Bone Marrow Adipocyte-Derived Free Fatty Acids Induce Gene Signature Linking Transcription with Metabolic Changes That Contribute to Survival of Acute Monocytic Leukemia Cells

Adipocytes are the prevalent stromal cell type in adult bone marrows (BM). With increasing age, BM stroma-resident mesenchymal stem cells (MSCs), increase their capacity to differentiate into adipocytes, which leads to the progressive accumulation of fat in the BM space. It is conceivable that the increased BM adipocyte content promotes leukemogenesis and negatively affects responsiveness to chemotherapy. We previously reported that free fatty acids (FFAs) promote the metabolic shift from pyruvate oxidation to fatty acid oxidation (FAO), which causes uncoupling of mitochondrial oxidative phosphorylation and promotes leukemia cell survival (Samudio, J Clin Invest. 2010). We further demonstrated the prominent antiapoptotic effects of BM-derived adipocytes co-cultured with cells from acute monocytic leukemia (AMoL), a poor-prognosis subtype of AML (Tabe ASH. 2013). Proteomic analysis with isobaric tags for relative and absolute quantification (iTRAQ) showed upregulation of protein folding pathways which increases the expression of antiapoptotic chaperone proteins HSP70 and HSP90, of integrin-mediated cell adhesion and migration pathways and downregulation of oxidative phosphorylation along with repression of cytochrome c.Metacore gene ontology (GO) analysis identified NF-kB, c-Jun, SP1, AP-1, and HMG as the potent relevant transcription factors that closely interact with and activate chaperone proteins, chromatin, and gene transcription.

In this study, we characterized a gene signature linking transcription with metabolic changes that contribute to AMoL cell survival under conditions mimicking aging BM with prevalent adipocytes. We confirmed the antiapoptotic role of FFAs produced by the primary BM MSC-derived adipocytes via pharmacologic inhibition of FAO by etomoxir (EX), which inhibits fatty acid entry into the mitochondria. EX (50mM) treatment reversed the prosurvival effects of adipocytes on serum-starved U937 monoblast cells (% Annexin V, -/+ EX: mono-culture, 30.1±9.0 / 31.5±4.6; co-culture with adipocytes, 8.9±2.1 / 29.6±9.2; P=0.02).

To assess the molecular links between metabolic pathways and gene expression triggered by BM adipocytes, we performed RNA-seq transcriptome analysis with a next generation sequencer system HiSeq1500 (Illumina) using TopHat software for alignment and Cufflinks software for identifying differential gene expression. RNA-Seq detected upregulation of 21 genes in U937 cells after co-culture with BM-derived adipocytes (false discovery rate, <0.05). Specifically, 10 of these upregulated genes were significantly decreased by EX treatment, including transcription factor–activating PPARg2 promoter KLF9, co-chaperone immunophilin protein FKBP5, chemokine CXCL12 receptor CXCR4, receptor tyrosine kinase FLT3, PI3K negative regulator PIK3IP1, and immunosuppressive transcriptional regulator TSC22D3. GO pathway analysis further revealed that co-culture with adipocytes induced upregulation of antioxidant thioredoxin peroxidase activity, which was reversed by EX. Together with the iTRAQ GO results, the antioxidative chaperone proteins might play critical roles in regulation of FAO with repression of oxidative phosphorylation in AMoL cells in adipocytes abundant BM.

DNA array (GeneSQUARE) analysis and quantitative RT-PCR detected upregulation of fatty acid binding protein 4 (FABP4), scavenger receptor CD36, nuclear receptor PPARG, and antiapoptotic Bcl-2 in U937 cells co-cultured with adipocytes. It is known that FFAs, the ligand of nuclear receptor PPARγ, activate PPARγ and promote FFA uptake through transcriptional induction of CD36 and FABP4 in monocytic cells. EX treatment, which blocks the entry of fatty acids into the mitochondria, induced prominent elevation of FABP4 in U937 cells co-cultured with adipocytes. These results indicate that the FABP4-mediated internalization and ligation of fatty acids to PPARγ facilitates transcriptional activation.

From the transcriptome analysis and the mitochondrial uncoupling metabolic changes, we conclude that the survival of AMoL cells depends on the cooperative interactions between lipid metabolism and transcriptional activation of factors associated with chaperones, chemokines, and integrins. Strategies targeting FAO warrant further exploration in patients with monocytic leukemia, which is highly dependent on altered lipid metabolism.