Background: In Acute Myeloid Leukemia (AML), a complete response to chemotherapy is usually obtained after conventional chemotherapy but overall patient survival is poor due to highly frequent relapses. As opposed to chronic myeloid leukemia, B lymphoma or multiple myeloma, AML is one of the rare malignant hemopathies the therapy of which has not significantly improved during the past 30 years despite intense research efforts. One promising approach is to determine metabolic dependencies in AML cells. Moreover, two key metabolic enzymes, isocitrate dehydrogenases (IDH1/2), are mutated in more than 15% of AML patient, reinforcing the interest in studying metabolic reprogramming, in particular in this subgroup of patients. Methods: Using a multi-omics approach combining proteomics, lipidomics, and isotopic profiling of [U-¹³C] glucose and [U-¹³C] glutamine cultures with more classical biochemical analyses, we studied the impact of the IDH1 R132H mutation in AML cells on lipid biosynthesis. Results: Global proteomic and lipidomic approaches showed a dysregulation of lipid metabolism, especially an increase of phosphatidylinositol, sphingolipids (especially few species of ceramide, sphingosine, and sphinganine), free cholesterol and monounsaturated fatty acids in IDH1 mutant cells. Isotopic profiling of fatty acids revealed that higher lipid anabolism in IDH1 mutant cells corroborated with an increase in lipogenesis fluxes. Conclusions: This integrative approach was efficient to gain insight into metabolism and dynamics of lipid species in leukemic cells. Therefore, we have determined that lipid anabolism is strongly reprogrammed in IDH1 mutant AML cells with a crucial dysregulation of fatty acid metabolism and fluxes, both being mediated by 2-HG (2-Hydroxyglutarate) production.

Mitochondria are more than just the powerhouse of cells; they dictate if a cell dies or survives. Mitochondria are dynamic organelles that constantly undergo fusion and fission in response to environmental conditions. We showed previously that mitochondria of cells in a low oxygen environment (hypoxia) hyperfuse to form enlarged or highly interconnected networks with enhanced metabolic efficacy and resistance to apoptosis. Modifications to the appearance and metabolic capacity of mitochondria have been reported in cancer. However, the precise mechanisms regulating mitochondrial dynamics and metabolism in cancer are unknown. Since hypoxia plays a role in the generation of these abnormal mitochondria, we questioned if it modulates mitochondrial function. The mitochondrial outer-membrane voltage-dependent anion channel 1 (VDAC1) is at center stage in regulating metabolism and apoptosis. We demonstrated previously that VDAC1 was post-translationally C-terminal cleaved not only in various hypoxic cancer cells but also in tumor tissues of patients with lung adenocarcinomas. Cells with enlarged mitochondria and cleaved VDAC1 were also more resistant to chemotherapy-stimulated cell death than normoxic cancer cells. Transcriptome analysis of mouse embryonic fibroblasts (MEF) knocked out for Vdac1 highlighted alterations in not only cancer and inflammatory pathways but also in the activation of the hypoxia-inducible factor-1 (HIF-1) signaling pathway in normoxia. HIF-1α was stable in normoxia due to accumulation of reactive oxygen species (ROS), which decreased respiration and glycolysis and maintained basal apoptosis. However, in hypoxia, activation of extracellular signal-regulated kinase (ERK) in combination with maintenance of respiration and increased glycolysis counterbalanced the deleterious effects of enhanced ROS, thereby allowing Vdac1 −/− MEF to proliferate better than wild-type MEF in hypoxia. Allografts of RAS-transformed Vdac1 −/− MEF exhibited stabilization of both HIF-1α and HIF-2α, blood vessel destabilization, and a strong inflammatory response. Moreover, expression of Cdkn2a, a HIF-1-target and tumor suppressor gene, was markedly decreased. Consequently, RAS-transformed Vdac1 −/− MEF tumors grew faster than wild-type MEF tumors. Metabolic reprogramming in cancer cells may be regulated by VDAC1 through vascular destabilization and inflammation. These findings provide new perspectives into the understanding of VDAC1 in the function of mitochondria not only in cancer but also in inflammatory diseases.

Dendrogenin A (DDA) is a mammalian cholesterol metabolite that displays potent antitumor properties on acute myeloid leukemia (AML). DDA triggers lethal autophagy in cancer cells through a biased activation of the oxysterol receptor LXRβ, and the inhibition of a sterol isomerase. We hypothesize that DDA could potentiate the activity of an anticancer drug acting through a different molecular mechanism, and conducted in vitro and in vivo combination tests on AML cell lines and patient primary tumors. We report here results from tests combining DDA with antimetabolite cytarabine (Ara-C), one of the main drugs used for AML treatment worldwide. We demonstrated that DDA potentiated and sensitized AML cells, including primary patient samples, to Ara-C in vitro and in vivo. Mechanistic studies revealed that this sensitization was LXRβ-dependent and was due to the activation of lethal autophagy. This study demonstrates a positive in vitro and in vivo interaction between DDA and Ara-C, and supports the clinical evaluation of DDA in combination with Ara-C for the treatment of AML.

Relapses and resistance to therapeutic agents are major barriers in the treatment of acute myeloid leukemia (AML) patients. These unfavorable outcomes emphasize the need for new strategies targeting drug-resistant cells. As IDH mutations are present in the preleukemic stem cells and systematically conserved at relapse, targeting IDH mutant cells could be essential to achieve a long-term remission in the IDH mutant AML subgroup. Here, using a panel of human AML cell lines and primary AML patient specimens harboring IDH mutations, we showed that the production of an oncometabolite (R)-2-HG by IDH mutant enzymes induces vitamin D receptor-related transcriptional changes, priming these AML cells to differentiate with pharmacological doses of ATRA and/or VD. This activation occurs in a CEBPα-dependent manner. Accordingly, our findings illuminate potent and cooperative effects of IDH mutations and the vitamin D receptor pathway on differentiation in AML, revealing a novel therapeutic approach easily transferable/immediately applicable to this subgroup of AML patients.

Dendrogenin A (DDA), a mammalian cholesterol metabolite with tumor suppressor properties, has recently been shown to exhibit strong anti-leukemic activity in acute myeloid leukemia (AML) cells by triggering lethal autophagy. Here, we demonstrated that DDA synergistically enhanced the toxicity of anthracyclines in AML cells but not in normal hematopoietic cells. Combination index of DDA treatment with either daunorubicin or idarubicin indicated a strong synergism in KG1a, KG1 and MV4-11 cell lines. This was confirmed in vivo using immunodeficient mice engrafted with MOLM-14 cells as well as in a panel of 20 genetically diverse AML patient samples. This effect was dependent on Liver X Receptor β, a major target of DDA. Furthermore, DDA plus idarubicin strongly increased p53BP1 expression and the number of DNA strand breaks in alkaline comet assays as compared to idarubicin alone, whereas DDA alone was non-genotoxic. Mechanistically, DDA induced JNK phosphorylation and the inhibition of AKT phosphorylation, thereby maximizing DNA damage induced by idarubicin and decreasing DNA repair. This activated autophagic cell death machinery in AML cells. Overall, this study shows that the combination of DDA and idarubicin is highly promising and supports clinical trials of dendrogenin A in AML patients.

The PEAK1 and Pragmin/PEAK2 pseudo-kinases have emerged as important components of the protein tyrosine kinase pathway implicated in cancer progression. They can signal using a scaffolding mechanism that involves a conserved split helical dimerization (SHED) module. We recently identified PEAK3 as a novel member of this family based on structural homology; however, its signaling mechanism remains unclear. In this study, we found that, although it can self-associate, PEAK3 shows higher evolutionary divergence than PEAK1/2. Moreover, the PEAK3 protein is strongly expressed in human hematopoietic cells and is upregulated in acute myeloid leukemia. Functionally, PEAK3 overexpression in U2OS sarcoma cells enhanced their growth and migratory properties, while its silencing in THP1 leukemic cells reduced these effects. Importantly, an intact SHED module was required for these PEAK3 oncogenic activities. Mechanistically, through a phosphokinase survey, we identified PEAK3 as a novel inducer of AKT signaling, independent of growth-factor stimulation. Then, proteomic analyses revealed that PEAK3 interacts with the signaling proteins GRB2 and ASAP1/2 and the protein kinase PYK2, and that these interactions require the SHED domain. Moreover, PEAK3 activated PYK2, which promoted PEAK3 tyrosine phosphorylation, its association with GRB2 and ASAP1, and AKT signaling. Thus, the PEAK1-3 pseudo-kinases may use a conserved SHED-dependent mechanism to activate specific signaling proteins to promote oncogenesis.