Geoffrey W. Krystal

Combination with vorinostat overcomes ABT-263 (navitoclax) resistance of small cell lung cancer

ABSTRACT Small cell lung cancer (SCLC) is an aggressive tumor type with high mortality. One promising approach for SCLC treatment would be to utilize agents targeting molecular abnormalities regulating resistance to apoptosis. BH3 mimetic antagonists, such as ABT-737 and its orally available derivative ABT-263 (navitoclax) have been developed to block the function of pro-survival BCL-2 family members. The sensitivity of SCLC to these drugs varies over a broad range in vitro and in clinical trials. We have previously shown that the expression of Noxa, a BH3-only pro-apoptotic BCL-2 family protein, is a critical determinant of sensitivity to ABT-737. Thus, pharmacological up-regulation of Noxa could enhance cell death induced by the BH3 mimetics. We find that the combination of ABT-263 and a HDAC inhibitor, vorinostat, efficiently induces apoptosis in a variety of SCLC cell lines, including ABT-263 resistant cell lines. Cell death induced by combined treatment is Noxa- and/or BIM-dependent in some cell lines but in others appears to be mediated by down-regulation of BCL-XL and release of BAK from BCL-XL and MCL-1. These results suggest that combination of HDAC inhibitors and BCL-2 inhibitors could be an alternative and effective regimen for SCLC treatment.

Venetoclax Is Effective in Small-Cell Lung Cancers with High BCL-2 Expression

Purpose: Small-cell lung cancer (SCLC) is an often-fatal neuroendocrine carcinoma usually presenting as extensive disease, carrying a 3% 5-year survival. Despite notable advances in SCLC genomics, new therapies remain elusive, largely due to a lack of druggable targets. Experimental Design: We used a high-throughput drug screen to identify a venetoclax-sensitive SCLC subpopulation and validated the findings with multiple patient-derived xenografts of SCLC. Results: Our drug screen consisting of a very large collection of cell lines demonstrated that venetoclax, an FDA-approved BCL-2 inhibitor, was found to be active in a substantial fraction of SCLC cell lines. Venetoclax induced BIM-dependent apoptosis in vitro and blocked tumor growth and induced tumor regressions in mice bearing high BCL-2–expressing SCLC tumors in vivo. BCL-2 expression was a predictive biomarker for sensitivity in SCLC cell lines and was highly expressed in a subset of SCLC cell lines and tumors, suggesting that a substantial fraction of patients with SCLC could benefit from venetoclax. Mechanistically, we uncover a novel role for gene methylation that helped discriminate high BCL-2–expressing SCLCs. Conclusions: Altogether, our findings identify venetoclax as a promising new therapy for high BCL-2–expressing SCLCs. Clin Cancer Res; 24(2); 360–9. ©2017 AACR.

Early FDG/PET Scanning as a Pharmacodynamic Marker of Anti-EGFR Antibody Activity in Colorectal Cancer

Panitumumab is an anti-EGF receptor (EGFR) antibody approved for use in treatment of chemotherapy-refractory colorectal cancers lacking K-RAS mutations. Despite overall response rates approximating 10%, no marker predictive of clinical benefit has been identified. We describe a chemotherapy-refractory patient whose clinical condition necessitated rapid identification of an effective agent in whom we used 18F-fluorodeoxyglucose-positron emission tomography (FDG-PET)/computed tomographic scanning 48 hours after an initial dose of panitumumab to document a pharmacodynamic response to the antibody. The initial 46% ± 2.7% drop in SUVmax of four target lesions correlated with a partial response by Response Evaluation Criteria in Solid Tumors and a >90% drop in serum carcinoembryonic antigen at 8 weeks, indicating that an early decrease in FDG uptake may predict subsequent clinical benefit in response to anti-EGFR antibody therapy in colorectal cancer. Mol Cancer Ther; 11(7); 1385–8. ©2012 AACR.

Abstract A23: Noxa determines localization and stability of MCL-1 and consequently ABT-737 sensitivity in small cell lung cancer.

Small cell lung cancer (SCLC) accounts for approximately 15% of lung cancers diagnosed in the US and causes a fatality rate of more than 90%. Over the past 25 years, progress has been made in limited stage SCLC, where the combination of concurrent radiation and chemotherapy has resulted in a long term survival rate of 20-25%. However, progress has been slow in extensive SCLC (70% of all cases), with long term survival rates of approximately 3% when treated with a variety of multi-agent chemotherapy regimens. Tumor cell death induced by chemotherapy or lack of appropriate cellular survival signals is mediated by the intrinsic apoptotic pathway. We and others have demonstrated that the anti-apoptotic member BCL-2, as well as BCL-XL and MCL-1, is overexpressed in SCLC. However, until recently, the precise role of these proteins in SCLC biology and therapeutic resistance was poorly understood. The breakthrough came with the development of BH3 mimetic antagonists that block the function of pro-survival BCL-2 family members. ABT-737, the prototype of this new drug class, binds to and blocks BCL-2 and BCL-XL, but not MCL-1 function. Surprisingly, the sensitivity to ABT-737 varies in a broad range in SCLC cells. We have previously shown that the expression of Noxa, a BH3-only pro-apoptotic BCL-2 family protein, is the critical determinant of ABT-737 sensitivity. We show here that Noxa regulates the localization and stability of MCL-1, an anti-apoptotic member, which results in modulating ABT-737 sensitivity. Mutations in Noxa within either the BH3 domain, the carboxyl terminus mitochondrial targeting domain, or of ubiquitinated lysines not only change the localization and stability of Noxa itself, but also affect the mitochondrial localization and phosphorylation/ubiquitination status of MCL-1 and consequently modulate sensitivity to ABT-737. Results of studies utilizing these mutant proteins indicate that Noxa recruits MCL-1 from the cytosol to the mitochondria. Translocation of MCL-1 initiates its phosphorylation and subsequent ubiquitination, which triggers proteasome-mediated degradation. The precise regulatory mechanisms of Noxa/MCL-1 expression and stability could provide alternative targets to modulate apoptosis induced by BH3 mimetic drugs or other chemotherapeutic reagents. Citation Format: Wataru Nakajima, Mark A. Hicks, Nobuyuki Tanaka, Geoffrey W. Krystal, Hisashi Harada. Noxa determines localization and stability of MCL-1 and consequently ABT-737 sensitivity in small cell lung cancer. [abstract]. In: Proceedings of the AACR-IASLC Joint Conference on Molecular Origins of Lung Cancer; 2014 Jan 6-9; San Diego, CA. Philadelphia (PA): AACR; Clin Cancer Res 2014;20(2Suppl):Abstract nr A23.

Abstract 208: Low-dose actinomycin D decreases Mcl-1 expression and acts synergistically with ABT-737 to induce apoptosis of SCLC cell lines

Proceedings: AACR 101st Annual Meeting 2010‐‐ Apr 17‐21, 2010; Washington, DC SCLC is initially a highly chemotherapy-responsive disease but relapse and progressive development of chemotherapy resistance is the rule. Overexpression of pro-survival members of the Bcl-2 family, including Bcl-2, Bcl-XL and Mcl-1 are thought to play a role in the pathogenesis and chemotherapy resistance of SCLC. ABT-737, a BH3-mimetic drug which blocks the function of Bcl-2 and Bcl-XL, has shown single agent activity in preclinical models of SCLC and enhances the activity of a variety of cytotoxic agents. Low levels of Mcl-1 expression and high levels of Noxa expression have been characterized as markers of sensitivity to ABT-737 in SCLC and other malignancies. We have also noted that Noxa-mediated downregulation of Mcl-1 expression is absent in 4/4 SCLC cell lines examined, suggesting roles for this defect in the pathogenesis of SCLC and in resistance to ABT-737. Because of the short half-life of Mcl-1 mRNA and protein, we studied the effects of combining ABT-737 and actinomycin D, a general transcriptional inhibitor used in the treatment of Wilms tumor, sarcomas, germ cell and trophoblastic tumors. Actinomycin D decreased Mcl-1 expression in a dose-dependent fashion in the low ng/ml range, 3 logs lower than that required for general transcriptional inhibition. While exposure to actinomycin resulted in a cell line-dependent increase in Noxa expression, Noxa was not required for the decrease in Mcl-1 expression, as it occurred in the H209 cell line, which does not express Noxa, as well as the H69, WBA and H526 cell lines which do. Concentrations of actinomycin D from 0.4-4 ng/ml showed single agent activity across a panel of SCLC cell lines in 72 hr MTT viability assays and specifically induced a high grade G2-M cell cycle blockade. When combined with low micromolar doses of ABT-737, near complete loss of viability was seen with combination indices in the 0.5 range, indicating synergy by Chou-Talalay analysis, without a significant effect on MRC-5 pulmonary fibroblasts. In addition to loss of Mcl-1 expression and enhanced Noxa expression, correlates of apoptosis induced by the combination included marked Bid cleavage. Exposure to 4 ng/ml actinomycin was only required for the first 24 hrs of the combined incubation but the presence of ABT-737 was required for the full 72 hrs to see maximum loss of viability and clonogenic potential. This actinomycin exposure mimics a clinically achievable AUC of 100 ng.hr/ml after bolus administration, suggesting the feasibility of combination studies utilizing bolus actinomycin D administration. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 208.