906 Immunogenomic evaluation of clear cell renal carcinoma uncovers HK3 as a myeloid specific metabolic enzyme

• Published in: Journal for immunotherapy of cancer 2021-11, Vol.9 (Suppl 2), p.A951-A951
• Main Authors: Reinfeld, Bradley, Madden, Matthew, Wolf, Melissa, Cubas, Agi de, Haake, Scott, Hongo, Rachel, Axelrod, Margaret, Bader, Jackie, Obradovic, Aleksandar, Greenwood, Dalton, Ye, Xiang, Balko, Justin, Beckermann, Katy, Vincent, Benjamin, Rini, Brian, Drake, Charles, Rathmell, Jeffrey, Rathmell, W
• Format: Article
• Language: English
• Subjects: Bone marrow; Consent; Enzymes; Genomes; Glucose; Immunotherapy; Kidney cancer; Metabolism; Regular and Young Investigator Award Abstracts; Tumors
• ISSN: 2051-1426
• DOI: 10.1136/jitc-2021-SITC2021.906

BackgroundGlucose fixation is a hallmark clear cell renal carcinoma (ccRCC).1 2 Our group has shown unique metabolic enzyme utilization between malignant cells and infiltrating cells. Additionally, we uncovered the glycolytic nature of tumor infiltrating myeloid cells.3 Therefore, we decided to investigate the role of the hexokinase isoforms (HK1,2/3, GCK, and HKDC1) in the ccRCC tumor microenvironment (TME).MethodsFor this study, we performed immunogenomic analyses across ccRCC samples available via The Cancer Genome Atlas (TCGA).4 5 Additionally, we examined the expression of hexokinases in the neoadjuvant VEGF inhibitor setting6 as well as correlation to a poor prognostic macrophage subset.7 Our group also performed single cell-ATAC seq on methocult cultures to further characterize the metabolic features of hematopoiesis. We additionally implemented qPCR on magnetically sorted bone marrow as well as myeloid cell culture to further interrogate the role of HK3 in macrophage biology and in-situ RNA hybridization (RNA-ISH) to describe the subpopulation of HK3+ cells in the ccRCC TME.ResultsGene set enrichment analysis confirmed HK1/2’s role in anabolic metabolism. GCK was barely detectable in these samples while HKDC1 expression decreased in ccRCC tumors. Intriguingly, patients with elevated expression of HK3 had an enrichment of interferon gamma response signature. In our evaluation of the TCGA, only HK3 expression correlated with poor outcome in ccRCC. CiberSortX demonstrated that HK3 expressing tumors correlated with the presence M2 macrophages while other HK family enzymes had marginal association with immune infiltrate. HK3 was the only hexokinase found to be significantly elevated with neoadjuvant pazopanib treatment in addition to being enriched in ccRCC patients with high levels of poor prognostic macrophages. RNA-ISH confirms HK3 expression is limited to myeloid cells in ccRCC tumors. The myeloid specific nature of HK3 is supported by transcript analysis from MC38 tumors, and qPCR on mouse bone marrow. Myeloid specificity for HK3 isoform expression is not restricted to malignancy; HK3 is one of a handful of genes that define myeloid identity from scATAC sequencing of in vitro differentiated CD34+ hematopoietic stem cells. Our ongoing in vitro studies indicate that M1 polarization (+LPS/IFNg) increases expression of HK1/2/3, consistent with the anabolic phenotype of activate macrophages. However, stimulation with IFNg alone only elevates the express