Oral Presentation Advances in Neuroblastoma Research Congress 2016

High MYCN, low MYC, low CERS4, and low anti-apoptotic BCL2 gene family expression are associated with sensitivity to fenretinide in neuroblastoma cell lines and PDXs (#100)

Michael M Song 1 , Rachel L Blaydes 1 , Lluis A Lopez-Barcons 1 , Min H Kang 1 , Patrick Reynolds 1
  1. Texas Tech University Health Sciences Center School of Medicine Cancer Center, Lubbock, Texas, United States

Introduction: Fenretinide (4-HPR) is a cytotoxic retinoid that acts via induction of reactive oxygen species (ROS) and dihydroceramide synthesis.  4-HPR-LXS oral powder has achieved durable complete responses in recurrent neuroblastoma.  We sought to identify molecular mechanisms of 4-HPR sensitivity and resistance in neuroblastoma.

 

Methods: Fenretinide cytotoxicity profiles were assessed in vitro in 150 STR-validated, tyrosine hydroxylase-positive, EBV-negative continuous NB cell lines (CL) and in vivo using 6 subcutaneous xenograft models (3 PDXs, 3 CL-xenografts) in nu/nu mice treated with 4-HPR-LXS oral powder + ketoconazole. Highly sensitive, intermediate, and resistant CL sub-groups were categorized via an algorithm (SENS-RES) employing maximum observed cytotoxicity and calculated IC50 values from DIMSCAN cytotoxicity assays.   ROS was by flow cytometry and RNA expression by RT-qPCR.

 

Results: Of the 150 CLs, 23 were classified as sensitive and 19 resistant to 4-HPR by SENS-RES.  Even in the most resistant CLs, 4-HPR demonstrated >1-log cytotoxicity at clinically achievable 30µM.  CLs with high multi-drug resistance to DNA damaging agents were not cross-resistant with 4-HPR.  Age, INSS stage, clinically documented MYCN status, prior therapy, p53-function, MDR1/MRP1 expression, or ROS induction by 5µM 4-HPR were not correlated with sensitivity status, although multi-fold increased ROS was observed in several sensitive CLs.  4-HPR-sensitive CLs demonstrated higher MYCN (p=0.043) and lower MYC (p=0.021) expression. Comparison of the most highly sensitive (3CLs, 1PDX) and resistant (3CLs, 1PDX) models also demonstrated lower ceramide synthase 4 (CERS4) (p<0.0001) and lower MYC (p<0.05) expression in the most sensitive models.  Ceramide-related genes CERS2, SPHK1, or SPHK2 expression levels were not different between the groups while BCL2 (p=0.011), MCL1 (p=0.021), and BCLxL/S (p<0.0001) expression was lower in the sensitive group.

 

Conclusion:  High MYCN and low MYC, CERS4, BCL2, MCL1, and BCLxL/S expression are characteristics of neuroblastoma CLs and PDXs that are highly sensitive to 4-HPR.