Abstract B67: Targeting glutamine metabolism in neuroblastoma and Ewing's sarcoma
Authors: Olsen Rachelle R. , Mary Michelle N. , Freeman Kevin W. 
Published: May 2015

Therapeutic targeting of aberrant cancer metabolism is currently an area of great interest in pediatric oncology. Previous reports have established that oncogenic Myc alters cellular metabolism resulting in cells dependent on exogenous glutamine. Therefore, we chose to target glutamine metabolism in the pediatric cancers Neuroblastoma and Ewing's Sarcoma. Neuroblastoma (NB) is a pediatric malignancy of the developing sympathetic nervous system, and approximately 20% of patients have amplified MYCN and very poor prognosis. Ewing's Sarcoma (EWS) is an aggressive tumor of the bone and soft tissue characterized by chromosome translocations resulting in EWSR1/ETS family fusion proteins. Approximately 85% of EWS cases contain an EWSR1/FLI fusion protein that targets c-Myc for overexpression and potentially cooperates with c-Myc in cell transformation.

We chose to use the glutamine antagonist DON (6-diazo-5-oxo-norleucine) as a tool to examine the effect of inhibiting glutamine metabolism in NB and EWS cells. DON was first explored as a cancer chemotherapeutic in the 1950s and 1960s, but severe nausea in patients following treatment limited its use as a cancer therapeutic. DON is a competitive inhibitor which irreversibly alkylates glutamine-utilizing enzymes, including those involved in nucleotide biosynthesis and glutaminolysis, two important Myc-regulated metabolic pathways in cancer. DON effectively inhibited a panel of six NB and three EWS cell lines, while having minimal effect on BJ fibroblast control cells. In addition, we found a strong correlation between sensitivity to glutamine addiction and susceptibility to DON (R2 = 0.73).

Next, we used human tumor xenografts in nude mice to examine the effect of inhibiting glutamine metabolism in the context of an established tumor. Following injection of neuroblastoma (SK-N-AS, SK-N-BE2) or Ewing's Sarcoma (SK-N-MC, SK-ES-1) cells, tumors were allowed to reach 200mm3 in size before being randomized into treatment groups. Mice were then treated with 100mg/kg DON or water control twice per week. DON treatment significantly inhibited both tumor growth and BrdU incorporation in all NB and EWS tumors tested, suggesting that targeting glutamine metabolism could be an effective therapeutic approach. Interestingly, although DON treatment was strongly cytostatic in all cell lines tested, only in the SK-N-BE2 cells did we detect increased cleavage of caspase-3 as a marker for apoptosis. These results suggest that perhaps combining glutamine inhibition with therapies to induce apoptosis may be more effective than targeting glutamine metabolism alone.

To further examine the mechanism by which DON and glutamine withdrawal induce cell death, we treated NB and EWS cell lines with the pan-caspase inhibitor QVD. Treatment with 20uM QVD for either 48hr or 72hr significantly rescued cell number after DON treatment. This effect was most pronounced in NB cell lines with NMyc amplification, such as Kelly and IMR-32.

Lastly, we performed an in vitro screen to identify pro-apoptotic compounds that increased the effects of DON on NB and EWS cell lines. We found three compounds which increased the effects of DON at clinically relevant concentrations: the synthetic retinoic acid derivative fenretinide, and the Bcl-2 family inhibitors obatoclax mesylate and navitoclax. Of these, navitoclax caused the greatest increase in DON activity across the entire panel of cell lines tested, suggesting that antagonizing Bcl-2 family members may be beneficial in combination with glutamine deprivation.

Our results strongly suggest that combining therapeutic approaches to inhibit glutamine metabolism and induce tumor cell apoptosis may be a promising strategy for treatment of both NB and EWS tumors.

Citation Format: Rachelle R. Olsen, Michelle N. Mary, Kevin W. Freeman. Targeting glutamine metabolism in neuroblastoma and Ewing's sarcoma. [abstract]. In: Proceedings of the AACR Special Conference on Pediatric Cancer at the Crossroads: Translating Discovery into Improved Outcomes; Nov 3-6, 2013; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2013;74(20 Suppl):Abstract nr B67.