Kinase Networks Regulate Metabolism: I’D(H1) Never Have Guessed! Sarah Horton and Brian J.P

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IN THE SPOTLIGHT Kinase Networks Regulate Metabolism: I’D(H1) Never Have Guessed! Sarah Horton and Brian J.P. Huntly Summary: Mutations in isoforms of isocitrate dehydrogenase (IDH) enzymes are described in multiple cancers and both mutant and wild-type IDH are important for the generation and maintenance of tumors, but how their activity is regulated is poorly understood. An article in this issue of Cancer Discovery identifi es a novel post- translational mechanism of IDH1 regulation involving phosphorylation of specifi c tyrosine residues by a network of kinases that alter the specifi city of substrate and cofactor binding, dimer formation, and ultimately enzyme activity.

See related article by Chen et al., p. 756 (8).

Cancer cells have increased requirements for energy and and an alteration of gene expression that contributes to the biomass production, and these requirements are served malignant phenotype ( 2, 5 ). Recently small-molecule inhibi- through alterations of their metabolic circuitry (1 ). Central tors of IDH1 and IDH2 have been developed that have dem- nodes within the circuitry of both normal and malignant cells onstrated considerable preclinical effi cacy ( 6, 7 ). This promise are the enzymes isocitrate dehydrogenase 1 and 2 (IDH1 and has been vindicated in early-phase trials and, within the last IDH2). These enzymes are located within the cytoplasm and two years, enosidenib (AG-221) and ivosidenib (AG-120) have mitochondria, respectively, and normally catalyze the oxida- been approved by the FDA for the treatment of IDH2- and tive decarboxylation of isocitrate to α-ketoglutarate (αKG, IDH1-mutant AML, respectively. However, how mutant and also known as 2-oxoglutarate) in the Krebs cycle, a stepwise wild-type (WT) IDH proteins are further regulated is poorly reaction that also produces CO2 and NADPH. αKG is an understood. intermediate in both the Krebs and glutaminolysis cycles, an In a very interesting article in this issue, Dong Chen and col- important nitrogen transporter for amino acid synthesis and leagues, from the laboratories of Jing Chen and Ross Levine ( 8 ), a cofactor for αKG-dependent dioxygenase enzymes, includ- describe and characterize a novel mechanism regulating IDH1 ing the TET DNA demethylases and JmJC histone demethy- activity. This occurs at the post-translational level and involves lases ( 2 ). NADPH provides reducing equivalents to generate phosphorylation of both the WT and mutant IDH1 proteins on and sustain reduced glutathione to maintain redox homeo- a specifi c tyrosine by a communicating network of kinases stasis within rapidly dividing cells, and additionally fuels ( Fig. 1 ). Their initial observation built on previous fi ndings macromolecular biosynthesis, including cholesterol synthesis of synergistic effi cacy in preclinical models when AG-221 was and fatty-acid chain elongation. Recently, cancer-associated combined with a FLT3 inhibitor ( 9 ). Interestingly, when the mutations in both IDH1 and IDH2 have been described authors assessed similar synergy between the IDH1 inhibitor across a number of malignancies, including glioma, glioblas- AG-120 and the FLT3 inhibitor quizartinib in primary AML toma multiforme, and acute myeloid leukemia (AML; refs. 3, blasts across a range of patient genotypes (WT or mutant IDH1 4 ). These mutations are hemizygous and alter specifi c argi- and either mutant or WT FLT3 ), to their surprise they noted nine residues (R132 in IDH1 and R140 and R172 in IDH2 ), that both AG-120 and quizartinib reduced the biochemical leading to a neomorphic enzymatic function that further activity of IDH1. The authors then linked this to tyrosine phos- reduces αKG to 2-hydroxyglutamate (2-HG) in an NADPH- phorylation of IDH1 using in vitro kinase assays, where they dependent manner. The so-called “oncometabolite” 2-HG could demonstrate a phosphorylation-dependent increase in is a competitive inhibitor of αKG-dependent dioxygenase enzymatic activity upon incubation with a number of recom- enzymes and leads to DNA and histone hypermethylation binant kinases. Performing the same assays with recombinant IDH1 systematically mutagenized to alter tyrosine residues (Y → F), they identifi ed critical residues to be Y42 and Y391 and could further link a group of tyrosine kinases (TK; which Wellcome Trust-MRC Cambridge Stem Cell Institute, Cambridge, United Kingdom. Department of Haematology, University of Cambridge, Cam- they called Group I kinases) including EGFR, JAK2, ABL1, bridge, United Kingdom. PDGFR, and FGFR1 to phosphorylation of Y42 and a separate Corresponding Author: Brian J.P. Huntly, University of Cambridge, Jeffrey group of kinases (Group II kinases), including Src and FLT3, Cheah Biomedical Centre, Hills Road, Cambridge CB2 0XY, United King- to Y391 phosphorylation. Moreover, biochemical and later dom. Phone: 4412-2333-1153; Fax: 4412-2376-2670; E-mail: bjph2@ chemical inhibition experiments could demonstrate a recipro- cam.ac.uk cal hierarchical/network relationship between the Group I and Cancer Discov 2019;9:699–701 Group II kinases, with evidence of Group I activating Group II doi: 10.1158/2159-8290.CD-19-0373 kinases (i.e., FGFR1/ABL1 activating Src kinases) or vice versa © 2019 American Association for Cancer Research. (i.e., FLT3 activating JAK2).

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A Group I TK Group II TK

EGFR, JAK2, FLT3, ABL1, PDGFR, Src kinases FGFR1 Y391 Y42

P Isocitrate/αKG

NADP+/H P Increased substrate binding P P Y391 Y42 P P Increased cofactor binding WT/mut IDH1

Increased dimer formation and stability

Activity

B Physiologic Pathologic

IDH1 WT IDH1R132H

Metabolic advantage to Metabolic advantage Metabolic advantage support normal proliferation Epigenetic dysfunction

Tumor formation and maintenance AML/glioma

Figure 1. Regulation of WT and mutant IDH1 function through Y42 and Y391 phosphorylation. A, Phosphorylation of WT and mutant IDH1 at residues Y42 and Y391 by the indicated Group I and Group II kinases leads to functional activation of IDH1. For Y42 phosphorylation, this involves increased dimer formation and stability and also increased binding of the reaction substrate, isocitrate for WT IDH1 and αKG for mutant IDH1. In contrast, Y391 phosphorylation increases cofactor binding—NADP+ for WT IDH1 and NADPH for mutant IDH1. Arrows between Group I and Group II kinases indicate that Group I and Group II kinases can activate each other. B, Functional consequences of IDH1 activation.

To understand the mechanisms of altered enzyme function their IDH1 and FLT3 mutational status, demonstrating that upon Y42 and Y391 phosphorylation, they next performed tyrosine phosphatase or FLT3 inhibitor treatment of either sophisticated in vitro biochemical analyses. These determined mutant or WT IDH1 cells reduced Y42 and Y391 levels, IDH1 that dimerization of IDH1 increased the enzymatic activity of dimerization, and enzymatic activity in all cells and 2-HG WT and mutant-containing dimers, and also that phospho- levels in mutant cells. They also corroborated their findings rylation of Y42 led to an increase in both dimer formation across tissue arrays from a wide range of human tumors, and stability. Y42 phosphorylated IDH1 (WT and mutant) demonstrating an increased Y42 phosphorylation of IDH1 also bound more avidly to labeled isocitrate and αKG, sub- protein in comparison with normal control tissue. strates for WT, and mutant IDH1, respectively. Conversely, On a basic level, these studies demonstrate a novel link Y391 phosphorylated IDH1 did not alter dimerization or between intracellular signaling and central metabolism. As this substrate binding, but instead demonstrated increased bind- communication requires neither mutant IDH1 nor mutant ing to NADP+ and NADPH, the cofactors for the oxidative kinases, it is likely that it represents a normal physiologic carboxylation reaction of WT IDH1 and the further reduc- response, coupling increased energy and biomass provision tion performed by mutant IDH1, respectively. Functionally, to proliferation, and it is not surprising that this process abrogating tyrosine phosphorylation at these critical tyrosine is hijacked during malignant transformation. It will be of residues led to growth retardation of both mutant IDH1 AML interest to determine whether other critical metabolic master and WT IDH1 lung cancer cells. This phenotype was linked regulators are similarly modulated in normal physiology, and to a decrease in IDH enzymatic activity, and also to reduced whether this process is co-opted in malignant transformation. 2-HG levels and histone hypermethylation in the context Of note, the mechanisms described seem highly specific for of IDH1-mutant AML. Finally, the authors confirmed their IDH1; they cannot occur at the corresponding residues in findings in primary AML cells from patients, irrespective of IDH2, where tyrosine residues are replaced by phenylalanines.

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This, in turn, asks the question of whether similar post- tyrosine signaling from both WT and oncogenic mutant TKs translational phosphorylation events occur as a regulatory has been shown to contribute to the malignant phenotype. mechanism for IDH2 and, if so, at which critical residues and by which kinases? The same authors, along with others, have Disclosure of Potential Conflicts of Interest previously shown that TKs do function in the mitochondria No potential conflicts of interest were disclosed. to alter the function of master regulators of metabolism such Published online June 3, 2019. as pyruvate dehydrogenase (10). However, although TKs such as ABL1 and some Src kinases have been demonstrated to be active in the mitochondria, general TK activity is less in this REFERENCES compartment. Therefore, the identity of any regulatory kinases . 1 Sullivan LB, Gui DY, Vander Heiden MG. Altered metabolite levels in is likely to differ between cytosol and mitochondria and will cancer: implications for tumour biology and cancer therapy. Nat Rev need to be investigated. Cancer 2016;16:680–93. These data also suggest that further studies are warranted 2. Dang L, White DW, Gross S, Bennett BD, Bittinger MA, Driggers EM, to determine the functional consequences of phosphoryla- et al. Cancer-associated IDH1 mutations produce 2-hydroxyglutarate. tion of other tyrosine residues within IDH1. For example, the Nature 2009;462:739–44. 3. Parsons DW, Jones S, Zhang X, Lin JC, Leary RJ, Angenendt P, et al. authors cite the proposed critical role of tyrosine 139 (Y139) An integrated genomic analysis of human glioblastoma multiforme. for full catalytic activity of IDH1. However, it is not currently Science 2008;321:1807–12. known whether this role involves direct phosphorylation of 4. Mardis ER, Ding L, Dooling DJ, Larson DE, McLellan MD, Chen K, Y139. Other important questions relate to the mechanisms et al. Recurring mutations found by sequencing an acute myeloid downstream of Y42 and Y391 phosphorylation and to their leukemia genome. N Engl J Med 2009;361:1058–66. exact stoichiometry. These include the following: What is the 5. Lu C, Ward PS, Kapoor GS, Rohle D, Turcan S, Abdel-Wahab O, et al. IDH mutation impairs histone demethylation and results in a block specific structural basis of increased dimer formation, dimer to cell differentiation. Nature 2012;483:474–8. stability, and increased substrate binding for Y42 phospho- 6. Wang F, Travins J, DeLaBarre B, Penard-Lacronique V, Schalm S, rylation? Similarly, what is the structural basis that explains Hansen E, et al. Targeted inhibition of mutant IDH2 in leukemia cells the increase in cofactor binding upon Y391 phosphorylation? induces cellular differentiation. Science 2013;340:622–6. Regarding stoichiometry, do both members of the dimer, or 7. Rohle D, Popovici-Muller J, Palaskas N, Turcan S, Grommes C, only one, need to be phosphorylated on Y42 and Y391, and Campos C, et al. An inhibitor of mutant IDH1 delays growth and promotes differentiation of glioma cells. Science 2013;340:626–30. what is the role for compound (Y42/391) phosphorylation? 8. Chen D, Xia S, Wang M, Lin R, Li Y, Mao H, et al. Mutant and To answer these questions, further detailed structural and wild-type isocitrate dehydrogenase 1 share enhancing mechanisms mechanistic studies will be required. The findings of Chen and involving distinct tyrosine kinase cascades in cancer. Cancer Discov colleagues also have therapeutic implications. They suggest 2019;9:756–77. that the efficacy of TK inhibitors in tumors may relate, at least 9. Shih AH, Meydan C, Shank K, Garrett-Bakelman FE, Ward PS, Intle- in part, to their modulation of IDH1 metabolic function. Fur- kofer AM, et al. Combination targeted therapy to disrupt aberrant thermore, they provide a further rationale for combining IDH1 oncogenic signaling and reverse epigenetic dysfunction in IDH2- and TET2-mutant acute myeloid leukemia. Cancer Discov 2017;7:494–505. inhibition with TK inhibitors in patients with IDH1-mutant 10. Hitosugi T, Fan J, Chung TW, Lythgoe K, Wang X, Xie J, et al. Tyrosine tumors, where both metabolic and epigenetic abnormalities phosphorylation of mitochondrial pyruvate dehydrogenase kinase 1 are targeted. This rationale is most compelling in AML, where is important for cancer metabolism. Mol Cell 2011;44:864–77.

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Sarah Horton and Brian J.P. Huntly

Cancer Discov 2019;9:699-701.

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