SUPPLEMENTARY METHODS

Compound PreparationFor in vivo administration 20 mg/ml of BAY1436032 were prepared in 2-(hydroxypropyl)-β-

cyclodextrin (HP-ß-CD, Sigma). In brief, 400 mg were added to 20 ml of 30% HP-ß-CD, pH was

made alkaline with 50-100 µl of 10 M NaOH, followed by sonication until the drug was

completely dissolved. Finally, the pH was readjusted to 7 with 2 M HCl. Fresh solution was

prepared every week for in vivo administration. For in vitro studies a 1 M stock solution of

BAY1436032 was prepared in DMSO and stored at -20°C for a maximal period of 30 days. All

dilutions were freshly prepared in cell culture medium and used immediately.

Patient Samples Diagnostic bone marrow or peripheral blood collected from AML patients registered for the

multicenter treatment trials AML-SHG 01/99, AML-SHG 02/95, SAL-DaunoDouble or the

AMLSG or SAG bioregistry studies were analyzed for mutations in IDH1 and IDH2 as previously

described.5,11 Mononuclear cells were isolated by ficoll density centrifugation, washed with PBS,

and red blood cells were lysed using ACK lysis buffer. Written informed consent was obtained

according to the Declaration of Helsinki, and the study was approved by the institutional review

boards of Hannover Medical School and University of Heidelberg.

Statistical AnalysisPairwise comparisons were performed by Student t test. The 2-sided level of significance was

set at P less than 0.05. Comparison of survival curves was performed using the log-rank test.

Calculations and graphs were prepared with Microsoft Excel 2010 (Microsoft), Graph Pad Prism

5.0 (GraphPad Software), and Adobe Illustrator 3.0 (Adobe Systems).

Gene Expression ProfilingFor gene expression profiling RNA was extracted using the RNeasy Plus mini kit (Qiagen) from

hCD45+ cells that were sorted from bone marrow of PDX1 mice 4 weeks after treatment with

either vehicle or 150 mg/kg BAY1436032. Quality and integrity of total RNA was controlled on an

Agilent Technologies 2100 Bioanalyzer (Agilent Technologies). 500 ng of total RNA were used

for biotin labelling according to the 3´ IVT Express Kit (Affymetrix). 7.5 µg of biotinylated cRNA

were fragmented and placed in a hybridization cocktail containing 4 biotinylated hybridization

controls (BioB, BioC, BioD, and Cre) as recommended by the manufacturer. Samples were

hybridized to an identical lot of Affymetrix GeneChip HG-U133 2.0 for 16 hours at 45°C. Steps

for washing and SA-PE staining were processed on the fluidics station 450 using the

recommended FS450 protocol (Affymetrix). Image Analysis was performed on GCS3000

Scanner and GCOS1.2 Software Suite (Affymetrix).

Analysis of microarray data was performed using the GeneSpring 11.5.1 software suite

(Agilent Technologies). Signal intensities (raw data) were background corrected, normalized and

summarized using RMA. Gene expression values per condition are given as log2 relative

expression to the mean expression value calculated from log2 intensities of all conditions (mean

centralization, normalized data). Significance of differential gene expression was calculated by

GeneSpring software using error corrected unpaired t-test. Hierarchical unsupervised cluster

analysis of the top differentially expressed genes was done using Euclidean distance for the

similarity metrics with Cluster 3.0 software38 and visualized with Java Treeview.39 The Broad

Institute GSEA software package was employed for gene set enrichment analysis using gene

ontology gene sets from the Molecular Signatures Database (http://www.broad.mit.edu/

gsea/msigdb/). Gene expression profiling data can be found at the gene expression omnibus

database under GEO accession no. GSE83485.

2-Hydroxyglutarate measurementMass spectrometric 2HG measurement was performed as recently described.40 In brief,

intracellular 2HG from AML cells or HoxA9 immortalized IDH1 mutant mouse bone marrow cells

treated with vehicle or BAY1436032 was extracted in HBSS by sonication for 10 minutes at

intervals of 30 seconds on, 30 seconds off at 40C using the Bioruptor (Diagenode) followed by

two rounds of freeze thaw cycles. Alternatively, 10 µl of serum was directly used from mice

transplanted with human IDH1mut AML cells treated with vehicle or BAY1436032. All analyses

were performed on an AB Sciex 4000 Q-trap triple quadruple mass spectrometer (Applied

Biosystems). LC was performed on a Waters Acquity UPLC BEH C18 analytical column [100 ×

2.1 mm (i.d.); 1.7-μm bead size with water–acetonitrile (96.5:3.5 by volume) containing 125 mg/L

ammonium formate (pH adjusted to 3.6 by addition of formic acid) as mobile phase as

described. The enzymatic R-2HG assay has been described previously.41 Briefly, probe material

was treated with a deproteinization kit (Biovision). Supernatants were then collected and stored

at -20°C. The total enzymatic reaction volume was 100 μl. Ten milliliter of assay solution was

freshly prepared for each 96-well plate subjected to R-2HG assay. The assay solution contained

100 mM HEPES pH 8.0, 100 μM NAD+ (Applichem), 0.1 μg HGDH, 5 μM resazurin (Applichem)

and 0.01 U/ml diaphorase (MP Biomedical). Immediately before use, 25 μl sample volume was

added to 75 μl of assay solution and incubated at room temperature for 30 min in black 96-well

plates (Thermo Fisher Scientific) in the dark. Fluorometric detection was performed in triplicate

with 25 μl deproteinized sample being analyzed in each reaction with excitation at 540 ± 10 nm

and emission of 610 ± 10 nm (FLUOstar Omega, BMG Labtech).

Transplantation and treatment of miceFor the PDX1-IDH1R132C and PDX3-IDH1-wild-type models 6-8 weeks old female NOD.Cg-

Prkdcscid Il2rgtm1Wjl/SzJ (NSG) mice were bred by the central animal laboratory of Hannover

Medical School and kept in pathogen-free conditions. One million patient-derived AML cells

collected from bone marrow and spleen of leukemic mice were injected intravenously in the tail

vein of sublethally (3 Gy) irradiated NSG mice. Randomly selected groups of transplanted mice

were treated once daily with BAY1436032 by oral gavage at doses of 45, 60, 90, 120 and 150

mg/kg or with 150 µl of vehicle wherever specified. One group was treated with 90 mg/kg every

alternate day (90 mg/kg, q2d). Treatment was initiated 17 days after transplantation, when

engraftment of human cells (hCD45+ cells) in peripheral blood had been confirmed. The

proportion of leukemic cells in peripheral blood of mice was measured with a human-specific

CD45 antibody every four weeks by tail vein bleeds and FACS analysis.28,42 Lineage distribution

was determined by FACS analysis (FACS Calibur, Becton Dickinson) using monoclonal

antibodies described above. Blood counts with differential WBC analysis were performed using

an ABC Vet Automated Blood counter (Scil animal care company GmbH). Cytospin preparations

were stained with Wright-Giemsa stain and morphologic analysis of bone marrow was done as

described above.

For limiting dilution transplantation, primary mice with high chimerism (60-70%) were

treated for 4 weeks with either vehicle or 150 mg/kg BAY1436032. After 4 weeks of treatment

bone marrow cells were obtained from primary mice and injected intravenously at doses of

200,000, 20,000, 2,000, 200 or 20 cells (n = 3 mice/dose) into irradiated secondary NSG

recipients. Eight weeks after transplantation, the presence of transplanted human cells in

peripheral blood was assessed by flow cytometry. Engraftment was determined positive when

more than 0.1% hCD45+ cells were detected. The frequency of stem cells was calculated by

applying Poisson statistics to the proportion of negative recipients at different dilutions using the

ELDA software.43

For the PDX2 model NSG mice were acquired from the DKFZ breeding facility and

housed in pathogen-free conditions at the animal laboratory of the DKFZ, Heidelberg.

Experimental procedures were approved by governmental authorities of the Regierungs-

präsidium Karlsruhe, Germany, and supervised by local animal welfare officials. After

confirmation of the presence of an IDH1 mutation, one million AML cells collected from bone

marrow or peripheral blood from AML patients were injected intravenously into the tail vein of 6-8

weeks old male NSG mice. Engraftment was constantly monitored by tail vein bleeds and flow

cytometry analysis for hCD45+ cells. Moribund mice were sacrificed by cardiocentesis. Samples

with confirmed IDH1 mutation and a percentage of at least 95% hCD45+ cells were

retransplanted into 2nd recipient NSG mice analogous to the first transplantation. Randomly

selected groups of transplanted mice were treated once daily with 150 mg/kg BAY1436032 or

similar volumes of HP-ß-CD by oral gavage. Treatment was initiated once engraftment of human

AML cells in peripheral blood reached a tumor burden of around 35% hCD45+ cells. WBC,

hemoglobin and platelets were determined using a Hemavet (Drew-Scientific) and the

immunophenotype at indicated time points was analyzed by flow cytometry analysis as

described above.

Pharmacokinetic analysis in miceFor short-term pharmacokinetic analysis, PDX1-IDH1R132C mice having 65-70% chimerism

were treated once with an oral dose of 45 or 150 mg/kg of BAY1436032 or vehicle. Plasma was

collected before and 3, 7 and 24 hours after treatment to determine R/S-2HG and BAY1436032

levels.31 For long-term pharmacokinetic analysis, 10µl of serum was collected at 55 days after

treatment start and subjected to R-2HG measurement.

Immunoblotting of histone modificationsCells were treated with either DMSO or 500 nM BAY1436032 for 14 days. For acid extraction of

histones 106 cells were collected, washed with PBS, resuspended in 100 µl 0.25 M HCl and

incubated overnight at 4°C. The supernatant was transferred to a new tube and the pellet was

resuspended in 50 µl 0.25 M HCl. After centrifugation the supernatants were combined.

Histones were precipitated by adding trichloroacetic acid to a final concentration of 33% and

samples were incubated for 30 min at 4°C. The pellet was washed twice with tert-butyl methyl

ether/ethanol (1:1), air dried and resuspended in water. Acid extracted histones were separated

by SDS PAGE, transferred to a PVDF membrane, blocked and immunoblotted with anti-

H3K4me3 (Abcam, ab8580), anti-H3K9me3 (Abcam, ab8898), anti-H3K36me3 (Abcam, ab9050)

or anti-H3K27me3 (Active Motive, 39155) antibodies. Reference H3 signals were obtained after

stripping a given histone modification blot and then reprobing with anti-H3 (Abcam, ab1791).

Anti-rabbit horseradish peroxidase (HRP)-linked antibody (Cell Signaling Technology, #7074)

and Clarity Western ECL Substrate (Bio-Rad) or SignalFire Plus ECL Reagent (Cell Signaling

Technology) were used for chemiluminescent protein detection with a ChemiDoc MP Imaging

System (Bio-Rad). Quantification of immunoblots was done with the Image Lab software (Bio-

Rad) by subtracting the background signal and calculating ratios of a given histone modification

to the H3 signal from the integrated signal intensities of corresponding bands. All ratios were

normalized to the ratio of the untreated IDH1 wild-type of the same blot and log-transformed

(base 2).

DNA methylation analysisThe Illumina Infinium HumanMethylation450 (450k) array was used to obtain the DNA

methylation status of 482,421 CpG sites (Illumina), according to the manufacturer’s instructions

at the Core Facility of the DKFZ. DNA methylation data was normalized by performing

background correction and dye bias correction (shifting of negative control probe mean intensity

to zero and scaling of normalization control probe mean intensity to 10,000, respectively).

Filtering of probes was performed as described previously.44 In total, 438370 probes were kept

for analysis. For the data of each patient an unsupervised agglomerative hierarchical clustering

analysis of the 450k methylation data was performed in R.45 The idat-files were processed using

the R-package minfi.46 25,000 CpGs exhibiting the highest standard deviation across the dataset

were included. The cluster distance is based on the Pearson correlation coefficient and clusters

are connected by average linkage. The unscaled methylation levels are shown in a heatmap

from unmethylated (blue) to methylated state (red). Boxplots show the mean methylation level of

all 438,370 CpGs (resp. all CpGs located in a specific promoter region, resp. all CpGs, that are

not associated to a promoter region). The whiskers extend from the edge of box to the highest

(resp. lowest) value that is within 1.5-fold of the inter-quartile-range. For calculation of P values a

two-sided t-test of the mean beta-values of the replicates for one group was performed.

Cell cycle analysisFor cell cycle analysis mice transplanted with human AML cells and treated with either vehicle or

BAY1436032 were injected i.p. with 100 µl BrdU (1 mM) at 36, 24 and 12 hours before harvest

of cells. 1x107 cells from peripheral blood at 1 week after treatment and bone marrow at 4 weeks

after treatment were stained with hCD45-FITC antibody followed by permeabilization, fixation,

and staining with an anti-BrdU antibody according to the manufacturers protocol (BD

Pharmingen Cat no. 559619). 5x105 cells were acquired per sample. Cell cycle phases were

determined on hCD45+ cells according to standard procedures where BrdU positive cells are in

the S phase of the cell cycle.

Apoptosis MeasurementFor apoptosis measurements 1x107 peripheral blood or bone marrow cells as described above

were stained with hCD45-FITC, Annexin V-APC and 7AAD according to the manufacturers

protocol (BD Pharmingen Cat no. 550474) and analyzed on a FACS Calibur flow cytometer

(Becton Dickinson).

Morphologic analysisCytospin preparations were stained with Wright-Giemsa stain. Morphology of AML cells was

examined using an Olympus BX51 microscope (Olympus) and a 100x/1.3 numerical aperture

objective with Zeiss immersol medium (Zeiss). OlympusXC50 (Olympus) and analySIS software

(Soft Imaging System) were used to capture images.

Supplementary Figure 1. BAY1436032 selectively inhibits R-2HG production and induces

myeloid differentiation in IDH1 mutant mouse hematopoietic and primary human AML cells. (A)

Ratio of R-2HG to S-2HG after 8 days of BAY1436032 treatment of HoxA9-immortalized mouse

bone marrow cells retrovirally transduced with IDH2R140Q or IDH2R172K. Intracellular R-

2HG/S-2HG ratios were calculated as percentage of DMSO (CTL) treatment (mean ± SEM, n =

3). (B) Ratio of R-2HG to S-2HG in primary human IDH2 mutant AML cells 24 hours after

BAY1436032 treatment. Intracellular R-2HG/S-2HG ratios were calculated as percentage of

DMSO (CTL) treatment (mean ± SEM, n = 3). (C) Representative FACS plots showing increased

expression of myelomonocytic differentiation markers CD14 and CD15 on primary AML cells

with different IDH1 mutations after ex vivo treatment with 1 M BAY1436032 for 19 days. DMSO

treatment was used as a control. Cells expressing CD14 and CD15 are marked by a gate and

percentages of CD14+/CD15+ cells are given in the graph. (c) Quantification of IDH1 wild-type

(wt) or IDH1 mutant primary AML cells expressing CD14 and CD15 after ex vivo treatment with

DMSO or BAY1436032 (mean ± SEM). Treatment durations and numbers of patient samples

examined are given in the graph. *, P < 0.05; **, P < 0.01.

Supplementary Figure 2. BAY1436032 reduces R-2HG serum levels in an AML PDX mouse

model in vivo. (A) BAY1436032 unbound plasma concentrations in PDX1-IDH1R132C mice

treated with a single oral dose of 45, 90 or 150 mg/kg BAY1436032 (mean ± SEM of 2-3

animals/time point) at 3, 7 and 24 hours after treatment. The dashed line indicates the in vitro R-

2HG/S-2HG ratio IC50 value in IDH1R132C mutant human primary AML cells. (B) R-2HG serum

levels of PDX1-IDH1R132C mice treated with a single oral dose of 45, 90 or 150 mg/kg

BAY1436032 at 3, 7 and 24 hours after treatment (mean ± SEM, n = 3). *, P < 0.05; ***, P <

0.001. (C) R-2HG serum levels of PDX1-IDH1R132C mice treated either daily with an oral dose

of 45, 60, 90, 120 or 150 mg/kg or every alternate day with 90 mg/kg (90 q2) BAY1436032 at 55

days after treatment start (mean ± SEM, n = 6-10 animals per treatment group). (D) Ratio of R-

2HG to S-2HG in serum of PDX1-IDH1R132C mice treated with BAY1436032. Animals were

treated with daily oral doses of 45, 60, 90, 120 or 150 mg/kg or every alternate day with 90mg/kg

BAY1436032 (indicated as 90 mg/kg, q2d in the graph). The R-2HG/S-2HG ratio was

determined at 55 days after treatment start.

Supplementary Figure 3. Peripheral blood counts for the PDX1- and PDX2-IDH1R132C

models. White blood cell counts (A), hemoglobin levels (B) and platelet counts (C) in peripheral

blood at different time points during treatment with either 45 or 150 mg/kg BAY1436032 or

vehicle of PDX1-IDH1R132C mice (mean ± SEM, n = 10). White blood cell counts (D),

hemoglobin levels (E) and platelet counts (F) in peripheral blood at different time points during

treatment with 150 mg/kg BAY1436032 or vehicle of PDX2-IDH1R132C mice (mean ± SD, n =

10). *, P < 0.05; **, P < 0.01; ***, P < 0.001; ns, not significant; nd, not determined; †, time of

death.

Supplementary Figure 4. Peripheral blood counts and immunophenotype for the PDX2-

IDH1R132C model after treatment stop. Percentages of total hCD45+ leukemic cells (A),

hCD45+/CD14+ cells (B), hCD45+/CD15+ cells (C), hCD45+/CD34+ cells (D) as well as white

blood cell counts (E), hemoglobin levels (F) and platelet counts (G) in peripheral blood of PDX2-

IDH1R132C mice at day 100 after treatment start with 150 mg/kg BAY1436032 (0) and 28 and

56 days after termination of treatment (mean, n = 6). *, P < 0.05; ***, P < 0.001; ns, not

significant.

Supplementary Figure 5. BAY1436032 neither has anti-leukemic activity nor induces myeloid

differentiation in an IDH1 wildtype AML PDX model in vivo (PDX3). (A) Engraftment of human

wildtype IDH1 AML patient cells in peripheral blood of NSG mice at different time points after

treatment with either vehicle or 150 mg/kg BAY1436032 (mean ± SEM, n = 4-5). (B) White blood

cell counts in peripheral blood of PDX3 mice at different time points after treatment with either

vehicle or 150 mg/kg BAY1436032 (mean ± SEM, n = 4-5). (C) Hemoglobin levels in peripheral

blood of PDX3 mice at different time points during treatment with either vehicle or 150 mg/kg

BAY1436032 (mean ± SEM, n = 4-5). (D) Platelet counts in peripheral blood of PDX3 mice at

different time points during treatment with either vehicle or 150 mg/kg BAY1436032 (mean ±

SEM, n = 4-5). (E) Immunophenotype of hCD45+ PDX3 cells in peripheral blood of mice after

treatment with vehicle at the indicated time points (mean ± SEM, n = 5). (F) Immunophenotype

of human PDX3 cells in peripheral blood of mice after treatment with 150 mg/kg BAY1436032 at

the indicated time points (mean ± SEM, n = 4). (G) Spleen weight at the time of death of PDX3

mice treated with vehicle or 150 mg/kg BAY1436032 until death. (H) Survival of PDX3 mice

treated with 150 mg/kg BAY1436032 or vehicle.

Supplementary Figure 6. BAY1436032 depletes LSC and impacts on gene expression of

IDH1R132C mutant AML cells in vivo. (A) Limiting dilution transplantation of bone marrow cells

from PDX1-IDH1R132C mice treated with 150 mg/kg BAY1436032 or vehicle for 4 weeks.

200.000, 20.000, 2.000, 200 or 20 cells per mouse were transplanted into 3 recipient mice per

group. LSC frequencies are shown (mean ± SEM, n = 3). (B) Unsupervised hierarchical

clustering using euclidean distance of cells from bone marrow of PDX1-IDH1R132C mice

treated with 150 mg/kg BAY1436032 or vehicle for 4 weeks. Gene expression profiling using

RNA was performed on Affymetrix Human HG_U133 Plus 2.0 microarrays (n = 3 per group). (C)

Top 15 enriched gene ontology gene sets from gene set enrichment analysis of cells from bone

marrow of PDX1-IDH1R132C mice treated with 150 mg/kg BAY1436032 or vehicle for 4 weeks.

NES, normalized enrichment score. (D) Enrichment plot for the gene sets “positive regulation of

PU.1” (left) and “negative regulation of E2F” (right) comparing PDX1-IDH1R132C mice treated

with 150 mg/kg BAY1436032 or vehicle. NES, normalized enrichment score. (E) Model of

transcription factor regulation during BAY1436032 treatment. While expression of E2F

transcription factors is downregulated, PU.1, NFkB and AP1 are upregulated. Proportion of

viable hCD45+ cells (F) and their cell cycle distribution (G) in peripheral blood (left panels) and

bone marrow (right panels) of PDX1-IDH1R132C mice treated with vehicle or 150 mg/kg

BAY1436032 for 1 or 4 weeks, respectively (mean ± SEM, n = 3). *, P < 0.05; **, P < 0.01; ns,

not significant.

Supplementary Figure 7. Cell cycle distribution of PDX1-IDH1R132C AML cells treated with

vehicle or BAY1436032 in vivo. A representative FACS plot from peripheral blood (left panels)

and bone marrow (right panels) of mice treated with either vehicle or 150 mg/kg BAY1436032.

Results are shown as percentages of hCD45+ cells.

Supplementary Figure 8. DNA methylation in primary IDH1R132C or IDH1R132H mutant AML

cells treated with 500 nM BAY1436032 or DMSO for 14 days ex vivo. (A) Hierarchical clustering

analysis of 450K DNA methylation data based on pearson correlation from primary IDH1R132C

or IDH1R132H mutant AML cells treated with 500 nM BAY1436032 or DMSO for 14 days ex

vivo. Heatmap scales from unmethylated (blue) to methylated (red). (B) Mean overall

methylation rates, (C) mean promoter methylation rates and (C) mean methylation rates without

promoter sites. Boxes represent medians and quartiles with wiskers ranging from the highest to

the lowest value. (D) PU.1 (69 promoter CpGs) and E2F1 (11 promoter CpGs) methylation in

primary IDH1R132C or IDH1R132H mutant AML cells treated with 500 nM BAY1436032 or

DMSO for 14 days ex vivo. Boxes represent medians and quartiles with wiskers ranging from the

highest to the lowest value.