(F-232) Elucidation of the mechanism of cancer cell death caused by oncolytic peptide CKS1

Oncolytic peptides are a group of peptides that induce cancer cell death with relatively weak toxicity to non-cancerous cells. They have an amphipathic structure and net positive charge. Due to differences in cell membrane components, cancer cells tend to be more anionic compared to normal cells. Oncolytic peptides therefore interact with the cancer cell membrane via electrostatic interactions, and then undergo a conformational change to disrupt the cell membrane and induce cell death. These peptides do not target proliferating cells, which mitigates the risk of side effects commonly seen in chemotherapy such as hair loss. As the mechanism of action does not depend on specific receptors or signaling pathways, oncolytic peptides may be applied to many cancer types with minimal risk of developing drug resistance. We have discovered a novel oncolytic peptide, CKS1. CKS1 induced cell death in multiple cancer cell types while showing minimal toxicity to normal endothelial cells and fibroblasts. Furthermore, CKS1 significantly inhibited tumor growth in murine colorectal and breast cancer models. Previous studies have reported differing models for the mechanism of cell death induced by oncolytic peptides. For example, the activity of 9-mer LTX-315 depends on the permeabilization of mitochondrial membrane, DIM-LF11-318 induces necrosis via rupture of the cell membrane and LTX-401 and R-DIM-P-LF11-322 are reported to accumulate in the Golgi apparatus. In the current study, we sought to reveal the mechanism of how CKS1 induces cancer cell death.

To analyze the morphology of cancer cells after CKS1 treatment, we observed 4T1 murine triple-negative breast cancer cells treated with CKS1 using electron microscopy. We further studied the events leading up to cell death by staining the cells with calcein-AM and subsequent live-cell imaging. To observe the dynamics of the peptide, we produced a FITC-tagged CKS1 and monitored its behavior when interacting with cancer cells via confocal microscopy. To study the involvement of programmed cell death pathways, we utilized pan-caspase inhibitor Z-VAD-FMK and receptor-interacting protein kinase 1 (RIPK1) inhibitor Nec-1s. After treating 4T1 cells with these inhibitors, we treated the cells with CKS1 and quantified the amount of lactate dehydrogenase (LDH) released in the culture media. We also quantified cleaved caspase-3 after CKS1 treatment using western blotting. To measure the activity of mitochondria during CKS1 treatment, we stained 4T1 cells with JC-1, a dye which emits green or red fluorescence depending on the mitochondrial membrane potential, and conducted live-cell imaging.

We discovered that the cell membrane of 4T1 cells is ruptured within 30 minutes of CKS1 treatment (Fig. 1). 4T1 cells treated with CKS1 for 6 hours showed an apoptotic morphology, indicating that CKS1 induces cell death in a 2-step process, rupturing of the cell membrane and induction of apoptosis at later time points. We observed that CKS1-treated 4T1 cells first undergo blebbing and swelling due to the influx of surrounding media through the pores created by CKS1 (Fig. 2). Eventually, the cell protrusions burst, leading to the release of cytosolic components. Inhibition of caspases or RIPK1 did not inhibit the release of LDH, but the amount of cleaved caspase-3 increased after 6 hours of CKS1 treatment. CKS1 treatment led to a rapid decrease in mitochondrial membrane potential (Fig. 3).

Understanding the mechanism of action is critical for developing CKS1 as a safe and effective therapeutic by anticipating safety hazards, enabling precise dosing, and minimizing treatment failure. We unveiled the mechanism by which CKS1 kills cancer cells, offering insight for future cancer therapies. Further studies are required to understand why different oncolytic peptides induce cancer cell death through different mechanisms. Oncolytic peptides can be applied clinically for treating tumors accessible endoscopically or transdermally. These tumors can receive direct intratumoral injection of the peptide, offering a targeted and localized approach. For example, CKS1 has potential to be utilized in neoadjuvant therapy for skin or breast cancer. Furthermore, for cancers where surgical intervention is undesirable like rectal cancer, combination of CKS1 with additional cancer therapies like chemotherapy and radiation may present a desirable alternative. This combination approach may be able to cure rectal cancer without the need for surgery.

We revealed the mechanism of cell death induced by CKS1. CKS1 targets malignant cells via a two-step process. First, it accumulates around the cell membrane and creates pores, disrupting the membrane. CKS1 then causes a subsequent decrease in the mitochondrial membrane potential, triggering the apoptotic pathway. Supported by NIH grant R01CA138264 (ASP).