Alice H. Berger

Mapping the Hallmarks of Lung Adenocarcinoma with Massively Parallel Sequencing

Lung adenocarcinoma, the most common subtype of non-small cell lung cancer, is responsible for more than 500,000 deaths per year worldwide. Here, we report exome and genome sequences of 183 lung adenocarcinoma tumor/normal DNA pairs. These analyses revealed a mean exonic somatic mutation rate of 12.0 events/megabase and identified the majority of genes previously reported as significantly mutated in lung adenocarcinoma. In addition, we identified statistically recurrent somatic mutations in the splicing factor gene U2AF1 and truncating mutations affecting RBM10 and ARID1A. Analysis of nucleotide context-specific mutation signatures grouped the sample set into distinct clusters that correlated with smoking history and alterations of reported lung adenocarcinoma genes. Whole-genome sequence analysis revealed frequent structural rearrangements, including in-frame exonic alterations within EGFR and SIK2 kinases. The candidate genes identified in this study are attractive targets for biological characterization and therapeutic targeting of lung adenocarcinoma.

A continuum model for tumour suppression

This year, 2011, marks the forty-year anniversary of the statistical analysis of retinoblastoma that provided the first evidence that tumorigenesis can be initiated by as few as two mutations. This work provided the foundation for the two-hit hypothesis that explained the role of recessive tumour suppressor genes (TSGs) in dominantly inherited cancer susceptibility syndromes. However, four decades later, it is now known that even partial inactivation of tumour suppressors can critically contribute to tumorigenesis. Here we analyse this evidence and propose a continuum model of TSG function to explain the full range of TSG mutations found in cancer.

Distinct patterns of somatic genome alterations in lung adenocarcinomas and squamous cell carcinomas

To compare lung adenocarcinoma (ADC) and lung squamous cell carcinoma (SqCC) and to identify new drivers of lung carcinogenesis, we examined the exome sequences and copy number profiles of 660 lung ADC and 484 lung SqCC tumor–normal pairs. Recurrent alterations in lung SqCCs were more similar to those of other squamous carcinomas than to alterations in lung ADCs. New significantly mutated genes included PPP3CA, DOT1L, and FTSJD1 in lung ADC, RASA1 in lung SqCC, and KLF5, EP300, and CREBBP in both tumor types. New amplification peaks encompassed MIR21 in lung ADC, MIR205 in lung SqCC, and MAPK1 in both. Lung ADCs lacking receptor tyrosine kinase–Ras–Raf pathway alterations had mutations in SOS1, VAV1, RASA1, and ARHGAP35. Regarding neoantigens, 47% of the lung ADC and 53% of the lung SqCC tumors had at least five predicted neoepitopes. Although targeted therapies for lung ADC and SqCC are largely distinct, immunotherapies may aid in treatment for both subtypes.

Functional analysis of receptor tyrosine kinase mutations in lung cancer identifies oncogenic extracellular domain mutations of ERBB2

We assessed somatic alleles of six receptor tyrosine kinase genes mutated in lung adenocarcinoma for oncogenic activity. Five of these genes failed to score in transformation assays; however, novel recurring extracellular domain mutations of the receptor tyrosine kinase gene ERBB2 were potently oncogenic. These ERBB2 extracellular domain mutants were activated by two distinct mechanisms, characterized by elevated C-terminal tail phosphorylation or by covalent dimerization mediated by intermolecular disulfide bond formation. These distinct mechanisms of receptor activation converged upon tyrosine phosphorylation of cellular proteins, impacting cell motility. Survival of Ba/F3 cells transformed to IL-3 independence by the ERBB2 extracellular domain mutants was abrogated by treatment with small-molecule inhibitors of ERBB2, raising the possibility that patients harboring such mutations could benefit from ERBB2-directed therapy.

Haplo-insufficiency: a driving force in cancer

It was originally proposed that tumour suppressor genes (TSGs) act in a recessive manner. Instead, numerous TSGs, including p53 and PTEN, exhibit haplo‐insufficiency as a consequence of the dose‐dependency of TSG function. Due to the challenges of identifying haplo‐insufficient TSGs by human genetics analysis alone, mouse models play a pivotal role in firmly establishing the haplo‐insufficiency of a gene, as in the recent identification of DOK2 as a haplo‐insufficient lung TSG. In many cases, TSGs exhibit conditional or compound haplo‐insufficiency, in which loss of one TSG allele is functionally important only in certain settings or after compound loss of other genes. The 5q deletion syndrome (5q−) is a paradigm of compound haplo‐insufficiency and demonstrates the importance of combinatorial interactions to elicit specific phenotypes. These concepts must be integrated into basic science studies to avoid delay in the identification of important TSGs. In the clinical realm, the challenges for molecular pathologists are the development of quantitative measures that can accurately and systematically ascertain the status of haplo‐insufficient genes in tumour biopsies, and the use of this information to accurately predict prognosis and response to therapy. Copyright

Identification of DOK genes as lung tumor suppressors

Genome-wide analyses of human lung adenocarcinoma have identified regions of consistent copy-number gain or loss, but in many cases the oncogenes and tumor suppressors presumed to reside in these loci remain to be determined. Here we identify the downstream of tyrosine kinase (Dok) family members Dok1, Dok2 and Dok3 as lung tumor suppressors. Single, double or triple compound loss of these genes in mice results in lung cancer, with penetrance and latency dependent on the number of lost Dok alleles. Cancer development is preceded by an aberrant expansion and signaling profile of alveolar type II cells and bronchioalveolar stem cells. In human lung adenocarcinoma, we identify DOK2 as a target of copy-number loss and mRNA downregulation and find that DOK2 suppresses lung cancer cell proliferation in vitro and in vivo. Given the genomic localization of DOK2, we propose it as an 8p21.3 haploinsufficient human lung tumor suppressor.

High-throughput Phenotyping of Lung Cancer Somatic Mutations

Recent genome sequencing efforts have identified millions of somatic mutations in cancer. However, the functional impact of most variants is poorly understood. Here we characterize 194 somatic mutations identified in primary lung adenocarcinomas. We present an expression-based variant-impact phenotyping (eVIP) method that uses gene expression changes to distinguish impactful from neutral somatic mutations. eVIP identified 69% of mutations analyzed as impactful and 31% as functionally neutral. A subset of the impactful mutations induces xenograft tumor formation in mice and/or confers resistance to cellular EGFR inhibition. Among these impactful variants are rare somatic, clinically actionable variants including EGFR S645C, ARAF S214C and S214F, ERBB2 S418T, and multiple BRAF variants, demonstrating that rare mutations can be functionally important in cancer.

Oncogenic RIT1 mutations in lung adenocarcinoma.

Lung adenocarcinoma is comprised of distinct mutational subtypes characterized by mutually exclusive oncogenic mutations in RTK/RAS pathway members KRAS, EGFR, BRAF and ERBB2, and translocations involving ALK, RET and ROS1. Identification of these oncogenic events has transformed the treatment of lung adenocarcinoma via application of therapies targeted toward specific genetic lesions in stratified patient populations. However, such mutations have been reported in only ∼55% of lung adenocarcinoma cases in the United States, suggesting other mechanisms of malignancy are involved in the remaining cases. Here we report somatic mutations in the small GTPase gene RIT1 in ∼2% of lung adenocarcinoma cases that cluster in a hotspot near the switch II domain of the protein. RIT1 switch II domain mutations are mutually exclusive with all other known lung adenocarcinoma driver mutations. Ectopic expression of mutated RIT1 induces cellular transformation in vitro and in vivo, which can be reversed by combined PI3K and MEK inhibition. These data identify RIT1 as a driver oncogene in a specific subset of lung adenocarcinomas and suggest PI3K and MEK inhibition as a potential therapeutic strategy in RIT1-mutated tumors.

BCR-ABL disrupts PTEN nuclear-cytoplasmic shuttling through phosphorylation-dependent activation of HAUSP

Chronic myeloid leukemia (CML) is a myeloproliferative disorder characterized by the t(9;22) translocation coding for the chimeric protein p210 BCR-ABL. The tumor suppressor phosphatase and tensin homolog (PTEN) has recently been shown to have a critical role in the pathogenesis of CML. Nuclear localization and proper nuclear-cytoplasmic shuttling are crucial for PTEN’s tumor suppressive function. In this study, we show that BCR-ABL enhances HAUSP-induced de-ubiquitination of PTEN in turn favoring its nuclear exclusion. We further demonstrate that BCR-ABL physically interacts with and phosphorylates HAUSP on tyrosine residues to trigger its activity. Importantly, we also find that PTEN delocalization induced by BCR-ABL does not occur in the leukemic stem cell compartment due to high levels of PML, a potent inhibitor of HAUSP activity toward PTEN. We therefore identify a new proto-oncogenic mechanism whereby BCR-ABL antagonizes the nuclear function of the PTEN tumor suppressor, with important therapeutic implications for the eradication of CML minimal residual disease.

Brain angiogenesis inhibitor 1 is expressed by gastric phagocytes during infection with Helicobacter pylori and mediates the recognition and engulfment of human apoptotic gastric epithelial cells

After Helicobacter pylori infection in humans, gastric epithelial cells (GECs) undergo apoptosis due to stimulation by the bacteria or inflammatory cytokines. In this study, we assessed the expression and function of brain angiogenesis inhibitor 1 (BAI1) in the engulfment of apoptotic GECs using human tissue and cells. After induction of apoptosis by H. pylori or camptothecin, there was a 5‐fold increase in the binding of apoptotic GECs to THP‐1 cells or peripheral blood monocyte‐derived macrophages as assayed by confocal microscopy or conventional and imaging flow cytometry. Binding was impaired 95% by pretreating apoptotic cells with annexin V, underscoring the requirement for phosphatidylserine recognition. The phosphatidylserine receptor BAI1 was expressed in human gastric biopsy specimens and gastric phagocytes. To confirm the role of BAI1 in apoptotic cell clearance, the functional domain of BAI1 was used as a competitive inhibitor or BAI1 expression was inhibited by small interfering RNA. Both approaches decreased binding and engulfment >40%. Exposing THP‐1 cells to apoptotic cells inhibited IL‐6 production from 1340 to <364 pg/ml; however, this decrease was independent of phagocytosis. We conclude that recognition of apoptotic cells by BAI1 contributes to their clearance in the human gastric mucosa and this is associated with anti‐inflammatory effects.—Das, S., Sarkar, A., Ryan, K. A., Fox, S., Berger, A. H., Juncadella, I. J., Bimczok, D., Smythies, L. E., Harris, P. R., Ravichandran, K. S., Crowe, S. E., Smith, P. D., Ernst, P. B. Brain angiogenesis inhibitor 1 is expressed by gastric phagocytes during infection with Helicobacter pylori and mediates the recognition and engulfment of human apoptotic gastric epithelial cells. FASEB J. 28, 2214–2224 (2014). www.fasebj.org