title: Mechanism of synergy of N-(4-hydroxyphenyl)retinamide and ABT-737 in acute lymphoblastic leukemia cell lines: Mcl-1 inactivation.
abstract: 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.
authors.affiliation: Division of Hematology-Oncology, USC-CHLA Institute for Pediatric Clinical Research, Children's Hospital Los Angeles, Los Angeles, CA 90027, USA. mkang@chla.usc.edu
authors.forename: Min H
authors.initials: MH
authors.lastname: Kang
authors.forename: Zesheng
authors.initials: Z
authors.lastname: Wan
authors.forename: Yun Hee
authors.initials: YH
authors.lastname: Kang
authors.forename: Richard
authors.initials: R
authors.lastname: Sposto
authors.forename: C Patrick
authors.initials: CP
authors.lastname: Reynolds
mesh_terms: D000970:Antineoplastic Agents / Q000494:pharmacology*; D017209:Apoptosis; D001713:Biphenyl Compounds / Q000494:pharmacology*; D020169:Caspases / Q000378:metabolism; D045744:Cell Line, Tumor; D045304:Cytochromes c / Q000378:metabolism; D004357:Drug Synergism; D004789:Enzyme Activation; D017313:Fenretinide / Q000494:pharmacology*; D015971:Gene Expression Regulation, Enzymologic / Q000187:drug effects; D015972:Gene Expression Regulation, Neoplastic / Q000187:drug effects; D020868:Gene Silencing; D006801:Humans; D015151:Immunoblotting; D048031:JNK Mitogen-Activated Protein Kinases / Q000235:genetics; D051336:Mitochondrial Membranes; D009596:Nitrophenols / Q000494:pharmacology*; D010766:Phosphorylation; D010879:Piperazines / Q000494:pharmacology; D054198:Precursor Cell Lymphoblastic Leukemia-Lymphoma / Q000188:drug therapy* / Q000378:metabolism; D019253:Proto-Oncogene Proteins c-bcl-2 / Q000187:drug effects* / Q000378:metabolism; D017382:Reactive Oxygen Species / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction; D013449:Sulfonamides / Q000494:pharmacology*
chemical_list: C501332:ABT-737; D000970:Antineoplastic Agents; D001713:Biphenyl Compounds; D009596:Nitrophenols; D010879:Piperazines; D019253:Proto-Oncogene Proteins c-bcl-2; D017382:Reactive Oxygen Species; D013449:Sulfonamides; D017313:Fenretinide; D045304:Cytochromes c; D048031:JNK Mitogen-Activated Protein Kinases; D020169:Caspases