Abstract 6396: CREM is a regulatory checkpoint of CAR and IL-15 signaling in NK cells

Abstract Chimeric antigen receptor (CAR) natural killer (NK) cell immunotherapy offers a promising approach against cancer. However, the molecular mechanisms governing CAR NK cell activity remain poorly understood. In this study, we identified the transcription factor cAMP response element modulator (CREM) as a pivotal regulator of CAR NK cell function. Using a Raji model, single-cell RNA sequencing revealed a marked upregulation of CREM in CAR NK cells during peak anti-tumor activity after adoptive transfer. CREM expression correlated with a transcriptional profile indicative of both activation and regulatory functions.In a CD70-targeting CAR NK cell model, CREM was induced following CAR ligation to its cognate antigen CD70, but only when CAR signaling via CD3ζ ITAMs was functional, underscoring its dependence on intact CAR signaling. Further experiments demonstrated that IL-15 induced CREM in a dose-dependent manner, with CREM expression abolished upon IL-15 blockade. The combination of CAR signaling and IL-15 stimulation synergistically enhanced CREM expression, highlighting the interplay between these pathways. Mass cytometry analysis revealed that CREM-high CAR/IL-15 NK cell clusters exhibited elevated activation markers (e.g., NKG2D, granzyme B) and inhibitory molecules (e.g., NKG2A, LAG3), suggesting an activation-induced exhaustion state.Mechanistically, we demonstrated that CREM is induced via the PKA-CREB axis, activated downstream of CAR and IL-15 signaling. Phosphorylated CREB, driven by PKA and calcium mobilization, directly promoted CREM transcription, as confirmed by ChIP-qPCR. IL-15 further amplified this pathway by upregulating PKA catalytic subunits.Notably, CRISPR/Cas9-mediated knockout (KO) of CREM in CAR NK cells significantly enhanced their cytotoxicity, cytokine production, resistance to tumor-induced immunosuppression across hematologic and solid tumor models, including CD70- and TROP2-targeting CAR NK cells. In vivo, CREM KO improved NK cell proliferation, persistence, tumor infiltration, tumor control, and survival of mice in multiple aggressive solid and hematologic tumor models including metastatic breast cancer, orthotopic pancreatic cancer, and Raji Burkitt lymphoma.Transcriptional and chromatin accessibility analyses (ChIP-seq and ATAC-seq) revealed that CREM acts as a dual-function transcriptional regulator. It represses critical genes involved in NK cell activation and cytotoxicity while upregulating exhaustion-associated genes. CREM KO enhanced chromatin accessibility at effector gene loci, enriching motifs for AP-1, CEBP, and STAT transcription factors, thereby promoting NK cell activation and function.These findings establish CREM as a critical inhibitory checkpoint in CAR NK cells. CREM KO boosts NK cell activation and cytotoxicity, positioning CREM as a compelling target for improving CAR NK cell-based cancer immunotherapies. Citation Format: Hind Rafei, Rafet Basar, Sunil Acharya, Yu-Sung Hsu, Pinghua Liu, Deqiang Zhang, Qingnan Liang, Vakul Mohanty, Ranjan Upadhyay, Ping Li, Pravin Phadatare, Merve Dede, Donghai Xiong, Corry Jones, May Daher, Ana Karen Nunez Cortes, Mayra Shanley, Bin Liu, Sadie M. Moseley, Patrick Zhang, Dexing Fang, Pinaki Banerjee, Nadima Uprety, Ye Li, Rejeena Shrestha, Xinhai Wan, Hong Shen, Vernikka Woods, April Gilbert, Seema Rawal, Jinzhuang Dou, Yukun Tan, Jeong-Min Park, Francia Reyes Silva, Alexander Biederstädt, Mecit Kaplan, Xin Ru Jiang, Inci Biederstädt, Silvia Tiberti, Madison Moore, Jingling Jin, Luis Muniz-Feliciano, Paul Lin, Gary M. Deyter, Natalie W. Fowlkes, Abhinav Jain, David Marin, Ken Chen, Elizabeth Shpall, Katayoun Rezvani. CREM is a regulatory checkpoint of CAR and IL-15 signaling in NK cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 6396.