Cap Analysis of Gene Expression (CAGE) Sequencing Reveals Alterations of the Transcript Signatures in Acute Monocytic Leukemia Cells By Fatty Acid Oxidation Inhibition

Adipocytes, differentiated from bone marrow (BM) stroma-resident mesenchymal stem cells (MSC), are the prevalent stromal cell type in adult BM that increase with aging and cause leukemia cell resistance to chemotherapy (Ehsanipour Cancer Res. 2013).

We previously reported that MSC-derived adipocytes prominently inhibited the spontaneous apoptosis of co-cultured acute monocytic leukemia (AMoL) cells, a poor-prognosis subtype of AML, and that a pharmacologic fatty acid oxidation (FAO) inhibitor Etomoxir (EX) reversed the prosurvival effects of adipocytes, indicating the importance of FAO dependent metabolic alterations in AMoL survival (Tabe ASH. 2014).

To assess the transcription factors responsible for AMoL cell survival in adipocyte co-culture condition and for apoptosis induction by FAO inhibition, the alterations of transcript signatures were examined by the cap analysis of gene expression (CAGE) sequencing utilizing second-generation sequencing platform (Illumina Genome Analyzer). CAGE identifies and quantifies the 5' ends of capped mRNA transcripts, which enables the identification of transcription start sites (TSS) and allows investigating promoter structures necessary for gene expression (Carninci et al. 1996). The TSS of genes altered in U937 and THP1 cells co-cultured with adipocytes in the presence or absence of EX were mapped, and the common alterations observed in both cell types were analyzed. CAGE detected upregulation of 366 genes and downregulation of 219 genes after co-culture with adipocytes (false discovery rate, < 0.05). Ingenuity Pathway Analysis (IPA) revealed that adipocyte co-culture activated the cancer associated transcription factors Myc and FOXM1, and inhibited the p53 transcription regulator IFI16 and FLT1 kinase, an upstream positive regulator of MAPK/ERK and PI3K/AKT signaling. After EX treatment, CAGE-IPA analysis implicated inhibition of the FAO initiation enzyme ACOX1, and activation of the transcription factor ATF4 (Activating Transcription Factor 4), the master coordinator of the integrated stress response (ISR), and of the nuclear receptor PPARG which controls the FAO pathway.

To narrow down the specific transcription factors responsible for EX induced apoptosis in AMoL cells co-cultured with adipocytes, CAGE-mapped TTS signature was integrated with the gene expression patterns detected by RNA-seq.

CAGE and RNA-seq detected 3 genes consistently upregulated by adipocyte co-culture (KLF9; a transcription factor that activates PPARg2 promoter, FKBP5; HSP90 interacting co-chaperone immunophilin protein, ATP13A2; a member of the P5 subfamily of ATPases) and downregulation of 2 genes (ANPEP; known as CD13 or Alanyl Aminopeptidase, SLC39A10; Zinc transporter which involves in lipid metabolism).

EX treatment under adipocyte co-culture condition specifically upregulated 12 genes including ISR mediator ATF4 and its target gene TRIB3. The upregulation of asparagine syntheses gene ASNS, known to be induced by ATF4, was also detected by RNA-seq. Concordant with CAGE-IPA results, EX treatment upregulated lipid accumulation marker PLIN2 and PPARG target of fatty acid binding protein FABP4 likely reflecting the direct feedback of FAO inhibition. Two genes were downregulated by EX treatment (SREBP1; the lipogenic transcription factor, P2RY2; G-protein coupled receptor activated by ATP).

Finally, capillary electrophoresis mass spectrometry (CE-MS) detected the upregulation of citric acid by adipocyte co-culture, which was significantly depleted after EX treatment. The EX treatment, however, increased lactic acid along with fructose 6-phosphate and glucose 6-phosphate upregulation, indicating that cellular metabolism shifts from oxidative phosphorylation and FAO to anaerobic glycolysis which is known to activate ISR.

Taken together, this study demonstrates that FAO inhibition by EX activates the pro-apoptotic transcriptional program of ISR through the up-regulation of ATF4 in adipocyte co-cultured AMoL cells. The strategies targeting FAO warrant further exploration in AMoL that survives in adipocyte abundant aged adult BM.