A multiplexed in vivo approach to identify driver genes in small cell lung cancer

Summary: Small cell lung cancer (SCLC) is a lethal form of lung cancer. Here, we develop a quantitative multiplexed approach on the basis of lentiviral barcoding with somatic CRISPR-Cas9-mediated genome editing to functionally investigate candidate regulators of tumor initiation and growth in genetically engineered mouse models of SCLC. We found that naphthalene pre-treatment enhances lentiviral vector-mediated SCLC initiation, enabling high multiplicity of tumor clones for analysis through high-throughput sequencing methods. Candidate drivers of SCLC identified from a meta-analysis across multiple human SCLC genomic datasets were tested using this approach, which defines both positive and detrimental impacts of inactivating 40 genes across candidate pathways on SCLC development. This analysis and subsequent validation in human SCLC cells establish TSC1 in the PI3K-AKT-mTOR pathway as a robust tumor suppressor in SCLC. This approach should illuminate drivers of SCLC, facilitate the development of precision therapies for defined SCLC genotypes, and identify therapeutic targets

Co-occurrence of targetable mutations in Non-small cell lung cancer (NSCLC) patients harboring MAP2K1 mutations

Background: MAP2K1 mutations are rare in non-small cell lung cancer (NSCLC) and considered to be mutually exclusive from known driver mutations. Activation of the MEK1-cascade is considered pivotal in resistance to targeted therapy approaches, and MAP2K1 K57 N mutation could be linked to resistance in preclinical models. We set out this study to detect MAP2K1 mutations and potentially targetable co-mutations using a molecular multiplex approach. Methods: Between 2012 and 2018, we routinely analyzed 14.512 NSCLC patients with two next-generation sequencing (NGS) panels. In a subset of patients, fluorescence in-situ hybridization was performed to detect rearrangements or amplifications. We assessed clinical parameters and co-occurring mutations and compared treatment outcomes of different forms of systemic therapy. Results: We identified 66 (0.5%) patients with MAP2K1 mutations. Both adenocarcinoma (n = 62) and squamous cell carcinoma (n = 4) histology. The presence of the mutations was linked to smoking, and transversions were more common than transitions. K57 N was the most frequent MAP2K1 mutation (n = 25). Additional mutations were found in 57 patients (86.4%). Mutations of TP53 were detected in 33 patients, followed by KEAP1 mutations in 28.1%. 24 patients (36.4%) had either MAP2K1-only or a co-occurring aberration considered targetable, including EGFR mutations, a BRAF V600E mutation and ROS1 rearrangements. Outcome analyses revealed a trend toward benefit from pemetrexed treatment. Conclusion: Our analysis shows that MAP2K1-mutated NSCLC patients might frequently present with potentially targetable aberrations. Their role in providing resistance in these subtypes and the possible therapeutic opportunities justify further analyses of this rare NSCLC subgroup

An Autochthonous Mouse Model of Myd88

Based on gene expression profiles, diffuse large B-cell lymphoma (DLBCL) is subdivided into germinal center B-cell-like (GCB) and activated B-cell-like (ABC) DLBCL. Two of the most common genomic aberrations in ABC-DLBCL are mutations in MYD88 as well as BCL2 copy-number gains. Here, we employ immune phenotyping, RNA sequencing, and wholeexome sequencing to characterize a Myd88- and BCL2-driven mouse model of ABC-DLBCL. We show that this model resembles features of human ABC-DLBCL. We further demonstrate an actionable dependence of our murine ABC-DLBCL model on BCL2. This BCL2 dependence was also detectable in human ABC-DLBCL cell lines. Moreover, human ABC-DLBCLs displayed increased PD-L1 expression compared with GCB-DLBCL. In vivo experiments in our ABC-DLBCL model showed that combined venetoclax and PD-1 blockade significantly increased the overall survival of lymphoma-bearing animals, indicating that this combination may be a viable option for selected human ABC-DLBCL cases harboring MYD88 and BCL2 aberrations. SIGNIFICANCE: Oncogenic Myd88 and BCL2 cooperate in murine DLBCL lymphomagenesis. The resulting lymphomas display morphologic and transcriptomic features reminiscent of human ABC-DLBCL. Data derived from our Myd88/BCL2-driven autochthonous model demonstrate that combined BCL2 and PD-1 blockade displays substantial preclinical antilymphoma activity, providing preclinical proof-of-concept data, which pave the way for clinical translation

An Autochthonous Mouse Model of Myd88- and BCL2-Driven Diffuse Large B-cell Lymphoma Reveals Actionable Molecular Vulnerabilities

Based on gene expression profiles, diffuse large B-cell lymphoma (DLBCL) is subdivided into germinal center B-cell-like (GCB) and activated B-cell-like (ABC) DLBCL. Two of the most common genomic aberrations in ABC-DLBCL are mutations in MYD88 as well as BCL2 copy-number gains. Here, we employ immune phenotyping, RNA sequencing, and wholeexome sequencing to characterize a Myd88- and BCL2-driven mouse model of ABC-DLBCL. We show that this model resembles features of human ABC-DLBCL. We further demonstrate an actionable dependence of our murine ABC-DLBCL model on BCL2. This BCL2 dependence was also detectable in human ABC-DLBCL cell lines. Moreover, human ABC-DLBCLs displayed increased PD-L1 expression compared with GCB-DLBCL. In vivo experiments in our ABC-DLBCL model showed that combined venetoclax and PD-1 blockade significantly increased the overall survival of lymphoma-bearing animals, indicating that this combination may be a viable option for selected human ABC-DLBCL cases harboring MYD88 and BCL2 aberrations. SIGNIFICANCE: Oncogenic Myd88 and BCL2 cooperate in murine DLBCL lymphomagenesis. The resulting lymphomas display morphologic and transcriptomic features reminiscent of human ABC-DLBCL. Data derived from our Myd88/BCL2-driven autochthonous model demonstrate that combined BCL2 and PD-1 blockade displays substantial preclinical antilymphoma activity, providing preclinical proof-of-concept data, which pave the way for clinical translation

K-ras Mutation Subtypes in NSCLC and Associated Co-occuring Mutations in Other Oncogenic Pathways

Introduction: Although KRAS mutations in NSCLC have been considered mutually exclusive driver mutations for a long time, there is now growing evidence that KRAS-mutated NSCLC represents a genetically heterogeneous subgroup. We sought to determine genetic heterogeneity with respect to cancer-related co-mutations and their correlation with different KRAS mutation subtypes. Methods: Diagnostic samples from 4507 patients with NSCLC were analyzed by next-generation sequencing by using a panel of 14 genes and, in a subset of patients, fluorescence in situ hybridization. Next-generation sequencing with an extended panel of 14 additional genes was performed in 101 patients. Molecular data were correlated with clinical data. Whole-exome sequencing was performed in two patients. Results: We identified 1078 patients with KRAS mutations, of whom 53.5% had at least one additional mutation. Different KRAS mutation subtypes showed different patterns of co-occurring mutations. Besides mutations in tumor protein p53 gene (TP53) (39.4%), serine/threonine kinase 11 gene (STK11) (19.8%), kelch like ECH associated protein 1 gene (KEAP1) (12.9%), and ATM serine/threonine kinase gene (ATM) (11.9%), as well as MNNG HOS Transforming gene (MET) amplifications (15.4%) and erb-b2 receptor tyrosine kinase 2 gene (ERBB2) amplifications (13.8%, exclusively in G12C), we found rare co-occurrence of targetable mutations in EGFR (1.2%) and BRAF (1.2%). Whole-exome sequencing of two patients with co-occurring phosphatidylinositol-4,5-bisphosphate 3-kinase catalytic subunit alpha gene (PIK3CA) mutation revealed clonality of mutated KRAS in one patient and subclonality in the second, suggesting different evolutionary backgrounds. Conclusion: KRAS-mutated NSCLC represents a genetically heterogeneous subgroup with a high frequency of co-occurring mutations in cancer-associated pathways, partly associated with distinct KRAS mutation subtypes. This diversity might have implications for understanding the variability of treatment outcome in KRAS-mutated NSCLC and for future trial design. (C) 2019 International Association for the Study of Lung Cancer. Published by Elsevier Inc