Mannose metabolism inhibition sensitizes acute myeloid leukemia cells to therapy by driving ferroptotic cell death

Published in Cancer
Mannose metabolism inhibition sensitizes acute myeloid leukemia cells to therapy by driving ferroptotic cell death
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Background

Acute myeloid leukaemia (AML) is a blood cancer characterised by the over-proliferation of immature white blood cells from the myeloid lineage. It has a particularly poor prognosis with a 5 year survival rate of around 30% and the biggest cause of treatment failure is either resistance to or relapse after chemotherapy, accelerating the need to find ways to understand how AML can be sensitised to therapies. It has been shown previously that metabolic adaptations in AML cells can drive resistance with cells that utilise oxidative phosphorylation or fatty acid oxidation rather than glycolysis for energy metabolism being more resistant to therapy1, so finding ways to target these pathways has come into focus.

The enzyme mannose phosphate isomerase (MPI) sits at a junction of glycolysis and interconverts fructose-6-phosphate and mannose-6-phosphate. Mannose is an essential building block of protein glycosylation and improper glycosylation can trigger endoplasmic reticulum (ER) stress and unfolded protein response (UPR) pathways. MPI has been studied in a range of solid tumours, where high levels of the protein can indicate a more aggressive and therapy resistant phenotype. In our previous work, we found loss of MPI sensitises AML blasts to therapies targeted against the common AML mutation FLT3-ITD in a genome wide CRISPR screen2.

In this work, we show that knocking out MPI or otherwise inhibiting it caused an increase in cell death when combined with both standard chemotherapies and newer FLT3-ITD inhibitors. To understand the mechanism behind this we used a number of techniques including RNA-seq, global and targeted metabolomics, real-time metabolic profiling and in vivo approaches. We found that removal of MPI causes ER stress which in turn leads to down-regulation of fatty acid oxidation (FAO). This is paired with an increase in the uptake of fatty acids leading to an accumulation of polyunsaturated fatty acids which combine with therapy induced reactive oxygen species (ROS) to trigger a form of iron dependent cell death known as ferroptosis.

Results

Using publicly available datasets we noticed that MPI levels in AML cells prognosticates patient outcome, correlated with gene signatures associated with therapy resistance and were higher at  relapse compared to diagnosis, indicating a role for MPI in AML resistance to chemotherapy. Furthermore, knocking out, knocking down or chemically inhibiting MPI sensitises AML cell lines and primary cells to both the standard chemotherapy cytarabine and the FLT3-ITD inhibitor quizartinib, a phenotype that is copied in vivo. 

In the search for the mechanism of this sensitisation, we used metabolomics and RNA sequencing to find that MPI knockout AML cells had higher levels of fatty acids within cells and reduced expression of genes involved in fatty acid oxidation. We found that MPI KO cells had a lower level of fatty acid oxidation, but that the increased levels of fatty acids within cells was also caused by an increase in the uptake of fatty acids from the environment, particularly polyunsaturated fatty acids.

The link between MPI and fatty acid oxidation is not immediately apparent, but it has been shown in other cell types that activation of ER stress and UPR can inhibit FAO, particularly the ATF6 arm of the UPR3. An upregulation of the ATF6 response upon MPI KO was seen in data from our RNA-seq and was confirmed using a reporter for ATF6 activation. Furthermore, inhibiting ATF6 activation was sufficient to rescue the effects of MPI KO on cell survival and fatty acid oxidation.

Our global metabolomics and RNA-seq data also pointed to activation of ferroptosis as a form of cell death, which is logical given that ferroptosis is triggered by a buildup of oxidised polyunsaturated fatty acids, MPI KO cells have high levels of polyunsaturated fatty acids and both chemotherapy and FLT3 inhibitors can lead to an increase in levels of ROS. We confirmed that MPI KO cells treated with both chemotherapy and FLT3 inhibitors had higher levels of lipid peroxidation. Moreover they underwent ferroptotic cell death as cell survival could be rescued by inhibiting ferroptosis both in vitro and in vivo but not when using inhibitors of other forms of cell death such as apoptosis and necroptosis.

Model of the effects of MPI knockout on AML cell metabolism and death upon exposure to chemotherapy.

Take home message

Therapy resistant AML cells rewire their metabolism, often moving from glycolysis to fatty acid oxidation, to allow them to survive. Our work uncovers a link between mannose metabolism, ER stress and fatty acid oxidation that could be exploited to sensitise AML cells to both novel and standard therapies through the induction of ferroptosis, a form of iron dependent cell death. Inducing ferroptotic cell death could be used in the future to eradicate resistant AML cell populations, as has been suggested in other cancer models4.

References

1. Bosc C, Saland E, Bousard A, Gadaud N, Sabatier M, Cognet G, Farge T, Boet E, Gotanègre M, Aroua N, Mouchel PL, Polley N, Larrue C, Kaphan E, Picard M, Sahal A, Jarrou L, Tosolini M, Rambow F, Cabon F, Nicot N, Poillet-Perez L, Wang Y, Su X, Fovez Q, Kluza J, Argüello RJ, Mazzotti C, Avet-Loiseau H, Vergez F, Tamburini J, Fournié JJ, Tiong IS, Wei AH, Kaoma T, Marine JC, Récher C, Stuani L, Joffre C, Sarry JE. Mitochondrial inhibitors circumvent adaptive resistance to venetoclax and cytarabine combination therapy in acute myeloid leukemia. Nat Cancer. 2021 Nov;2(11):1204-1223. 

2. Gallipoli P, Giotopoulos G, Tzelepis K, Costa ASH, Vohra S, Medina-Perez P, Basheer F, Marando L, Di Lisio L, Dias JML, Yun H, Sasca D, Horton SJ, Vassiliou G, Frezza C, Huntly BJP. Glutaminolysis is a metabolic dependency in FLT3ITD acute myeloid leukemia unmasked by FLT3 tyrosine kinase inhibition. Blood. 2018 Apr 12;131(15):1639-1653. 

3. Jao TM, Nangaku M, Wu CH, Sugahara M, Saito H, Maekawa H, Ishimoto Y, Aoe M, Inoue T, Tanaka T, Staels B, Mori K, Inagi R. ATF6α downregulation of PPARα promotes lipotoxicity-induced tubulointerstitial fibrosis. Kidney Int. 2019 Mar;95(3):577-589. doi: 10.1016/j.kint.2018.09.023. Epub 2019 Jan 11. PMID: 30639234.

4. Hassannia B, Vandenabeele P, Vanden Berghe T. Targeting Ferroptosis to Iron Out Cancer. Cancer Cell. 2019 Jun 10;35(6):830-849

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