Targeting cell death pathways to prevent KRAS inhibitor resistance - Project Title: Targeting cell death pathways to prevent KRAS inhibitor resistance Abstract Pancreatic ductal adenocarcinoma (PDAC) remains one of the most lethal malignancies, with over 90% of cases driven by KRAS mutations. Although recently developed KRAS-G12C inhibitors have shown promise in other cancers, their applicability to PDAC is limited, as most patients harbor non-G12C KRAS mutations. New pan- KRAS inhibitors broaden therapeutic reach and often arrest tumor growth, but cancer cell subpopulations do not undergo adequate cell death, enabling the cells to survive and acquire resistance. Our preliminary studies indicate that KRAS inhibitor-resistant PDAC cells upregulate several death receptors, suggesting that cell death regulation plays a key role in KRAS inhibitor resistance. We found that combining TRAIL with pan-KRAS inhibitors synergistically promotes tumor cell death and disrupts proteostasis—a process essential for maintaining protein homeostasis and cell survival. While recombinant TRAIL effectively induces apoptosis, its efficacy is limited by its rapid clearance and by potential pro-survival signaling. To overcome these challenges, we will utilize two novel TRAIL-based approaches: a chimeric Fc-TRAIL fusion protein with enhanced stability, and natural killer (NK) cells engineered to secrete trimerized TRAIL, which will be combined with pan-KRAS inhibitors to enhance therapeutic efficacy. Our central hypothesis is that KRAS inhibition disrupts cell death signaling in PDAC, creating a therapeutic vulnerability that novel TRAIL-based strategies can exploit by disrupting proteostasis, restoring apoptotic pathways, and preventing resistance to KRAS inhibitors and TRAIL. In Aim 1, we will dissect how combining pan-KRAS inhibitors with Fc-TRAIL or TRAIL-secreting NK cells reactivate extrinsic apoptosis, disrupts proteostasis, and counters pro-survival signals. We will validate these findings in patient-derived PDAC primary cells. In Aim 2, we will assess the therapeutic efficacy of this combination in peritoneal metastasis models of PDAC, monitoring tumor progression, survival outcomes, and the tumor microenvironment using single-cell RNA sequencing and machine-learning analyses. Additionally, we will enhance NK cell specificity by engineering chimeric antigen receptors that recognize the immune checkpoint proteins PD-L1 or B7-H4, which are expressed on pancreatic cancer cells, while concurrently secreting TRAIL. By integrating pan-KRAS inhibition, targeted cell death pathways, and NK cell immunotherapy, this project aims to elucidate the response and resistance mechanisms to pan-KRAS inhibitors and develop a more effective, multifaceted strategy against KRAS-driven PDAC.
