Author Manuscript Published OnlineFirst on March 12, 2019; DOI: 10.1158/2159-8290.CD-18-1040 Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. Mutant and wild-type isocitrate dehydrogenase 1 share enhancing mechanisms involving distinct tyrosine kinase cascades in cancer Running title: Tyrosine phosphorylation activates IDH1 and R132H mutant Dong Chen1,4,14, Siyuan Xia1,4,14, Mei Wang1,4,5,14, Ruiting Lin1,4, Yuancheng Li2,4, Hui Mao2,4, Mike Aguiar6, Christopher A. Famulare7, Alan H. Shih7, Cameron W. Brennan7, Xue Gao1,4, Yaozhu Pan1,8, Shuangping Liu1,9, Jun Fan3,4, Lingtao Jin1,4, Lina Song10, An Zhou10, Joydeep Mukherjee11, Russell O. Pieper11, Ashutosh Mishra12, Junmin Peng12, Martha Arellano1,4, William G. Blum1,4, Sagar Lonial1,4, Titus J. Boggon13, Ross L. Levine7, and Jing Chen1,4 1Department of Hematology and Medical Oncology 2Department of Radiology and Imaging Sciences 3Department of Radiation Oncology 4Winship Cancer Institute Emory University School of Medicine, Atlanta, GA 30322, USA 5Department of Pharmacy, Children’s Hospital of Soochow University, Suzhou, China, 215000 6Cell Signaling Technology, Inc. (CST), Danvers, MA 01923, USA 7Memorial Sloan-Kettering Cancer Center, New York, NY 10065, USA 8General Hospital of Lanzhou Military Region, Lanzhou, China, 730050 9Department of Pathology, Medical College, Dalian University, Dalian, China, 116622 10Department of Neurobiology, Morehouse School of Medicine, Atlanta, GA 30310, USA 11Department of Neurological Surgery, University of California, San Francisco, CA 94143, USA 1 Downloaded from cancerdiscovery.aacrjournals.org on September 29, 2021. © 2019 American Association for Cancer Research. Author Manuscript Published OnlineFirst on March 12, 2019; DOI: 10.1158/2159-8290.CD-18-1040 Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. 12Department of Structural Biology & Developmental Neurobiology, St. Jude Children's Research Hospital, Memphis, TN 38105, USA 13Department of Pharmacology, Yale University School of Medicine, New Haven, CT 06520, USA 14These authors contributed equally Corresponding Authors: Ross L. Levine, Memorial Sloan Kettering Cancer Center, 415 East 68th Street, RRL429, New York, NY 10065. Phone: 646-888-2796; Fax: 646-422-0856; E-mail: [email protected]; and Jing Chen, Department of Hematology and Medical Oncology, Emory University School of Medicine, Winship Cancer Institute of Emory, 1365-C Clifton Road NE, Suite C3002, Atlanta, GA 30322. Phone: 404-778-5274; Fax: 404-778-4755; E-mail: [email protected]. Conflict of Interest Statement: R.L.L. is on the supervisory board of Qiagen and is a scientific advisor to Loxo, Imago, C4 Therapeutics and Isoplexis, which each include an equity interest. He receives research support from and consulted for Celgene and Roche, he has received research support from Prelude Therapeutics, and he has consulted for Incyte, Novartis, Astellas, Morphosys and Janssen. He has received honoraria from Lilly and Amgen for invited lectures and from Gilead for grant reviews. ABSTRACT Isocitrate dehydrogenase 1 (IDH1) is important for reductive carboxylation in cancer cells, and IDH1 R132H mutant plays a pathogenic role in cancers including acute myeloid leukemia (AML). However, the regulatory mechanisms modulating IDH1 mutant and/or wild-type (WT) function remain unknown. Here we show that two groups of tyrosine kinases (TKs) enhance the activation of IDH1 mutant and WT through preferential Y42 or Y391 phosphorylation. Mechanistically, Y42 2 Downloaded from cancerdiscovery.aacrjournals.org on September 29, 2021. © 2019 American Association for Cancer Research. Author Manuscript Published OnlineFirst on March 12, 2019; DOI: 10.1158/2159-8290.CD-18-1040 Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. phosphorylation occurs in IDH1 monomers, which promotes dimer formation with enhanced substrate (isocitrate or α-ketoglutarate) binding, while Y42-phosphorylated dimers show attenuated disruption to monomers. Y391 phosphorylation occurs in both monomeric and dimeric IDH1, which enhances cofactor (NADP+ or NADPH) binding. Diverse oncogenic TKs activate Src to achieve Y42 and Y391 phosphorylation of IDH1, respectively, which contributes to reductive carboxylation and tumor growth, while FLT3 or FLT3-ITD mutant activate JAK2 to enhance IDH1 mutant activity through phosphorylation of Y391 and Y42, respectively, in AML cells. SIGNIFICANCE We demonstrated an intrinsic connection between oncogenic TKs and activation of WT and mutant IDH1, which involves distinct tyrosine kinase cascades in related cancers. In particular, these results provide an additional rationale supporting the combination of FLT3 and mutant IDH1 inhibitors as a promising clinical treatment of mutant IDH1-positive AML. INTRODUCTION The terms metabolic “reprogramming” and “rewiring” have emerged to describe the increasingly better understood metabolic changes observed in cancer cells (1,2). From a definitional perspective, “metabolic reprogramming” represents “software changes” in cancer cells and describes metabolic alterations that are normally induced by growth factors in proliferating cells but are hijacked by oncogenic signals; while "metabolic rewiring" represents “hardware changes” and describes metabolic alterations due to neo-functions of oncogenic mutants, which are not found in normal cells (3). For example, oncogenic signals reprogram cancer cells in an acute manner involving diverse post-translational modifications of metabolic enzymes that also exist in proliferating normal 3 Downloaded from cancerdiscovery.aacrjournals.org on September 29, 2021. © 2019 American Association for Cancer Research. Author Manuscript Published OnlineFirst on March 12, 2019; DOI: 10.1158/2159-8290.CD-18-1040 Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. cells (4). The identification of mutations in isocitrate dehydrogenase (IDH) 1 and 2 in glioma and acute myeloid leukemia (AML) represents a rewiring because the mutations confer a neo-function to IDH1/2 to produce the oncometabolite 2-hydroxyglutamate (2-HG) to regulate cancer epigenetics, which is not found in normal cells harboring wild type (WT) IDH1/2 (5-8). We previously reported that oncogenic BRAF V600E rewires the ketogenic pathway to allow cancer cells to benefit from ketone body acetoacetate-promoted BRAF V600E-MEK1 binding, which is not found in cells expressing BRAF WT (3). Thus, clearly distinguishing and characterizing metabolic “reprogramming” and “rewiring” in cancer cells offers apparent advantages to inform therapy development because targeting rewiring (e.g. IDH mutant inhibitors) in cancer cells will have minimal toxicity to normal cells. IDH1 and IDH2 are two highly homologous members of the IDH family of metabolic enzymes, and are located in the cytoplasm and mitochondria, respectively. IDH1/2 form homodimers and convert isocitrate to α-ketoglutarate (αKG) with the reduction of NADP+ to NADPH (9). αKG is a key intermediate in the Krebs cycle and glutaminolysis, an important nitrogen transporter, and a ligand for αKG-dependent enzymes including histone demethylases such as Jhd1 and methylcytosine dioxygenase enzyme TET2 (10). NADPH not only fuels macromolecular biosynthesis such as lipogenesis but also functions as a crucial antioxidant to quench the reactive oxygen species (ROS) produced during rapid proliferation of cancer cells, which is important for the maintenance of cellular redox homeostasis to protect against toxicity of ROS and oxidative DNA damage (11). Thus, IDH1/2 are important for many metabolic processes in cells including bioenergetics, biosynthesis, and redox homeostasis. Moreover, recent evidence demonstrates that IDH1/2 play an important role in reductive carboxylation that is enhanced in cells under hypoxia, allowing the generation of isocitrate/citrate from αKG and glutamine, which is in particular 4 Downloaded from cancerdiscovery.aacrjournals.org on September 29, 2021. © 2019 American Association for Cancer Research. Author Manuscript Published OnlineFirst on March 12, 2019; DOI: 10.1158/2159-8290.CD-18-1040 Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. important in cancer cells for producing citrate and acetyl-CoA that are essential for lipid synthesis during tumorigenesis, as well as reducing mitochondrial ROS to sustain redox homeostasis during anchorage-independent growth (12,13). Missense mutations of R132 in the enzyme active site of IDH1 were identified in patients with glioblastoma (GBM) and AML cases (5-7,14,15), and corresponding IDH2 R172 mutations as well as a novel R140Q mutant repeatedly occur in AML patients (14,16,17). Overall, IDH1/2 mutations are identified in >75% of grade 2/3 glioma and secondary GBM cases and >20% of AML cases. IDH mutations were also identified in other cancer types such as chondrosarcoma and cholangiocarcinoma (9). IDH mutations are heterozygous events, resulting in loss-of-function of wild type IDH1 enzyme activity but a gain-of-function to mutant IDH1, allowing NADPH- dependent reduction of αKG to produce the oncometabolite 2-HG. 2-HG competitively inhibits the function of αKG-dependent enzymes such as TET2, which in turn causes epigenetic dysregulation including DNA hypermethylation in both GBM and AML, and consequent block of differentiation (18). In AML, IDH1 R132,