Abstract 1646: IL-23R is a regulator of the mitotic spindle and is critical for cell viability in AML
Authors: Duong Nathan , Khan Dilshad H. , Thomas Geethu E. , Hurren Rose , Lee Jong Bok , St-Germain Jonathan , Drimmer Lily , Yan Yongran , Maclean Neil , Gronda Marcela , Rondeau Vincent , Brown Brandon D. , Jones Courtney L. , Chang Hong , Arruda Andrea , Minden Mark D. , Zhang Li , Kornblau Steven M. , Raught Brian , Spadavecchio Vito , Schimmer Aaron D. 
Published: March 2024

Abstract
To identify novel biological vulnerabilities in AML, we searched for gene ontologies that were upregulated in AML compared to normal hematopoietic cells. Of the upregulated processes, the mitotic spindle ontology was a top hit. Unexpectedly, expression of the IL-23 receptor (IL-23R) showed a strong positive correlation with the mitotic spindle ontology. IL-23R is a cell surface cytokine receptor that is canonically expressed on T cells, but not known to have a role in AML. In 7 of 7 tested AML cell lines and 15 of 20 primary AML patient samples, IL-23R protein was expressed &gt;2 fold higher compared to mean IL-23R in normal mononuclear hematopoietic cells (n=5) and CD34+ cells (n=3). IL-23R is classically a T cell surface receptor and we confirmed localization to the cell surface in T cells. However, only small amounts of IL-23R were present on the cell surface of AML cell lines and primary AML samples. Rather, in AML cells, primary AML samples, and AML stem cells, IL-23R was located intracellularly as determined by 4 different modalities and 4 different antibodies directed against 4 different epitopes of the receptor. Of note, only the full-length IL-23R protein was detected by immunoblotting. We also detected IL-23R’s heterodimeric partner, IL12Rβ1, in AML cells. To understand the function of intracellular IL-23R, we used BioID mass spectrometry to identify proteins that interacted with IL-23R. Pathway analysis of interacting proteins identified mitotic spindle formation as the top pathway. Using Proximity Ligation Assays, we validated interactions with mitotic spindle proteins, and BioID hits, NUMA, TMEM201, TACC1, and BAG6 in AML cells. By confocal microscopy, we demonstrated that IL-23R colocalized with the mitotic spindle and centrosomes in AML cells and primary AML samples. IL-23R interacted with the mitotic spindle via its (S/T)x(I/L)P motif (amino acids 588-591). IL-12Rβ1 was also found in complex with IL-23R at the mitotic spindle in AML cells. Additionally, it was discovered that the cytokine, IL-23, promoted translocation of IL-23R to the mitotic spindle through clathrin-mediated endocytosis. Knockdown and knockout of IL-23R in AML cells led to dysregulation of the mitotic spindle with multipolarity, lagging chromosomes, and other spindle defects. Knockdown of IL-23R also reduced proliferation, clonogenic growth, and marrow engraftment of AML cells and primary AML samples. In contrast, knockdown of IL-23R in normal CD34+ cells did not impair engraftment. Likewise, constitutive homozygote IL-23R knockout mice had no difference in complete blood counts and stem cell number/function compared to wild type. In summary, IL-23R interacts with the mitotic spindle and centrosome where it regulates mitotic spindle formation and is critical for AML cell viability. Thus, we discover a novel function for IL-23R and a potential therapeutic target for this disease.
Citation Format: Nathan Duong, Dilshad H. Khan, Geethu E. Thomas, Rose Hurren, Jong Bok Lee, Jonathan St-Germain, Lily Drimmer, Yongran Yan, Neil Maclean, Marcela Gronda, Vincent Rondeau, Brandon D. Brown, Courtney L. Jones, Hong Chang, Andrea Arruda, Mark D. Minden, Li Zhang, Steven M. Kornblau, Brian Raught, Vito Spadavecchio, Aaron D. Schimmer. IL-23R is a regulator of the mitotic spindle and is critical for cell viability in AML [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 1646.