Bozepinib, a novel small antitumor agent, induces PKR-mediated apoptosis and synergizes with IFNα triggering apoptosis, autophagy and senescence

Juan Antonio Marchal,^1,2 Esther Carrasco,¹ Alberto Ramirez,^1,3 Gema Jiménez,^1,2 Carmen Olmedo,⁴ Macarena Peran,^1,3 Ahmad Agil,⁵ Ana Conejo-García,⁶ Olga Cruz-López,⁶ Joaquin María Campos,⁶ María Ángel García<sup>4,7

1</sup>Biopathology and Regenerative Medicine Institute, Centre for Biomedical Research, ²Department of Human Anatomy and Embryology, Faculty of Medicine, University of Granada, Granada, ³Department of Health Sciences, University of Jaén, Jaén, ⁴Experimental Surgery Research Unit, Virgen de las Nieves University Hospital, Granada, ⁵Department of Pharmacology and Neurosciences Institute, Faculty of Medicine, ⁶Department of Pharmaceutical and Organic Chemistry, Faculty of Pharmacy, University of Granada, Granada, ⁷Department of Oncology, Virgen de las Nieves University Hospital, Granada, Spain

Abstract: Bozepinib [(RS)-2,6-dichloro-9-[1-(p-nitrobenzenesulfonyl)-1,2,3,5-tetrahydro-4,1- benzoxazepin-3-yl]-9H-purine] is a potent antitumor compound that is able to induce apoptosis in breast cancer cells. In the present study, we show that bozepinib also has antitumor activity in colon cancer cells, showing 50% inhibitory concentration (IC₅₀) values lower than those described for breast cancer cells and suggesting great potential of this synthetic drug in the treatment of cancer. We identified that the double-stranded RNA-dependent protein kinase (PKR) is a target of bozepinib, being upregulated and activated by the drug. However, p53 was not affected by bozepinib, and was not necessary for induction of apoptosis in either breast or colon cancer cells. In addition, the efficacy of bozepinib was improved when combined with the interferon-alpha (IFNα) cytokine, which enhanced bozepinib-induced apoptosis with involvement of protein kinase PKR. Moreover, we report here, for the first time, that in combined therapy, IFNα induces a clear process of autophagosome formation, and prior treatment with chloroquine, an autophagy inhibitor, is able to significantly reduce IFNα/bozepinib-induced cell death. Finally, we observed that a minor population of caspase 3-deficient MCF-7 cells persisted during long-term treatment with lower doses of bozepinib and the bozepinib/IFNα combination. Curiously, this population showed ß-galactosidase activity and a percentage of cells arrested in S phase, that was more evident in cells treated with the bozepinib/IFNα combination than in cells treated with bozepinib or IFNα alone. Considering the resistance of some cancer cells to conventional chemotherapy, combinations enhancing the diversity of the cell death outcome might succeed in delivering more effective and less toxic chemotherapy.

Keywords: seven-member heterocycles, purines, IC₅₀, interferon cytokine, cell death, breast and colon cancer cells