De novo pyrimidine synthesis is a targetable vulnerability in IDH mutant glioma

. Author manuscript; available in PMC: 2022 Sep 28.

Published in final edited form as: Cancer Cell. 2022 Aug 18;40(9):939–956.e16. doi: 10.1016/j.ccell.2022.07.011

KEY RESOURCES TABLE

REAGENT or RESOURCE	SOURCE	IDENTIFIER

Antibodies

Mouse monoclonal anti-IDH1 R132H antibody (IHC)	Dianova	Cat# DIA-H09, RRID:AB_2335716
Rabbit monoclonal anti-Cleaved Caspase 3	Cell Signaling Technology	Cat# 9661, RRID:AB_2341188
Mouse monoclonal anti-Akt (pan)	Cell Signaling Technology	Cat# 2920, RRID:AB_1147620
Rabbit monoclonal anti-pAkt	Cell Signaling Technology	Cat# 2965S, RRID:AB_2255933
Rabbit polyclonal anti-GFAP	Abcam	Cat# ab7260, RRID:AB_305808
Rabbit polyclonal anti-Sox2	Abcam	Cat# ab97959, RRID:AB_2341193
Rabbit polyclonal anti-Olig2	Ligon et al. (2004)	N/A
Mouse monoclonal anti-Nestin	Abcam	Cat# ab6142, RRID:AB_305313
Mouse monoclonal anti-γH2A.X	Cell Signaling Technology	Cat# 9718S, RRID:AB_2118009
Mouse monoclonal anti-FLAG	Sigma-Aldrich	Cat# F1804, RRID:AB_262044
Mouse monoclonal anti-HA	Covance	Cat# MMS-101P RRID: AB_2314672
Mouse monoclonal, anti-Vinculin	Sigma-Aldrich	Cat# V9131, RRID:AB_477629
Anti-53BP1 antibody	Novus	Cat# NB100–304, RRID:AB_10003037
Rabbit polyclonal anti-DHODH	Proteintech	Cat#14877 RRID: AB_2091723
Donkey anti-rabbit IgG, Alexa Fluor 488-conjugated secondary antibody	Invitrogen	Cat# A-21206 RRID:AB_2535792
Goat anti-mouse IgG, HRP-linked secondary antibody	Thermo Fisher	Cat# 31430 RRID:AB_228307
Goat anti-rabbit IgG, HRP-linked secondary antibody	Thermo Fisher	Cat#31460 RRID:AB_228341

Bacterial and virus strains

Adeno-associated virus (pAAV2-sgTrp53-sgAtrx-EFS-Cre)	This paper, AAV packaging performed by Vigene Biosciences	https://www.vigenebio.com/aav-packaging/
HB101 Competent Cells	Promega	Cat# L2015

Biological samples

UTSW6577 glioma specimen	Abdullah et al. (2022)	N/A
UTSW1382 glioma specimen	Abdullah et al. (2022)	N/A
UTSW9647 glioma specimen	Abdullah et al. (2022)	N/A
UTSW2243 glioma specimen	Abdullah et al. (2022)	N/A
UTSW2846 glioma specimen	This paper	N/A
UTSW2294 glioma specimen	This paper	N/A
UTSW9698 glioma specimen	This paper	N/A
UTSW9501 glioma specimen	This paper	N/A

Chemicals, peptides, and recombinant proteins

Metabolic inhibitor library	This paper	N/A
Anticancer drug library	Selleck Chemicals	N/A (purchased in 2015)
Brequinar	Sigma-Aldrich	Cat# SML0113
6-Azauridine	Sigma-Aldrich	Cat# A1882
Pyrazofurin	Sigma-Aldrich	Cat# SML1502
Uridine	Sigma-Aldrich	Cat# U3750
Lometrexol	Cayman Chemical	Cat# 18049
AnnexinV-FITC	Fisher Scientific	Cat# BD556547
AnnexinV-PE	Fisher Scientific	Cat# BD559763
AnnexinV-APC	Fisher Scientific	Cat# BD550475
DAPI	Cayman Chemical	Cat# 14285
AGI-5198 (IDH-C35)	Xcess Bio	Cat# M60068
Deoxythymidine	Sigma-Aldrich	Cat# 3416–05-5
Deoxycytidine	Sigma-Aldrich	Cat# 951–77-9
Palbociclib (PD 0332991)	Cayman Chemical	Cat# 16273
BAY 2402234	Bayer	N/A
(R)-2HG-TFMB	Provided by Ryan E. Looper	N/A
amide-15N-glutamine	Cambridge Isotope Laboratories	Cat# NLM-557
15N-uracil	Cambridge Isotope Laboratories	Cat# NLM-637
15N-uridine	Cambridge Isotope Laboratories	Cat# NLM-812
15N-hypoxanthine	Cambridge Isotope Laboratories	Cat# NLM-8500
Tunicamycin	Sigma-Aldrich	Cat# T7765
4-hydroxytamoxifen	Sigma-Aldrich	Cat# H7904
L-dihydroorotic acid	Sigma-Aldrich	Cat# D7128
Decylubiquinone	Sigma-Aldrich	Cat# D7911
2,6-dichloroindophenol	Sigma-Aldrich	Cat# D1878
Sodium malonate	Sigma-Aldrich	Cat# 63409
Potassium cyanide	Sigma-Aldrich	Cat# 60178

Critical commercial assays

KOD Xtreme Hot Start DNA Polymerase Kit	Millipore Sigma	Cat# 71975
DNeasy Blood and Tissue kit	Qiagen	Cat# 695045
TissueLyser	Qiagen	Cat# 85300
Neural Tissue Dissociation Kit	Miltenyi	Cat# 130–095-942
RNeasy Mini Kit	Qiagen	Cat# 74004
AffinityScript qPCR cDNA Synthesis Kit	Agilent	Cat# 600559
NEB Monarch kit	New England BioLabs	Cat# T1030
Qiagen gel extraction kit	Qiagen	Cat# 28706
Mouse genotyping	Transnetyx	https://www.transnetyx.com/
Bond Polymer Refine Detection Kit	Leica Biosystems	Cat# DS9800
BP Clonase	Invitrogen	Cat# 11789100
LR Clonase	Invitrogen	Cat# 11791020

Deposited data

TCGA Brain Lower Grade Glioma dataset	GDAC Firehose, Broad Institute	http://gdac.broadinstitute.org/runs/analyses__2016_01_28/reports/cancer/LGG-TP/index.html
Metabolomics Analysis of HOG-EV and HOG-R132H Cells with and without BAY 2402234 Treatment	National Metabolomics Data Repository	NMDR: https://doi.org/10.21228/M81X41

Experimental models: Cell lines

BT054	Kelly et al. (2010)	N/A
BT260	Koivunen et al. (2012)	N/A
TS516	Rohle et al. (2013)	N/A
TS603	Rohle et al. (2013)	N/A
HK213	Laks et al. (2016)	N/A
HK211	Laks et al. (2016)	N/A
HK157	Laks et al. (2016)	N/A
HK252	Laks et al. (2016)	N/A
HK308	Laks et al. (2016)	N/A
MGG152	Wakimoto et al. (2014)	N/A
HOG	Provided by Pablo Paez	N/A
NHA (immortalized with HPV E6 and E7 and hTERT)	Provided by Russell Pieper	N/A
NHA (EV or IDH1-R132H)	Koivunen et al. (2012)
UTSW6577 glioma organoids	Abdullah et al. (2022)	N/A
UTSW1382 glioma organoids	Abdullah et al. (2022)	N/A
UTSW9647 glioma organoids	Abdullah et al. (2022)	N/A
UTSW2243 glioma organoids	Abdullah et al. (2022)	N/A
UTSW2846 glioma organoids	This paper	N/A
UTSW2294 glioma organoids	This paper	N/A
UTSW9698 glioma organoids	This paper	N/A
UTSW9501 glioma organoids	This paper	N/A
DF-AA27 GEMM GSC	This paper	N/A
MEFs, irradiated	Life Technologies	Cat# A34181
IC (H11^{LSL-Cas9+/−};Idh1^tm1Mak/WT) MEFs	This paper	N/A
HEK293T	ATCC	Cat# CRL-11268
Primary human astrocytes	Lonza	Cat# CC-3187
Human neural stem cells	Fisher Scientific	Cat# 10419428

Experimental models: Organisms/strains

NCr Nude mice, 6–10-week-old females	Taconic	Cat# NCRNU-F
NCr Nude mice, 4–5-week-old females	Taconic	Cat# NCRNU-F
ICR SCID mice, 6–12-week-old females	Taconic	Cat# ICRSC-F
R26-Pik3ca^H1047R (FVB.129S6-Gt(ROSA)26Sor^{tm1(Pik3ca*H1047R)Egan}/J mouse	Jackson Laboratories	Cat# 016977
Idh1^tm1Mak mouse	Provided by Tak W. Mak	N/A
H11^LSL-Cas9 (B6; 129-Igs2^{tm1(CAG-cas9*)Mmw}/J mouse	Jackson Laboratories	Cat# 026816
*PIC* (H11^{LSL-Cas9+/−};Idh1^tm1Mak/WT;R26-Pik3ca^H1047R+/−) mouse	This paper	N/A
PC (H11^{LSL-Cas9+/−};Idh1^WT/WT;R26-Pik3ca^H1047R+/−) mouse	This paper	N/A
IC (H11^{LSL-Cas9+/−};Idh1^tm1Mak/WT) mouse	This paper	N/A
C (H11^{LSL-Cas9+/−};Idh1^WT/WT) mouse	This paper	N/A

Oligonucleotides

sgATRX: ATAAGGACTTTGAGTGAA	This paper	N/A
sgTrp53: GTGTAATAGCTCCTGCATG	Platt et al. (2014)	N/A
sgControl: GGAGGCTAAGCGTCGCAA	Provided by Feng Zhang	N/A
sgDHODH: CATCTTATAAAGTCCGTCCA	This paper	N/A
Trp53 outer fwd ATAGAGACGCTGAGTCCGGTTC	Oser et al. (2019)	N/A
Trp53 outer rev CCTAAGCCCAAGAGGAAACAGA	Oser et al. (2019)	N/A
Trp53 inner fwd TGCAGGTCACCTGTAGTGAGGTAGG	Oser et al. (2019)	N/A
Trp53 inner rev GAAACAGGCAGAAGCTGGGGAAGAAAC	Oser et al. (2019)	N/A
ATRX outer fwd GCTATCTGAAACTCAATCCACG	This paper	N/A
ATRX outer rev GACTTGGTTTCTCCTTTGCCATG	This paper	N/A
ATRX inner fwd GCTTCCTGTAAGCTCATAAGTAC	This paper	N/A
ATRX inner rev CTAATGCCATATGAGTGTAACTC	This paper	N/A
ATRX second inner fwd CTCTTACATAATGGCCATTCTC	This paper	N/A
ATRX second inner rev CTGTGAGTCATGATCATTCTTTGC	This paper	N/A
Recombined IDH1 fwd GATTGATTCTGCCGCCATGATCCTAGT	Sasaki et al. (2012b)	N/A
Recombined IDH1 rev CCTGGTCATTGGTGGCATCACGATTCTC	Sasaki et al. (2012b)	N/A
Actb fwd (genomic DNA for Idh1-R132H assay) TGACCCAGGT CAGTATCCCGGGT	This paper	N/A
Actb actin rev (genomic DNA for Idh1-R132H assay) GAACACAGCTAAGTTCAGTGTGCTGGGA	This paper	N/A
PIK3CA H1047R cDNA fwd CTAGGTAGGGGATCGGGACTCT	This paper	N/A
PIK3CA H1047R rev AATTTCTCGATTGAGGATCTTTTCT	This paper	N/A
Actb fwd (cDNA for Pik3ca-H1047R assay) TAGGCACCAGGGTGTGATG	This paper	N/A
Actb rev (cDNA for Pik3ca-H1047R assay) CATGGCTGGGGTGTTGAAGG	This paper	N/A

Recombinant DNA

pLX304-Ubc-GATE-IRES-GFP	Gift from Vidyasagar Koduri	N/A
pDONR223	Invitrogen	N/A
pLX304-Ubc-EV-IRES-GFP	EV provided as a gift by Vidyasagar Koduri	N/A
pLX304-Ubc-DHODH-WT-IRES-GFP	DHODH WT cDNA provided as a gift by Laura Evans & Julie Losman	N/A
pLX304-Ubc-DHODH-A58T-IRES-GFP	DHODH A58T cDNA provided as a gift by Laura Evans & Julie Losman	N/A
pLX304-CMV-BFP	Gift from Hilary Nicholson	N/A
pLX304-CMV-GFP	Gift from Hilary Nicholson	N/A
pLXSN-H-Ras_V12	Addgene	Cat# 39516
pLenti-Flag-P85	Addgene	Cat# 40219
pLenti4-Flag-P85-DKRMNS560del	Addgene	Cat# 40225
pLenti-Flag-P85-R574fs	Addgene	Cat# 40227
pLenti4-Flag-T576del	Addgene	Cat# 40228
p1321 HPV-16 E6/E7	Addgene	Cat# 8641
pAAV2-sgTrp53-sgAtrx-EFS-Cre	This paper	Addgene, Cat# 189977
pLBCX-Large-T-antigen^{K1 mutant}	This paper	Addgene, Cat# 189978
pMSCV-MerCreMer-hygro	This paper	Addgene, Cat# 188982
LeGO-iG2-FLuc	McBrayer et al. (2018)	N/A
pBABE-HA-hygro	Koivunen et al. (2012)	N/A
pBABE-IDH1-R132H-HA-hygro	Koivunen et al. (2012)	N/A
plenti-Ubc-IRES-hygro empty vector	McBrayer et al. (2018)	N/A
plenti-Ubc-IDH1-R132H-HA-IRES-hygro	McBrayer et al. (2018)	N/A
pMD2.G	Provided by Didier Trono	Addgene, Cat# 12259
Gag/pol	Provided by Tannishtha Reya	Addgene, Cat# 14887
psPAX2	Provided by Didier Trono	Addgene, Cat# 12260
VSV.G	Provided by Tannishtha Reya	Addgene, Cat# 14888
pX551	Provided by Feng Zhang	Addgene Cat# 60957

Software and Algorithms

MATLAB	MathWorks	https://www.mathworks.com/products/matlab.html
Metran	Yoo et al. (2008)	N/A
Multi-Quant 2.1 software	Sciex	https://sciex.com/products/software/multiquant-software
GraphPad Prism	GraphPad	https://www.graphpad.com/scientific-software/prism/
Living Image	PerkinElmer	http://www.perkinelmer.com/product/li-software-for-spectrum-1-seat-add-on-128113
R	The R Project for Statistical Computing	https://www.r-project.org/
AccuCor R package (version 0.2.3)	Su et al. (2017)	https://github.com/lparsons/accucor
El-Maven 0.12.0	Elucidata	https://elucidata.io/el-maven/
TraceFinder 5.1 SP2	Thermo Fisher	Cat# OPTON-31001
Freestyle 1.7 SP	Thermo Fisher	Cat# XCALI-98282
MetaboAnalyst	Publicly available	https://www.metaboanalyst.ca/
FlexControl, version 4.0	Bruker	https://www.bruker.com/en/products-and-solutions/mass-spectrometry/maldi-tof/autoflexmax.html
FlexImaging, version 5.0 and SCiLS Lab	Bruker Daltonics	https://www.bruker.com/en/products-and-solutions/mass-spectrometry/ms-software/scilslab.html
ImageJ	NIH	https://imagej.net/
QuPath software (version 0.2.3)	Publicly available	https://qupath.github.io/

Other

Geltrex	Life Technologies	Cat# A1413202
AGM SingleQuots Supplements	Lonza	Cat# CC-4123
NeuroCult Basal Medium (Mouse and Rat)	StemCell Technologies	Cat# 05702
NeuroCult NS-A Basal Medium (Human)	StemCell Technologies	Cat# 05750
Neurobasal Medium (MGG152 GSCs)	Gibco	Cat# 21103049
Neurobasal Medium (organoids)	Thermo Fisher	Cat# 21103049
DMEM-F12 Medium	Gibco	Cat# 11320033
DMEM Medium	Gibco	Cat# 11995–065
Accutase	StemCell Technologies	Cat# 07922
RBC lysis buffer	Thermo Fisher	Cat# 00433357
2-mercaptoethanol	Thermo Fisher	Cat# BP176–100
Glutamax	Thermo Fisher	Cat# 35050061
N-2 supplement	Thermo Fisher	Cat# 17502048