Abstract 4528: Quantitative mass spectrometry to interrogate proteomic heterogeneity in metastatic lung adenocarcinoma and validate a novel somatic mutation CDK12-G879V
Authors: Xhang Xu , Nguyen Khoa Dang P. , Rudnick Paul , Roper Nitin , Kawaler Emily , Maity Tapan K. , Awasthi Shivangi , Gao Shaojian , Biswas Romi , Venugopalan Abhilash , Cultraro Constance , Fenyo David , Guha Udayan 
Published: August 2019

Abstract
Lung cancer is the leading cause of cancer mortality. Tumor heterogeneity is a major cause of treatment failure. Intra- and inter-metastatic tumor heterogeneity has been demonstrated by next generation sequencing (NGS) studies. However, heterogeneity in the proteome and phosphoproteome has been less studied. Integrated proteogenomics is essential to understanding the intricacies of tumor heterogeneity affecting treatment response. Here, we performed integrated mass spectrometry-based proteogenomics to characterize spatial and temporal heterogeneity of an exceptional responder lung adenocarcinoma patient who survived with metastatic disease for more than 7 years while on combination treatment with HER2-targeted and chemotherapy.
We employed Super-SILAC and TMT labeling strategies to quantify the proteome and phosphoproteome of a lung metastatic site and ten different metastatic progressive lymph nodes from our patient collected across a span of seven years, including at autopsy. To further interrogate the mass spectrometry data, patient-specific database was built to incorporate all the somatic variants identified by NGS. An extensive validation pipeline was built for confirmation of variant peptides. CRISPR-Cas9-mediated gene knockout, cell viability assays, and confocal microscopy were used for further validation of novel variants.
A total of 6214 and 4061 proteins were identified from Super-SILAC and TMT experiments, respectively. 3648 proteins were identified and quantified in both experiments. More than 2000 proteins had catalytic activity, including kinases, phosphatases and metabolic enzymes. We identified 78 and 23 mutant peptides from Super-SILAC and TMT experiments, respectively. Three somatic variants, CDK12-G879V, FASN-R1439Q and HNRNPF-A105T, were confirmed using our variant peptide detection pipeline. Multiple reaction monitoring in a triple quadrupole mass spectrometer successfully identified and relatively quantified two of the variant tryptic peptides harboring the mutations, CDK12-G879V and FASN-R1439Q from the lung and lymph node metastatic sites, respectively. We investigated the consequences of loss of CDK12 function, as predicted from the novel CDK12-G879V mutant, in chemotherapy sensitivity. A549 lung adenocarcinoma cells, upon knockdown of CDK12, exhibited greater chemotherapy sensitivity that was rescued by wild type CDK12, but not by CDK12-G879V mutant.
We demonstrate the importance of integrated proteogenomic analyses to identify variant peptides in mass spectrometry data and studying proteomic heterogeneity affecting treatment response. CDK12-G879V mutation results in a nonfunctional CDK12 kinase and chemotherapy susceptibility in lung metastatic sites, likely explaining the “cure” of lung metastatic sites in this patient.
Citation Format: Xu Xhang, Khoa Dang P. Nguyen, Paul Rudnick, Nitin Roper, Emily Kawaler, Tapan K. Maity, Shivangi Awasthi, Shaojian Gao, Romi Biswas, Abhilash Venugopalan, Constance Cultraro, David Fenyo, Udayan Guha. Quantitative mass spectrometry to interrogate proteomic heterogeneity in metastatic lung adenocarcinoma and validate a novel somatic mutation CDK12-G879V [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 4528.