Abstract A05: Molecular, immunologic, metabolic, and radiomic associations of oxidative phosphorylation (OXPHOS) in melanoma brain metastases (MBMs)
Authors: Fischer Grant M. , Kotrotsou Aikaterini , Ahmed Sara , O’Neal Pilar , Sahnoune Iman , Gopal Y.N. Vashisht , Hudgens Courtney , Knighton Barbara , Colen Rivka , Tetzlaff Michael T. , Davies Michael A. 
Published: June 2022

Abstract
Background: OXPHOS mediates resistance to BRAF and MEK inhibitors in melanoma. Recently, we showed that MBMs have higher expression of OXPHOS genes than patient-matched extracranial metastases (ECMs). The induction of, and dependence upon, OXPHOS in MBMs was confirmed in mouse xenograft models. Notably, our studies demonstrated that OXPHOS varies among MBMs. In order to improve our understanding of the High-OXPHOS phenotype, we report here the molecular, immune, metabolic, and radiomic features that correlate with OXPHOS levels in MBMs.
Methods: We applied an OXPHOS gene signature to RNA-sequencing (RNA-seq) data from surgically resected MBMs (n=88). Clustering by these genes identified MBMs with significant enrichment (High-OXPHOS; n=21) and depletion (Low-OXPHOS; n=25) of OXPHOS genes. The EdgeR/limma/voom pipeline was used to perform differential gene expression (DGE) analysis between High- and Low-OXPHOS MBMs. Pathway analyses were performed via Ensemble of Gene Set Enrichment Analyses (EGSEA). The ESTIMATE and MCP-Counter R packages were used to assess immune infiltrates from voom-transformed counts. Quantitative analysis of P-S6 and P-PRAS40 was performed by immunohistochemistry (IHC). Tumor phenotypes were segmented on conventional magnetic resonance (MR) images, and radiomic features were extracted using an in-house radiome-sequencing pipeline. Radiomic features were utilized to predict High- vs. Low-OXPHOS status. To evaluate the robustness of the estimates, Leave One Out cross-validation (LOOCV) was conducted on the patient set. Direct metabolite analysis was performed via liquid chromatography mass spectrometry (LC-MS) of Low-OXPHOS A375 and High-OXPHOS, MEKi-resistant A375-R1 brain xenografts.
Results: RNA-seq analysis identified that High-OXPHOS MBMs are characterized by significant enrichment of PGC1α (FDR q-val=0.0002) and mTOR (FDR q-val=0.001) signaling and significant depletion of ImmuneScores, T cells, cytotoxic lymphocytes, B lineage cells, and NK cells (p&lt;0.01 for all analyses) vs. Low-OXPHOS MBMs. We observed significantly higher P-S6 (p=0.019) in High-OXPHOS MBMs but did not observe differences in P-PRAS40 staining. Furthermore, we identified radiomic features that accurately predicted OXPHOS status (AUC: 98.4%, Sensitivity/Specificity: 100%/97.87%). Direct metabolite analysis identified significant enrichment (FDR q-val&lt;0.25) of the glutamine derivatives alanine, aspartate, and glutamate in High-OXPHOS A375-R1 brain xenografts.
Conclusions: Significant differences in mTOR (enriched) and immune (depleted) signaling were identified in High-OXPHOS clinical MBMs, and enrichment of glutamine signaling was observed in High-OXPHOS MBM xenografts. Further, our results demonstrated that changes in MR radiomic features can noninvasively discriminate between High- and Low-OXPHOS MBMs. Additional studies are ongoing to evaluate the clinical, prognostic, and predictive values of OXPHOS status in melanoma.
Citation Format: Grant M. Fischer, Aikaterini Kotrotsou, Sara Ahmed, Pilar O’Neal, Iman Sahnoune, Y.N. Vashisht Gopal, Courtney Hudgens, Barbara Knighton, Rivka Colen, Michael T. Tetzlaff, Michael A. Davies. Molecular, immunologic, metabolic, and radiomic associations of oxidative phosphorylation (OXPHOS) in melanoma brain metastases (MBMs) [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 A05.