Abstract A014: Targeting the KDEL receptor 2 confers tumor regression in the absence of T cells
Authors: Pattanayak Shakti P , Tirosh Boaz 
Published: October 2024

Abstract
In the connection between cancer and inflammation is complex. Inflammatory conditions in the tumor microenvironment (TME) are predicted to improve the anti-cancer immune response. Current paradigms suggest that the main effect is the activation of the adoptive immune responses by improving antigen presentation and stimulates immunotherapy. Hence, in situ inflammation is desirable. The KDEL receptors are critical to retain KDEL proteins in the endoplasmic reticulum (ER). One is calreticulin, a potent inducer of Immunogenic cell death (ICD). We found that the silencing of the R2 isoform is sufficient to cause the extracellular release of KDEL and KDEL-associated proteins to the extracellular matrix. Our secretome analysis revealed the presence of calreticulin, BiP, Thrombospondin-1, PDIs etc. We developed a mouse colon cancer model (Tet-On-Inducible CRISPR/Cas9-MC38) in which KDELR2 is silenced in a doxycycline(DOX)-dependent manner. In vitro analysis confirmed the presence of ER-protein calreticulin on the cell surface after knock-out (KO) induction in Tet-On-MC38 cells. In vitro Tet-on-MC38 cells die within 4-5 days after DOX-treatment. Remarkably, when the KDEL-R2 cells are mixed with the parental cells at a 1:10 ratio, parental tumors shrunk in a syngeneic murine model in the presence of DOX within few weeks after induction. Upon analyzing the TME (by flowcytometry, IHC & IF), we observed that the cold tumor had become hot, characterized by significant infiltration of dominant neutrophils, M1 macrophages, and PMNs. We confirmed the tumor phagocytosis and macrophagic migration on the KDEL-R2 silenced Tet-On-MC38 cells in vitro. In a macrophagic polarization study, we found 46.02% of the M0-macrophages polarize to M1 in the presence of the cellular secretions from MC38 KO-cells in comparison to their wild-type controls. To detect the intensity of ICD in the TME, we generated a probe that covalently labels extracellular PDIs with biotin on the cell surface following the KO. In another athymic (Nu/Nu) mouse model of syngeneic tumor we also found a regression of tumor justifying the role of innate immunity and ICD after the knockdown of the KDEL-R2 receptor. A re-challenge experiment, conducted after the initial tumor regression, demonstrated the priming of T-cells and infiltrating immune cells within the TME. The predominant ICD in the tumor involves the multifaceted roles of calreticulin (as an "eat-me" signal), Thrombospondin-1 (for immune polarization), and other PDIs, which are activated following the silencing of KDEL-R2. This study reveals a significant advancement in cancer immunotherapy and embraces the potential for synergistic blending with current treatments for example CAR-T therapy and checkpoint inhibitors.
Citation Format: Shakti P Pattanayak, Boaz Tirosh. Targeting the KDEL receptor 2 confers tumor regression in the absence of T cells [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Tumor Immunology and Immunotherapy; 2024 Oct 18-21; Boston, MA. Philadelphia (PA): AACR; Cancer Immunol Res 2024;12(10 Suppl):Abstract nr A014.