Showing papers by "Todd R. Golub published in 2016"

Single-cell RNA-seq supports a developmental hierarchy in human oligodendroglioma

Abstract: Although human tumours are shaped by the genetic evolution of cancer cells, evidence also suggests that they display hierarchies related to developmental pathways and epigenetic programs in which cancer stem cells (CSCs) can drive tumour growth and give rise to differentiated progeny. Yet, unbiased evidence for CSCs in solid human malignancies remains elusive. Here we profile 4,347 single cells from six IDH1 or IDH2 mutant human oligodendrogliomas by RNA sequencing (RNA-seq) and reconstruct their developmental programs from genome-wide expression signatures. We infer that most cancer cells are differentiated along two specialized glial programs, whereas a rare subpopulation of cells is undifferentiated and associated with a neural stem cell expression program. Cells with expression signatures for proliferation are highly enriched in this rare subpopulation, consistent with a model in which CSCs are primarily responsible for fuelling the growth of oligodendroglioma in humans. Analysis of copy number variation (CNV) shows that distinct CNV sub-clones within tumours display similar cellular hierarchies, suggesting that the architecture of oligodendroglioma is primarily dictated by developmental programs. Subclonal point mutation analysis supports a similar model, although a full phylogenetic tree would be required to definitively determine the effect of genetic evolution on the inferred hierarchies. Our single-cell analyses provide insight into the cellular architecture of oligodendrogliomas at single-cell resolution and support the cancer stem cell model, with substantial implications for disease management.

Single-cell RNA-seq supports a developmental hierarchy in human oligodendroglioma

Abstract: Although human tumours are shaped by the genetic evolution of cancer cells, evidence also suggests that they display hierarchies related to developmental pathways and epigenetic programs in which cancer stem cells (CSCs) can drive tumour growth and give rise to differentiated progeny. Yet, unbiased evidence for CSCs in solid human malignancies remains elusive. Here we profile 4,347 single cells from six IDH1 or IDH2 mutant human oligodendrogliomas by RNA sequencing (RNA-seq) and reconstruct their developmental programs from genome-wide expression signatures. We infer that most cancer cells are differentiated along two specialized glial programs, whereas a rare subpopulation of cells is undifferentiated and associated with a neural stem cell expression program. Cells with expression signatures for proliferation are highly enriched in this rare subpopulation, consistent with a model in which CSCs are primarily responsible for fuelling the growth of oligodendroglioma in humans. Analysis of copy number variation (CNV) shows that distinct CNV sub-clones within tumours display similar cellular hierarchies, suggesting that the architecture of oligodendroglioma is primarily dictated by developmental programs. Subclonal point mutation analysis supports a similar model, although a full phylogenetic tree would be required to definitively determine the effect of genetic evolution on the inferred hierarchies. Our single-cell analyses provide insight into the cellular architecture of oligodendrogliomas at single-cell resolution and support the cancer stem cell model, with substantial implications for disease management.

Genomic Copy Number Dictates a Gene-Independent Cell Response to CRISPR/Cas9 Targeting

Abstract: The CRISPR-Cas9 system enables genome editing and somatic cell genetic screens in mammalian cells. We performed genome scale loss-of-function screens in 33 cancer cell lines to identify genes essential for proliferation/survival and found a strong correlation between increased gene copy number and decreased cell viability after genome editing. Within regions of copy number gain, CRISPR-Cas9 targeting of both expressed and unexpressed genes, as well as intergenic loci, led to significantly decreased cell proliferation through induction of a G2 cell cycle arrest. By examining single guide RNAs that map to multiple genomic sites, we found that this cell response to CRISPR-Cas9 editing correlated strongly with the number of target loci. These observations indicate that genome targeting by CRISPR-Cas9 elicits a gene-independent anti-proliferative cell response. This effect has important practical implications for interpretation of CRISPR-Cas9 screening data and confounds the use of this technology for identification of essential genes in amplified regions.

Open Source Drug Discovery with the Malaria Box Compound Collection for Neglected Diseases and Beyond.

Abstract: A major cause of the paucity of new starting points for drug discovery is the lack of interaction between academia and industry. Much of the global resource in biology is present in universities, whereas the focus of medicinal chemistry is still largely within industry. Open source drug discovery, with sharing of information, is clearly a first step towards overcoming this gap. But the interface could especially be bridged through a scale-up of open sharing of physical compounds, which would accelerate the finding of new starting points for drug discovery. The Medicines for Malaria Venture Malaria Box is a collection of over 400 compounds representing families of structures identified in phenotypic screens of pharmaceutical and academic libraries against the Plasmodium falciparum malaria parasite. The set has now been distributed to almost 200 research groups globally in the last two years, with the only stipulation that information from the screens is deposited in the public domain. This paper reports for the first time on 236 screens that have been carried out against the Malaria Box and compares these results with 55 assays that were previously published, in a format that allows a meta-analysis of the combined dataset. The combined biochemical and cellular assays presented here suggest mechanisms of action for 135 (34%) of the compounds active in killing multiple life-cycle stages of the malaria parasite, including asexual blood, liver, gametocyte, gametes and insect ookinete stages. In addition, many compounds demonstrated activity against other pathogens, showing hits in assays with 16 protozoa, 7 helminths, 9 bacterial and mycobacterial species, the dengue fever mosquito vector, and the NCI60 human cancer cell line panel of 60 human tumor cell lines. Toxicological, pharmacokinetic and metabolic properties were collected on all the compounds, assisting in the selection of the most promising candidates for murine proof-of-concept experiments and medicinal chemistry programs. The data for all of these assays are presented and analyzed to show how outstanding leads for many indications can be selected. These results reveal the immense potential for translating the dispersed expertise in biological assays involving human pathogens into drug discovery starting points, by providing open access to new families of molecules, and emphasize how a small additional investment made to help acquire and distribute compounds, and sharing the data, can catalyze drug discovery for dozens of different indications. Another lesson is that when multiple screens from different groups are run on the same library, results can be integrated quickly to select the most valuable starting points for subsequent medicinal chemistry efforts.

MTAP deletion confers enhanced dependency on the PRMT5 arginine methyltransferase in cancer cells.

Abstract: The discovery of cancer dependencies has the potential to inform therapeutic strategies and to identify putative drug targets. Integrating data from comprehensive genomic profiling of cancer cell lines and from functional characterization of cancer cell dependencies, we discovered that loss of the enzyme methylthioadenosine phosphorylase (MTAP) confers a selective dependence on protein arginine methyltransferase 5 (PRMT5) and its binding partner WDR77. MTAP is frequently lost due to its proximity to the commonly deleted tumor suppressor gene, CDKN2A. We observed increased intracellular concentrations of methylthioadenosine (MTA, the metabolite cleaved by MTAP) in cells harboring MTAP deletions. Furthermore, MTA specifically inhibited PRMT5 enzymatic activity. Administration of either MTA or a small-molecule PRMT5 inhibitor showed a modest preferential impairment of cell viability for MTAP-null cancer cell lines compared with isogenic MTAP-expressing counterparts. Together, our findings reveal PRMT5 as a potential vulnerability across multiple cancer lineages augmented by a common "passenger" genomic alteration.

Open Source Drug Discovery with the Malaria Box Compound Collection for Neglected Diseases and Beyond

Abstract: A major cause of the paucity of new starting points for drug discovery is the lack of interaction between academia and industry. Much of the global resource in biology is present in universities, whereas the focus of medicinal chemistry is still largely within industry. Open source drug discovery, with sharing of information, is clearly a first step towards overcoming this gap. But the interface could especially be bridged through a scale-up of open sharing of physical compounds, which would accelerate the finding of new starting points for drug discovery. The Medicines for Malaria Venture Malaria Box is a collection of over 400 compounds representing families of structures identified in phenotypic screens of pharmaceutical and academic libraries against the Plasmodium falciparum malaria parasite. The set has now been distributed to almost 200 research groups globally in the last two years, with the only stipulation that information from the screens is deposited in the public domain. This paper reports for the first time on 236 screens that have been carried out against the Malaria Box and compares these results with 55 assays that were previously published, in a format that allows a meta-analysis of the combined dataset. The combined biochemical and cellular assays presented here suggest mechanisms of action for 135 (34%) of the compounds active in killing multiple life-cycle stages of the malaria parasite, including asexual blood, liver, gametocyte, gametes and insect ookinete stages. In addition, many compounds demonstrated activity against other pathogens, showing hits in assays with 16 protozoa, 7 helminths, 9 bacterial and mycobacterial species, the dengue fever mosquito vector, and the NCI60 human cancer cell line panel of 60 human tumor cell lines. Toxicological, pharmacokinetic and metabolic properties were collected on all the compounds, assisting in the selection of the most promising candidates for murine proof-of-concept experiments and medicinal chemistry programs. The data for all of these assays are presented and analyzed to show how outstanding leads for many indications can be selected. These results reveal the immense potential for translating the dispersed expertise in biological assays involving human pathogens into drug discovery starting points, by providing open access to new families of molecules, and emphasize how a small additional investment made to help acquire and distribute compounds, and sharing the data, can catalyze drug discovery for dozens of different indications. Another lesson is that when multiple screens from different groups are run on the same library, results can be integrated quickly to select the most valuable starting points for subsequent medicinal chemistry efforts.

High-throughput identification of genotype-specific cancer vulnerabilities in mixtures of barcoded tumor cell lines

Abstract: Hundreds of genetically characterized cell lines are available for the discovery of genotype-specific cancer vulnerabilities. However, screening large numbers of compounds against large numbers of cell lines is currently impractical, and such experiments are often difficult to control. Here we report a method called PRISM that allows pooled screening of mixtures of cancer cell lines by labeling each cell line with 24-nucleotide barcodes. PRISM revealed the expected patterns of cell killing seen in conventional (unpooled) assays. In a screen of 102 cell lines across 8,400 compounds, PRISM led to the identification of BRD-7880 as a potent and highly specific inhibitor of aurora kinases B and C. Cell line pools also efficiently formed tumors as xenografts, and PRISM recapitulated the expected pattern of erlotinib sensitivity in vivo.

Systematic functional interrogation of rare cancer variants identifies oncogenic alleles

Abstract: Cancer genome characterization efforts now provide an initial view of the somatic alterations in primary tumors. However, most point mutations occur at low frequency, and the function of these alleles remains undefined. We have developed a scalable systematic approach to interrogate the function of cancer-associated gene variants. We subjected 474 mutant alleles curated from 5,338 tumors to pooled in vivo tumor formation assays and gene expression profiling. We identified 12 transforming alleles, including two in genes ( PIK3CB, POT1 ) that have not been shown to be tumorigenic. One rare KRAS allele, D33E, displayed tumorigenicity and constitutive activation of known RAS effector pathways. By comparing gene expression changes induced upon expression of wild-type and mutant alleles, we inferred the activity of specific alleles. Because alleles found to be mutated only once in 5,338 tumors rendered cells tumorigenic, these observations underscore the value of integrating genomic information with functional studies. Significance: Experimentally inferring the functional status of cancer-associated mutations facilitates the interpretation of genomic information in cancer. Pooled in vivo screen and gene expression profiling identified functional variants and demonstrated that expression of rare variants induced tumorigenesis. Variant phenotyping through functional studies will facilitate defining key somatic events in cancer. Cancer Discov; 6(7); 714–26. ©2016 AACR. See related commentary by Cho and Collisson, [p. 694][1] . This article is highlighted in the In This Issue feature, [p. 681][2] [1]: /lookup/volpage/6/694?iss=7 [2]: /lookup/volpage/6/681?iss=7

Genetic interrogation of circulating multiple myeloma cells at single-cell resolution.

Abstract: Multiple myeloma (MM) remains an incurable disease, with a treatment-refractory state eventually developing in all patients. Constant clonal evolution and genetic heterogeneity of MM are a likely explanation for the emergence of drug-resistant disease. Monitoring of MM genomic evolution on therapy by serial bone marrow biopsy is unfortunately impractical because it involves an invasive and painful procedure. We describe how noninvasive and highly sensitive isolation and characterization of circulating tumor cells (CTCs) from peripheral blood at single-cell resolution recapitulate MM in the bone marrow. We demonstrate that CTCs provide the same genetic information as bone marrow MM cells and even reveal mutations with greater sensitivity than bone marrow biopsies in some cases. Single CTC RNA sequencing enables classification of MM and quantitative assessment of genes that are relevant for prognosis. We propose that the genomic characterization of CTCs should be included in clinical trials to follow the emergence of resistant subclones after MM therapy.

The landscape of chromosomal aberrations in breast cancer mouse models reveals driver-specific routes to tumorigenesis

Abstract: Aneuploidy and copy-number alterations (CNAs) are a hallmark of human cancer. Although genetically engineered mouse models (GEMMs) are commonly used to model human cancer, their chromosomal landscapes remain underexplored. Here we use gene expression profiles to infer CNAs in 3,108 samples from 45 mouse models, providing the first comprehensive catalogue of chromosomal aberrations in cancer GEMMs. Mining this resource, we find that most chromosomal aberrations accumulate late during breast tumorigenesis, and observe marked differences in CNA prevalence between mouse mammary tumours initiated with distinct drivers. Some aberrations are recurrent and unique to specific GEMMs, suggesting distinct driver-dependent routes to tumorigenesis. Synteny-based comparison of mouse and human tumours narrows critical regions in CNAs, thereby identifying candidate driver genes. We experimentally validate that loss of Stratifin (SFN) promotes HER2-induced tumorigenesis in human cells. These results demonstrate the power of GEMM CNA analysis to inform the pathogenesis of human cancer.

The Metastatic Breast Cancer Project: A national direct-to-patient initiative to accelerate genomics research.

Abstract: LBA1519Background: The challenge in studying tumors from patients (pts) with metastatic breast cancer (MBC) has been that most tumors are not available for research, largely because most pts are cared for in community settings where genomics studies are not conducted. To address this, we launched a nationwide study, The Metastatic Breast Cancer Project, which seeks to empower patients to accelerate research by sharing their samples and clinical information. Methods: In collaboration with pts and advocacy groups, we developed a website to allow MBC pts to participate across the U.S. Enrolled pts are sent a saliva kit and asked to mail back a saliva sample, which is used to extract germline DNA. We contact participants’ medical providers and obtain medical records and part of their tumor biopsy. Whole exome and transcriptome sequencing is performed on tumor and germline. Clinically annotated genomic data are used to identify mechanisms of response and resistance to therapies. The database is shared widely w...

Abstract 4368: High-throughput phenotyping of lung cancer somatic mutations

Abstract: Recent cancer genome sequencing and analysis has identified millions of somatic mutations in cancer. However, the functional impact of most variants is poorly understood, limiting the use of this genetic knowledge for clinical decision-making. Here we describe a new high-throughput approach, expression-based variant impact phenotyping (eVIP), which uses gene expression changes to infer somatic mutation impact. We generated a lentiviral expression library representing 53 genes and 194 somatic mutations identified in primary lung adenocarcinomas. Next, we introduced this library into A549 lung adenocarcinoma cells and 96 hours later performed gene expression profiling using Luminex-based L1000 profiling. We built a computational pipeline, eVIP, to compare mutant and wild-type expression signatures to infer whether variants were gain-of-function, change-of-function, loss-of-function, or neutral. Overall, eVIP identified 69% of mutations as impactful whereas 31% appeared functionally neutral. A very high rate, 92%, of missense mutations in the KEAP1 and STK11 tumor suppressor genes were found to inactivate or diminish protein function. As a complementary approach, we assessed which mutations are epistatic to EGFR or capable of initiating xenograft tumor formation in vivo. A subset of the impactful mutations identified by eVIP could induce xenograft tumor formation in mice and/or confer resistance to cellular EGFR inhibition. Among these mutations were 20 rare or non-canonical somatic variants in clinically-actionable or -relevant oncogenes including EGFR S645C, ARAF S214C and S214F, ERBB2 S418T, and PIK3CA E600K. eVIP can, in principle, characterize any genetic variant, independent of prior knowledge of gene function. Further application of eVIP should significantly advance the pace of functional characterization of mutations identified from genome sequencing. Citation Format: Alice H. Berger, Angela N. Brooks, Xiaoyun Wu, Yashaswi Shrestha, Candace Chouinard, Federica Piccioni, Mukta Bagul, Atanas Kamburov, Marcin Imielinski, Larson Hogstrom, Cong Zhu, Xiaoping Yang, Sasha Pantel, Ryo Sakai, Nathan Kaplan, David Root, Rajiv Narayan, Ted Natoli, David Lahr, Itay Tirosh, Pablo Tamayo, Gad Getz, Bang Wong, John Doench, Aravind Subramanian, Todd R. Golub, Matthew Meyerson, Jesse S. Boehm. High-throughput phenotyping of lung cancer somatic mutations. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 4368.

Antibodies, compounds and screens for identifying and treating cachexia or pre-cachexia

Abstract: The invention provides therapeutic, engineered protein/peptide compositions comprising e.g., RAGE antibodies, T-cell receptors to target a RAGE receptor (including soluble forms thereof) directly and/or via differential competition with one or more pre-cachexia and/or cachexia-associated RAGE ligands or markers.

Corrigendum: The genomic landscape of juvenile myelomonocytic leukemia.

Abstract: Nat. Genet. 47, 1326–1333 (2015); published online 12 October 2015; corrected after print 7 December 2015 In the version of this article initially published, two patients were stated on page 5 to have been excluded owing to insufficient follow-up data. These patients were included in the final analysis, but two additional patients were excluded owing to the presence of Noonan syndrome.

Compositions and methods for treating cardiac dysfunction

Abstract: The invention provides compositions and methods for treating cardiac dysfunction, particularly cachexia-associated or RAGE-associated cardiac dysfunction, using an anti-RAGE agent. The invention also provides compositions and methods for identifying therapeutic agents useful for disrupting (slowing, reducing, reversing, or preventing). The methods comprise designing or identifying agents that bind to functional sites identified on the RAGE polypeptide, wherein binding of agents to the functional site(s) inhibit RAGE-mediated cachetogenic signaling.