Inducing Targeted, Caspase-Independent Apoptosis with New Chimeric Proteins for Treatment of Solid Cancers.

Background: Most newly developed anticancer treatments trigger tumor cell death through apoptosis, for which involvement of caspases activity is essential. However, numerous mutations in apoptotic pathways that lead to cancer and favor resistance to apoptosis are known; most are related to caspase-dependent apoptosis pathways and thus have low efficacy. To overcome these limitations, we constructed a novel chimeric protein, GnRH-AIF, using a gonadotropin-releasing hormone (GnRH) analog as a targeting moiety and the apoptosis-inducing factor (AIF) in its cleaved form as a killing moiety, fused at the cDNA level. AIF has a crucial role in the caspase-independent apoptotic pathway. A wide variety of solid tumors overexpress GnRH-receptors (GnRH-R) that are targeted by the new GnRH-AIF chimeric protein. Methods and Results: In this study, we constructed, expressed, and highly purified GnRH-AIF chimeric proteins. We demonstrated the ability of the chimera to enter and specifically and very efficiently kill solid cancer cell lines overexpressing GnRH-R. Most importantly, upon its entry, GnRH-AIFs translocate to the nucleus where it causes DNA fragmentation leading to a direct caspase-independent apoptotic death. As AIFs lack nuclease activity, our findings also emphasize that cell death induced by GnRH-AIF is dependent on the presence of the ENDOG and PPIA proteins, known to participate in the formation of a DNA-degradosome complex. Finally, we demonstrated the high anti-tumor efficacy of the GnRH-AIF ex vivo, in a human, colon cancer organoid model. Conclusions: Our study shows the potential of using a GnRH-AIF chimeric protein as a novel approach to treat solid cancers that overexpress GnRH-R, via a caspase-independent apoptotic pathway.