Title: Identification and characterisation of metabolic pathways as determinants of drug resistance in acute myeloid leukaemia
Funder: Cancer Research UK

Abstract: BACKGROUND Clinical and biological evidence suggest that current therapies, including novel targeted agents, are unlikely to achieve cure in Acute Myeloid Leukaemia(AML) mostly as a result of disease relapse. Therefore an improved understanding of the molecular mechanisms of relapse is required. Rewired metabolism has been shown as essential for the malignant phenotype and resistance to therapies in multiple cancers, however a comprehensive characterisation of metabolic adaptations to AML therapies is lacking. Starting from an unbiased CRISPR/Cas9 drop-out screen, we recently demonstrated that adaptive metabolic responses, particularly increased glutaminolysis, tricarboxylic acid cycle(TCA) activity and oxidative phosphorylation, play a key role in resistance to FLT3-tyrosine kinase inhibitors(TKI) in the aggressive FLT3ITD-mutant AML subtype. I hypothesise that metabolic adaptations to external selective pressures play a key role in the resistance to multiple therapies across different AML subtypes AIMS 1) To study mannose metabolism(MM) as a resistance mechanism to FLT3-TKI. 2) Test metabolic adaptations as a means of resistance to isocitrate dehydrogenase(IDH)2 inhibitors. 3) To determine how the high tricarboxylic acid(TCA) cycle activity of leukaemic stem cells(LSC) regulates leukaemic transcriptional programmes to favour therapeutic resistance. METHODS I will use standard in vitro(apoptosis, cell cycle, proliferation, colony forming cell) and in vivo (transplantation in syngeneic recipient followed by survival and leukaemic burden assessment) assays, metabolomics by Liquid chromatography/Mass Spectrometry(LC/MS), proteomics by MS, and transcriptional analysis(RNA and ChIP-Seq) to test if: 1) Targeting the key enzyme in MM, mannose phosphoisomerase(MPI) (highly depleted in our CRISPR/Cas9 screen), sensitises FLT3ITD-mutant cells to FLT3-TKI. I will test if MPI depletion redirects glucose metabolism towards the production of N-acetylglucosamine(GlcNAc) via the hexosamine biosynthetic pathway, leading to changes in O-GlcNAcylation modification of transcription factors/histones necessary for leukaemic cells survival. 2) Upregulation of a-KG-consuming enzymes re-establishes inhibition of target demethylases/dioxygenases, by reducing the levels of their activating cofactor a-KG, thus inducing resistance to AG221(Enasidenib), a specific IDH2-mutant protein inhibitor approved for clinical use in IDH2-mutant patients. 3) Modulation of specific TCA cycle intermediates, involved in epigenetic modifications, by candidate enzymes, chosen based on differential metabolite and gene expression levels between normal and LSC in complementary models, subverts leukaemic transcriptional programmes thus contributing to therapy resistance. EXPECTED OUTCOMES I will unveil metabolic states leading to therapy resistance in several AML subtypes at the level of the LSC and identify mechanistic links between these and AML transcriptional programmes. The findings will support preclinical studies aimed to develop novel inhibitors of aberrant metabolism in AML.

Lay abstract: BACKGROUND Clinical and biological evidence suggest that current therapies, including novel targeted agents, are unlikely to achieve cure in Acute Myeloid Leukaemia(AML) mostly as a result of disease relapse. Therefore an improved understanding of the molecular mechanisms of relapse is required. Rewired metabolism has been shown as essential for the malignant phenotype and resistance to therapies in multiple cancers, however a comprehensive characterisation of metabolic adaptations to AML therapies is lacking. Starting from an unbiased CRISPR/Cas9 drop-out screen, we recently demonstrated that adaptive metabolic responses, particularly increased glutaminolysis, tricarboxylic acid cycle(TCA) activity and oxidative phosphorylation, play a key role in resistance to FLT3-tyrosine kinase inhibitors(TKI) in the aggressive FLT3ITD-mutant AML subtype. I hypothesise that metabolic adaptations to external selective pressures play a key role in the resistance to multiple therapies across different AML subtypes AIMS 1) To study mannose metabolism(MM) as a resistance mechanism to FLT3-TKI. 2) Test metabolic adaptations as a means of resistance to isocitrate dehydrogenase(IDH)2 inhibitors. 3) To determine how the high tricarboxylic acid(TCA) cycle activity of leukaemic stem cells(LSC) regulates leukaemic transcriptional programmes to favour therapeutic resistance. METHODS I will use standard in vitro(apoptosis, cell cycle, proliferation, colony forming cell) and in vivo (transplantation in syngeneic recipient followed by survival and leukaemic burden assessment) assays, metabolomics by Liquid chromatography/Mass Spectrometry(LC/MS), proteomics by MS, and transcriptional analysis(RNA and ChIP-Seq) to test if: 1) Targeting the key enzyme in MM, mannose phosphoisomerase(MPI) (highly depleted in our CRISPR/Cas9 screen), sensitises FLT3ITD-mutant cells to FLT3-TKI. I will test if MPI depletion redirects glucose metabolism towards the production of N-acetylglucosamine(GlcNAc) via the hexosamine biosynthetic pathway, leading to changes in O-GlcNAcylation modification of transcription factors/histones necessary for leukaemic cells survival. 2) Upregulation of a-KG-consuming enzymes re-establishes inhibition of target demethylases/dioxygenases, by reducing the levels of their activating cofactor a-KG, thus inducing resistance to AG221(Enasidenib), a specific IDH2-mutant protein inhibitor approved for clinical use in IDH2-mutant patients. 3) Modulation of specific TCA cycle intermediates, involved in epigenetic modifications, by candidate enzymes, chosen based on differential metabolite and gene expression levels between normal and LSC in complementary models, subverts leukaemic transcriptional programmes thus contributing to therapy resistance. EXPECTED OUTCOMES I will unveil metabolic states leading to therapy resistance in several AML subtypes at the level of the LSC and identify mechanistic links between these and AML transcriptional programmes. The findings will support preclinical studies aimed to develop novel inhibitors of aberrant metabolism in AML.

Research activity codes: 5.1 PHARMACEUTICALS
Health categories: CANCER AND NEOPLASMS