Journal: Cancer letters

Article Title: Human papillomavirus insertions identify the PIM family of serine/threonine kinases as targetable driver genes in head and neck squamous cell carcinoma

doi: 10.1016/j.canlet.2020.01.012

Figure Lengend Snippet: (A) HPV integrant-mediated genomic amplification of the PIM1 locus in UPCI:SCC090 HNSCC cells. (Top) Histograms show (y-axis) depth of WGS coverage by (blue) well-aligned sequence reads, and (middle) counts of (red) HPV insertional and (gray) host-host breakpoint reads in UPCI:SCC090 cells, mapped to the (bottom) reference human genome (hg19) at (x-axis) the PIM1 locus on Chr. 6 (bottom, gene schematics, chromosomal coordinates in Mb). Reproduced from Akagi et al. with permission [4]. See also Supp. Fig. S1. (B) RNA-seq expression levels (y-axis; FPKM) of PIM1, PIM2 and PIM3 transcripts expressed in HNSCC cell lines (i.e. UD-SCC-2, UM-SCC-47, UPCI:SCC090, CAL 27, D562). (C) Western blot analysis of PIM1 (L, long and S, short isoforms), PIM2, and PIM3 expressed in panel of HNSCC cell lines, with beta-tubulin as loading control. Arrows, expected protein mass.

Article Snippet: Dual inhibition of PIM and EGFR pathways To assay effects of PIM pathway inhibition on cell viability, cell lines were treated with pan-PIM inhibitor INCB053914 (Incyte Corp.) [ 18 ], or the PIM1 inhibitors Quercetagetin (Santa Cruz Technologies) or PIM1 Inhibitor 2 (Santa Cruz Technologies), or a negative control, DMSO, as described in Supplementary Methods .

Techniques: Amplification, Sequencing, RNA Sequencing, Expressing, Western Blot, Control

Journal: Cancer letters

Article Title: Human papillomavirus insertions identify the PIM family of serine/threonine kinases as targetable driver genes in head and neck squamous cell carcinoma

doi: 10.1016/j.canlet.2020.01.012

Figure Lengend Snippet: (A) UPCI:SCC090 cells were transduced with lentivirus expressing shRNAs with indicated targets of scrambled control (scr. control), FOXE1 or PIM1, or no lentivirus. Cell viability was determined by trypan blue exclusion using a hemacytometer in light microscopy. Barplots, mean of duplicate cell counts; error bars, range of data. (B) HPV-positive (UD-SCC-2, UM-SCC-47, UPCI:SCC090) and HPV negative (CAL 27, D562) OSCC cell lines were transduced with lentivirus expressing shRNAs with indicated targets of scrambled control, PIM1 or PIM3, or no lentivirus. Barplots, mean of duplicate cell counts; error bars, range.

Article Snippet: Dual inhibition of PIM and EGFR pathways To assay effects of PIM pathway inhibition on cell viability, cell lines were treated with pan-PIM inhibitor INCB053914 (Incyte Corp.) [ 18 ], or the PIM1 inhibitors Quercetagetin (Santa Cruz Technologies) or PIM1 Inhibitor 2 (Santa Cruz Technologies), or a negative control, DMSO, as described in Supplementary Methods .

Techniques: Transduction, Expressing, Control, Light Microscopy

Journal: Cancer letters

Article Title: Human papillomavirus insertions identify the PIM family of serine/threonine kinases as targetable driver genes in head and neck squamous cell carcinoma

doi: 10.1016/j.canlet.2020.01.012

Figure Lengend Snippet: (A) Western blot analysis of PIM1 expression and (B) cell proliferation curves (y-axis, Log10 average cell count) of parental UPCI:SCC090 cell line, 2 negative controls (empty vector mock-edited by CRISPR, clones C1, C2), and 5 PIM1 knockout clones (gene edited by CRISPR, clones C9, C2, C5, C4, C11) cultured over time (x-axis, days). Key: colors, symbols: cell clones. Slopes of linear regression lines indicative of growth rates are: parental, 0.157; empty C1,0.156; empty C2, 0.157; PIM1 C9, 0.135; PIM1 C2, 0.148; PIM1 C5, 0.104; PIM1 C4, 0.121; and PIM1 C11, 0.066. Error bars, range of cell counts in duplicate wells. (C) Western blot analysis of downstream proteins and phosphorylation targets of PIM1, in a selected PIM1 knockout clone derived from UPCI:SCC090 cells by CRISPR gene editing, compared with parental UPCI:SCC090 control cells. (D) Schematic of possible mechanism of resistance in response to PIM inhibition in HNSCC cells such as CAL 27 cells, through feedback loops involving increased expression of PIM protein through STAT activation. Downstream targets of PIM kinases are displayed showing activating (green) and inactivating (orange) phosphorylation, and biological responses associated with these signaling interactions. GF, growth factor ligand, e.g. EGF, epidermal growth factor; RTK, receptor tyrosine kinase, e.g. EGFR, epidermal growth factor receptor; STAT, signal transducer and activator of transcription. See also Supp. Figs. S6 and S11.

Article Snippet: Dual inhibition of PIM and EGFR pathways To assay effects of PIM pathway inhibition on cell viability, cell lines were treated with pan-PIM inhibitor INCB053914 (Incyte Corp.) [ 18 ], or the PIM1 inhibitors Quercetagetin (Santa Cruz Technologies) or PIM1 Inhibitor 2 (Santa Cruz Technologies), or a negative control, DMSO, as described in Supplementary Methods .

Techniques: Western Blot, Expressing, Cell Counting, Plasmid Preparation, CRISPR, Clone Assay, Knock-Out, Cell Culture, Phospho-proteomics, Derivative Assay, Control, Inhibition, Activation Assay

Journal: Cancer letters

Article Title: Human papillomavirus insertions identify the PIM family of serine/threonine kinases as targetable driver genes in head and neck squamous cell carcinoma

doi: 10.1016/j.canlet.2020.01.012

Figure Lengend Snippet: (A-C) Various cell lines (legend, color symbols) were treated with (A) INCB053914; (B) PIM1 inhibitor 2; or (C) quercetagetin at indicated concentrations (x-axis) for 72 h. Cell viability was assessed using neutral red assay (y-axis, normalized to DMSO control). Data represent averages of triplicate wells normalized to DMSO control. Error bars, standard deviation. (D) Nude mice injected with UPCI:SCC090 cells (day 0) were treated twice daily with either vehicle or INCB053914 via oral gavage, starting on day 20. Mean tumor volume (y-axis) was assessed for 8 days (x-axis); error bars, standard deviation (n = 7 mice).

Article Snippet: Dual inhibition of PIM and EGFR pathways To assay effects of PIM pathway inhibition on cell viability, cell lines were treated with pan-PIM inhibitor INCB053914 (Incyte Corp.) [ 18 ], or the PIM1 inhibitors Quercetagetin (Santa Cruz Technologies) or PIM1 Inhibitor 2 (Santa Cruz Technologies), or a negative control, DMSO, as described in Supplementary Methods .

Techniques: Neutral Red Assay, Control, Standard Deviation, Injection

Journal: Cancer letters

Article Title: Human papillomavirus insertions identify the PIM family of serine/threonine kinases as targetable driver genes in head and neck squamous cell carcinoma

doi: 10.1016/j.canlet.2020.01.012

Figure Lengend Snippet: The histogram shows the distribution of median gene expression levels in 151 primary human HNSCC tumors for each of 18,640 genes [3]. After normalization and batch correction of RNA-seq data, genes expressed in at least in one sample (log2(TPM + 1) > 0) were compared. Gencode v18 genes were used as a reference set of annotated genes for comparison of expression. X-axis, median of transcript levels for each gene across all HNSCC samples (transformed as log2 TPM+1), ranging from 0.014 to 13.7; y-axis, number of genes. Blue vertical lines, log2 transformed median expression levels of PIM1 = 6.51 (top 6.5th percentile), PIM2 =4.94 (21.4th percentile), PIM3 =8.43 (1.3rd percentile) and EGFR = 6.29 (7.7th percentile).

Article Snippet: Dual inhibition of PIM and EGFR pathways To assay effects of PIM pathway inhibition on cell viability, cell lines were treated with pan-PIM inhibitor INCB053914 (Incyte Corp.) [ 18 ], or the PIM1 inhibitors Quercetagetin (Santa Cruz Technologies) or PIM1 Inhibitor 2 (Santa Cruz Technologies), or a negative control, DMSO, as described in Supplementary Methods .

Techniques: Gene Expression, RNA Sequencing, Comparison, Expressing, Transformation Assay