Abstract 4545: Development of a synthetic organic peroxide, RA1, that overcomes p53 and Bcl-2 resistance mechanisms
Authors: Abrams Rachel P. , Woerpel K. A. , Carroll William L. 
Published: August 2015

Mutations in p53 and elevated expression of Bcl-2 proteins are associated with therapy resistance in many human tumors. A lead compound capable of bypassing these resistance pathways, RA1, was identified from several synthetic peroxides tested at the National Cancer Institute through the In Vitro Cell Line Screening Project. The 1,2-dioxolane scaffold of RA1 differs from that of the peroxide containing anti-malarial agent artemisinin. The lowest concentration of RA1 to cause 50% growth inhibition (GI50) was observed in an ovarian cancer line, IGROV-1 (GI50 435 nM), and the highest in a lung cancer line, NCI-H322M (GI50 42 μM). Eighteen out of the fifty-nine cell lines tested showed a GI50 below 2.5 μM. In cell lines with altered p53 status, RA1 exhibited a potent effect (GI50 for NCI-H522, HOP-92, and HL-60: 1.2 μM, 1.4 μM, and 2.2 μM, respectively). RA1 showed a high selectivity index, inducing &gt;90% cell death at 40 μM in a transformed cancerous cell line (BJ-ELR: LC50 30 μM); while only ∼20% cell death was observed in non-transformed cells of the same origin (BJ-hTERT: LC50 55 μM).

Using a B-lymphoblastic cell leukemia line, RS4:11 (LC50 ∼7 μM; wild type p53), as a model system, we investigated the mechanism of action of RA1. No increase in p53 protein expression was observed upon treatment with RA1. Unlike conventional chemotherapy drugs, overexpression of the Bcl-2 protein to prevent mitochondrial outer membrane permeabilization (MOMP) had no effect on RA1's ability to induce cell death. The LC50 of GFP control and Bcl-2 overexpressing cells with RA1 is 7.7 and 7.4 μM, respectively, compared to the control (etoposide: LC50 0.6 μM and &gt;4 μM). No change in cell cycle distribution was observed. No activation of the executioner caspases was seen by Western Blot; however, pretreatment with a caspase-2 inhibitor (z-VDVAD-FMK) prevented cell death. Electron microscopy images showed no morphological features characteristic of apoptosis, necrosis, or autophagy. Ferroptosis, an iron and reactive oxygen species (ROS) dependent form of cell death also lacks these morphological characteristics. While no organic peroxides are known to induce ferroptosis, the use of an iron chelator and Ferrostatin (a lipophillic anti-oxidant known to inhibit ferroptosis) inhibited cell death induced by RA1. Similar to some ferroptosis inducing compounds, a water soluble anti-oxidant had no effect on cell death; however, use of a ROS detection kit indicated oxidative stress is occurring.

RA1 demonstrates potential therapeutic activity for the treatment of cancer. It is capable of overcoming two common chemo-resistance pathways: p53 mutation and inhibition of MOMP by modulation of the Bcl-2 family of proteins. RA1 targets cancer cells by a unique mechanism distinct from other peroxide compounds, as well as conventional chemotherapies. Current experimental data suggests RA1 is inducing a form of ferroptotic cell death.

Citation Format: Rachel P. Abrams, K. A. Woerpel, William L. Carroll. Development of a synthetic organic peroxide, RA1, that overcomes p53 and Bcl-2 resistance mechanisms. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 4545. doi:10.1158/1538-7445.AM2015-4545