response will benefit the clinical development of ABT-263. We identified the expression of Bcl-2 family genes that correlated best with sensitivity to ABT-263 in a panel of 36 SCLC and 31 leukemia/lymphoma cell lines. In cells sensitive to ABT-263, expression of Bcl-2 and Noxa is elevated, whereas expression of Mcl-1 is higher in resistant cells. We also examined global expression differences to identify gene signature sets that correlated with sensitivity to ABT-263 to generate optimal signature sets predictive of sensitivity to ABT-263. Independent cell lines were used to verify the predictive power of the gene sets and to refine the optimal gene signatures. When comparing normal lung tissue and SCLC primary tumors, the expression pattern of these genes in the tumor tissue is most similar to sensitive SCLC lines, whereas normal tissue is most similar to resistant SCLC lines. Most of the genes identified using global expression patterns are related to the apoptotic pathway; however, all but Bcl-rambo are distinct from the Bcl-2 family. This study leverages global expression data to identify key gene expression patterns for sensitivity to ABT-263 in SCLC and leukemia/lymphoma and may provide guidance in the selection of patients in future clinical trials.

Purpose: The purpose of this study was to characterize the activity of the Bcl-2 protein family inhibitor ABT-263 in a panel of small cell lung cancer (SCLC) xenograft models., Experimental Design: A panel of 11 SCLC xenograft models was established to evaluate the efficacy of ABT-263. Single agent activity was examined on a continuous dosing schedule in each of these models. The H146 model was used to further evaluate dose and schedule, comparison to standard cytotoxic agents, and induction of apoptosis., Results: ABT-263 exhibited a range of antitumor activity, leading to complete tumor regression in several models. Significant regressions of tumors as large as 1 cc were also observed. The efficacy of ABT-263 was also quite durable; in several cases, minimal tumor regrowth was noted several weeks after the cessation of treatment. Antitumor effects were equal or superior to that of several clinically approved cytotoxic agents. Regression of large established tumors was observed through several cycles of therapy and efficacy was retained in a Pgp-1 overexpressing line. Significant efficacy was observed on several dose and therapeutic schedules and was associated with significant induction of apoptosis., Conclusions: ABT-263 is a potent, orally bioavailable inhibitor of Bcl-2 family proteins that has recently entered clinical trials. The efficacy data reported here suggest that SCLC is a promising area of clinical investigation with this agent.

ABT-737 is a novel and potent Bcl-2 antagonist with single-agent activity against small-cell lung cancer (SCLC) cell lines. Here, we evaluated the contribution of Bcl-2 family members to the in vitro cellular response of several SCLC cell lines to ABT-737. Relatively higher levels of Bcl-2, Bcl-X(L), Bim and Noxa, and lower levels of Mcl-1 characterized naïve SCLC cell lines that were sensitive to ABT-737. Conversely, a progressive decrease in the relative levels of Bcl-2 and Noxa and a progressive increase in Mcl-1 levels characterized the increased resistance of H146 cells following chronic exposure to ABT-737. Knockdown of Mcl-1 with small interfering RNA sensitized two resistant SCLC cell lines H196 and DMS114 to ABT-737 by enhancing the induction of apoptosis. Likewise, up-regulation of Noxa sensitized H196 cells to ABT-737. Combination treatment with DNA-damaging agents was extremely synergistic with ABT-737 and was associated with the down-regulation of Mcl-1 and the up-regulation of Noxa, Puma, and Bim in H196 cells. Thus, SCLC cells sensitive to ABT-737 expressed the target proteins Bcl-2 and Bcl-X(L), whereas Mcl-1 and factors regulating Mcl-1 function seem to contribute to the overall resistance of SCLC cells to ABT-737. Overall, these observations provide further insight as to the mechanistic bases for ABT-737 efficacy in SCLC and will be helpful for profiling patients and aiding in the rational design of combination therapies.

Inhibition of the prosurvival members of the Bcl-2 family of proteins represents an attractive strategy for the treatment of cancer. We have previously reported the activity of ABT-737, a potent inhibitor of Bcl-2, Bcl-X(L), and Bcl-w, which exhibits monotherapy efficacy in xenograft models of small-cell lung cancer and lymphoma and potentiates the activity of numerous cytotoxic agents. Here we describe the biological activity of A-385358, a small molecule with relative selectivity for binding to Bcl-X(L) versus Bcl-2 (K(i)'s of 0.80 and 67 nmol/L for Bcl-X(L) and Bcl-2, respectively). This compound efficiently enters cells and co-localizes with the mitochondrial membrane. Although A-385358 shows relatively modest single-agent cytotoxic activity against most tumor cell lines, it has an EC(50) of <500 nmol/L in cells dependent on Bcl-X(L) for survival. In addition, A-385358 enhances the in vitro cytotoxic activity of numerous chemotherapeutic agents (paclitaxel, etoposide, cisplatin, and doxorubicin) in several tumor cell lines. In A549 non-small-cell lung cancer cells, A-385358 potentiates the activity of paclitaxel by as much as 25-fold. Importantly, A-385358 also potentiated the activity of paclitaxel in vivo. Significant inhibition of tumor growth was observed when A-385358 was added to maximally tolerated or half maximally tolerated doses of paclitaxel in the A549 xenograft model. In tumors, the combination therapy also resulted in a significant increase in mitotic arrest followed by apoptosis relative to paclitaxel monotherapy.