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

Abstract

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<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<0.05) and bone marrow (P<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

PRMT1 promotes neuroblastoma cell survival through ATF5

“…This notion is supported by our current work revealing downregulation of ATF5 in PRMT1 knockdown cells regardless of MYCN amplification status, and similar sensitivity in MYCNnon-amplified neuroblastoma cells to PRMT1 inhibition as that in MYCN-amplified cells, as well as by initial studies of ATF5 in neuroblastoma 11 . Yamashiro's group found that suppression of ATF5 activity with an inhibitory peptide led to a concentration-dependent decrease of cell viability and increase of cell apoptosis in a panel of human neuroblastoma cell lines regardless of MYCN amplification 11 . Therefore, it is tempting to speculate that ATF5 may act as a general downstream effector of PRMT1mediated survival signaling in neuroblastoma.…”

“…Yamashiro's group identified ATF5 as a potential synthetic lethal gene to MYCN-amplified neuroblastoma 10 . Their initial studies demonstrate that ATF5 is highly expressed in MYCNamplified tumors, and that silencing of ATF5 inhibits cell proliferation and induces cell apoptosis 10,11 . They further show that an ATF5-targeting peptide has profound in vitro and in vivo cytotoxic and apoptotic effects on neuroblastoma 11 .…”