Overexpressing PHD3 limits FAO via regulation of ACC2 and consequently impedes leukemia cell proliferation

Overexpressing PHD3 limits FAO via regulation of ACC2 and consequently impedes leukemia cell proliferation. our understanding of tumor metabolism is usually incomplete because numerous tumors are FDG-PET unfavorable (Long and Smith, 2011; Ono et al., 2007), suggesting many cancers utilize alternate carbon sources. Multiple malignancy types have been suggested to rely on FAO for survival (Carracedo et al., 2013), highlighting a need to identify specific lipid metabolic programs that may go awry in malignancy. Post-translational modifying enzymes are key components of metabolic reprogramming (German and Haigis, 2015; Hitosugi and Chen, 2013). PHDs (also called EGLN1-3) are one class of enzymes poised to coordinate metabolism in response to changing cellular conditions. PHDs are a conserved family of oxygen- and -ketoglutarate dependent enzymes that are well known to regulate glycolytic metabolism through hydroxylation of hypoxia inducible factor (HIF) (Gorres and Raines, 2010). Hypoxia and a number of mutations in malignancy repress activity of some PHDs, stabilizing HIF and triggering a transcriptional program to increase glycolysis and anabolism while limiting mitochondrial bioenergetics (Masson and Ratcliffe, 2014). Recent reports suggest that PHDs are also responsive to cellular nutrient status (Kaelin and Ratcliffe, 2008). This may be linked to the use of -ketoglutarate during prolyl hydroxylation (Durn et al., 2012). PHD3 is usually notable for its particular sensitivity to -ketoglutarate, or perhaps more generally to the high nutrient state that may be achieved by addition of -ketoglutarate. Along these lines, treating AZD8835 mouse xenografts with cell-permeable -ketoglutarate inhibited growth by a PHD3-dependent mechanism (Tennant and Gottlieb, 2010). This raises the question of whether PHD3 is usually responsive to fluctuations in the AZD8835 nutrient state. We hypothesized that PHD3 might link nutrient status with implementation of metabolic adaptations. Therefore, we aimed to identify metabolic pathways regulated by PHD3. In this study, we identify acetyl-CoA carboxylase 2 (ACC2), the gatekeeper of FAO, as a PHD3 substrate. By activating ACC2, PHD3 represses oxidation of long chain fatty acids. Fatty acid catabolism is usually a dynamic cellular process that responds to metabolic imbalances and restores homeostasis (Gerhart-Hines et al., 2007). We show that PHD3 represses FAO during nutrient large quantity, and that cells with low PHD3 have prolonged FAO regardless of external nutrient cues. In AML, expression is dramatically decreased, contributing to a boost in fatty acid consumption that drives AZD8835 AML cell proliferation and disease severity. RESULTS PHD3 binds and modifies ACC by prolyl hydroxylation To probe for PHD3 substrates, we performed immunoprecipitation of PHD3 followed by liquid chromatography tandem mass spectrometry (LC-MS2) and detected an conversation with CD33 acetyl-CoA carboxylase (ACC). 21 ACC peptides were recognized in the PHD3 immunoprecipitation, while no ACC peptides were recognized in PHD2 or unfavorable control samples (Table S1). IP-Western blots confirmed that ACC interacted with PHD3 but not PHD1, PHD2 or anti-HA affinity resin alone (Physique 1A). ACC converts acetyl-CoA to malonyl-CoA, which serves as a precursor for excess fat synthesis and an inhibitor of FAO (Abu-Elheiga et al., 2003). Hence, ACC is usually a key regulator of fatty acid homeostasis that determines whether cells catabolize or synthesize fatty acids (Brownsey et al., 2006). Open in a separate window Physique 1 ACC interacts with PHD3 and is altered by hydroxylation at Pro450(A) HA-tagged PHD1-3 or vacant vector was transfected into 293T cells and immunoprecipitated with HA affinity resin. ACC co-immunoprecipitated with PHD3, as detected by immunoblot. (BCC) Immunoblot to detect ACC hydroxylation. ACC was immunoprecipitated from 293T cells overexpressing HA-PHD3, vector, or catalytically inactive PHD3 mutants (R206K and H196A). Cells had been treated in serum-free, low glucose medium for 12 h prior to immunoprecipitation (IP). WT PHD3 increased hydroxylation, as detected by immunoblot with hydroxyproline (OH-Pro) antibody. (D) Immunoblot to measure hydroxylation of ACC1 versus ACC2 in 293T cells overexpressing vector or PHD3. ACC1 and ACC2 were immunoprecipitated using isoform-specific antibodies. Cells were treated 12 h with serum-free, low glucose medium.