Poster Presentation Advances in Neuroblastoma Research Congress 2016

Targeting activating transcription factor 5 (ATF5) in neuroblastoma with a novel dominant negative inhibitor (#338)

Debarshi Banerjee 1 , Shunpei Okochi 2 , Sunjay M Barton 3 , Eileen P Connolly 3 , James M Angelastro 4 , Markus D Siegelin 5 , Lloyd A Greene 5 , Angela Kadenhe-Chiweshe 2 , Darrell J Yamashiro 1
  1. Pediatrics, Columbia University, New York, NY, USA
  2. Surgery, Columbia University, New York, USA
  3. Radiation Oncology, Columbia University, New York, USA
  4. Molecular Biosciences, University of California, Davis, Davis, California, USA
  5. Pathology, Columbia University, New York, USA

Background: Activating transcription factor 5 (ATF5), a member of the ATF/CREB family of transcription factors, is a potential target for neuroblastoma (NB) therapeutics. ATF5 mediates resistance to apoptotic NB cell death. ATF5 has significantly higher expression in Stage 4 MYCN-amplified tumors compared to Stage 1 tumors, and inversely correlates with survival. We utilized a recently developed novel drug CP-d/n-ATF5, a cell penetrating, dominant negative inhibitor that targets ATF5.

Methods: A panel of nine NB cell lines was treated with 50uM, 100uM and 200uM of CP-d/n-ATF5 and cell viability was assessed by WST-8 assay. To investigate the anti-tumor effects of CP-d/n-ATF5 in vivo, the human NB cell line, SK-N-Be2c, was implanted into the left kidney of nude mice to generate xenograft tumors. Tumors were then treated with CP-d/n-ATF5 or vehicle at Day 8, with a dose of 50mg/kg IP daily for 3 days, then twice per week. Tumor growth was monitored by bioluminescence. Mice were sacrificed when tumors reached a target flux. Kaplan-Meier survival analysis was performed. Ex vivo bioluminescence imaging was performed on liver, spleen, kidney to assess the metastatic burden. Bone marrow metastasis was evaluated by harvesting cells and measuring bioluminescence with a lumiometer. TUNEL immunostaining was performed to assess the apoptosis.

Results: CP-d/n-ATF5-S1 significantly decreased cell viability in vitro in a of panel nine NBL cell lines in a dose dependent manner. CP-d/n-ATF5 significantly prolonged survival of SK-N-Be2c tumor-bearing mice compared to vehicle treated mice (P=0.0013). Most notably, metastasis to bone marrow, liver, spleen, and kidney were all significantly suppressed by CP-d/nATF5 treatment. TUNEL staining showed a significant increase (P<0.05) of apoptotic cells in the tumors treated with drug.

Conclusion: Our data shows ATF5 is a novel target in NB and the drug CP-d/n-ATF5 is a potential anti-NB therapeutic agent in the clinic. Our data also shows CP-d/n-ATF5 exerts anti-tumor effects by inducing apoptosis and reducing organ metastasis.