Journal of the National Cancer Institute Advance Access originally published online on April 8, 2008
JNCI Journal of the National Cancer Institute 2008 100(8):580-595; doi:10.1093/jnci/djn076
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ARTICLES |
Mechanism of Synergy of N-(4-Hydroxyphenyl)Retinamide and ABT-737 in Acute Lymphoblastic Leukemia Cell Lines: Mcl-1 Inactivation
Affiliations of authors: Developmental Therapeutics Program, Division of Hematology–Oncology, USC-CHLA Institute for Pediatric Clinical Research, Children's Hospital Los Angeles, Los Angeles, CA; Department of Pediatrics, University of Southern California School of Medicine, Los Angeles, CA
Correspondence to: Min H. Kang, PharmD, Division of Hematology–Oncology, Children's Hospital Los Angeles, 4650 Sunset Blvd, MS57, Los Angeles, CA 90027 (e-mail: mkang{at}chla.usc.edu).
Background: ABT-737 is a pan-Bcl-2 inhibitor that has a wide range of single-agent activity against acute lymphoblastic leukemia (ALL) cell lines and xenografts. A relationship between expression of myeloid cell leukemia 1 (Mcl-1), an antiapoptotic member of the Bcl-2 family of proteins, and resistance to ABT-737 has been reported for various cancers. The synthetic cytotoxic retinoid N-(4-hydroxyphenyl)retinamide (4-HPR) is known to generate reactive oxygen species (ROS), and ROS have been shown to activate c-Jun kinase (JNK), which in turn phosphorylates and inhibits Mcl-1. Thus, we investigated whether 4-HPR–mediated inactivation of Mcl-1 could act synergistically with ABT-737 to promote leukemia cell death.
Methods: Cytotoxicity was determined using the fluorescence-based DIMSCAN assay. Synergy was defined as a combination index (CIN) less than 1. The expression of Bcl-2 family messenger RNAs was measured by real-time reverse transcription–polymerase chain reaction, and caspase activity was measured enzymatically. Changes in Bcl-2 family proteins and release of mitochondrial cytochrome c were detected by immunoblotting. ROS, apoptosis, mitochondrial membrane depolarization, and phospho-JNK were measured by flow cytometry. Gene silencing was by small interfering RNA (siRNA). All statistical tests were two-sided.
Results: ABT-737 decreased Mcl-1 protein expression in ABT-737–sensitive ALL cell lines but not in ABT-737–resistant lines. Using the antioxidant ascorbic acid and siRNA-mediated knockdown of JNK, we showed that 4-HPR decreased Mcl-1 via ROS generation (that phosphorylates JNK) in ABT-737-resistant cell lines. Combining ABT-737 with 4-HPR enhanced the mitochondrial apoptotic cascade (percentage of cells with depolarized mitochondrial membrane at 6 hours, ABT-737 vs ABT-737 plus 4-HPR: 24.5% vs 45.5%, difference = 20.1%, 95% CI = 18.9% to 13.9%; P < .001) and caused caspase-dependent, synergistic multilog cytotoxicity in all seven ALL cell lines examined (mean CIN = 0.57, 95% CI = 0.37 to 0.87), with minimal cytotoxicity for normal lymphocytes.
Conclusions: An increase of Mcl-1 protein in response to ABT-737 is one mechanism of ABT-737 resistance that can be overcome by 4-HPR, resulting in synergistic cytotoxicity of ABT-737 combined with 4-HPR in ALL cell lines.
| CONTEXT AND CAVEATS Prior knowledge ABT-737 is a pan-Bcl-2 inhibitor that is cytotoxic for acute lymphoblastic leukemia (ALL) cell lines and xenografts. Resistance to ABT-737 is associated with expression of myeloid cell leukemia 1 (Mcl-1), an antiapoptotic Bcl-2 family member, in some cancers. The synthetic retinoid N-(4-hydroxyphenyl)retinamide (4-HPR) acts via generation of reactive oxygen species (ROS) and may inhibit Mcl-1 expression. Study design Molecular studies in seven human ALL cell lines to study the mechanisms of resistance to ABT-737 and the synergistic effects of 4-HPR on the cytotoxicity of ABT-737. Contribution In ABT-737–resistant ALL cell lines, Mcl-1 protein levels increased with ABT-737 treatment and decreased with 4-HPR treatment. 4-HPR–induced inhibition of Mcl-1 expression occurred via c-Jun kinase phosphorylation downstream of ROS generation. Treatment with ABT-737 plus 4-HPR enhanced the mitochondrial apoptotic cascade and caused caspase-dependent, synergistic multilog cytotoxicity in all seven ALL cell lines examined but had minimal cytotoxicity for normal lymphocytes. Implications Clinical trials of ABT-737 in combination with 4-HPR are warranted. Limitations The small sample size (n = 7 cell lines) was not sufficient to determine a statistically significant correlation between the expression of Bcl-2 family members and ABT-737 sensitivity. There was no direct demonstration of Mcl-1 phosphorylation by 4-HPR. The mechanisms underlying changes in Mcl-1 expression in response to ABT-737 remain to be elucidated.
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Manuscript received July 27, 2007; revised January 28, 2008; accepted February 27, 2008.
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J Natl Cancer Inst 2008 100: 519.