Abstract 1542: Metabolic dysregulations of aged bone marrow mesenchymal cells in Acute Myeloid Leukemia reveal novel therapeutic targets

Abstract Background: Despite recent efforts to elucidate the dependence of acute myeloid leukemia (AML) on the bone marrow microenvironment, the contribution of aged bone marrow mesenchymal stem cells (BMMSCs) to AML patient survival and treatment response remains understudied. Deregulated nutrient sensing is an established hallmark of aging and previous works have identified age-dependent metabolic dysregulations in cancer. The dependence of AML on oxidative phosphorylation (OXPHOS) has been well established, as AML adapts to fluctuations of nutrient and oxygen availability in the bone marrow microenvironment. We hypothesized that metabolic pathways, such as oxidative phosphorylation, are critical in aged AML-BMMSCs as they drive pro-tumorigenic properties and drug resistance. Methods: Our group expanded primary Normal Donor (ND)-BMMSCs and AML-BMMSCs (n=9 CBF-AML, n=11 CK-AML, n=7 ND-BMMSCs) under conditions that mimic the bone marrow microenvironment (91% nitrogen, 4% oxygen, and 5% CO2). We isolated DNA and RNA from early passaged cells to perform methylation analyses using 850k Illumina DNA methylation arrays and gene expression analyses using mRNA-seq. We utilized the Horvath Skin and Blood clock to calculate epigenetic age based on DNA methylation. qPCR assessing mRNA expression of OXPHOS-related genes in ND- and AML-BMMSCs was performed to validate sequencing results. Western blot analyses of Total OXPHOS and its complexes were performed to assess expression levels of OXPHOS-related proteins in ND- and AML-BMMSCs. Seahorse assays were performed to obtain live and repeated metabolic measurements of Oxygen Consumption Rate (OCR) and Extracellular Acidification Rate (ECAR) in ND- and AML-BMMSCs. Lastly, 2D co-culture experiments were performed to assess cell viability of AML cell lines with BMMSCs treated ± OXPHOS inhibitors (IACS-010759 and Tamoxifen) utilizing AO/PI staining. Results: Our integrative omics approach showed that with increased epigenetic age, as measured by the Horvath Skin and Blood clock, AML-BMMSCs reveal upregulations in hallmark gene sets for MYC and OXPHOS when compared to ND-BMMSCs. Western blot analyses confirmed upregulation of MYC and OXPHOS complexes in AML-BMMSCs when compared to controls. Results from seahorse assay revealed an increase in OCR and ATP production in AML-BMMSCs when compared to controls. Lastly, our 2D co-culture experiments in vitro revealed a decrease in cell viability on AML cells that were co-cultured with AML-BMMSCs treated with IACS-010759 and/or Tamoxifen. Conclusions: Our results elucidate age-dependent metabolic dysregulations within the bone marrow microenvironment in AML and provide novel targetable approaches. Future portions of testing aim to identify the mechanism of action of AML-BMMSCs and OXPHOS. Citation Format: Mary E. Fuentes, Julia K. Christian, Amy Kovacs, James R. Lerma, Liang Niu, Alice S. Mims, Christopher C. Oaks, John C. Byrd, Erin K. Hertlein, Amina Abdul-Aziz. Metabolic dysregulations of aged bone marrow mesenchymal cells in Acute Myeloid Leukemia reveal novel therapeutic targets [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 1542.