FAK and BCL2/BCL-XL inhibitors induced apoptosis.

<p>A, OCCC cell lines were treated with 1 µM ABT-737 (A), 1 µM BEZ235 (B), or 5 µM PF271 (P), individually or in combinations (AB indicates ABT-737/BEZ235; AP, ABT-737/PF271; BP, BEZ235/PF271; ABP, ABT-737/BEZ235/PF271) at the same drug concentrations for 6 hr. Cell lysates were prepared and subjected to Western blot analysis using the indicated antibodies. Cleavage of caspase-3 (CASP3), caspase-8 (CASP8), PARP, and FAK indicated that apoptosis had occurred. BCL-2 was not expressed in OVMANA and OVISE. B, TOV21G, RMGI and OVISE cells were treated individually or in combination with 1 µM ABT-737, 1 µM BEZ235 and/or 5 µM PF271 for 6 hr. The cells were fixed with 70% ethanol, were stained with propidium iodide (PI) and analyzed using a FACS Calibur flow cytometer.</p

FAK overexpression was associated with an increased copy number in OCCCs.

<p>A, Paraffin-embedded tumor sections were immunohistochemically stained using an anti-FAK antibody. The FAK staining results were scored into four categories based on the signal intensity: 0, no detection; 1, weak; 2, moderate; 3, strong. B, Variation in the FAK copy number was assessed with quantitative PCR. Copy number log ratios higher than 0.32 and 1.00 were considered evidence of copy number gain and gene amplification, respectively. The X-axis represents the samples. C, FAK overexpression (IHC score 3) correlated with the gains in copy number (copy number log ratio >0.32). Weak expressions of FAK (IHC score 1) resulted in normal or reduced FAK copy numbers (copy number log ratio <0.32). D, Samples with a mutation in <i>PIK3CA</i> (<i>PIK3CA</i>_Mut) are marked as red diamonds on the plot. The vertical dotted line indicates a copy number log ratio of 0.32.</p

PF271 enhanced the lethality of ABT-737.

<p>A-upper panels, Ovarian cancer cell lines were exposed to various concentrations of ABT-737 (⋄: A), sorafenib (△: S), ABT-737 in combination with 1 µM BEZ235 and 5 µM PF271 (: ABP), or sorafenib in combination with 1 µM BEZ235 and 5 µM PF271 (▴: SBP) for 72 hr. Cell viability (%) was calculated and dose response curves were predicted with a three-parameter log-logistic function. A-lower panel, The EC₅₀ of the anti-apoptotic inhibitors alone or in combination. This represents the result of one experiment; two additional studies also exhibited equivalent results. B, The protein expression patterns of the anti-apoptotic proteins and the phosphorylation status of AKT in nine ovarian cancer cell lines. Immunoblots were done to assess the basal expression levels of BCL-2, BCL-XL, MCL1, and phosphorylated AKT. C, The standard mRNA expression levels of BCL-XL and BCL-2 in forty ovarian cancer cell lines (CCLE database).</p

FAK inhibitors down-regulate MCL1.

<p>A, MCL1 was transiently down-regulated by treatment with PF271 or PF228. RMGI and OVISE cells were incubated in the presence of PF271 or PF228 at 5 µM for RMGI and 10 µM for OVISE. The cells were harvested at the indicated time points and lysed. D indicates the dimethyl sulfoxide (DMSO) vehicle control. Immunoblots were performed for p-FAK, FAK, MCL1, AKT, and p-AKT. B, A proposed schematic model by how the combined pharmacological inhibition of FAK/PYK2 and BCL-XL/BCL-2 induces apoptosis.</p

Drug responses of ovarian cancer cell lines to the FAK inhibitors.

<p>A, The chemical structure of the FAK inhibitors PF-573,228 (PF228) and PF-562,271 (PF271). B. OVISE cells were incubated for 24 hr at the indicated concentrations of the FAK inhibitors. Immunoblots were performed to assess inhibition of auto-phosphorylation by the FAK inhibitors. A vehicle control, containing dimethyl sulfoxide (DMSO), was performed. C, The viability of the ovarian cancer cells was determined after exposure to PF228 and PF271 for 72 hr. The results from only one experiment are shown; two additional studies also exhibited equivalent results.</p

<i>NTRK1</i> Fusion in Glioblastoma Multiforme

<div><p>Glioblastoma multiforme (GBM) is the most aggressive form of brain tumor, yet with no targeted therapy with substantial survival benefit. Recent studies on solid tumors showed that fusion genes often play driver roles and are promising targets for pharmaceutical intervention. To survey potential fusion genes in GBMs, we analysed RNA-Seq data from 162 GBM patients available through The Cancer Genome Atlas (TCGA), and found that 3′ exons of neurotrophic tyrosine kinase receptor type 1 (<i>NTRK1</i>, encoding TrkA) are fused to 5′ exons of the genes that are highly expressed in neuronal tissues, neurofascin (<i>NFASC</i>) and brevican (<i>BCAN</i>). The fusions preserved both the transmembrane and kinase domains of <i>NTRK1</i> in frame. <i>NTRK1</i> is a mediator of the pro-survival signaling of nerve growth factor (NGF) and is a known oncogene, found commonly altered in human cancer. While GBMs largely lacked <i>NTRK1</i> expression, the fusion-positive GBMs expressed fusion transcripts in high abundance, and showed elevated <i>NTRK1</i>-pathway activity. Lentiviral transduction of the <i>NFASC-NTRK1</i> fusion gene in NIH 3T3 cells increased proliferation <i>in vitro</i>, colony formation in soft agar, and tumor formation in mice, suggesting the possibility that the fusion contributed to the initiation or maintenance of the fusion-positive GBMs, and therefore may be a rational drug target.</p></div

Molecular consequences of <i>NTRK1</i>-fusion.

<p>(<b>A</b>) <i>NTRK1</i> expression in 170 TCGA GBM samples (from 162 patients) with RNA-Seq data. Samples bearing <i>NTRK1</i>-fusion genes are marked and labeled. (<b>B</b>) Relationship between <i>NTRK1</i> expression and NGF/TrkA-downstream pathway activity in 526 TCGA GBM samples (from 526 patients) with microarray gene expression data. Samples with <i>NTRK1</i>-fusion are marked with red circles. Two other samples with outlier <i>NTRK1</i> expression are marked with blue circles (TCGA-32-4209, TCGA-19-5947).</p

<i>NFASC-NTRK1</i> fusion.

<p>(<b>A</b>) Per-nucleotide read coverage (expression) of genomic regions along <i>NFASC</i> and <i>NTRK1</i>. The dotted line marks the DNA-level break-points in the two genes, as instructed by the fusion-point mapping result in panel B. (<b>B</b>) A schematic of pre-mRNAs of the <i>NFASC-NTRK1</i> fusion gene. Top and bottom sequences in black are the reads that map onto the DNA-level fusion-point. The fusion-point is mapped with slight ambiguity due to 2-nt-long micro-homology between the two break-points in the involved genes. (<b>C</b>) A schematic of spliced transcripts of the fusion gene. Bottom sequences in black are the reads that map onto the chimeric exon-exon splicing junction.</p

Top-20 potential gene fusions predicted by discordant read pair analysis.

a<p>Fusion type: intra, intra-chromosomal; inter, inter-chromosomal; read-through, the involved genes are adjacent and on the same strand; cis, the involved genes are adjacent and on the opposite strands.</p>b<p>For the fusions that were not excluded by indicated reasons, gene 1 and gene 2 correspond to the 5′- or 3′-partner of each fusion.</p>c<p>Sample IDs are abbreviated.</p>d<p>RESPER is FusionSeq-reported scores for prioritization. The fusions that were not excluded are indicated in bold font.</p

<i>BCAN-NTRK1</i> fusion.

<p>(<b>A</b>) Per-nucleotide read coverage of genomic regions along <i>BCAN</i> and <i>NTRK1</i>. The dotted line marks approximate positions where the fusion has occurred. (<b>B</b>) A schematic of spliced transcripts of the fusion gene. Bottom sequences in black are the reads that map onto the chimeric exon-exon splicing junction.</p