www.sciencemag.org/cgi/content/full/science.1229259/DC1

Supplementary Material for

Highly Recurrent TERT Promoter Mutations in Human Melanoma

Franklin W. Huang, Eran Hodis, Mary Jue Xu, Gregory V. Kryukov, Lynda Chin, Levi A. Garraway*

*To whom correspondence should be addressed. E-mail: levi_garraway@dfci.harvard.edu

Published 24 January 2013 on Science Express DOI: 10.1126/science.1229259

This PDF file includes:

Materials and Methods

Fig. S1

Table S1

References (12, 13)

Other Supplementary Material for this manuscript includes the following: (available at www.sciencemag.org/cgi/content/full/science.1229259/DC1)

Table S1 as an Excel file

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Materials and Methods: Clinical samples. All melanoma samples analyzed in this study were collected and

sequenced under Institution-Review-Board-approved protocols

Identification and validation of TERT promoter mutations. Whole-genome sequencing data

corresponding to 25 melanoma tumor-normal pairs (1) were interrogated for somatic mutations highly

recurrent at the nucleotide level. 6 of 25 discovery pairs did not have adequate locus coverage, reducing

the discovery set to 19 pairs. Polymerase chain reaction (PCR) was performed on genomic DNA

followed by direct sequencing on amplified PCR products using an ABI 3730 DNA Sequence Analyzer

on a subset of these tumor-normal pairs to verify the individual TERT promoter mutations as well as on

an additional 51 pairs for further confirmation. Additionally the mutations were subcloned directly from

tumor DNA using the TOPO (Invitrogen) cloning kit according to manufacturer’s instructions followed

by Sanger sequencing (Fig. S1B). Oligonucleotide primers were synthesized according to the hg19

genomic reference sequence of TERT (genome.ucsc.edu). Primer sequences are available upon request.

The 70 tested melanomas represented 28 metastatic tumor samples, 11 primary tumor samples, and

31 tumor-derived cultures. Massively parallel sequencing was performed on cell lines as described

previously (1) and the TERT promoter was interrogated for recurrent mutations at coordinates chr 5:

1,295,228 and chr 5: 1,295,250 using MuTect (3). Chromatograms were viewed and screenshots taken

with the Geneious software program (www.geneious.com). P-values and confidence intervals were

calculated using standard packages in R (www.r-project.org).

Plasmids. Using normal germline DNA, a portion of the TERT core promoter (-132 to +5) or the full

core promoter (-200 to +73) were cloned into the multiple cloning site of pGL3-Basic (Promega)

upstream of the firefly luciferase gene with primers flanked with Mlu1 or Xho1 sites to create pGL3-

TERT132wt, a wild-type TERT-luc promoter construct. Using this strategy, the C228T mutation was also

cloned directly from tumor DNA into pGL3-Basic. Site-directed mutagenesis using QuikChange

Lightning (Agilent) was performed on the wild-type promoter construct to produce the promoter

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mutations. Constructs were verified by Sanger sequencing. All primer sequences are available upon

request.

Reporter assays. A375 melanoma cells, RPMI 7951 melanoma cells, UACC-62 melanoma cells, T24

bladder cancer cells, and HepG2 hepatocellular carcinoma cells were seeded at a density of 3 x 105 cells

in a 6-well format. T24 cells were cultured in McCoy’s 5A Medium (Life Technologies), HepG2 cells

were cultured in Dulbecco’s Modified Eagle Medium (Life Technologies), A375 cells were cultured in

Minimal Essential Medium (Life Technologies), RPMI cells and UACC-62 cells were cultured in RPMI

media, all containing 10% fetal bovine serum. Cells were transfected the following day using Fugene 6

(Promega) with 2.25μg of the TERT–luc promoter construct and 0.25μg of pRL-TK (Promega), a control

Renilla luciferase vector (12, 13). 48 hours later cells were lysed and luciferase activity was assayed with

the Dual Luciferase Reporter (Promega) assay in a 96-well format according to manufacturer instructions.

Experiments were performed in triplicate wells. Relative luciferase activity was calculated as the ratio of

firefly to Renilla luciferase activity, to control for transfection efficiency. Control is the relative

luciferase activity of cells transfected with promoterless reporter alone (pGL3-Basic).

Figure S1. Identification of TERT promoter mutations in melanoma

(A) Representative screenshot of TERT promoter mutations chr 5: 1,295,228 C>T (C228T) and chr 5:

1,295,250 C>T (C250T) from Integrative Genomics Viewer. Average depth of coverage in the 19

melanoma tumor-normal pairs with whole genome sequence coverage at the relevant loci was 58x in the

tumor and 30x in the normal at chr 5: 1,295,228 and 61x in the tumor and 30x in the normal at chr 5:

1,295,250, with minimum base quality score of 30 and minimum read mapping quality of 60.

(B) Additional sequence chromatograms of matched tumor and normal DNA representing somatic

mutations C228T and C250T in the TERT promoter locus.

(C) Subcloning of TERT core promoter mutations C228T and C250T. Sequence chromatograms depict

the reverse complement G>A transition.

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(D) Luciferase reporter assays for transcriptional activity from a portion of the TERT core promoter (-132

to +5) with either the C228T or C250T mutation compared to wild-type promoter in A375, T24 or HepG2

cell lines. The results depicted are the average of at least 3 independent experiments. Values are mean ±

s.d. * P < 0.01.

Table S1. Recurrent TERT promoter mutation status of melanomas and CCLE cell lines

Melanoma samples and cell lines are listed with indicated TERT promoter mutation. Two samples had a

dinucleotide mutation CC>TT causing C228T and C229T, were counted as C228T samples, and also

generated a consensus ETS motif.

Supplementary References:

12. I. Horikawa et al., Cancer Research 59, 826 (1999).

13. J.L. Babitt et al., Nature Genetics 38, 531 (2006).

References and Notes 1. M. F. Berger et al., Melanoma genome sequencing reveals frequent PREX2 mutations. Nature

485, 502 (2012). Medline

2. Materials and methods are available as supplementary materials on Science Online.

3. J. Barretina et al., The Cancer Cell Line Encyclopedia enables predictive modelling of anticancer drug sensitivity. Nature 483, 603 (2012). doi:10.1038/nature11003 Medline

4. E. Hodis et al., A landscape of driver mutations in melanoma. Cell 150, 251 (2012). doi:10.1016/j.cell.2012.06.024 Medline

5. M. Krauthammer et al., Exome sequencing identifies recurrent somatic RAC1 mutations in melanoma. Nat. Genet. 44, 1006 (2012). doi:10.1038/ng.2359 Medline

6. M. Imielinski et al., Mapping the Hallmarks of Lung Adenocarcinoma with Massively Parallel Sequencing. Cell 150, 1107 (2012). doi:10.1016/j.cell.2012.08.029

7. D. C. Bennett, Pigment Cell. Melanoma Res. 21, 27 (2008).

8. C. Michaloglou et al., BRAFE600-associated senescence-like cell cycle arrest of human naevi. Nature 436, 720 (2005). doi:10.1038/nature03890 Medline

9. A. Zhang et al., Cancer Res. 22, 6320 (2000).

10. C. Pirker et al., Chromosomal imbalances in primary and metastatic melanomas: over-representation of essential telomerase genes. Melanoma Res. 13, 483 (2003). doi:10.1097/00008390-200310000-00007 Medline

11. J. L. Rutter et al., A single nucleotide polymorphism in the matrix metalloproteinase-1 promoter creates an Ets binding site and augments transcription. Cancer Res. 58, 5321 (1998). Medline

12. I. Horikawa, P. L. Cable, C. Afshari, J. C. Barrett, Cloning and characterization of the promoter region of human telomerase reverse transcriptase gene. Cancer Res. 59, 826 (1999). Medline

13. J. L. Babitt et al., Bone morphogenetic protein signaling by hemojuvelin regulates hepcidin expression. Nat. Genet. 38, 531 (2006). doi:10.1038/ng1777 Medline

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