title: Select Bcl-2 antagonism restores chemotherapy sensitivity in high-risk neuroblastoma.
abstract: BACKGROUND
  Pediatric patients with high-risk neuroblastoma (HR NB) often fail to respond to upfront intensive multimodal therapy. Tumor-acquired suppression of apoptosis contributes to therapy resistance. Many HR NB tumors depend on the anti-apoptotic protein Bcl-2 for survival, through Bcl-2 sequestration and inhibition of the pro-apoptotic protein, Bim. Bcl-2 dependent xenografts derived from aggressive human NB tumors are cured with a combination of cyclophosphamide and ABT-737, a Bcl-2/Bcl-XL/Bcl-w small molecule antagonist. The oral analogue to ABT-737, Navitoclax (ABT-263), clinically causes an immediate drop in peripheral platelet counts as mature platelets depend on Bcl-xL for survival. This led to the creation of a Bcl-2 selective inhibitor, ABT-199 (Venetoclax). A Phase I trial of ABT-199 in CLL showed remarkable antitumor activity and stable patient platelet counts. Given Bcl-XL does not play a role in HR NB survival, we hypothesized that ABT-199 would be equally potent against HR NB.
  
  
  METHODS
  Cytotoxicity and apoptosis were measured in human derived NB cell lines exposed to ABT-199 combinations. Co-Immunoprecipitation evaluated Bim displacement from Bcl-2, following ABT-199. Murine xenografts of NB cell lines were grown and then exposed to a 14-day course of ABT-199 alone and with cyclophosphamide.
  
  
  RESULTS
  Bcl-2 dependent NB cell lines are exquisitely sensitive to ABT-199 (IC50 1.5-5 nM) in vitro, where Mcl-1 dependent NBs are completely resistant. Treatment with ABT-199 displaces Bim from Bcl-2 in NB to activate caspase 3, confirming the restoration of mitochondrial apoptosis. Murine xenografts of Mcl-1 and Bcl-2 dependent NBs were treated with a two-week course of ABT-199, cyclophosphamide, or ABT-199/cyclophosphamide combination. Mcl-1 dependent tumors did not respond to ABT-199 alone and showed no significant difference in time to tumor progression between chemotherapy alone or ABT-199/cyclophosphamide combination. In contrast, Bcl-2 dependent xenografts responded to ABT-199 alone and had sustained complete remission (CR) to the ABT-199/cyclophosphamide combination, with one recurrent tumor maintaining Bcl-2 dependence and obtaining a second CR after a second course of therapy.
  
  
  CONCLUSIONS
  HR NB patients are often thrombocytopenic at relapse, raising concerns for therapies like ABT-263 despite its HR NB tumor targeting potential. Our data confirms that Bcl-2 selective inhibitors like ABT-199 are equally potent in HR NB in vitro and in vivo and given their lack of platelet toxicity, should be translated into the clinic for HR NB.
authors.affiliation: Division of Hematology/Oncology, Aflac Cancer and Blood Disorders Center, Children's Healthcare of Atlanta, Atlanta, GA, 30322, USA.
authors.forename: Rachel
authors.initials: R
authors.lastname: Tanos
authors.affiliation: Division of Hematology/Oncology, Aflac Cancer and Blood Disorders Center, Children's Healthcare of Atlanta, Atlanta, GA, 30322, USA.
authors.forename: Dipan
authors.initials: D
authors.lastname: Karmali
authors.affiliation: Division of Hematology/Oncology, Aflac Cancer and Blood Disorders Center, Children's Healthcare of Atlanta, Atlanta, GA, 30322, USA.
authors.forename: Srilatha
authors.initials: S
authors.lastname: Nalluri
authors.affiliation: Division of Hematology/Oncology, Aflac Cancer and Blood Disorders Center, Children's Healthcare of Atlanta, Atlanta, GA, 30322, USA. kgoldsm@emory.edu.|Department of Pediatrics, Emory University School of Medicine, Atlanta, GA, 30322, USA. kgoldsm@emory.edu.
authors.forename: Kelly C
authors.initials: KC
authors.lastname: Goldsmith
mesh_terms: D000814:Aniline Compounds / Q000009:adverse effects; D017209:Apoptosis / Q000187:drug effects; D051017:Apoptosis Regulatory Proteins / Q000235:genetics*; D000072224:Bcl-2-Like Protein 11; D019086:Bridged Bicyclo Compounds, Heterocyclic / Q000008:administration & dosage* / Q000009:adverse effects; D053148:Caspase 3 / Q000235:genetics; D045744:Cell Line, Tumor; D003520:Cyclophosphamide / Q000008:administration & dosage / Q000009:adverse effects; D019008:Drug Resistance, Neoplasm / Q000235:genetics; D006801:Humans; D008565:Membrane Proteins / Q000235:genetics*; D009447:Neuroblastoma / Q000188:drug therapy* / Q000235:genetics / Q000473:pathology; D010976:Platelet Count; D011518:Proto-Oncogene Proteins / Q000235:genetics*; D019253:Proto-Oncogene Proteins c-bcl-2 / Q000037:antagonists & inhibitors / Q000096:biosynthesis / Q000235:genetics*; D018570:Risk Assessment; D013449:Sulfonamides / Q000008:administration & dosage* / Q000009:adverse effects; D023041:Xenograft Model Antitumor Assays; D051020:bcl-X Protein / Q000235:genetics
chemical_list: D000814:Aniline Compounds; D051017:Apoptosis Regulatory Proteins; C000606775:BCL2L11 protein, human; D000072224:Bcl-2-Like Protein 11; D019086:Bridged Bicyclo Compounds, Heterocyclic; D008565:Membrane Proteins; D011518:Proto-Oncogene Proteins; D019253:Proto-Oncogene Proteins c-bcl-2; D013449:Sulfonamides; D051020:bcl-X Protein; D003520:Cyclophosphamide; D053148:Caspase 3; C579720:venetoclax; C528561:navitoclax