Idh1 mutations contribute to the development of T-cellmalignancies in genetically engineered miceZhenyue Haoa,1, Rob A. Cairnsa,1, Satoshi Inouea, Wanda Y. Lia, Yi Shengb, François Lemonniera,c, Andrew Wakehama,Bryan E. Snowa, Carmen Dominguez-Brauera, Jing Yea, Dana M. Larsend, Kimberly S. Straleye, Erica R. Tobine,Rohini Narayanaswamye, Philippe Gaulardc, and Tak W. Maka,f,2

aThe Campbell Family Institute for Breast Cancer Research at Princess Margaret Cancer Centre, University Health Network, Toronto, ON, M5G 2C1, Canada;bDepartment of Biology, York University, Toronto, ON, M3J 1P3, Canada; cInserm U955, Université Paris Est and Département de Pathologie, GroupeHenri-Mondor Albert-Chenevier, Assistance Publique–Hôpitaux de Paris, Créteil, 94000 France; dMbed Pathology, Toronto, Ontario, M5N 2G4, Canada;eAgios Pharmaceuticals, Cambridge, MA, 02139; and fDepartment of Medical Biophysics, University of Toronto, Toronto, ON, M5G 1L7, Canada

Contributed by Tak W. Mak, December 23, 2015 (sent for review December 14, 2015; reviewed by Navdeep S. Chandel and Jorge Moscat)

Gain-of-function mutations in isocitrate dehydrogenase 1 (IDH1) arekey drivers of hematopoietic malignancies. Although these mutationsare most commonly associated with myeloid diseases, they also occurin malignancies of the T-cell lineage. To investigate their role in thesediseases and provide tractable disease models for further investiga-tion, we analyzed the T-cell compartment in a conditional knock-in(KI) mouse model of mutant Idh1. We observed the development of aspontaneous T-cell acute lymphoblastic leukemia (T-ALL) in these an-imals. The disease was transplantable and maintained expression ofmutant IDH1. Whole-exome sequencing revealed the presence of aspontaneous activating mutation in Notch1, one of the most commonmutations in human T-ALL, suggesting Idh1 mutations may have thecapacity to cooperate with Notch1 to drive T-ALL. To further investi-gate the Idh1mutation as an oncogenic driver in the T-cell lineage, wecrossed Idh1-KI mice with conditional Trp53 null mice, a well-charac-terized model of T-cell malignancy, and found that T-cell lymphoma-genesis was accelerated in mice bearing both mutations. Becauseboth IDH1 and p53 are known to affect cellular metabolism, we com-pared the requirements for glucose and glutamine in cells derivedfrom these tumors and found that cells bearing the Idh1 mutationhave an increased dependence on both glucose and glutamine. Thesedata suggest that mutant IDH1 contributes to malignancy in the T-celllineage and may alter the metabolic profile of malignant T cells.

isocitrate dehydrogenase | T-ALL | lymphoma | p53

Somatic mutations in isocitrate dehydrogenase 1 (IDH1) arefrequently observed in a number of malignancies, including

glioma, cholangiocarcinoma, chondrosarcoma, and several hema-tological malignancies (1). IDH1 is a cytoplasmic enzyme thatcatalyzes the NADP-dependent conversion of isocitrate toα-ketoglutarate (αKG). Mutations in IDH1 at arginine 132 (R132)cause an enzymatic gain of function that results in the NADPH-dependent conversion of αKG to D-2-hydroxyglutarate (2HG) (2).This metabolite is normally maintained at very low levels in cellsand tissues and is not part of any known productive metabolicpathway. However, in cells and tissues of patients with IDH1 mu-tant tumors, 2HG builds up to high levels and is thought to con-tribute to tumorigenesis by inhibiting a class of αKG-dependentenzymes (1). The precise effects important for driving tumorigenesisdownstream of IDH1 mutations are not fully understood and maydiffer between disease states.In the hematopoietic system, IDH1 mutations are most often

associated with myeloid diseases, where they are commonly foundin myelodysplastic syndrome and acute myeloid leukemia (3).However, IDH1 mutations are also found in a small proportion ofadult T-cell acute lymphoblastic leukemia (T-ALL) (4, 5). T-ALL isan aggressive malignancy of developing T cells and is responsible for∼25% of adult ALL (6, 7). T-ALL is thought to arise via a multistepprocess of oncogenic mutation that leads to the transformation ofimmature T cells. The genetic landscape of the disease has beencharacterized, and a large number of driver mutations have been

identified (6). The most common genetic feature of T-ALL is thepresence of activating mutations in Notch1, which are present inmore than 50% of patients (8). Interestingly, IDH1 mutations seemto be confined to a subset of adult patients with T-ALL bearing animmature T-cell gene expression signature and harboring otheroncogenic mutations in genes more commonly associated withmyeloid malignancy, including Flt3 andDNMT3A (4, 9). This subsetof T-ALL has recently been recognized as a distinct disease entitycalled early T-cell precursor T-ALL and is associated with therapyresistance and a particularly poor outcome (10). The role of IDH1mutations in this subset of T-ALL is not understood.Using a myeloid lineage-specific conditional Idh1-R132-KI

mouse model, we previously showed that mutant IDH1 partiallyblocks differentiation and produces a hematopoietic phenotypesimilar to human myelodysplastic syndrome (11). In this study, wecrossed the Idh1-R132-KI mouse with Vav-cre animals to introducethe IDH1 R132 mutation into the entire hematopoietic system toinvestigate the role of Idh1 mutations in T-cell malignancy.

ResultsDevelopment of T-ALL in a Vav-Idh1-KI Mouse. To investigate effectsof Idh1 mutations in the lymphoid system, Idh1-R132-KI micewere crossed with Vav-cre mice to produce Vav-Idh1-KI animals

Significance

Isocitrate dehydrogenase 1 (IDH1) mutations are drivers of he-matological malignancy. Although these mutations are mostoften associated with myeloid disease, they are also found inlymphoid malignancies, including T-cell acute lymphoblasticleukemia (T-ALL). Treatment strategies targeting these muta-tions are currently being devised, including small-molecule in-hibitors of the mutant IDH1 enzyme. A better understanding ofthe role of these mutations in tumorigenesis and their effects ontumor cells will allow these treatment strategies to be effec-tively translated to the clinic. Here we show that Idh1 mutationscan contribute to the development of T-cell malignancies, in-cluding T-ALL, using a conditional knock-in mouse model. Thesemouse models provide a platform for further evaluation oftreatment strategies for T-cell malignancy.

Author contributions: Z.H., R.A.C., S.I., Y.S., and T.W.M. designed research; Z.H., R.A.C.,S.I., W.Y.L., Y.S., F.L., A.W., B.E.S., C.D.-B., J.Y., D.M.L., K.S.S., E.R.T., R.N., and P.G. per-formed research; Z.H., R.A.C., S.I., Y.S., F.L., J.Y., D.M.L., K.S.S., E.R.T., R.N., and P.G. ana-lyzed data; and R.A.C. and T.W.M. wrote the paper.

Reviewers: N.S.C., Northwestern University; and J.M., Sanford Burnham Prebys Institute.

The authors declare no conflict of interest.

Freely available online through the PNAS open access option.1Z.H. and R.A.C. contributed equally to this work.2To whom correspondence should be addressed. Email: tmak@uhnresearch.ca.

This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10.1073/pnas.1525354113/-/DCSupplemental.

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that express the mutant protein throughout the hematopoieticsystem (12). As expected, these mice develop the myeloid phe-notypes previously described (11). However, in addition, weobserved the development of an aggressive, peripheral lymph-adenopathy accompanied by splenomegaly in a Vav-Idh1-KImouse (Fig. 1A). Histologically, neoplastic lymphocytes effacedmore than 80% of the bone marrow, and diffuse sheets of thesecells completely obliterated normal splenic and lymph node ar-chitecture (Fig. 1A). Flow cytometry showed that the malignantlymphocytes in the spleen, lymph nodes, and bone marrow wereCD8-positive T cells (Fig. 1B). On the basis of expression of thesurface markers CD44 and CD24, these cells resembled an ab-normal population of early, immature T lymphocytes (Fig. 1C).As a result of these findings, the neoplastic disease in this mousewas characterized as T-ALL.To further define this disease, T-ALL cells and tissue frag-

ments isolated from the mutant mouse were transplanted intosyngeneic C57BL6/J mice s.c. or into the mammary fat pad.Recipient animals developed a similar aggressive T-ALL phe-notype within 3–6 wk that involved the bone marrow, secondarylymphoid organs, and other tissues, including the kidney andliver (Fig. 1A). The disease could be serially passaged (fivepassages have been performed), indicating a fully transformed

phenotype. Interestingly, malignant cells could not be grown insuspension culture under the media conditions tested, which isconsistent with previous difficulties encountered when attempt-ing to maintain Idh1 mutant cells in culture. To determinewhether the activity of the mutant IDH1 enzyme was retained intumor cells on serial passage, 2HG levels were measured in se-rum and urine. In both cases, levels of 2HG were increasedbetween 10- and 100-fold compared with wild-type animals, andthe concentrations observed in transplanted animals were similarto those observed in the initial T-ALL mouse (Fig. 1D).

Whole-Exome Sequencing Identifies a Spontaneous Notch1 Mutationin Idh1-KI T-ALL. Because the T-ALL phenotype is not normallyobserved in these animals (penetrance of 1/110 mutant mice an-alyzed), we hypothesized that a spontaneous cooperating mutationcontributed to this malignancy. To investigate this possibility, weconducted whole-exome sequencing on cells derived from theinitial tumor and a tumor after the third serial passage. Bonemarrow samples from a wild-type C57BL/6J mouse and a healthyage-matched Vav-Idh1-KI mouse were sequenced as controls.Genomic variants (single nucleotide variants and short indels)relative to the mm9 reference genome were identified usingHaplotype caller. We next removed variants present in dbSNP or

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Fig. 1. Development of T-ALL in a Vav-Idh1-KI mouse. (A) Gross pathology and H&E-stained histological sections of the indicated tissues from a Vav-Idh1-KImouse diagnosed with T-ALL, and from a T-ALL-bearing animal after serial transplantation of the disease (fourth serial passage). (D) 2HG concentration inurine and serum from the indicated mice (wild-type, n = 4; T-ALL, n = 1; Transplant, n = 4). Error bars represent SD. *Significant difference compared withwild-type (P < 0.01). (B) Flow cytometry characterizing the cell surface phenotype of the malignant T cells isolated from spleen, lymph node (LN), and bonemarrow (BM) of the T-ALL mouse compared with cells isolated from a wild-type animal. (C) Flow cytometry characterizing the malignant T-ALL cells relative toT cells obtained from the lymph node of a wild-type animal.

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in the Sanger Mouse Genome Project SNP database (13). Thisstep eliminates many false-positive calls resulting from strainvariation, as well as deficiencies in the reference sequence. Tofurther focus on likely somatic mutations, we eliminated variantsidentified in the wild-type C57BL/6J mouse sample or present insamples from two other C57BL/6J mice from our breeding colonythat had been previously subjected to whole-exome sequencing,using the same analysis pipeline to eliminate alignment artifactsand strain background differences. Finally, we restricted ouranalysis to nonsynonymous exonic variants (Table 1). Interest-ingly, the number of exonic variants and the variant allele fre-quency distribution in the healthy Vav-Idh1-KI bone marrowsample was similar to that found in the two T-ALL tumor samples(Table 1; Fig. 2A). This suggests that the development of thisT-ALL was not associated with a dramatic increase in genomicinstability, which is consistent with observations in humanT-ALL. There was a high concordance in the identities of thealtered genes in the initial T-ALL sample and the third passagetumor, further suggesting the genome of this tumor was relativelystable during serial passage (Fig. 2B). There was also a significantoverlap between the genes altered in the tumor samples and thosefound altered in the healthy Vav-Idh1-KI control bone marrow(Fig. 2B). This may be a result of false-positive identification of

variants caused by misalignment of short-read sequences in highlyrepetitive genes.Upon inspection of the list of altered genes (Dataset S1), a

mutation in Notch1 was identified. This mutation was present inboth the initial T-ALL tumor and the transplanted tumor, wherethe variant allele frequencies were 0.46 (51/95 reads) and 0.49(37/72 reads), respectively, suggesting a heterozygous mutation.This mutation was not present in the healthy Vav-Idh1-KI bonemarrow sample. The mutation is an 11-base pair deletion causinga frameshift at amino acid Q2415 (Q2415fs), in the PEST do-main of the Notch1 protein, which is one of two regions com-monly found mutated in human T-ALL (Fig. 2C). Mutationsin the proline, glutamate, serine, and threonine rich (PEST)domain cause increased stability and increased nuclear re-tention of the intracellular domain of Notch, and thereby in-crease the transcription of a number of Notch target genes,including Myc, which is thought to contribute to T-ALL tu-morigenesis (14, 15). Consistent with this mechanism of ac-tion, MYC levels were increased in T-ALL cells relative tothymic T cells from wild-type mice (Fig. 2D), suggesting theQ2415fs mutation identified in this tumor causes Notch pathwayactivation and may have contributed to the development ofT-ALL in this animal.

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Fig. 2. Exome sequencing identifies a T-ALL-associated Notch1 mutation in Vav-Idh1-KI T-ALL cells. (A) The allele frequency distribution of exonic variantsidentified by whole-exome sequencing of malignant cells from the initial T-ALL-bearing mouse, an animal bearing T-ALL after serial transplant of the disease(fourth passage), and bone marrow from a healthy, age-matched Vav-Idh1-KI mouse. (B) A Venn diagram illustrating the unique altered genes in eachsample. (C) Schematic of the Notch1 protein (ECD, extracellular domain; ICD, intracellular domain) illustrating the major protein domains. ANK, ankyrinrepeats; LNR, LIN12/Notch repeats; PEST, PEST domain; RAM, RAM domain; TAD, transactivation domain; TM, transmembrane domain. Hotspots for T-ALL-associated mutations are shown in red, and the position of the Q2415fs mutation identified in the Vav-KI-IDH1 T-ALL is indicated. (D) Western blot showinglevels of MYC in cells isolated from wild-type thymus, and Vav-Idh1-KI T-ALL cells (two exposures are shown). Tubulin is shown as a loading control.

Table 1. Identification of coding exonic variants from whole-exome sequencing

Sample All variants* Excluding SNPs†Excluding wild-type

variants Exonic variantsUnique genes

altered

Wild-type B6/J bone marrow 5,379 3,744 0 0 0Idh1-KI bone marrow 17,806 5,748 2,483 315 91T-ALL 21,454 6,123 2,773 229 92T-ALL transplant 21,080 6,061 26,84 237 100

*Relative to the mm9 reference genome.†Compared with dbSNP128 and the Sanger Mouse Genomes Project SNP database.

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Idh1Mutations Accelerate Development of Thymic Lymphoma in Vav-p53fl/fl Mice. To further investigate the effect of Idh1mutations ontumorigenesis in the T-cell lineage, we crossed Vav-Idh1-KI micewith conditional Trp53 null mice (p53fl/fl). Loss of p53 in thiscontext is known to cause thymic T-cell lymphoma (16, 17). Asexpected, Vav-p53fl/fl mice developed thymic lymphoma with amedian latency of ∼200 d (Fig. 3A). The addition of the Idh1mutation significantly accelerated the development of thymiclymphoma and decreased the overall survival of double-mutantanimals (Fig. 3A). Vav-Idh1-KI mice also displayed a shortenedlife span compared with wild-type animals, but this was not aresult of abnormalities in the lymphoid system (Fig. 3A). Theprecise cause of the shortened life span in these animals is notfully understood, but appears to be related to the myelodys-plastic syndrome-like phenotype, which includes splenomegalyand anemia.Histological examination of the tumors that arose in Vav-p53fl/fl

and Vav-Idh1-KI p53fl/fl animals showed no differences between thetwo diseases. Both sets of animals developed large localized thymicmasses composed of sheets of neoplastic lymphocytes (Fig. 3B).Flow cytometric analysis confirmed that these were T-cell lympho-mas, although the cells from double-mutant animals displayed a cellsurface phenotype more similar to activated CD8 T-cells, withhigher expression of CD44 and lower expression of CD62Lcompared with cells from Vav-p53fl/fl lymphomas (Fig. 3B). Thus,introduction of an Idh1mutation accelerated the development ofT-cell malignancy in this well-characterized mouse model.

Idh1 Mutations Render p53 Null Lymphoma Cells More Sensitive toNutrient Withdrawal in Vitro. Both Vav-p53fl/fl and Vav-Idh1-KIp53fl/fl double-mutant lymphoma cells could be cultured in vitro.Because both IDH1 and p53 are known to affect cellular me-tabolism, we took this opportunity to determine whether theintroduction of mutant IDH1 altered the nutrient requirementsof these T-cell lymphoma cells. In a short-term nutrient depri-vation assay, we found that double-mutant cells were more

sensitive to withdrawal of glucose or glutamine from the growthmedia (Fig. 4). These data suggest that although the presence ofmutant IDH1 accelerates tumorigenesis in p53 null T-cell pre-cursors, these cells may be more dependent on both glucose andglutamine for maintenance of viability.

DiscussionTaken together, these data support the concept that Idh1 mu-tations can cooperate with other oncogenic events to drive ma-lignant transformation in the T-cell lineage. Although this studyfocused exclusively on mutations in Idh1, it should be noted thatIDH2 mutations are also observed in T-cell malignancies, in-cluding T-ALL. IDH2 is the mitochondrial counterpart of IDH1,and similar mutations in critical arginine residues of IDH2 leadto the production of 2HG. Although mutations in both genesresult in gain of enzymatic function and production of 2HG,there are significant differences in the frequencies of IDH1 andIDH2 mutations in different diseases (1). Even among T-cellmalignancies, this is the case. Whereas both IDH1 and IDH2mutations are observed in adult early T-cell precursor T-ALL, asignificant proportion of angioimmunoblastic T-cell lymphomasharbor exclusively IDH2 mutations (18). A better understandingof how these mutations affect T-cell development and malignanttransformation may lead to an understanding of the differenteffects caused by mutations in these two very similar genes.The development of a T-ALL-like disease in a Vav-Idh1-KI

mouse is intriguing. The identification of a spontaneous, canonicalT-ALL-associated Notch1 mutation in this tumor suggests Idh1mutations can cooperate with Notch1 mutations to drive the de-velopment of this disease. Previous work using mouse models ofcommon human Notch1 mutations, including those in the PESTdomain similar to the mutation characterized here, have shownthat similar to mutant Idh1, these Notch1 mutations alone are notsufficient to give rise to T-ALL (19). However, these mutationscan cooperate with other oncogenic mutations, including those in

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Fig. 3. Mutant Idh1 accelerates development of thymic lymphoma in Vav-p53fl/fl mice. (A) Kaplan-Meier curve showing overall survival of the indicatedgenotypes. All animals carry the Vav-cre allele. All curves are significantly different (P < 0.01, log-rank test). (B) Histology of representative thymic lymphomasin Vav-p53fl/fl and Vav-p53fl/fl Idh1-KI animals at two levels of magnification. (C) Representative flow cytometry characterization of the cell surface markerprofiles of cells derived from the thymus (wild-type and Vav-Idh1-KI) or thymic T-cell lymphomas (Vav-p53fl/fl and Vav-Idh1-KI p53fl/fl) of the indicatedgenotypes.

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Kras, to accelerate the development of lymphoid malignancy (19).A number of studies have identified Myc as the Notch target mostlikely to be responsible for its effects on the development of T-ALL(14, 15, 20). Given that MYC levels were elevated in the Vav-Idh1-KI T-ALL cells, it will be important to investigate whether mutantIDH1 can cooperate with MYC in driving tumorigenesis in thehematopoietic system. Future work will seek to formally investigatethe contribution of Idh1 and Idh2mutations to the development ofT-ALL, in combination with other T-ALL-associated mutations.This may be particularly relevant for the newly recognized subset ofearly T-cell precursor T-ALL that appears to harbor other muta-tions more often associated with myeloid neoplasia.The fact that Idh1 mutations accelerate the development of

T-cell lymphoma in a well-characterized model driven by loss ofp53 further illustrates the capacity of mutant IDH1 to contributeto tumorigenesis in the T-cell lineage. In this context, Idh1mutations result in accelerated thymic T-cell lymphomagenesis,rather than disseminated T-ALL, or a more severe myeloid phe-notype, reinforcing the concept that Idh1 mutations can con-tribute to different disease states, depending on the identity ofthe cooperating mutations.Metabolic pathways and nutrient requirements of T cells are

remarkably dynamic and can be rapidly remodeled during dif-ferentiation and activation and in response to extracellularconditions (21). Because mutant IDH1 alters core metabolicpathways involving the NADP:NADPH redox couple and αKG,and p53 has been shown to have effects on metabolic regulation(22), we investigated the nutrient requirements of malignant Tcells isolated from thymic lymphomas, taking advantage of theopportunity to examine the effects of mutant Idh1 in a controlledin vitro system. The finding that mutant IDH1 renders cells moresensitive to nutrient deprivation suggests core metabolic path-ways are altered by the presence of this mutant enzyme. Furtherwork will be required to define the precise mechanisms re-sponsible for these effects and to determine whether they mayrepresent vulnerabilities that may be exploited for therapy.There is very little known regarding the effects of IDHmutations

on tumorigenesis in the T-cell lineage. First, it is unclear whetherthe same mechanisms responsible for malignant transformation inthe myeloid lineage, or in other tissues, are also important for

development and progression of T-cell malignancies. Second, it isunclear whether IDH mutations are early initiating events or actlater in the multistep process of tumorigenesis in the lymphoidsystem. Finally, it is unclear whether the presence of IDH muta-tions in T-cell diseases may offer opportunities to personalizetherapy. The mouse models described here will provide platformsto answer some of these questions and provide opportunities toevaluate treatment strategies in these disease states.

Materials and MethodsAnimals. Conditional Idh1-R132-KI (11, 23) and conditional Trp53 knockout(p53fl/fl) (24) mice have been described previously. Idh1-KI mice and p53fl/fl

mice were bred with Vav-cre mice (Jackson Laboratories; cat. no. 008610) toproduce Vav-Idh1-KI, Vav-p53fl/fl, and Vav-Idh1-KI p53fl/fl double-mutantanimals. For the data shown in Fig. 3, the strain background was C57BL/6 ×129Ola9; F3, whereas for all other experiments, the strain background wasC57BL/6 (F10). To serially transplant tumors, 2–3 mm3 tumor fragments, orsingle-cell suspensions in 50% PBS:50% (vol/vol) Matrigel (Corning) weresurgically implanted s.c. or in the mammary fat pad, as previously described(25). All animal experiments were approved by the University Health NetworkAnimal Care Committee.

Histology. Tissues were fixed in 10% (wt/vol) formalin for 24 h, embedded inparaffin, and sectioned for histological evaluation. Sternums were rinsed andneutralized in an ammonium hydroxide solution, washed in PBS, and thenfixed in 10% (wt/vol) formalin overnight. Sternums were then treated asother tissues for processing and embedding in paraffin. Sections were stainedwith H&E, using a standard protocol. Histological sections were examined bya board-certified veterinary anatomic pathologist.

Flow Cytometry. Flow cytometry was performed as previously described (26).Single-cell suspensions were prepared from spleen, lymph nodes, thymus, orbone marrow preparations treated to lyse red blood cells. Cells (1–2 × 106)were preincubated with Fc block for 15 min at 4 °C and immunostained withantibodies recognizing the following: CD3 (145-2C-11), CD4 (GK1.5), CD5(53-7.3), CD8 (53-6.7), CD13 (R3-242), CD16/CD32 (2.4G2), CD19 (1D3), CD24(30F1), CD25 (PC61), CD44 (IM7), CD137 (BST2, PDCA-1), CD62L (MEL-14), andTCRb (H57-597), all from BioLegend, BD Biosciences, or eBioscience unlessotherwise specified. Protein detection by intracellular staining was per-formed as described previously (26). Briefly, cells were fixed with 1.6% (wt/vol)paraformaldehyde and incubated for 30 min at room temperature. Data wereacquired using BD FCMCanto flow cytometer and analyzed with the FlowJoanalysis program (Tree Star Inc.).

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)Fig. 4. Mutant Idh1 increases sensitivity to in vitro nutrient deprivation in p53 null T-cell lymphoma cells. For all experiments, cells were resuspended inglucose and glutamine-free RPMI 1640 media containing 10% (vol/vol) dialyzed FBS, 1% FBS, and the indicated nutrient conditions, and viability wasmeasured by flow cytometry, using annexin V/PI at the indicated times. (A) 20 mM glucose, 10 mM glutamine; (B) glucose-free, 10 mM glutamine; (C)glutamine-free, 20 mM glucose.

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Cell Culture. Jurkat cells (ATCC) and cells derived from Vav-p53fl/fl and Vav-Idh1-KI p53fl/fl T-cell thymic lymphomas were cultured in nonadherent tissueculture plates in RPMI 1640 supplemented with 10% (vol/vol) FBS. Cells weremaintained at 37 °C and 5% partial pressure CO2 in a humidified incubator.For nutrient deprivation experiments, cells were grown in glucose and glu-tamine-free RPMI 1640 media (Life Technologies) supplemented with theindicated concentrations of glucose and glutamine, 10% (vol/vol) dialyzedFBS, and 1% (vol/vol) FBS. Cell viability was measured by flow cytometryusing Annexin V and propidium iodide, as previously described (27).

2HG Measurement. For 2HG measurement, metabolites were extracted using80% (vol/vol) aqueous methanol, as previously described (28). Extracts weresubjected to ion-paired reverse-phase LC coupled to negative mode elec-trospray triple-quadrupole mass spectrometry, using multiple reactionmonitoring. Integrated elution peaks were compared with metabolitestandard curves for absolute quantification.

Exome Sequencing.Genomic DNAwas isolated using DNeasy Blood and TissueKit (Qiagen) according to the manufacturer’s protocol. Whole-exome se-quencing and data analysis were performed at the University Health Net-work Genomics Centre. Two hundred nanograms DNA was used to generatelibraries after Agilent SureSelect XT target enrichment kit, according to themanufacturer’s protocol. A 750-ng library from each sample was hybridizedfor 24 h for mouse all-exon capture. Captured enriched libraries were sizevalidated, using Agilent Bioanalyzer, and concentration validated by quantitativePCR. All libraries were normalized to 10 nM and pooled. Next, 10 pM of

pooled libraries were loaded onto Illumina cBot for cluster generation. Theclustered flow cell was then sequenced using paired-end 100 cycles on anIllumina HighSEq. 2000. Read quality was checked using FASTQC (v. 0.11.2),and raw sequence data were aligned to the mouse genome (mm9), usingBWA-MEM (v. 0.7.10). Alignment quality was assessed using Qualimap (v. 2.0).Bam files were preprocessed using Picard (v. 1.124) to mark duplicates andGATK (v. 3.2–2) to perform local realignment around indels and to performbase quality score recalibration. HaplotypeCaller (Broad Institute) was used toidentify variants relative to the mm9 reference genome. To eliminate variantsthat have a low likelihood of being somatic events, we removed variants pre-sent in the mouse dbSNP database (v128), and eliminated common variantsidentified by the Sanger Institute Mouse Genomes Project (13). To furtheridentify potential somatic variants of interest, we eliminated variants present inthe control wild-type C57BL/6J mouse from Jackson Laboratories, or in DNAfrom two C57BL/6J mice from our breeding colony. We further focused onexonic coding mutations. This approach should eliminate most variants calledas a result of misalignment artifacts, sequencing artifacts, and errors in theunderlying reference sequence.

ACKNOWLEDGMENTS. We are grateful for the administrative assistance ofMs. Irene Ng and for technical assistance from the Princess Margaret CancerCenter flow cytometry facility, genotyping facility, and animal resource center,and the University Health Network Genomics Centre. This work was supportedby a grant from the Canadian Institutes of Health Research (to T.W.M, R.A.C,and Z.H.) and by a grant from the Leukemia and Lymphoma Society (to T.W.M.).F.L. received a grant from the Institut National du Cancer.

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