Abstract 333: Interleukin 23 receptor is a critical regulator of the mitotic spindle and necessary for AML viability
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 , Brown Brandon D. , Raught Brian , Arruda Andrea , Chang Hong , Minden Mark D. , Zhang Li , Kornblau Steven M. , Spadavecchio Vito , Schimmer Aaron D. 
Published: June 2023

Classically, the interleukin-23 receptor (IL-23R) is expressed on the surface of T cells where activation by its ligand, interleukin-23 (IL-23) leads to T cell activation through the JAK/STAT pathway. Here, we identify a novel localization and function for IL-23R in which intracellular IL-23R is necessary for AML viability by regulating mitotic spindle and centrosome formation.

IL-23R was identified as a potential target through InterLinked Therapeutics’ ITX machine learning platform. IL-23R was identified through this unbiased in silico screen as a top hit and a potential regulator of the G2/M checkpoint and mitotic spindle. We confirmed that IL-23R protein was overexpressed &gt;2-fold in 6 of 8 primary AML patient samples compared to mean IL-23R expression in normal hematopoietic cells.

Consistent with prior studies, we confirmed cell surface localization of IL-23R in double negative T cells by flow cytometry and confocal microscopy. In contrast, only small amounts of IL-23R were detected on the surface of AML cells and primary AML patient samples. Rather, IL-23R was located intracellularly as demonstrated by flow cytometry, confocal microscopy, and immunoblotting of subcellular fractions. We confirmed intracellular localization of IL-23R in AML cells using 4 different antibodies targeted against 4 different epitopes of the receptor.

We used BioID mass spectrometry to identify novel protein interactors with intracellular IL-23R and identified 61 interacting proteins. 36 of those 61 were nuclear or cytoplasmic in localization and the largest subset was associated with mitotic spindle regulation. By Proximity Ligation Assay, we confirmed the interactions between endogenous IL-23R with known mitotic spindle proteins NUMA, TMEM201, BAG6, and TACC1 in OCI-AML2 cells and primary AML samples.

By confocal microscopy, we discovered that IL-23R co-localized with the mitotic spindle and centrosomes in AML cells. Genetic knockout of IL-23R increased spindle defects with multipolarity, lagging chromosomes, and spindle orientation errors. Interestingly, heterodimeric partner of IL-23R, IL12Rβ1 was also detected to be co-localized with mitotic spindles but the cytokine, IL-23, could not be detected.

Knockdown of IL-23R also reduced cell growth in OCI-AML2, TEX, K562, NB4, and U937 cells. IL-23R knockdown also reduced the clonogenic growth and engraftment efficiency of TEX leukemia cells into murine bone marrow.

In summary, intracellular IL-23R is a critical regulator of mitosis in AML and is necessary for AML viability. This study highlights a novel function and localization of IL-23R in AML and identifies a potential therapeutic target.

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, Brandon D. Brown, Brian Raught, Andrea Arruda, Hong Chang, Mark D. Minden, Li Zhang, Steven M. Kornblau, Vito Spadavecchio, Aaron D. Schimmer. Interleukin 23 receptor is a critical regulator of the mitotic spindle and necessary for AML viability [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 333.