Applied diagnostics in liver cancer. Efficient combinations of sorafenib with targeted inhibitors blocking AKT/mTOR

Hepatocellular carcinoma (HCC) is the third most common cause of cancer-related deaths worldwide. There is increasing interest in developing specific markers to serve as predictors of response to sorafenib and to guide targeted therapy. Using a sequencing platform designed to study somatic mutations in a selection of 112 genes (HepatoExome), we aimed to characterize lesions from HCC patients and cell lines, and to use the data to study the biological and mechanistic effects of case-specific targeted therapies used alone or in combination with sorafenib. We characterized 331 HCC cases in silico and 32 paired samples obtained prospectively from primary tumors of HCC patients. Each case was analyzed in a time compatible with the requirements of the clinic (within 15 days). In 53% of the discovery cohort cases, we detected unique mutational signatures, with up to 34% of them carrying mutated genes with the potential to guide therapy. In a panel of HCC cell lines, each characterized by a specific mutational signature, sorafenib elicited heterogeneous mechanistic and biological responses, whereas targeted therapy provoked the robust inhibition of cell proliferation and DNA synthesis along with the blockage of AKT/mTOR signaling. The combination of sorafenib with targeted therapies exhibited synergistic anti-HCC biological activity concomitantly with highly effective inhibition of MAPK and AKT/mTOR signaling. Thus, somatic mutations may lead to identify case-specific mechanisms of disease in HCC lesions arising from multiple etiologies. Moreover, targeted therapies guided by molecular characterization, used alone or in combination with sorafenib, can effectively block important HCC disease mechanisms.


SUPPLEMENTARY MATERIALS Enrichment library design, preparation and sequencing
Targeted enrichment sequencing was performed on human FF tumor and non-tumor specimen and, when indicated, on blood samples. The custom probe design was constructed with SureDesign (Agilent Technologies) enriching for the exons of 112 genes of interest (Design ID: 37503-1413372517). The design focused on the coding regions of a group of 112 genes known to be mutated in HCC, and which were selected based on the following criteria: i) genes of known relevance in HCC, ii) genes that may be associated with pharmacological inhibitors with potential clinical use and iii) genes shown mutated in HCC independently of the population frequency.
DNA libraries were prepared with the HaloPlex Target Enrichment System, following the manufacturer's instructions. Briefly, 400 ng of genomic DNA was digested with the specific cocktail of restriction enzymes provided in the kit. Digested DNA was then hybridized to a probe for target enrichment, indexed and captured. Each DNA was then amplified by PCR at Tm = 60ºC, for 18 cycles, using a Herculase II Fusion Enzyme kit (Agilent Technologies). Next, amplified target libraries were purified using an Agencourt AMPure XP Kit (Beckman Coulter Genomics), following the manufacturer's instructions, and quantified with Qubit 2.0 fluorimeter (Life Technologies), using the Qubit® dsDNA HS Assay Kit (Life Technologies). They were also analyzed in parallel by capillary electrophoresis in a 2100 Bioanalyzer (Agilent Technologies), using High Sensitivity DNA reagents and chip Kits (Agilent Technologies). Libraries were sequenced at the Instituto de Medicina Genómica (IMEGEN, Valencia University, Spain) with a MiSeq Personal Sequencer (Illumina).

Somatic mutation identification
Somatic mutation identification was done by using Agilent Sure Call 2.1.1.13 software and IGV 2.3.46 software. In parallel Sequencing data were aligned against the human reference genome (hg19) using the BWA aligner [1]. The alignment was refined using SAMTOOLS fixmate and PICARD TOOLS cleanSam tools [2], (http:// broadinstitute.github.io/picard.). Local realignment of insertions and deletions (indels) was then performed using the GATK suite [2] before final sorting and indexing. The RAMSES application, written in-house, was used to detect nucleotide substitutions [3]. Small indels were identified using Pindel in paired tumor-normal mode [4]. For greater specificity, only simple insertion and deletion events of fewer than 10 bp were selected. An in-house perl script filter was used to extract high-quality indels: considering the high sequence coverage obtained in these samples, only those indels with a minimum coverage of 20 reads in both tumor and normal samples, and with a minimum frequency of 10% of the reads and a minimum of five independent reads supporting the event in the tumor sample, and with no evidence in the normal sample, were considered. All potential somatic mutations were filtered using the dbSNP132 and 1000 Genomes Project mutation databases and the functional consequence at the protein level was annotated according to the Ensembl database using an in-house perl script based on the Ensembl database API.

Validation
Genomic DNA was amplified using the specific oligonucleotides described in Supplementary Table 5. All amplicons from the same patient were mixed in a tube and each sample was quantified by Qubit 2.0 (Life Technologies), using the Qubit ® dsDNA BR Assay Kit (Life Technologies). 500 ng of each DNA sample was repaired using NEBNext: Ultra End Repair/dA Tailing Module kit (Biolabs) and linked to a pair of adapters; 3'-end and 5'-end, respectively. Then, a pair of indexing primers was bound to the adapters to allow subsequent identification of each sample. DNA was purified with Agencourt AMPure XP beads (Beckman Coulter) and 4 ng of each DNA was sequenced by Next Generation Sequencing, using a MiSeq Personal platform (Illumina).

Cell viability and synergism analyses
Cells were seeded in a 96-well plate at a density of 1,000 cells (for Hep-G2, SNU-423, SNU-449 and SNU-475 cells), 2,000 cells (for HUH-7 cells) or 3,000 cells (for SNU-182 cells) per well overnight at 37ºC (with 10% CO 2 and at 96% RH) unless otherwise stated. After that time, cells were attached and exponentially grown to approximately 50% confluence, and the appropriate concentrations of inhibitors were added in each case to the medium. Cellular proliferation was evaluated using CellTiter-Glo ® Luminescent Cell Viability Assay (Promega) and luminometric changes were quantified using the Synergy™ HTX Multi-Mode Microplate Reader (Biotek).
IC 50 dose was estimated by using increasing concentrations of each drug and analyzing those data with GraphPad Prism 5 software (GraphPad Software Inc., La Jolla, CA, USA).
CalcuSyn software (version 2.11, Biosoft, Cambridge, UK) was used to analyze and generate combination index (CI) values, as previously described [5]. This software assesses whether a combination of two drugs results in a synergistic (CI < 1), additive (CI = 1) or antagonistic effect (CI > 1). This method considers the fraction of affected cells of both monotherapies and compares this with the fraction of affected cells of the combination therapies.

DNA synthesis assays
To assess the effects on DNA synthesis, cells were grown in 12mm coverslips to approximately 70% confluence. After 24h of treatment with indicated inhibitors, cells were incubated for a further 2 h with Click-iT® EdU (Alexa Fluor ® 594 Imaging Kit; Life Technologies, C10339). Immediately afterwards, cells were fixed using 3.7% formaldehyde in PBS and permeabilized with 0.5% Triton X-100. Finally, DNA was stained using Hoechst 33342 1:2000 in PBS. Cell images were captured with a Nikon A1R confocal microscope with Plan Apo 10x/0.45NA and Plan ApoVC 60x/1.40NA objectives. A 405-nm laser diode was used to excite Hoechst 33342 and a 561-nm laser diode was used to excite Alexa Fluor 594. Images were processed and analyzed using the object count tool of Nis elements software. Briefly, red and blue nuclei were segmented, channels were separated, and all images thresholded with the same parameters. Finally, the fluorescence of the segmented nuclei was measured. The median of fluorescence intensity of all images was set up as threshold. All measurements below this threshold were considered low EdU whereas all measurements above were considered high EdU.

Western blot
Cells growing exponentially at approximately 70% confluence were treated under the desired conditions. Cells were starved overnight, treated with the appropriate inhibitor and lysed with RIPA buffer enriched with phosphatase and protease inhibitors. Whole cell lysates were subjected to acrylamide SDS-PAGE, using standard procedures, then were transferred onto a nitrocellulose support membrane (Immobilon, Millipore) and western blotted. The primary antibodies at 1:1000 dilution were: P-ERK1/2 (Cell Signaling, Ref

PathScan intracellular signaling array assay
To detect the activation of intracellular signaling, SNU-449, Hep-G2, SNU-182, SNU-475, SNU-423 and HUH-7 cells were starved 3h and treated with the indicated IC 50 concentrations of the inhibitors or the control vehicle (DMSO). After 1h of treatment, cells were collected and lysed. Intracellular signaling was detected using a PathScan ® Intracellular Signaling Array Kit (Cell Signaling Technology, Ref. #7744) following the manufacturer's instructions. Data were collected using an Odyssey Infrared imaging system (Li-Cor Biosciences) and quantified using Image J software.