Blocking the PI3K pathway does not influence cell growth or signaling.

<p>A) CHP-212 and SK-N-AS cells were treated with 500nM of the MEK inhibitor MEK162 or the PI3K inhibitor BKM120 for 1 hour and next lysed and subjected to Western blot. Phosphorylation levels of AKT, ERK and S6 were detected by specific anti-phospho antibodies. Loading was verified by specific antibodies to total AKT, ERK and anti—tubulin. B) CHP-212 and SK-N-AS cells were treated with 500nM of MEK162 or BKM120 or combinations thereof as indicated for 96h. Then, cell growth was assessed by Cell titer Glo. C) Same as B) but the AKT1/2/3 inhibitor GSK690693 was used instead of BKM120. D) Same as C) but the AKT1/2/3 inhibitor MK2206 was used instead of GSK690693.</p

MEK inhibition blocks cell growth in NRAS mutant cells.

<p>A-C) All indicated cell lines were kept under equal conditions. Cell lines were left untreated or treated with indicated concentrations of the 3 MEK inhibitors AZD6244, MEK162 and PD0325901 for 96 hours. Then, cell growth was measured by ATP changes by Cell Titer Glo according to the manufacturer’s instructions. IC50 were calculated with GraphPad Prims and statistical difference calculated with t-test. *** p < 0.0001. D) All cell lines were kept under equal conditions, then treated with 500nM of the MEK inhibitors AZD6244, MEK162 and PD0325901 for 1 hour and next lysed and subjected to Western blot. Phosphorylation levels of ERK and MEK were detected by specific anti-phospho antibodies. Loading was verified by specific antibodies to total ERK, MEK and anti—tubulin. E) Crystal violet stain of CHP-212 and SK-N-AS cells from a clonogenic assay after 10 d treatment with DMSO (ctr), 0.001μM, 0.01 μM, 0.1μM and 1μM MEK162.</p

Inhibition of mTOR reduces cell growth in NRAS mutant cell lines.

<p>A) CHP-212 and SK-N-AS cells were treated with 5nM of the mTOR inhibitors Everolimus or 250nM AZD8055 for 1 hour. Then, cells were lysed and analysed by Western blot. B) All indicated cell lines were left untreated or treated with indicated concentrations of Everolimus for 96 hours. Next, cell growth was measured by Cell Titer Glo according to the manufacturer’s instructions C) Same as B) but the mTOR inhibitor AZD8055 was used instead. D) NRAS mutant and wild-type cell lines were incubated with indicated concentrations of Everolimus for 72 hours. Then, apoptosis was determined by Annexin V and PI staining.</p

Combined blockage of mTOR and MEK pathways reduces cell growth synergistically.

<p>A) CHP-212 and SK-N-AS cells were treated with indicated concentrations of AZD6244, MEK162, Everolimus or AZD8055 or combinations thereof as indicated for 1 hour. Then, cells were lysed and analysed by Western blot. Phosphorylation levels of AKT, ERK and S6 were detected by specific anti-phospho antibodies. Loading was verified by specific antibodies to total AKT, ERK and anti—tubulin. B) CHP-212 cells were treated with indicated concentrations of AZD6244, MEK162 or Everolimus or combinations thereof for 96h. Then, cell growth was measured by Cell Titer Glo. Combination index (CI) values with CalcuSyn Software (Biosoft). C) Same as B) but AZD8055 was used instead of Everolimus. D) SK-N-AS cells were treated with indicated concentrations of AZD6244 or Everolimus or combinations thereof for 96h. Then, cell growth was measured by Cell Titer Glo. E) Same as D) but AZD8055 was used instead of Everolimus.</p

Enrichment of Targetable Mutations in the Relapsed Neuroblastoma Genome - Fig 1

<p><b>Study cohort overview</b> A) Tabulation of Children’s Oncology Group (COG) risk classification and treatment time points of biopsy for 151 samples. (Intermed. = intermediate risk group) B) Number of samples taken at each treatment time point for nine patients with serial biopsies. (HR = high risk, IR = intermediate risk, LR = low risk at time of biopsy; further information in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1006501#pgen.1006501.s003" target="_blank">S2 Table</a>) C) Tabulation of all variants identified (VUS: variants of unknown significance) D) Total number of variants identified per sample, stratified by COG risk group. Inset shows a similar calculation for suspected driver variants only. Heavy line represents the median of the data. “n” indicates the number of patients in each risk group. E) Total number of variants in each sample. Each bar represents an individual sample; color corresponds to risk group (red = high, blue = intermediate, green = low).</p

Figure 3

<p>LOH and copy number changes on chromosomes 11, 3, 4, 1, 17, and 7. Global view of common areas of LOH (left) and copy number change (right) in 22 primary neuroblastomas. Each sample is depicted as a series of vertical bars in both the LOH and copy number panels. Blue areas represent regions of LOH, while yellow denotes retention of heterozygosity. Copy number is marked by shades of red, with ≤1 copy in light red and ≥3 copies in dark red (see scale at the bottom of the panel).</p

Association of CHD5 expression with risk factors and outcome in primary neuroblastomas

) Normalized CHD5 expression in 101 primary neuroblastomas stratified based on the presence (n = 26) or absence (n = 75) of 1p deletion (two-sample test, < .001). ) Association of normalized CHD5 expression with the risk group (low, intermediate, high, and ultrahigh) as defined above and in (). Briefly, for this study, low-risk patients were defined as infants (<p><b>Copyright information:</b></p><p>Taken from ", a Tumor Suppressor Gene Deleted From 1p36.31 in Neuroblastomas"</p><p></p><p>JNCI Journal of the National Cancer Institute 2008;100(13):940-949.</p><p>Published online 2 Jul 2008</p><p>PMCID:PMC2483574.</p><p></p

Patient disposition.

<p>Patient disposition.</p

Histology and CHD5 expression in xenografts derived from NLF and IMR5 cells transfected with either CHD5 sense or CHD5 antisense constructs

) Histology of NLF and IMR5 xenograft tumors (see ) after 5 weeks of growth. NLF--AS tumors were composed of undifferentiated cells with scant cytoplasm (top left), whereas NLF- tumors showed areas of necrosis () and differentiation (; top right). Cells in the IMR5--AS tumors were undifferentiated (bottom left), whereas cells in the IMR5- tumors had a more elongated appearance (bottom right). Bar = 20 μm. ) Relative expression of CHD5, normalized to glyceraldehyde-3-phosphate dehydrogenase (GAPDH), was determined by real-time reverse transcription–polymerase chain reaction in parental NLF and IMR5 cell lines, as well as the CHD5 sense and CHD5 antisense transfected lines used for the xenograft experiments. The normalized values indicated by each bar graph represent the mean of three measurements. Replicate measurements were within 10% of the mean for each bar shown. ) Expression of CHD5 protein, as detected by immunoblotting, is shown for the NLF and IMR5 parental lines and the corresponding sense- and antisense-transfected cells used in these experiments.<p><b>Copyright information:</b></p><p>Taken from ", a Tumor Suppressor Gene Deleted From 1p36.31 in Neuroblastomas"</p><p></p><p>JNCI Journal of the National Cancer Institute 2008;100(13):940-949.</p><p>Published online 2 Jul 2008</p><p>PMCID:PMC2483574.</p><p></p

Common regions of LOH in high-risk neuroblastoma and corresponding copy number change (A), and areas of copy number gain (B) identified by 10K SNP array analysis

<p>Common regions of LOH in high-risk neuroblastoma and corresponding copy number change (A), and areas of copy number gain (B) identified by 10K SNP array analysis</p