Wild‐type IDH1: A molecular target in IDH1 mutant cancers?

Wild‐type IDH1: A molecular target in IDH1 mutant cancers?
Julie A. Wickenden, Paul Russell, Amy Smith, Tom Henley, Jane Elliott, Dan Gitterman, Mark Stockdale, Christine Schofield, Chris Torrance, Jonathan D. Moore.
Horizon Discovery Ltd, Cambridge, UK
Introduction
Neomorphic mutations targeting R132 of the TCA cycle enzyme, IDH1, have been identified in
multiple cancer types and lead to a build up of (R)‐2‐hydroxyglutarate (R)‐2HG. Several
mechanisms have been proposed to account for mutant‐IDH1‐mediated transformation: (R)‐2HG
may compete with alpha‐ketoglutarate (α‐KG) dependent enzymes that act as tumour suppressors
such as TET2 or EGLN, (R)‐2HG might inhibit electron transport chain function, or rapid (R)‐2HG
generation may deplete the cellular pool of α‐KG leading to depletion of NADPH. According to
some reports in the literature, heterodimer formation between mutant and wild‐type alleles of
IDH1 is important for the production of high levels of (R)‐2HG1.
We were interested in exploring novel ways to target tumour cells bearing mutant IDH1 alleles
that were distinct from the obvious opportunity available to identify mutant‐specific IDH1
inhibitors. The potential metabolic vulnerabilities of mutant IDH1 cancers raised the possibility
that wild‐type IDH1 might be essential for tumourigenesis or tumour maintenance in this context.
We therefore employed Horizon’s rAAV‐mediated homologous recombination gene engineering
technology in an attempt to generate conditional knockouts of the IDH1+ or IDH1R132C alleles in the
fibrosarcoma cell line, HT1080.
(A)
(C)
Cat#
Clone
Genotype
HD PAR‐020
PAR
IDH1 (+/R132C)
HD 148‐001
1C9
IDH1 (+cKO/R132C)
HD 148‐001
11B4
IDH1 (+cKO/R132C)
HT-1080
Parental
(B)
(A)
(B)
(C)
(D)
(D)
HT-1080
11B4
Isogenic Cell Line Generation
HT-1080
1C9
Figure 2. HT‐1080 IDH1 (+cKO/R132C) X‐MAN™ isogenic cell line fail to express wild type IDH1. (A) The
two clones derived from HT‐1080 “parental” cells with the selection marker targeted to the wild type
allele. (B) Droplet Digital™ PCR‐SNP assay of genomic DNA from each of the clones confirms the presence
of wild‐type and mutant alleles in equal ploidy. (C) Sequencing of cDNA across the mutation revealed no
expression of the wild‐type allele. (D) Further analysis of cDNA by ddPCR using Taqman expression and
SNP assays demonstrated that IDH1 mRNA expression levels are maintained in both clones with loss of
expression of the wild type allele.
(A)
# of Colonies Screened
Approx. 20,000
# of +ve wells identified
8
Assay Configuration
58 x 96‐well plates
Targeting Efficiency
Results
0.04%
Of the 8 PCR positive wells identified from two separate infections:
• 2 wells contained targeted cells (pools) with the correct insertion, none of which were clonal. These pools were targeted on the wild type R132 allele.
• 2 targeted pools were single cell diluted on HT1080 feeder layers.
• Multiple clones were obtained for each pool which were targeted on the R132 wild type allele.
• 2 clones from separate pools were isolated.
Figure 1. Generation of HT‐1080 IDH1 (+cKO/R132C) X‐MAN™ isogenic cell line. HT‐1080 cells with
endogenous heterozygous mutation (R132C) in IDH1 were targeted via recombinant adeno‐associated
virus (rAAV) technology2. The gene for IDH1 was targeted so as to incorporate LoxP sites either side of
exon 5. The targeted allele should remain active until the addition of cre recombinase which will excise
exon 5 from the allele and generate a knock‐out of IDH1. The strategy did not show any targeting to the
mutant allele (R132C) which would have expected to be present in equal amounts. Further analysis was
performed to confirm expression of the wild‐type and mutant alleles.
(B)
Figure 4. IDH1 mutant specific compound inhibit the ability of IDH1 mutant cells to grow under serum
starved conditions. (A) MCF10A parental cells fail to grow under restricted growth conditions. Introduction
of an IDH1(R132H/+) (HD 101‐013) or a BRAF(V600E/+) (HD 101‐012) mutation using Horizon’s rAAV‐
mediated homologous recombination gene engineering technology confers the ability of MCF10A to grow
under restricted growth conditions. (B) Treatment of MCF‐10A cells with AGI‐5198 reduces the ability of
the IDH1(R132H/+) clone to grow under restricted growth conditions. (C) Treatment of IDH1(R132H/+)
cells with AGI‐5198 starts to inhibits growth from just 48 hours after addition of drug despite alternative
mutant IDH over‐expression models requiring extended periods in culture before robust phenotypes
emerge 3‐6. (D) Treatment of IDH1(R132H/+) cells with 50μM (R)‐2‐HG reverses the growth inhibition of
100nM AGI‐5198 under restricted growth conditions.
Conclusions
•
•
•
•
Figure 3. HT‐1080 IDH1 (+cKO/R132C) X‐MAN™ isogenic cell lines are able to grow in xenograft models in
the absence of wild type allele expression. (A) 4x106 cells of the parental and the two clone lines were
implanted into NCr Nude mice (n=8). Bi‐weekly monitoring of tumour growth with calipers and
bodyweights was performed. (B) Tumours were excised and RNA extracted. Analysis of cDNA by ddPCR
using Taqman expression and SNP assays demonstrated that IDH1 mRNA expression levels are maintained
in all tumours derived from the HT‐1080 cell lines. Tumours derived from IDH1(+cKO/R132C) X‐MAN™ isogenic
cell line clones do not express the wild type allele.
Horizon Discovery Group plc, 7100 Cambridge Research Park, Waterbeach, Cambridge, CB25 9TL United Kingdom
Generated HT‐1080 IDH1(+cKO/R132C) X‐MAN™ isogenic cell line that lack expression of the wild type
allele. Cre‐mediated excision of exon 5 was not necessary to silence expression of targeted allele.
Cells lacking expression of the wild type allele are able to undergo tumorigenesis in xenograft
models.
Inhibition of the mutant protein prevents the IDH1(R132H/+) mediated growth of MCF10A cells.
MCF10A IDH1(R132H/+) cells enable facile proliferation assays to track activity of IDH1 inhibitors .
Wild type IDH1 is dispensable for tumour growth in IDH1 mutant cancer cells.
References:
1. Jin, G. et al. Disruption of wild‐type IDH1 suppresses D‐2‐hydroxyglutarate production in IDH1‐mutated gliomas. Cancer Res. 73, 496‐501 (2013)
2. Kohli, M., et al. Facile methods for generating human somatic cell gene knockouts using recombinant adeno‐associated viruses. Nucleic Acids Res. 32, e3 (2004)
3. Lu, C. et al. IDH mutation impairs histone demethylation and results in a block to cell differentiation. Nature 483, 474‐78 (2012)
4. Koivunen, P. et al. Transformation by the (R)‐enantiomer of 2‐hydroxyglutarate linked to EGLN activation. Nature 483, 484‐88 (2012)
5. Turcan, S. et al. IDH1 mutation is sufficient to establish the glioma hypermethylator phenotype. Nature 483, 479‐83 (2012)
6. Grassian, A.R. et al. Isocitrate dehydrogenase (IDH) mutations promote a reversible ZEB1/microRNA (miR)‐200‐dependent epithelial‐
mesenchymal transition (EMT). J Biol Chem 287, 42180‐94 (2012)
Email: [email protected] Web: www.horizondiscovery.com

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