Objectives/Goals: This study evaluates the significance of PU.1 upregulation and the PU.1-mediated regulation of the anti-inflammatory miR-146a on pro-inflammatory NF-kB signaling in RCC-derived macrophages. The downstream effects of PU.1 targeting on key phenotypes related to RCC immunity and how this impacts RCC progression will also be examined. Methods/Study Population: Bioinformatics data identifying PU.1 upregulation within RCC tumors, correlation with prognostic clear cell RCC markers, and survival analysis were obtained using The Cancar Genome Atlas (TCGA) RNA-sequencing data. Murine RCC cell line Renca possessing a Vhl deletion are used to recapitulate a common deletion in human clear cell RCC tumors. This study utilizes two methods to target PU.1 in orthotopic RCC, a conditional PU.1 deletion model and a pharmacologic PU.1 inhibitor model. Quantitative real-time PCR, western blotting, and immunofluorescent staining will evaluate NF-kB signaling in PU.1-targeted macrophages and murine RCC tumors. Flow cytometry and single-cell RNA-sequencing will reveal the effects of PU.1 targeting on key immune cell phenotypes and key metrics of RCC outcomes. Results/Anticipated Results: PU.1 mRNA and protein expression are upregulated in human clear cell RCC tumors compared to normal kidney tissue. A 10-year overall survival analysis in clear cell RCC tumors indicates that increased PU.1 mRNA expression is unfavorable (p = 0.0298; HR = 1.24; N = 133/133). PU.1 inhibition in orthotopic RCC reduces tumor volume, compared to control (p = 0.0328; N = 5/5). NF-kB signaling effectors are upregulated in PU.1-inhibited macrophages, including canonical miR-146a targets. We anticipate that PU.1-targeted macrophages and RCC tumors will yield significant increases in NF-kB signaling, pro-inflammatory macrophage phenotypes, increased antitumor immunity, and reduced tumor volume. Discussion/Significance of Impact: This is the first study to address PU.1 upregulation in RCC and use multiple methods to examine a potential function of PU.1 in immunosuppressive macrophages and RCC immunity. This work also assesses the relevance of miR-146a on canonical, pro-inflammatory NF-kB signaling in the novel context of RCC tumor immunity.
Echols et al. (Wed,) studied this question.