Abstract 702: A novel cell-penetrating ATF5 antagonist peptide CP-d/n-ATF5 exerts in vitro and in vivo anti-tumor effects in a broad spectrum of pediatric cancers
Authors: Banerjee Debarshi , Boboila Shuobo , Street Cherease , Okochi Shunpei , Cruz Filemon S. Dela , Connolly Eileen , Kadenhe-Chiweshe Angela , Yamashiro Darrell 
Published: July 2017

Purpose: Activating transcription factor 5 (ATF5), a member of the ATF/CREB family transcription factor, has been implicated in the pathogenesis of glioblastoma and other adult tumors. Recently, a novel cell penetrating (CP-d/n-ATF5) peptide has been developed to antagonize ATF5 function. The goal of the current study is to test the efficacy of CP-d/n-ATF5 in several children tumors including neuroblastoma, hepatoblastoma, Ewing sarcoma, and rhabdoid tumor, in vitro and in vivo.

Methods: A panel of neuroblastoma cell lines: SK-N-Be(2)C, SK-N-DZ, NGP, IMR-32, NGP, SHEP-21N, KELLY, CHP-212, CHLA-20, CHLA-15 and SK-N-SH; hepatoblastoma cell lines HUH 6 and Hep-G2; Ewing sarcoma cell lines A673, SKNMC, SKNEP1, and TC32; and the rhabdoid cell G401, were treated with vehicle or 50, 100 and 200 μM of CP-d/n-ATF5. Cell viability and apoptosis were assessed after 72 hr by WST-8 and TUNEL assays, respectively. To test in vivo efficacy, SK-N-Be(2)C kidney xenograft tumors were treated with the peptide at dose 50mg/kg, IP injection once per day for first three days and then twice per week. Tumor growth was monitored by bioluminescence imaging and mice were sacked when flux reached a threshold value. Organ metastases were determined by ex vivo imaging. A patient-derived xenograft (PDX) model of rhabdoid tumors was employed where PDX tumors, at 150-200 mm3 size, were enrolled in the penetratin (control) and CP-d/n-ATF5 treatment (same dose as above). Tumors were measured biweekly with a calipers and mice were sacrificed when tumor volume reached a threshold (1500 mm3).

Results: CP-d/n-ATF5 exerted cytotoxicity or apoptosis, in a dose dependent manner, across a wide panel of pediatric tumor cell lines. In vitro, tumor cell viabilities were reduced 40-70% (P&lt;0.05) and apoptosis was increased 50-80% at 200 μM of CP-d/n-ATF5. In vivo, CP-d/n-ATF5 significantly inhibited SK-N-Be(2)C xenograft growth in nude mice, with a median survival of 35 days for control against 21 days for CP-d/n-ATF5, P=0.0013. CP-d/n-ATF5 also reduced SK-N-Be(2)C metastatic burden in the liver (P&lt;0.05) and bone marrow (P&lt;0.01). In the rhabdoid PDX model, there was a significant inhibition of tumors treated with CP-d/n-ATF5 as compared to Penetratin treatment, with a mean tumor volume of control 1283 ± 266.6 mm3 (n=5) vs CP-d/n-ATF5 234.2 ± 50.66 mm3 (n=6), at day 10 post treatment. Penetratin treated tumors showed a median post-treatment time of 13 days to reach threshold volume. None of the CP-d/n-ATF5 treated tumors reached the threshold after 28 days of treatment with some tumors demonstrating regression, indicating a profound anti-tumor effect of the peptide.

Conclusion: Our study shows that a novel ATF5-targeting peptide CP-d/n-ATF5 has broad and profound cytotoxic and apoptotic effects in several pediatric tumors in vitro and in vivo. Our study also indicates that CP-d/n-ATF5 has the potential to act as an anti-metastatic agent.

Citation Format: Debarshi Banerjee, Shuobo Boboila, Cherease Street, Shunpei Okochi, Filemon S. Dela Cruz, Eileen Connolly, Angela Kadenhe-Chiweshe, Darrell Yamashiro. A novel cell-penetrating ATF5 antagonist peptide CP-d/n-ATF5 exerts in vitro and in vivo anti-tumor effects in a broad spectrum of pediatric cancers [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 702. doi:10.1158/1538-7445.AM2017-702