Abstract PR11: Understanding the role of myddosome dynamics in melanoma using live cell imaging

Abstract

Abstract Melanoma is a complex and devastating disease that impacts patients all over the world. There are many factors that play a role in melanoma, and current studies have suggested TLR signaling involving IRAK4 can be a contributing element. Signal transduction by Toll-like receptors (TLR) was originally studied in the context of the innate immune response by recognizing pathogen-associated molecular patterns and triggering expansion of the immune cell population. Melanocytes express TLRs and can use this pathway to trigger cellular proliferation. In this study we demonstrate the importance of TLR signaling in melanoma using live cell imaging to track the dynamics of the myddosome. Activation of TLRs induces the recruitment of the death domain (DD) adaptor protein MyD88, which assembles into an oligomeric complex called the myddosome containing IRAK4. The myddosome complex mediates NF-κB activation leading to cell growth and survival. The sequential, multicomponent assembly of the myddosome and its transient nature provides a unique opportunity to study myddosome biology using live cell fluorescence resonance energy transfer (FRET) microscopy. Melanoma cells were engineered to stably express fluorescently tagged forms of MyD88 and IRAK4 to interrogate real-time myddosome dynamics using confocal microscopy to resolve individual complexes responding to TLR ligands and pyrrolopyrimidine IRAK4 inhibitors. Cells treated with LPS, a TLR activator, have more assembled myddosomes as read out by the FRET signal, while cells treated with IRAK4 inhibitors have fewer. I have also recapitulated this pattern by mutating MyD88 and found that the activated form of MyD88 leads to an 8-fold increase in the number of myddosomes, while the negative mutant prevents any FRET signal from being detected. Studies have shown that the majority of melanoma tumor samples have elevated levels of phosphorylated IRAK4, and our own findings suggest that inhibition of IRAK4, in combination with a topoisomerase inhibitor, increases the survival of mice after implantation of melanoma cells with a hazard ratio of 0.25. Additionally, human melanoma samples display a subcellular localization of active IRAK4 similar to cells stably expressing an active myddosome complex, while normal skin samples correspond to cells expressing a nonfunctional myddosome. Melanoma can progress rapidly and is known to develop resistance to current therapies; hence there is a great need to develop alternative therapeutic targets. The work presented here hopes to address this need by illuminating the role of the myddosome in melanoma and how targeting this complex, via IRAK4, presents an attractive therapeutic strategy. This abstract is also being presented as Poster B23. Citation Format: Bridget Kreger, Adam Hendricks. Understanding the role of myddosome dynamics in melanoma using live cell imaging [abstract]. In: Proceedings of the AACR Special Conference on Melanoma: From Biology to Target; 2019 Jan 15-18; Houston, TX. Philadelphia (PA): AACR; Cancer Res 2020;80(19 Suppl):Abstract nr PR11.