Abstract 1025: Aberrant TCR activation and altered metabolism drive decreased T cell cytokine production in IDH mutated acute myeloid leukemia
Authors: Li Zhuoyan , Reinfeld Bradley I. , Reisman Benjamin J. , Madden Matthew Z. , Potts Chad R. , Rathmell Wendy K. , Rathmell Jeffrey C. , Ferrell Paul B. 
Published: September 2020

Isocitrate dehydrogenase 1 or 2 (IDH1/2) mutations are recurrent in AML and introduce neomorphic function in this metabolic enzyme. The resulting accumulation of the oncometabolite R-2-hydroxyglutarate (2-HG) in leukemic cells leads to epigenetic changes, abnormal gene expression, proliferation, and differentiation arrest. Moreover, studies in solid tumors have shown that 2-HG impacts T cells in the microenvironment. In the context of IDH1 mutated gliomas, elevated levels of 2-HG interfere with T cell activation and suppress chemotaxis. We therefore hypothesized that 2-HG directly decreases TCR activation via disruption of metabolic homeostasis leading to T cell dysfunction in AML.

Using sorted healthy T cells, we studied the effect of 2-HG on T cell cytokine expression, proliferation, and phenotype. TNF-α expression was significantly decreased after 48-hour treatment with 10 mM 2-HG versus control. Using a co-culture assay with TCR-stimulated T cells and gene-edited AML cell line expressing the IDH2R140Q mutation (E:T 1:1), there was no change in proliferation of T cells compared with T cells expanded without co-culture. However, there was greater expansion of effector memory T cells (CCR7-CD45RA-) cells and decreased expansion of naïve T cells (CCR7+CD45RA+) in the co-culture system compared with T cells in culture alone. Next, we evaluated the effect of 2-HG on activation of NFAT and NF-κB, transcription factors involved in cytokine expression. We created an NFAT-GFP reporter Jurkat cell line that showed significantly decreased NFAT activation after treatment with 2-HG versus control using both PMA/ionomycin and direct TCR stimulation. This was similarly seen in 2-HG treated healthy T cells with decreased nuclear NFAT by Western blot. NF-κB was shown to alter TNF-α expression as treatment with IKK16, an inhibitor of the IKK complex, showed a decrease in TNF-α expression and log fold decrease at pNF-kB. However, treatment with 2-HG showed no difference in response at pNF-κB compared with control. Lastly, we examined alterations of metabolism in the presence of 2-HG. Treatment with 2-HG reduced oxidative phosphorylation in vitro while increasing fatty acid uptake. Contrary to previous reports, 2-HG did not inhibit ATP synthase activity with acute exposure to 2-HG and assay against purified yeast F1 ATPase. Moreover, there was minimal effect on ATP production in isolated mouse liver mitochondria.

Our data showed that 2-HG produced in IDH-mutated AML caused decreased cytokine expression by inhibiting NFAT activation without affecting NF-κB phosphorylation. Additionally, 2-HG decreased oxidative phosphorylation in T cells which may directly affect T cell differentiation and TCR activation. This novel immunosuppressive mechanism of IDH-mutated AML demonstrates how oncogenic mutations contribute to immune evasion and has large implications for the use of immune-directed therapies in AML.

Citation Format: Zhuoyan Li, Bradley I. Reinfeld, Benjamin J. Reisman, Matthew Z. Madden, Chad R. Potts, Wendy K. Rathmell, Jeffrey C. Rathmell, Paul B. Ferrell. Aberrant TCR activation and altered metabolism drive decreased T cell cytokine production in IDH mutated acute myeloid leukemia [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 1025.