Abstract 4366: Loss of EP300 triggers IL-1α signaling and subsequent activation of the IL-6/JAK/STAT3 axis to drive oncogenesis in bladder cancer

Abstract

Abstract Bladder Cancer (BLCA) is the sixth most common cancer in the United States with 81,180 new cases and 17,100 deaths in 2022. Next Generation Sequencing of BLCA has revealed an enrichment of alterations in genes classified as chromatin modifiers compared to other solid tumors. Among these is EP300, the gene encoding the p300 histone acetyltransferase, which is mutated in ~12% of BLCA with the majority putative loss of function frameshift and nonsense mutations. To investigate the biological role of EP300 loss-of-function in BLCA pathogenesis, we generated isogenic EP300 null RT112 bladder cancer cells using a 2-step CRISPR knockout (KO) and knockin strategy. EP300 KO cells demonstrated increased proliferation, anchorage independent cell growth, tumor formation in mice, and enhanced invasive potential. To investigate the mechanism(s) whereby EP300 loss-of-function enhances oncogenicity, we performed an unbiased Tandem Tag Mass Spectrometry (TMT MS) screen to identify pathways dysregulated in EP300 KO versus parental cells. EP300 loss-of-function was found to induce IL-6/JAK/STAT3 signaling via upregulated expression of IL-1α, a canonical pro-inflammatory cytokine. Knock down of IL1A gene expression was sufficient to downregulate STAT3 activation in EP300 KO cells, whereas ectopic stimulation using IL-1α increased STAT3 activation in parental RT112 cells, suggesting that IL-1α was the predominant upstream activator of JAK/STAT3 signaling in our model. Pharmacological inhibition of downstream targets of IL-1α signaling such as IRAK1/4 were also able to inhibit STAT3 signaling in EP300 KO cells in a dose and time dependent manner. Further investigations indicated that activated IL-1α signaling not only drove transcriptional upregulation of IL-6 but also the production of soluble IL-6R, activating IL-6 trans signaling. Finally, to elucidate how IL-1α upregulation is triggered by EP300 loss-of-function, we performed immunoprecipitation and immunofluorescence assays, which revealed that IL-1α binds directly to p300. Additionally, ectopic p300 expression in EP300 KO clones dramatically reduced IL-1α protein expression, indicating that p300 acts as a negative regulator of IL-1α in a bladder cancer context. In summary, EP300 loss-of-function relieves p300’s transcriptional and/or physical-tethering inhibition on IL-1α signaling, subsequently activating the IL-6/JAK/STAT3 pathway to drive oncogenesis in BLCA. Citation Format: James A. Rodrigues, Jiaqian Luo, Amanda R. Sabel, Ziyu Chen, Xinran Tang, Fengshen Kuo, Hikmat Al-Ahmadie, Kwanghee Kim, Eugene Pietzak, Gopa Iyer, David B. Solit, Sizhi P. Gao. Loss of EP300 triggers IL-1α signaling and subsequent activation of the IL-6/JAK/STAT3 axis to drive oncogenesis in bladder cancer [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 4366.