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SUPPLEMENTARY INFORMATION
Supplementary Tables
Supplementary Table S1. Samples Analyzed for AURKA Copy Number by SNP-Array and qPCR (related
to Figure 2)
Sample
Name
STS 26T
S462
ST 8814
S462 TY
T265
MPNST-A
MPNST-B
MPNST-C
MPNST-D
MPNST-E
MPNST-F
MPNST-G
MPNST-H
MPNST-I
MPNST-J
MPNST-K
MPNST-L
MPNST-M
MPNST-N
NF-A
NF-B
NF-C
NF-D
NF-E
NF-F
NF-G
NF-H
Control
Control
Control
Control
Control
Control
Control
Control
Control
Control
Control
Control
SNPa copy
number
qPCR copy
number
3n
3n
3n
3n
4n
3n
2n
2n
2n
2n
2n
3n
3n
2n
2n
2n
2n
2n
2n
2n
2n
NA
NA
NA
NA
NA
NA
2n
2n
2n
2n
2n
2n
NA
NA
NA
NA
NA
NA
4n
3n
4n
3n
4n
3n
3n
2n
2n
2n
2n
NA
4n
3n
3n
3n
3n
2n
3n
3n
2n
2n
2n
2n
2n
2n
2n
2n
2n
2n
2n
2n
2n
2n
2n
2n
2n
2n
3n
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Supplementary Table S1 (related to Figure 2): AURKA copy number analysis results obtained by applying
qPCR-DNA and SNP-array techniques. The results indicate a clear amplification of AURKA locus in MPNST
cell lines and also in a proportion of primary MPNST tumors. Divergences on copy number results between
both techniques were probably due to the presence of a high percentage of normal cells within primary MPNST
samples (data not shown). As a rule of thumb, qPCR is a more sensitive technique, being able to detect locus
copy number changes in the presence of high percentages of normal cells, but with a greater variability (less
accuracy), while SNPa shows more robustness against false positives, but is more sensible to false negatives
due to the presence of high amounts of normal cells within tumors (less precision). NA=not analyzed.
Supplementary Table S2: Primers for qPCR DNA Analysis (related to Figure 2) Primer sequences used
for qPCR analysis of the AURKA locus in human NF1 tumor and control DNA samples.
Primer name Sequence
AURKA-L
AURKA-R
LINE1-L
LINE1-R
L1PA-L
L1PA-R
Amplicon size UPL
probe
111 bp
#55
5'-ggacggttcttctggagctt-3'
5'-gcgagacttcgtctcaaaaca-3'
5'-gcgctaaacatggaaaggaa-3'
66 bp
5'-tggtctttacaatttggcatgt-3'
5'-aaaaagtcaggaaacaacaggtg-3' 71 bp
#28
#55
5'-tcccaccaacagtgtaaaagtg-3'
Supplementary Figures
Supplementary Figure S1: 339 Ras-driven Genes Differentially expressed in Mouse or Human
Neurofibromas or MPNSTs (see separate Excel File)
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Supplementary Figure S2. Gene Interaction Network of 339 Ras-Driven Genes Differentially Expressed
in Mouse or Human Neurofibromas or MPNSTs.
Supplementary Figure S2: Gene interaction network shows direct interactions of up- and down-regulated
genes from latent factor regression modeling. Green lines indicate a positive effect, red lines a negative effect;
arrows direction of effect. Yellow highlights are targets of AURKA (Aurora-A), highlighted in grey. Grey outlines
indicate additional drug targets. Red circles indicate up-regulated gene expression while blue circles indicate
down-regulation of gene expression. The intensity of blue and red in circles corresponds to degree of change
in gene expression. (See also Supplementary Figure S1).
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Supplementary Figure S3. Dot plot showing qPCR DNA-copy number results for AURKA gene locus in 37
samples analyzed (see Supplementary Table S1). A calculated relative copy number of 1.0 indicates a diploid
status (2 copies) of AURKA locus, whereas 1.5, or above, indicates an amplification event. Dashed lines
represent calculated threshold values of 0.74, 1.22 and 1.71, which delimitate 4 areas of locus copy number: 1
copy, 2 copies, 3 copies and 4 or more copies. As it can be observed, a significant proportion of primary
MPNST tumors exhibit AURKA locus amplification (mostly 3 copies), and all MPNST cell lines exhibit AURKA
locus amplification with 3 or even more copies.
Supplementary Figure S4. Expressional down regulation or inhibition of Aurora Kinase Reduces
Survival in MPNST Cells.
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Supplementary Figure S4. (A) % survival of MPNST cell line T265, STS26T and ST8814 post down
regulation of AurkA and AurkB using three lenti-viral shRNA per gene. (B) Aurora Kinase inhibitors, VX-680
and ZM447439 reduce survival in all 5 MPNST cell lines.
Supplementary Experimental Procedures
Gene expression microarray data generation
Total RNA was isolated from mouse nerves and tumors using the Qiagen fibrous tissue protocol
(Qiagen, Inc. Valencia, CA). The murine PNST mouse models included T-P0T-P0CreB; Nf2 flox/flox (3), TP0CreB; Nf2 flox/+; Nf1 flox/flox (5), T-P0CreB; Nf2 flox/flox; Nf1 flox/+ (3), T-P0CreB; Nf2 flox/flox; p53+/- (3)
T-NPcis (4). Expression profiles were generated using Affymetrix HG-U133 plus 2 and MOE430 2.0
oligonucleotide microarrays. Affymetrix Microarray Suite 5.0 was used to generate ‘CEL’ files for each sample
that were normalized using the Robust Multichip Analysis (RMA) algorithm as implemented in Bioconductor/R
(1). Affymetrix probes were remapped to the latest annotated RefSeq genes (version 11.0.1) (2).
Ortholog gene mapping
Two data sources were used for orthogonal mapping between mouse and human genes Affymetrix probe
sequence data (Human affx_probe.fasta downloaded on 2009-07-16 and Mouse affx_probe.fasta downloaded
on 2009-07-16) and Refseq RNA sequence data (human.rna.gbff.gz downloaded on 2009-07-16 and
mouse.rna.gbff.gz downloaded on 2009-07-16). These files were used to extract transcript sequences, probe
sequences and probe sets. We validated probe sets by verifying that probes match the Refseq gene
sequence, are unique across all genes and have a valid probe ratio ≥ 50%. Validated probe sets were then
used to form the Orthogonal Mapping for the Human and Mouse species, utilizing The Jackson Laboratory’s
Human vs. Mouse Orthology (HMD_Human5.rpt generated 2009-07-16). Once the data was integrated, we
focused on 8,974 gene orthologs statistically different in at least one of the two species and present in both
mouse and human data sets.
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To identify inversely expressed transcripts, expression level filtering on log scale normalized intensities
was used to identify and pool orthologous genes up-regulated > 1.2 or down-regulated < 0.8 in 18 of 26 human
neurofibroma samples but down-regulated < 0.8 or up-regulated > 1.2 in 11 of 15 GEM model neurofibroma
samples, and genes up-regulated > 1.2 or down-regulated < 0.8 in 4 of 6 human MPNST samples but downregulated < 0.8 or up-regulated > 1.2 in 13 of 18 GEM model MPNST samples.
To identify uniquely regulated human transcripts, we isolated those gene orthologs statistically different
from the human data set but not differentially expressed in the mouse data set or in the list of gene orthologs
similarly or inversely expressed. For mouse, we isolated those gene orthologs statistically different from the
mouse data set but not differentially expressed in the human data set or in the list of gene orthologs similarly or
inversely expressed.
AURKA locus copy number analysis
Extracted BAF and LRR were analyzed, and copy number (CN) calls made using R and genoCN (3). When
available, normal tissue matching tumor samples was also analyzed and used to help the algorithm with the
copy number alteration callings. Like most SNP array analysis algorithms, genoCNA tends to perform poorly
when: 1) too much variation is found in the data, or 2) when cell populations not carrying the genetic alteration
present in the tumor represents more than 50% of the cells. Thus, a manual curation of the data was
performed to refine the analysis. Copy number alterations (CNA) with an extension shorter than 1 Mb are not
reported. For PCR analysis, DNA amount was normalized to that of LINE1 and L1PA, which are genomic
repetitive elements for which copy number per haploid genome is similar among all human tissues (normal or
tumoral) (4). The UPL Assay Design Center webpage from Roche (https://www.roche-appliedscience.com/sis/rtpcr/upl/index.jsp?id=uplct_030000) was used to design a primer pair and choose a suitable
UPL probe for each gene (Supplementary Table S2). Conditions for amplification were as follows: one cycle
of 95°C for 10 min, followed by 45 cycles of 95°C for 10 sec, 60°C for 30 sec and 72°C for 1 sec, and a final
cycle of 40°C for 30s. Threshold cycle (Ct) numbers were obtained using LightCycler® 480 software. PCRs for
each primer set and sample were performed in triplicate and threshold cycle numbers were averaged. Fold
changes for AURKA, L1 and L1PA genes were calculated for each sample according to Pfaffl method (5) and
those for L1 and L1PA were then averaged. The relative copy number value for each sample was calculated
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as the ratio Fold Change (AURKA)/mean Fold Change (L1-L1PA) (Figure 2C). A calculated relative copy
number of 1.0 indicates a diploid status (2 copies) of AURKA locus, whereas 1.5, or above, indicates an
amplification event. Dashed lines represent calculated threshold values of 0.74, 1.22 and 1.71, which
delimitate 4 areas of locus copy number: 1 copy, 2 copies, 3 copies and 4 or more copies. A significant
proportion of primary MPNST tumors exhibit AURKA locus amplification (mostly 3 copies), and all MPNST cell
lines exhibit AURKA locus amplification with 3 or even more copies.
Immunolabeling
Teased sciatic nerves from adult wild type or CNP-HRAS12v mice were prepared for immunolabeling as
described (6). Schwann cells were labeled with anti-S100 antibodies (Dako, Carpenteria, CA) followed by
FITC-conjugated secondary antibodies (green); expression of the HA-tagged CNP-HRAS12V transgene was
detected with anti-HA antibodies (Santa Cruz Biotechnology, Inc., Santa Cruz, CA) followed by TRITCconjugated secondary antibodies (red).
Electron microscopy
Saphenous nerves from wild type or CNP-HRAS12V mice were prepared for electron microscopy as described
(7). We counted numbers of unmyelinated axons in each Remak bundle and compared wild type versus CNPHRAS12V nerves using Student’s t-test.
Supplementary References
1.
Irizarry RA, Hobbs B, Collin F, Beazer-Barclay YD, Antonellis KJ, Scherf U, et al. Exploration,
normalization, and summaries of high density oligonucleotide array probe level data. Biostatistics
2003;4:249-64.
2.
Dai M, Wang P, Boyd AD, Kostov G, Athey B, Jones EG, et al. Evolving gene/transcript definitions
significantly alter the interpretation of GeneChip data. Nucleic Acids Res 2005;33:e175.
3.
Sun W, Wright FA, Tang Z, Nordgard SH, Van Loo P, Yu T, et al. Integrated study of copy number
states and genotype calls using high-density SNP arrays. Nucleic Acids Res 2009;37:5365-77.
4.
Wang TL, Maierhofer C, Speicher MR, Lengauer C, Vogelstein B, Kinzler KW, et al. Digital karyotyping.
Proc Natl Acad Sci U S A 2002;99:16156-61.
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5.
Pfaffl MW. A new mathematical model for relative quantification in real-time RT-PCR. Nucleic Acids
Res 2001;29:e45.
6.
Daston MM, Scrable H, Nordlund M, Sturbaum AK, Nissen LM, and Ratner N. The protein product of
the neurofibromatosis type 1 gene is expressed at highest abundance in neurons, Schwann cells, and
oligodendrocytes. Neuron 1992;8:415-28.
7.
Ling BC, Wu J, Miller SJ, Monk KR, Shamekh R, Rizvi TA, et al. Role for the epidermal growth factor
receptor in neurofibromatosis-related peripheral nerve tumorigenesis. Cancer Cell 2005;7:65-75.
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