Indicators of Performance in the Primary Screen.

<p>A. Scatter plots comparing the percent S6-P positive cells in replicate plates for the entire screen. For each gene, the % S6-P positive cells in one plate is plotted against the value observed in a replicate plate (AvsB, AvsC, BvsC). Blue and red squares are values of %S6-P positive cells after transfection of scramble and mTOR siRNAs respectively, whereas aqua squares correspond to all other genes. The correlation coefficient (R^2 value) range from ~0.62 to ~0.67. B. Distribution of averaged z scores for all genes across the entire screen. Scatter plot comparing the z scores (y axis), averaged for all three replicates plates (numbered on the x-axis) for all genes across the entire screen. Color coding as in A. The cutoff of z+/- 2 is highlighted. Individula plates whose z’ was greatly inferior to replicates were eliminated from scoring (see Text and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0116096#sec002" target="_blank">methods</a>). C. Rank order plot. The averaged z-score for all replicates of all genes screened; primary positives were considered those exhibiting a z score exceeding +/- 2 in 2 or more replicates (~75% of genes) or on one plate chosen because either it was the only plate recovered or it exhibited a z’ of >0.4 over the replicates (~25% of genes). The position of selected genes is shown in the rectangular box.</p

siRNA-mediated depletion of QARS inhibits mTORC1 signaling.

<p>A. The effect of mTOR, QARS and LARS siRNA upon polypeptide knockdown and S6K-Thr³⁸⁹ phosphorylation. U2OS and Hela cells were transfected with siRNA oligos against mTOR, QARS (Q1,Q2), LARS(L1,L2) and a scramble control. After 72h amino acids were withdrawn for 2 hours and added back for 15 minutes as indicated. Cells extracts were subjected to SDS-PAGE and membranes were immunoblotted with the antibodies as indicated. The bar graphs display the combined results of three experiments (mean-/+ 1 S.D.; ** = p<0.0001 and * = p<0.002 vs scramble) The experiment shown in the bottom panels compares the effects of Thapsigargin (10M) with the indicated siRNAs on S6K-P (fourth from top), eIF2α(Ser51-P) (third from bottom) and the abundance of REDD1 (bottom). B. siRNA-mediated depletion of QARS inhibits global protein synthesis. Graphical representation of the combined results from three experiments (mean-/+ 1 S.D.) examining the effect, relative to scramble siRNA, of siRNA against mTOR, QARS and LARS on the abundance of the target polypeptides (upper), the relative phosphorylation of S6K-P (middle; ** = p<0.0001 and * = p<0.002 vs scramble) and on overall protein synthesis (bottom) in nutrient and serum replete U2OS cells. Analyses were carried out three days after transfection. ³⁵S[Methionine+cysteine] was added two hours before harvest; cycloheximide (CHX, 100μM) or carrier was added 30′ prior to ³⁵S. C. The effect of inhibiting translation for three days on mTORC1 signaling. Graphical representation of a dose response of cycloheximide (CHX) on global protein synthesis in U2OS cells. U2OS cells were plated in DMEM with 10% FCS +/- CHX and fresh media containing carrier or CHX was added every 24 hours for 72 hours. The cell were harvested at 72 hours; protein content expressed as a fraction of carrier control is plotted in the upper graph. Immunoblots of cell extracts for the proteins indicated are shown in the middle and the ratio of S6K-P/S6K (mean-/+ 1 S.D.) is shown in the bar graph at the bottom.</p

Genes in Tsc1 null MEFs scoring positive in one of three replicates.

<p>Genes in Tsc1 null MEFs scoring positive in one of three replicates.</p

Classification of the “Confirmed S6-P positives” into functional groups.

<p>Categorization of “Confirmed S6-P positives” using the PANTHER classification system into A. Protein Class; B. Molecular Function; C. Biological Processes. D. Manual reclassification by molecular function of the non-redundant “Confirmed S6-P positives” (listed in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0116096#pone.0116096.s009" target="_blank">Supp Table 7</a>) comprising the subcategories of “Biological Processes overrepresented in comparison to the whole genome (shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0116096#pone.0116096.s001" target="_blank">S1 Fig</a>.).”</p

High-throughput image-based screens for genes regulating the phosphorylation of rpS6.

<p>A. Immunofluorescence analysis (IF) of rpS6Ser(235/236) phosphorylation. Mia-Paca 2 cells were transfected in 384-well plates with a control, nonspecific RNAis NS1 (upper panel) and (NS2) (lower panel), and RNAi pools directed at S6K, TOR and Raptor, TSC1, TSC2 and PTEN. After 72 hours they were fixed, permeabilized and stained by using a rabbit monoclonal anti-S6-P(Ser 235/236) primary antibody, detected with secondary anti-rabbit Alexa 488 antibody (green). Nuclei are stained with DAPI (blue). Representative images are shown. B. Quantitation of cytoplasmic S6-P levels. The bars indicate the % of total MIA PaCa-2 cells (estimated by nuclear count) that exhibit cytoplasmic S6-P immunofluorescence at an intensity above an arbitrary threshold (% S6-P positive cells; see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0116096#sec002" target="_blank">methods</a>). The z’ is 0.31 for the combined use of NS1 and NS2; because NS2 gave consistently higher z’ than NS1 (e.g., 0.45 vs 0.32 for the experiment shown) NS2 was used exclusively in the primary screen; error bars represent 1S.D. * = p<0.01. C. Flow chart of the primary screen: Summary of the screening, hit analysis and hit selection. 21,121 genes were tested using RNAis composed of pools of 4 RNAi oligos (Dharmacon Library); 72 384-well plates were screened in triplicate (See <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0116096#pone.0116096.t001" target="_blank">Table 1</a>). The criteria for a “primary positive” are described in the text (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0116096#pone.0116096.t002" target="_blank">Table 2</a> lists genes not scored due to severe inhibition of proliferation). D. Results of the confirmation screen. From the 1046 “primary positives”, 870 genes, including all 161 positive kinases and the top 709 ranked by Q (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0116096#pone.0116096.s005" target="_blank">S3 Table</a>), were examined in a confirmation screen wherein each of the four RNAis was tested individually. The pie chart indicates how many of the potential positive hits were confirmed by 0–4 individual siRNAs (listed in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0116096#pone.0116096.s006" target="_blank">S4 Table</a>).</p

Confirmed S6-P positives whose depletion affects the viability of a panel of cell lines similar to that of mTOR depletion.

<p>This heat map shows the standardized essentiality profiles (blue more essential, red less essential) of 43 S6-P positives genes that are also significantly associated (FDR < 0.05) with mTOR essentiality (left). The association is determined by the normalized mutual information (IC score, nominal p value and FDR are shown at the top).</p

Table_2_Clinical and genomic characterization of chemoradiation-resistant HPV-positive oropharyngeal squamous cell carcinoma.xlsx

IntroductionMost patients with HPV-positive oropharyngeal squamous cell carcinoma (OPSCC) have an excellent response to chemoradiation, and trials are now investigating de-escalated treatment. However, up to 25% of patients with HPV-positive OPSCC will experience recurrence, and up to 5% will even progress through primary treatment. Currently, there are no molecular markers to identify patients with poor prognosis who would be harmed by de-escalation. Herein we report the clinical and genomic characteristics of persistent HPV-positive OPSCC after definitive platinum-based chemoradiation therapy.MethodsPatients with HPV-positive OPSCC treated with curative intent platinum-based chemoradiation between 2007 and 2017 at two institutions and with a persistent locoregional disease were included. We evaluated clinical characteristics, including smoking status, age, stage, treatment, and overall survival. A subset of five patients had tissue available for targeted exome DNA sequencing and RNA sequencing. Genomic analysis was compared to a previously published cohort of 47 treatment-responsive HPV+ OPSCC tumors after batch correction. Mutational landscape, pathway activation, and OncoGPS tumor states were employed to characterize these tumors.ResultsTen patients met the inclusion criteria. The tumor and nodal stages ranged from T1 to T4 and N1 to N2 by AJCC 8th edition staging. All patients were p16-positive by immunohistochemistry, and eight with available in situ hybridization were confirmed to be HPV-positive. The 1-year overall survival from the time of diagnosis was 57%, and the 2-year overall survival was 17%. TP53 mutations were present in three of five (60%) persistent tumors compared to 2% (one of 47) of treatment-responsive HPV-positive tumors (p = 0.008). Other genes with recurrent mutations in persistent HPV-positive OPSCC tumors were NF1, KMT2D, PIK3C2B, and TFGBR2. Compared to treatment-responsive HPV-positive tumors, persistent tumors demonstrated activation of DNA Repair and p53, EMT, MYC, SRC, and TGF-beta signaling pathways, with post-treatment samples demonstrating significant activation of the PI3K-EMT-Stem pathways compared to pretreatment samples.ConclusionChemoradiation-resistant HPV-positive OPSCC occurs infrequently but portends a poor prognosis. These tumors demonstrate higher rates of p53 mutation and activation of MYC, SRC, and TGF-beta pathways. A comparison of tumors before and after treatment demonstrates PI3K-EMT-Stem pathways post-treatment in HPV-positive tumors with persistent disease after platinum-based chemoradiation.</p

Table_1_Clinical and genomic characterization of chemoradiation-resistant HPV-positive oropharyngeal squamous cell carcinoma.xlsx

IntroductionMost patients with HPV-positive oropharyngeal squamous cell carcinoma (OPSCC) have an excellent response to chemoradiation, and trials are now investigating de-escalated treatment. However, up to 25% of patients with HPV-positive OPSCC will experience recurrence, and up to 5% will even progress through primary treatment. Currently, there are no molecular markers to identify patients with poor prognosis who would be harmed by de-escalation. Herein we report the clinical and genomic characteristics of persistent HPV-positive OPSCC after definitive platinum-based chemoradiation therapy.MethodsPatients with HPV-positive OPSCC treated with curative intent platinum-based chemoradiation between 2007 and 2017 at two institutions and with a persistent locoregional disease were included. We evaluated clinical characteristics, including smoking status, age, stage, treatment, and overall survival. A subset of five patients had tissue available for targeted exome DNA sequencing and RNA sequencing. Genomic analysis was compared to a previously published cohort of 47 treatment-responsive HPV+ OPSCC tumors after batch correction. Mutational landscape, pathway activation, and OncoGPS tumor states were employed to characterize these tumors.ResultsTen patients met the inclusion criteria. The tumor and nodal stages ranged from T1 to T4 and N1 to N2 by AJCC 8th edition staging. All patients were p16-positive by immunohistochemistry, and eight with available in situ hybridization were confirmed to be HPV-positive. The 1-year overall survival from the time of diagnosis was 57%, and the 2-year overall survival was 17%. TP53 mutations were present in three of five (60%) persistent tumors compared to 2% (one of 47) of treatment-responsive HPV-positive tumors (p = 0.008). Other genes with recurrent mutations in persistent HPV-positive OPSCC tumors were NF1, KMT2D, PIK3C2B, and TFGBR2. Compared to treatment-responsive HPV-positive tumors, persistent tumors demonstrated activation of DNA Repair and p53, EMT, MYC, SRC, and TGF-beta signaling pathways, with post-treatment samples demonstrating significant activation of the PI3K-EMT-Stem pathways compared to pretreatment samples.ConclusionChemoradiation-resistant HPV-positive OPSCC occurs infrequently but portends a poor prognosis. These tumors demonstrate higher rates of p53 mutation and activation of MYC, SRC, and TGF-beta pathways. A comparison of tumors before and after treatment demonstrates PI3K-EMT-Stem pathways post-treatment in HPV-positive tumors with persistent disease after platinum-based chemoradiation.</p

Image_1_Clinical and genomic characterization of chemoradiation-resistant HPV-positive oropharyngeal squamous cell carcinoma.jpeg

IntroductionMost patients with HPV-positive oropharyngeal squamous cell carcinoma (OPSCC) have an excellent response to chemoradiation, and trials are now investigating de-escalated treatment. However, up to 25% of patients with HPV-positive OPSCC will experience recurrence, and up to 5% will even progress through primary treatment. Currently, there are no molecular markers to identify patients with poor prognosis who would be harmed by de-escalation. Herein we report the clinical and genomic characteristics of persistent HPV-positive OPSCC after definitive platinum-based chemoradiation therapy.MethodsPatients with HPV-positive OPSCC treated with curative intent platinum-based chemoradiation between 2007 and 2017 at two institutions and with a persistent locoregional disease were included. We evaluated clinical characteristics, including smoking status, age, stage, treatment, and overall survival. A subset of five patients had tissue available for targeted exome DNA sequencing and RNA sequencing. Genomic analysis was compared to a previously published cohort of 47 treatment-responsive HPV+ OPSCC tumors after batch correction. Mutational landscape, pathway activation, and OncoGPS tumor states were employed to characterize these tumors.ResultsTen patients met the inclusion criteria. The tumor and nodal stages ranged from T1 to T4 and N1 to N2 by AJCC 8th edition staging. All patients were p16-positive by immunohistochemistry, and eight with available in situ hybridization were confirmed to be HPV-positive. The 1-year overall survival from the time of diagnosis was 57%, and the 2-year overall survival was 17%. TP53 mutations were present in three of five (60%) persistent tumors compared to 2% (one of 47) of treatment-responsive HPV-positive tumors (p = 0.008). Other genes with recurrent mutations in persistent HPV-positive OPSCC tumors were NF1, KMT2D, PIK3C2B, and TFGBR2. Compared to treatment-responsive HPV-positive tumors, persistent tumors demonstrated activation of DNA Repair and p53, EMT, MYC, SRC, and TGF-beta signaling pathways, with post-treatment samples demonstrating significant activation of the PI3K-EMT-Stem pathways compared to pretreatment samples.ConclusionChemoradiation-resistant HPV-positive OPSCC occurs infrequently but portends a poor prognosis. These tumors demonstrate higher rates of p53 mutation and activation of MYC, SRC, and TGF-beta pathways. A comparison of tumors before and after treatment demonstrates PI3K-EMT-Stem pathways post-treatment in HPV-positive tumors with persistent disease after platinum-based chemoradiation.</p

Image_2_Clinical and genomic characterization of chemoradiation-resistant HPV-positive oropharyngeal squamous cell carcinoma.jpeg

IntroductionMost patients with HPV-positive oropharyngeal squamous cell carcinoma (OPSCC) have an excellent response to chemoradiation, and trials are now investigating de-escalated treatment. However, up to 25% of patients with HPV-positive OPSCC will experience recurrence, and up to 5% will even progress through primary treatment. Currently, there are no molecular markers to identify patients with poor prognosis who would be harmed by de-escalation. Herein we report the clinical and genomic characteristics of persistent HPV-positive OPSCC after definitive platinum-based chemoradiation therapy.MethodsPatients with HPV-positive OPSCC treated with curative intent platinum-based chemoradiation between 2007 and 2017 at two institutions and with a persistent locoregional disease were included. We evaluated clinical characteristics, including smoking status, age, stage, treatment, and overall survival. A subset of five patients had tissue available for targeted exome DNA sequencing and RNA sequencing. Genomic analysis was compared to a previously published cohort of 47 treatment-responsive HPV+ OPSCC tumors after batch correction. Mutational landscape, pathway activation, and OncoGPS tumor states were employed to characterize these tumors.ResultsTen patients met the inclusion criteria. The tumor and nodal stages ranged from T1 to T4 and N1 to N2 by AJCC 8th edition staging. All patients were p16-positive by immunohistochemistry, and eight with available in situ hybridization were confirmed to be HPV-positive. The 1-year overall survival from the time of diagnosis was 57%, and the 2-year overall survival was 17%. TP53 mutations were present in three of five (60%) persistent tumors compared to 2% (one of 47) of treatment-responsive HPV-positive tumors (p = 0.008). Other genes with recurrent mutations in persistent HPV-positive OPSCC tumors were NF1, KMT2D, PIK3C2B, and TFGBR2. Compared to treatment-responsive HPV-positive tumors, persistent tumors demonstrated activation of DNA Repair and p53, EMT, MYC, SRC, and TGF-beta signaling pathways, with post-treatment samples demonstrating significant activation of the PI3K-EMT-Stem pathways compared to pretreatment samples.ConclusionChemoradiation-resistant HPV-positive OPSCC occurs infrequently but portends a poor prognosis. These tumors demonstrate higher rates of p53 mutation and activation of MYC, SRC, and TGF-beta pathways. A comparison of tumors before and after treatment demonstrates PI3K-EMT-Stem pathways post-treatment in HPV-positive tumors with persistent disease after platinum-based chemoradiation.</p