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34


Sources of Error in Mammalian Genetic Screens

Sack, Laura Magill; Davoli, Teresa; Xu, Qikai; Li, Mamie Z; Elledge, Stephen J
Genetic screens are invaluable tools for dissection of biological phenomena. Optimization of such screens to enhance discovery of candidate genes and minimize false positives is thus a critical aim. Here, we report several sources of error common to pooled genetic screening techniques used in mammalian cell culture systems, and demonstrate methods to eliminate these errors. We find that reverse transcriptase-mediated recombination during retroviral replication can lead to uncoupling of molecular tags, such as DNA barcodes (BCs), from their associated library elements, leading to chimeric proviral genomes in which BCs are paired to incorrect ORFs, shRNAs, etc This effect depends on the length of homologous sequence between unique elements, and can be minimized with careful vector design. Furthermore, we report that residual plasmid DNA from viral packaging procedures can contaminate transduced cells. These plasmids serve as additional copies of the PCR template during library amplification, resulting in substantial inaccuracies in measurement of initial reference populations for screen normalization. The overabundance of template in some samples causes an imbalance between PCR cycles of contaminated and uncontaminated samples, which results in a systematic artifactual depletion of GC-rich library elements. Elimination of contaminating plasmid DNA using the bacterial endonuclease Benzonase can restore faithful measurements of template abundance and minimize GC bias.
PMCID:5015935
PMID: 27402361
ISSN: 2160-1836
CID: 2667882

How aneuploidy drives cancer [Meeting Abstract]

Davoli, Teresa; Uno, Hajima; Xu, Andrew; Mengwasser, Kristen; Sack, Laura; Park, Peter J; Elledge, Stephen J
ISI:000389941703036
ISSN: 1538-7445
CID: 2668002

A primary melanoma and its asynchronous metastasis highlight the role of BRAF, CDKN2A, and TERT [Case Report]

Hosler, Gregory A; Davoli, Teresa; Mender, Ilgen; Litzner, Brandon; Choi, Jaehyuk; Kapur, Payal; Shay, Jerry W; Wang, Richard C
BACKGROUND: Alterations in pathways including BRAF, CDKN2A, and TERT contribute to the development of melanoma, but the sequence in which the genetic alterations occur and their prognostic significance remains unclear. To clarify the role of these pathways, we analyzed a primary melanoma and its metastasis. METHODS: Immunohistochemistry for BRAF-V600E, Sanger sequencing of BRAF and the TERT promoter, fluorescence in-situ hybridization, and telomere analyses were performed on a primary melanoma and its asynchronous cerebellar metastasis. Using the log-rank test and Cox-proportional model, the cancer genome atlas (TCGA) cohort of melanomas was analyzed for the effect of BRAF mutation and CDKN2A loss on survival. RESULTS: The primary melanoma expressed mutant BRAF-V600E and possessed a homozygous deletion of CDKN2A. In addition to these early defects, the metastatic lesion also possessed evidence of aneuploidy and an activating mutation of the TERT promoter. In the TCGA melanoma cohort, there was a non-significant trend toward poor prognosis in early stage cutaneous melanoma patients with concomitant BRAF mutation and CDKN2A loss. CONCLUSION: BRAF mutation and CDKN2A loss occurred early and TERT promoter mutation later in a case of lethal metastatic melanoma. The effects of these pathways on survival warrant further investigation in early stage cutaneous melanoma patients.
PMCID:4470704
PMID: 25407517
ISSN: 1600-0560
CID: 2667892

Comprehensive identification of host modulators of HIV-1 replication using multiple orthologous RNAi reagents

Zhu, Jian; Davoli, Teresa; Perriera, Jill M; Chin, Christopher R; Gaiha, Gaurav D; John, Sinu P; Sigiollot, Frederic D; Gao, Geng; Xu, Qikai; Qu, Hongjing; Pertel, Thomas; Sims, Jennifer S; Smith, Jennifer A; Baker, Richard E; Maranda, Louise; Ng, Aylwin; Elledge, Stephen J; Brass, Abraham L
RNAi screens have implicated hundreds of host proteins as HIV-1 dependency factors (HDFs). While informative, these early studies overlap poorly due to false positives and false negatives. To ameliorate these issues, we combined information from the existing HDF screens together with new screens performed with multiple orthologous RNAi reagents (MORR). In addition to being traditionally validated, the MORR screens and the historical HDF screens were quantitatively integrated by the adaptation of an established analysis program, RIGER, for the collective interpretation of each gene's phenotypic significance. False positives were addressed by the removal of poorly expressed candidates through gene expression filtering, as well as with GESS, which identifies off-target effects. This workflow produced a quantitatively integrated network of genes that modulate HIV-1 replication. We further investigated the roles of GOLGI49, SEC13, and COG in HIV-1 replication. Collectively, the MORR-RIGER method minimized the caveats of RNAi screening and improved our understanding of HIV-1-host cell interactions.
PMCID:4926641
PMID: 25373910
ISSN: 2211-1247
CID: 2667902

Cumulative dosage effect of TSGs and OGs drives aneuploidy patterns in cancer [Meeting Abstract]

Davoli, Teresa; Xu, Andrew Wei; Mengwasser, Kristen E; Sack, Laura M; Yoon, John C; Park, Peter J; Elledge, Stephen J
ISI:000349906903131
ISSN: 1538-7445
CID: 2667982

How aneuploidy drives tumorigenesis [Meeting Abstract]

Davoli, Teresa; Xu, Wei; Park, Peter; Elledge, Stephen J
ISI:000349910205478
ISSN: 1538-7445
CID: 2667992

Cumulative haploinsufficiency and triplosensitivity drive aneuploidy patterns and shape the cancer genome

Davoli, Teresa; Xu, Andrew Wei; Mengwasser, Kristen E; Sack, Laura M; Yoon, John C; Park, Peter J; Elledge, Stephen J
Aneuploidy has been recognized as a hallmark of cancer for more than 100 years, yet no general theory to explain the recurring patterns of aneuploidy in cancer has emerged. Here, we develop Tumor Suppressor and Oncogene (TUSON) Explorer, a computational method that analyzes the patterns of mutational signatures in tumors and predicts the likelihood that any individual gene functions as a tumor suppressor (TSG) or oncogene (OG). By analyzing >8,200 tumor-normal pairs, we provide statistical evidence suggesting that many more genes possess cancer driver properties than anticipated, forming a continuum of oncogenic potential. Integrating our driver predictions with information on somatic copy number alterations, we find that the distribution and potency of TSGs (STOP genes), OGs, and essential genes (GO genes) on chromosomes can predict the complex patterns of aneuploidy and copy number variation characteristic of cancer genomes. We propose that the cancer genome is shaped through a process of cumulative haploinsufficiency and triplosensitivity.
PMCID:3891052
PMID: 24183448
ISSN: 1097-4172
CID: 2667912

Haploinsufficiency in cancer [Meeting Abstract]

Davoli, Teresa; Solimini, Nicole L; Pavlova, Natalya N; Xu, Qikai; Mengwasser, Kristen; Sack, Laura M; Liang, Anthony C; Schlabach, Michael R; Luo, Ji; Burrows, Anna E; Anselmo, Anthony N; Li, Mamie Z; Elledge, Stephen J
ISI:000209496600171
ISSN: 1538-7445
CID: 2667962

Genetic Approaches to Cancer [Meeting Abstract]

Elledge, Steve; Pavlova, Natasha; Davoli, Teresa; Solimini, Nicole
ISI:000319883500556
ISSN: 0892-6638
CID: 2667972

STOP gene Phactr4 is a tumor suppressor

Solimini, Nicole L; Liang, Anthony C; Xu, Chunxiao; Pavlova, Natalya N; Xu, Qikai; Davoli, Teresa; Li, Mamie Z; Wong, Kwok-Kin; Elledge, Stephen J
Cancer develops through genetic and epigenetic alterations that allow unrestrained proliferation and increased survival. Using a genetic RNAi screen, we previously identified hundreds of suppressors of tumorigenesis and/or proliferation (STOP) genes that restrain normal cell proliferation. Our STOP gene set was significantly enriched for known and putative tumor suppressor genes. Here, we report a tumor-suppressive role for one STOP gene, phosphatase and actin regulator 4 (PHACTR4). Phactr4 is one of four members of the largely uncharacterized Phactr family of protein phosphatase 1 (PP1)-and actin-binding proteins. Our work suggests that Phactr4 restrains normal cell proliferation and transformation. Depletion of Phactr4 with multiple shRNAs leads to increased proliferation and soft agar colony formation. Phactr4 acts, in part, through an Rb-dependent pathway, because Rb phosphorylation is maintained upon growth factor withdrawal in Phactr4-depleted cells. Examination of tumor copy number analysis and sequencing revealed that PHACTR4 is significantly deleted and mutant in many tumor subtypes. Furthermore,cancer cell lines with reduced Phactr4 expression exhibit tumor suppressor hypersensitivity upon Phactr4 complementation,leading to reduced proliferation, transformation, and tumor formation. Thus, Phactr4 acts as a tumor suppressor that is deleted and mutant in several cancers.
PMCID:3562831
PMID: 23319639
ISSN: 1091-6490
CID: 2269842