Julien Sage

Integrative genome analyses identify key somatic driver mutations of small-cell lung cancer

Small-cell lung cancer (SCLC) is an aggressive lung tumor subtype with poor prognosis. We sequenced 29 SCLC exomes, 2 genomes and 15 transcriptomes and found an extremely high mutation rate of 7.4 ± 1 protein-changing mutations per million base pairs. Therefore, we conducted integrated analyses of the various data sets to identify pathogenetically relevant mutated genes. In all cases, we found evidence for inactivation of TP53 and RB1 and identified recurrent mutations in the CREBBP, EP300 and MLL genes that encode histone modifiers. Furthermore, we observed mutations in PTEN, SLIT2 and EPHA7, as well as focal amplifications of the FGFR1 tyrosine kinase gene. Finally, we detected many of the alterations found in humans in SCLC tumors from Tp53 and Rb1 double knockout mice. Our study implicates histone modification as a major feature of SCLC, reveals potentially therapeutically tractable genomic alterations and provides a generalizable framework for the identification of biologically relevant genes in the context of high mutational background.

Cellular mechanisms of tumour suppression by the retinoblastoma gene

The retinoblastoma (RB) tumour suppressor gene is functionally inactivated in a broad range of paediatric and adult cancers, and a plethora of cellular functions and partners have been identified for the RB protein. Data from human tumours and studies from mouse models indicate that loss of RB function contributes to both cancer initiation and progression. However, we still do not know the identity of the cell types in which RB normally prevents cancer initiation in vivo, and the specific functions of RB that suppress distinct aspects of the tumorigenic process are poorly understood.

Acute mutation of retinoblastoma gene function is sufficient for cell cycle re-entry

Cancer cells arise from normal cells through the acquisition of a series of mutations in oncogenes and tumour suppressor genes. Mouse models of human cancer often rely on germline alterations that activate or inactivate genes of interest. One limitation of this approach is that germline mutations might have effects other than somatic mutations, owing to developmental compensation. To model sporadic cancers associated with inactivation of the retinoblastoma (RB) tumour suppressor gene in humans, we have produced a conditional allele of the mouse Rb gene. We show here that acute loss of Rb in primary quiescent cells is sufficient for cell cycle entry and has phenotypic consequences different from germline loss of Rb function. This difference is explained in part by functional compensation by the Rb-related gene p107. We also show that acute loss of Rb in senescent cells leads to reversal of the cellular senescence programme. Thus, the use of conditional knockout strategies might refine our understanding of gene function and help to model human cancer more accurately.

Discovery and Preclinical Validation of Drug Indications Using Compendia of Public Gene Expression Data

A systematic computational method predicts new uses for existing drugs by integrating public gene expression signatures of drugs and diseases. Greening Drug Discovery Recycling is good for the environment—and for drug development too. Repurposing existing, approved drugs can speed their adoption in the clinic because they can often take advantage of the existing rigorous safety testing required by the Food and Drug Administration and other regulatory agencies. In a pair of papers, Sirota et al. and Dudley et al. examined publicly available gene expression data and determined the genes affected in 100 diseases and 164 drugs. By pairing drugs that correct abnormal gene expression in diseases, they confirm known effective drug-disease pairs and predict new indications for already approved agents. Experimental validation that an antiulcer drug and an antiepileptic can be reused for lung cancer and inflammatory bowel disease reinforces the promise of this approach. The authors scrutinized the data in Gene Expression Omnibus and identified a disease signature for 100 diseases, which they defined as the set of mRNAs that reliably increase or decrease in patients with that disease compared to normal individuals. They compared each of these disease signatures to each of the gene expression signatures for 164 drugs from the Connectivity Map, a collection of mRNA expression data from cultured human cells treated with bioactive small molecules that is maintained at the Broad Institute at Massachusetts Institute of Technology. A similarity score calculated by the authors for every possible pair of drug and disease ranged from +1 (a perfect correlation of signatures) to −1 (exactly opposite signatures). The investigators suggested that a similarity score of −1 would predict that the drug would ameliorate the abnormalities in the disease and thus be an effective therapy. This proved to be true for a number of drugs already on the market. The corticosteroid prednisolone, a common treatment for Crohn’s disease and ulcerative colitis, showed a strong similarity score for these two diseases. The histone deacetylase inhibitors trichostatin A, valproic acid, and vorinostat were predicted to work against brain tumors and other cancers (esophagus, lung, and colon), and there is experimental evidence that this is indeed the case. But in the ultimate test of method, the authors confirmed two new predictions in animal experiments: Cimetidine, an antiulcer drug, predicted by the authors to be effective against lung cancer, inhibited tumor cells in vitro and in vivo in mice. In addition, the antiepileptic topiramate, predicted to improve inflammatory bowel disease by similarity score, improved damage in colon tissue of rats treated with trinitrobenzenesulfonic acid, a model of the disease. These two drugs are therefore good candidates for recycling to treat two diseases in need of better therapies—lung cancer and inflammatory bowel disease—and we now have a way to mine available data for fast routes to new disease therapies. The application of established drug compounds to new therapeutic indications, known as drug repositioning, offers several advantages over traditional drug development, including reduced development costs and shorter paths to approval. Recent approaches to drug repositioning use high-throughput experimental approaches to assess a compound’s potential therapeutic qualities. Here, we present a systematic computational approach to predict novel therapeutic indications on the basis of comprehensive testing of molecular signatures in drug-disease pairs. We integrated gene expression measurements from 100 diseases and gene expression measurements on 164 drug compounds, yielding predicted therapeutic potentials for these drugs. We recovered many known drug and disease relationships using computationally derived therapeutic potentials and also predict many new indications for these 164 drugs. We experimentally validated a prediction for the antiulcer drug cimetidine as a candidate therapeutic in the treatment of lung adenocarcinoma, and demonstrate its efficacy both in vitro and in vivo using mouse xenograft models. This computational method provides a systematic approach for repositioning established drugs to treat a wide range of human diseases.

Comprehensive genomic profiles of small cell lung cancer

We have sequenced the genomes of 110 small cell lung cancers (SCLC), one of the deadliest human cancers. In nearly all the tumours analysed we found bi-allelic inactivation of TP53 and RB1, sometimes by complex genomic rearrangements. Two tumours with wild-type RB1 had evidence of chromothripsis leading to overexpression of cyclin D1 (encoded by the CCND1 gene), revealing an alternative mechanism of Rb1 deregulation. Thus, loss of the tumour suppressors TP53 and RB1 is obligatory in SCLC. We discovered somatic genomic rearrangements of TP73 that create an oncogenic version of this gene, TP73Δex2/3. In rare cases, SCLC tumours exhibited kinase gene mutations, providing a possible therapeutic opportunity for individual patients. Finally, we observed inactivating mutations in NOTCH family genes in 25% of human SCLC. Accordingly, activation of Notch signalling in a pre-clinical SCLC mouse model strikingly reduced the number of tumours and extended the survival of the mutant mice. Furthermore, neuroendocrine gene expression was abrogated by Notch activity in SCLC cells. This first comprehensive study of somatic genome alterations in SCLC uncovers several key biological processes and identifies candidate therapeutic targets in this highly lethal form of cancer.

PDGF signalling controls age-dependent proliferation in pancreatic β-cells

Determining the signalling pathways that direct tissue expansion is a principal goal of regenerative biology. Vigorous pancreatic β-cell replication in juvenile mice and humans declines with age, and elucidating the basis for this decay may reveal strategies for inducing β-cell expansion, a long-sought goal for diabetes therapy. Here we show that platelet-derived growth factor receptor (Pdgfr) signalling controls age-dependent β-cell proliferation in mouse and human pancreatic islets. With age, declining β-cell Pdgfr levels were accompanied by reductions in β-cell enhancer of zeste homologue 2 (Ezh2) levels and β-cell replication. Conditional inactivation of the Pdgfra gene in β-cells accelerated these changes, preventing mouse neonatal β-cell expansion and adult β-cell regeneration. Targeted human PDGFR-α activation in mouse β-cells stimulated Erk1/2 phosphorylation, leading to Ezh2-dependent expansion of adult β-cells. Adult human islets lack PDGF signalling competence, but exposure of juvenile human islets to PDGF-AA stimulated β-cell proliferation. The discovery of a conserved pathway controlling age-dependent β-cell proliferation indicates new strategies for β-cell expansion.

Targeted point mutations of p53 lead to dominant-negative inhibition of wild-type p53 function

The p53 tumor suppressor gene is the most frequently mutated gene in human cancers, and germ-line p53 mutations cause a familial predisposition for cancer. Germ-line or sporadic p53 mutations are usually missense and typically affect the central DNA-binding domain of the protein. Because p53 functions as a tetrameric transcription factor, mutant p53 is thought to inhibit the function of wild-type p53 protein. Here, we studied the possible dominant-negative inhibition of wild-type p53 protein by two different, frequently occurring point mutations. The R270H and P275S mutations were targeted into the genome of mouse embryonic stem cells to allow the analysis of the effects of the mutant proteins expressed in normal cells at single-copy levels. In embryonic stem cells, the presence of a heterozygous point-mutated allele resulted in delayed transcriptional activation of several p53 downstream target genes on exposure to γ irradiation. Doxorubicin-induced apoptosis was severely affected in the mutant embryonic stem cells compared with wild-type cells. Heterozygous mutant thymocytes had a severe defect in p53-dependent apoptotic pathways after treatment with γ irradiation or doxorubicin, whereas p53-independent apoptotic pathways were intact. Together these data demonstrate that physiological expression of point-mutated p53 can strongly limit overall cellular p53 function, supporting the dominant-negative action of such mutants. Also, cells heterozygous for such mutations may be compromised in terms of tumor suppression and response to chemotherapeutic agents.

Novel roles for A-type lamins in telomere biology and the DNA damage response pathway

A‐type lamins are intermediate filament proteins that provide a scaffold for protein complexes regulating nuclear structure and function. Mutations in the LMNA gene are linked to a variety of degenerative disorders termed laminopathies, whereas changes in the expression of lamins are associated with tumourigenesis. The molecular pathways affected by alterations of A‐type lamins and how they contribute to disease are poorly understood. Here, we show that A‐type lamins have a key role in the maintenance of telomere structure, length and function, and in the stabilization of 53BP1, a component of the DNA damage response (DDR) pathway. Loss of A‐type lamins alters the nuclear distribution of telomeres and results in telomere shortening, defects in telomeric heterochromatin, and increased genomic instability. In addition, A‐type lamins are necessary for the processing of dysfunctional telomeres by non‐homologous end joining, putatively through stabilization of 53BP1. This study shows new functions for A‐type lamins in the maintenance of genomic integrity, and suggests that alterations of telomere biology and defects in DDR contribute to the pathogenesis of lamin‐related diseases.

A Drug Repositioning Approach Identifies Tricyclic Antidepressants as Inhibitors of Small Cell Lung Cancer and Other Neuroendocrine Tumors

UNLABELLED Small cell lung cancer (SCLC) is an aggressive neuroendocrine subtype of lung cancer with high mortality. We used a systematic drug repositioning bioinformatics approach querying a large compendium of gene expression profiles to identify candidate U.S. Food and Drug Administration (FDA)-approved drugs to treat SCLC. We found that tricyclic antidepressants and related molecules potently induce apoptosis in both chemonaïve and chemoresistant SCLC cells in culture, in mouse and human SCLC tumors transplanted into immunocompromised mice, and in endogenous tumors from a mouse model for human SCLC. The candidate drugs activate stress pathways and induce cell death in SCLC cells, at least in part by disrupting autocrine survival signals involving neurotransmitters and their G protein-coupled receptors. The candidate drugs inhibit the growth of other neuroendocrine tumors, including pancreatic neuroendocrine tumors and Merkel cell carcinoma. These experiments identify novel targeted strategies that can be rapidly evaluated in patients with neuroendocrine tumors through the repurposing of approved drugs. SIGNIFICANCE Our work shows the power of bioinformatics-based drug approaches to rapidly repurpose FDA-approved drugs and identifies a novel class of molecules to treat patients with SCLC, a cancer for which no effective novel systemic treatments have been identified in several decades. In addition, our experiments highlight the importance of novel autocrine mechanisms in promoting the growth of neuroendocrine tumor cells.

From Fly Wings to Targeted Cancer Therapies: A Centennial for Notch Signaling

Since Notch phenotypes in Drosophila melanogaster were first identified 100 years ago, Notch signaling has been extensively characterized as a regulator of cell-fate decisions in a variety of organisms and tissues. However, in the past 20 years, accumulating evidence has linked alterations in the Notch pathway to tumorigenesis. In this review, we discuss the protumorigenic and tumor-suppressive functions of Notch signaling, and dissect the molecular mechanisms that underlie these functions in hematopoietic cancers and solid tumors. Finally, we link these mechanisms and observations to possible therapeutic strategies targeting the Notch pathway in human cancers.