Figure 4.

CDK12 and ERBB2 are coamplified and overexpressed but display a different mutation pattern in both metastatic sites. (A) Region with high-copy-number amplification in the lung tumor (L; red) and metastatic lymph node (LN1; blue) as assessed by copy-number variation sequencing (CNV-seq) analysis of WGS data. Arrows indicate the approximate locations for ERBB2 and CDK12. (B) CDK12 and ERBB2 expression levels in the lung tumor, lymph nodes, and HPL1D lung cell line control were determined by quantitative reverse transcription–polymerase chain reaction (RT-PCR) with POLR2A mRNA as an endogenous control. Samples were analyzed in quadruplicate, and values expressed as the mean ± SD. (C) Chromatogram obtained from Sanger sequencing of cDNA shows that ERBB2 with a somatic T>G (CtG to CgG: leucine to arginine) mutation is expressed in the metastatic lymph node. (D) Chromatogram obtained from Sanger sequencing of cDNA shows that CDK12 with a somatic G>T (GgA to GtA: glycine to valine) mutation is expressed in the lung tumor. (E) Effects of G879V mutation on the active conformation of CDK12. (Top) Active conformation of CDK12 with cyclin K (PDB ID 4CXA chains A, B) and mutated structure are superimposed in the same orientation and are colored as follows: αC-helix, activation loop, and ATP-like compound are highlighted in pink, green, and cyan, respectively; the residue G879 and its mutated version V879 are shown in red and blue, respectively; the side chains of amino acids that interact with the G879V site are shown in yellow. Sequence representation is shown below. Region of CDK12 sequence (760–900): Residues interacting with the point mutation site are highlighted by yellow; the mutation site is highlighted in red. (F) Expression of CDK12-regulated DNA repair genes in the lung tumor, metastatic lymph nodes, and HPL1D lung cell line control were determined by quantitative RT-PCR with POLR2A mRNA as an endogenous control. Samples were analyzed in quadruplicate, and values expressed as the mean ± SD.