Comprehensive kinome NGS targeted expression profiling by KING-REX

Abstract

Background: Protein kinases are enzymes controlling different cellular functions. Genetic alterations often result in kinase dysregulation, making kinases a very attractive class of druggable targets in several human diseases. Existing approved drugs still target a very limited portion of the human 'kinome', demanding a broader functional knowledge of individual and co-expressed kinase patterns in physiologic and pathologic settings. The development of novel rapid and cost-effective methods for kinome screening is therefore highly desirable, potentially leading to the identification of novel kinase drug targets.

Results: In this work, we describe the development of KING-REX (KINase Gene RNA EXpression), a comprehensive kinome RNA targeted custom assay-based panel designed for Next Generation Sequencing analysis, coupled with a dedicated data analysis pipeline. We have conceived KING-REX for the gene expression analysis of 512 human kinases; for 319 kinases, paired assays and custom analysis pipeline features allow the evaluation of 3'- and 5'-end transcript imbalances as readout for the prediction of gene rearrangements. Validation tests on cell line models harboring known gene fusions demonstrated a comparable accuracy of KING-REX gene expression assessment as in whole transcriptome analyses, together with a robust detection of transcript portion imbalances in rearranged kinases, even in complex RNA mixtures or in degraded RNA.

Conclusions: These results support the use of KING-REX as a rapid and cost effective kinome investigation tool in the field of kinase target identification for applications in cancer biology and other human diseases.

Keywords: Kinome gene expression; NGS RNA targeted panel.

Fig. 1

Flowchart of kinome assay selection. a Selection process of ASSAY IN, targeting the kinase domain; b Selection process of ASSAY OUT, outside the kinase domain; definition of ASSAY ADD upon re-selection of ASSAY IN. All the selected pre-designed assays were derived from Illumina DesignStudio Custom Assay Design Tool for use with the TruSeq Targeted RNA Expression (TREx) kit (Illumina, San Diego, CA, USA)

Fig. 2

Clustering of cancer cell lines before and after the normalization step. Cluster analysis of a heterogeneous panel of 5 cancer cell lines, tested in duplicate with KING-REX, before (a) and after (b) the second normalization step in the pipeline for potential kinase fusion event detection. ASSAY_IN expression values for each sample are annotated in green, while ASSAY_OUT in orange. The blue shading indicates the Euclidean distance between the expression values of two samples (cell lines), ranging from dark blue (high similarity) to light blue (low similarity)

Fig. 3

Comparison between measured and theoretical kinase gene expression values. Kinase gene expression values measured with KING-REX within a serially diluted sample set of KARPAS 299 and U-118 MG RNAs, mixed in different proportions (87.5–12.5%; 75–25%, 50–50%, 25–75%, 12.5–87.5%,), plotted vs. ‘theoretical’ expression values, calculated for each kinase and for each dilution factor as described in M&M; the respective correlation R² value is displayed

Fig. 4

Detection of gene fusion events in diluted samples. a KING-REX log₂(NC) expression values for ROS1 ASSAY IN (light blue) and ASSAY OUT (dark blue) in U-118 MG, diluted in different percentages of KARPAS 299 sample background; b KING-REX log₂(NC) expression values for ALK ASSAY IN (light blue) and ASSAY OUT (dark blue) in KARPAS 299, diluted in different percentages of U-118 MG sample background. The yellow and red bar outline indicates the duplicate A and B of the ASSAY expression value, respectively