Intracellular IL-23 Receptor (IL-23R) Is Necessary for AML Viability and Regulates Mitotic Spindle and Centrosome Formation

The IL-23 receptor (IL-23R) is a cell surface receptor classically expressed on T cells. In response to inflammatory stimuli, such as microbial infection, dendritic cells and macrophages secrete the cytokine IL-23, which stimulates IL-23R leading to increased T cell activation and proliferation. Here, we identify a novel function for IL-23R in which intracellular IL-23R is necessary for AML viability by regulating mitotic spindle and centrosome formation.

An unbiased in silico screen was performed using the Interlinked Therapeutics ITX machine learning platform to identify novel targets and mechanisms in AML. This analysis identified IL-23R as a top hit and a potential regulator of the G2/M checkpoint and mitotic spindle. We confirmed IL-23R protein was over-expressed >2-fold in 6 of 8 primary AML patient samples compared to mean IL-23R expression (n=3) in normal hematopoietic cells.

IL-23R is classically a cell surface receptor, and we confirmed cell surface localization in DNT (Double Negative T) cells by flow cytometry and confocal microscopy. In contrast, only small amounts of IL-23R were present on the cell surface of AML cells and primary AML patient samples. Rather, IL-23R was located intracellularly in the cytoplasm and nucleus as demonstrated by flow cytometry, confocal microscopy, and immunoblotting of subcellular fractions. To validate that we were indeed detecting IL-23R, we confirmed intracellular localization in AML cells using 4 different antibodies directed against 4 different epitopes of the receptor.

To understand the function of intracellular IL-23R, we used BioID mass spectrometry to identify proteins that interact with IL-23R. Compared to the BioID controls, we identified 61 proteins that preferentially interacted with IL-23R. Of the 61 proteins, 36 are known cytoplasmic or nuclear proteins. Pathway analysis of interacting proteins identified mitotic spindle formation as the top pathway. Using a Proximity Ligation Assay and confocal microscopy, we validated the interaction of endogenous IL-23R with the known mitotic spindle proteins, NUMA, TMEM201, TACC1, and BAG6 in OCI-AML2 cells. In addition, by confocal microscopy, we demonstrated that IL-23R co-localizes with the mitotic spindle and centrosomes in AML. Knockdown and knockout of IL-23R in AML cells led to dysregulation of the mitotic spindle with multipolarity, lagging chromosomes, and spindle orientation errors.

We then evaluated the effects of IL-23R knockdown on AML growth and viability. Knockdown or knockout of IL-23R reduced AML growth in OCI-AML2, TEX, K562, NB4, and U937 cells. In addition, knockdown of IL-23R led to a significant reduction in engraftment efficiency of TEX cells into murine bone marrow.

To evaluate the effects of IL-23R depletion on normal hematopoiesis, we evaluated constitutive homozygote IL-23R knockout mice. IL23R-/- mice were bred in normal ratios and grew similar to wild type mice. IL-23R was associated with the mitotic spindle and centrosomes in hematopoietic cells of wild type mice and no IL-23R was detected in the knockout mice. Complete blood counts did not differ compared to wild type mice. The abundance of HSC (hematopoietic stem cell) and LSK (Lin⁻Sca-1⁺c-Kit⁺) hematopoietic stem cells were also similar between IL-23R -/- and wild type mice.

In summary, we discovered that IL-23R is necessary for AML growth and viability by regulating the formation of the mitotic spindle and centrosome. Thus, we identified a new function and localization for IL-23R and highlight a new therapeutic target for this disease.

Zhang:WYZE Biotech Co. Ltd: Consultancy, Current holder of stock options in a privately-held company, Honoraria. Spadevecchio:Interlinked Therapeutics, LLC: Current Employment. Schimmer:UHN: Patents & Royalties: the use of DNT cells to treat AML; Astra Zeneca: Consultancy, Honoraria; BMS: Consultancy, Honoraria, Research Funding; Novartis: Consultancy, Honoraria; Medivir AB: Research Funding; Takeda Pharmaceuticals: Consultancy, Honoraria, Research Funding; Jazz: Consultancy, Honoraria; Otsuka Pharmaceuticals: Consultancy, Honoraria.