Securinine Targets Cancer Cells through an Irak and JNK Dependent Pathway
Authors: Gupta Kalpana , Parizher Gary , De Lima Marcos , Wald David N. 
Published: October 2019

Abstract
Securinine is a plant-derived alkaloid from the securinega plant that has been used clinically as a therapeutic for primarily neurological related diseases. We and others have previously identified securinine as a promising therapeutic candidate for Acute Myeloid Leukemia and colon cancer. In particular, securinine was found to induce the differentiation and cell death of AML cells. In addition, securinine was observed to lead to preferential killing of p53 deficient cells, a clinically beneficial strategy as only cancer cells exhibit p53 deficiency. Despite the efficacy of securinine in cell and animal model systems, the mechanisms of action of securinine in inducing cancer cell death and differentiation remain largely unknown. Here we report significant insights into the mechanisms through which securinine leads to cancer cell death. Not only are these observations important in furthering the development of securinine as a cancer therapeutic, but also as they shed light on novel mechanisms of cancer therapeutics in general.
Our strategy involved a global genetic screen to identify genes required for securinine-mediated killing. We used a pooled library of shRNA to knockdown the expression of 27,000 genes in cultured cells, and then screened the population of cells for resistance to securinine-mediated death. This screen identified interleukin-1 receptor associated kinase 1 (IRAK1) as an important player in securinine-mediated cancer cell killing. IRAK1 is a kinase acting in toll-like receptor signaling, a key innate immune inflammatory pathway that ultimately activates the transcription factors AP1 and NFkB. The fact that loss of IRAK1 could protect against securinine-mediated killing was quite unexpected as NFkB activation provides a pro-survival signal.
To confirm the shRNA screen, IRAK1−/− cells were utilized and reconstituted with wild-type IRAK1 or kinase-deficient IRAK1. These studies confirmed the ability of securinine to preferentially kill cells expressing wild-type IRAK. Interestingly, currently used chemotherapeutics did not exhibit a similar reduced sensitivity when tested on IRAK deficient cells demonstrating the unique mechanisms of action of securinine.As TRAF6 is an adapter protein downstream of IRAK signaling, we also demonstrated that dominant negative TRAF6 impaired securinine-mediated cell killing. To further delineate how securinine induces cell death, the two major pathways downstream of IRAK, NFkB and JNK/AP1 were investigated. We observed that both pathways were rapidly induced in cancer cells through the use of gene reporter assays (NFkB) or western blot (JNK/AP1). To assess the importance of these pathways in the cell killing effects of securinine, they were inhibited using chemical inhibitors. While blockade of NFkB had no impact upon securinine-mediated killing, inhibition of JNK dramatically impaired the ability of securinine to kill cancer cells. Overall, this study provides us with a greater mechanistic insight into a promising new therapeutic candidate and provides a framework for new pathways that can be targeted for cancer drug development.

Disclosures
Wald: Invenio Therapeutics: Equity Ownership.