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De novo assembly and delivery to mouse cells of a 101 kb functional human gene

Mitchell, Leslie A; McCulloch, Laura H; Pinglay, Sudarshan; Berger, Henri; Bosco, Nazario; Brosh, Ran; Bulajić, Milica; Huang, Emily; Hogan, Megan S; Martin, James A; Mazzoni, Esteban O; Davoli, Teresa; Maurano, Matthew T; Boeke, Jef D
Design and large-scale synthesis of DNA has been applied to the functional study of viral and microbial genomes. New and expanded technology development is required to unlock the transformative potential of such bottom-up approaches to the study of larger mammalian genomes. Two major challenges include assembling and delivering long DNA sequences. Here we describe a workflow for de novo DNA assembly and delivery that enables functional evaluation of mammalian genes on the length scale of 100 kilobase pairs (kb). The DNA assembly step is supported by an integrated robotic workcell. We demonstrate assembly of the 101 kb human HPRT1 gene in yeast from 3 kb building blocks, precision delivery of the resulting construct to mouse embryonic stem cells, and subsequent expression of the human protein from its full-length human gene in mouse cells. This workflow provides a framework for mammalian genome writing. We envision utility in producing designer variants of human genes linked to disease and their delivery and functional analysis in cell culture or animal models.
PMID: 33742653
ISSN: 1943-2631
CID: 4821992

Immune evasion in HPV- head and neck precancer-cancer transition is driven by an aneuploid switch involving chromosome 9p loss

William, William N; Zhao, Xin; Bianchi, Joy J; Lin, Heather Y; Cheng, Pan; Lee, J Jack; Carter, Hannah; Alexandrov, Ludmil B; Abraham, Jim P; Spetzler, David B; Dubinett, Steven M; Cleveland, Don W; Cavenee, Webster; Davoli, Teresa; Lippman, Scott M
An aneuploid-immune paradox encompasses somatic copy-number alterations (SCNAs), unleashing a cytotoxic response in experimental precancer systems, while conversely being associated with immune suppression and cytotoxic-cell depletion in human tumors, especially head and neck cancer (HNSC). We present evidence from patient samples and cell lines that alterations in chromosome dosage contribute to an immune hot-to-cold switch during human papillomavirus-negative (HPV-) head and neck tumorigenesis. Overall SCNA (aneuploidy) level was associated with increased CD3+ and CD8+ T cell microenvironments in precancer (mostly CD3+, linked to trisomy and aneuploidy), but with T cell-deficient tumors. Early lesions with 9p21.3 loss were associated with depletion of cytotoxic T cell infiltration in TP53 mutant tumors; and with aneuploidy were associated with increased NK-cell infiltration. The strongest driver of cytotoxic T cell and Immune Score depletion in oral cancer was 9p-arm level loss, promoting profound decreases of pivotal IFN-γ-related chemokines (e.g., CXCL9) and pathway genes. Chromosome 9p21.3 deletion contributed mainly to cell-intrinsic senescence suppression, but deletion of the entire arm was necessary to diminish levels of cytokine, JAK-STAT, and Hallmark NF-κB pathways. Finally, 9p arm-level loss and JAK2-PD-L1 codeletion (at 9p24) were predictive markers of poor survival in recurrent HPV- HNSC after anti-PD-1 therapy; likely amplified by independent aneuploidy-induced immune-cold microenvironments observed here. We hypothesize that 9p21.3 arm-loss expansion and epistatic interactions allow oral precancer cells to acquire properties to overcome a proimmunogenic aneuploid checkpoint, transform and invade. These findings enable distinct HNSC interception and precision-therapeutic approaches, concepts that may apply to other CN-driven neoplastic, immune or aneuploid diseases, and immunotherapies.
PMID: 33952700
ISSN: 1091-6490
CID: 4866502

Immune evasion in HPV-head and neck precancer-cancer transition is driven by an aneuploid switch involving chromosome 9p loss

William Jr, William N.; Zhao, Xin; Bianchi, Joy J.; Lin, Heather Y.; Cheng, Pan; Lee, J. Jack; Carter, Hannah; Alexandrov, Ludmil B.; Abraham, Jim P.; Spetzler, David B.; Dubinett, Steven M.; Cleveland, Don W.; Cavenee, Webster; Davoli, Teresa; Lippman, Scott M.
ISI:000651328500003
ISSN: 0027-8424
CID: 4893812

Not all cancers are created equal: Tissue specificity in cancer genes and pathways

Bianchi, Joy J; Zhao, Xin; Mays, Joseph C; Davoli, Teresa
Tumors arise through waves of genetic alterations and clonal expansion that allow tumor cells to acquire cancer hallmarks, such as genome instability and immune evasion. Recent genomic analyses showed that the vast majority of cancer driver genes are mutated in a tissue-dependent manner, that is, are altered in some cancers but not others. Often the tumor type also affects the likelihood of therapy response. What is the origin of tissue specificity in cancer? Recent studies suggest that both cell-intrinsic and cell-extrinsic factors play a role. On one hand, cell type-specific wiring of the cell signaling network determines the outcome of cancer driver gene mutations. On the other hand, the tumor cells' exposure to tissue-specific microenvironments (e.g. immune cells) also contributes to shape the tissue specificity of driver genes and of therapy response. In the future, a more complete understanding of tissue specificity in cancer may inform methods to better predict and improve therapeutic outcomes.
PMID: 32092639
ISSN: 1879-0410
CID: 4323162

De novo assembly, delivery and expression of a 101 kb human gene in mouse cells

Mitchell, Leslie A; McCulloch, Laura H; Pinglay, Sudarshan; Berger, Henri; Bosco, Nazario; Brosh, Ran; Bulajic, Milica; Huang, Emily; Hogan, Megan S; Martin, James A; Mazzoni, Esteban O; Davoli, Teresa; Maurano, Matthew T; Boeke, Jef D
Design and large-scale synthesis of DNA has been applied to the functional study of viral and microbial genomes. New and expanded technology development is required to unlock the transformative potential of such bottom-up approaches to the study of larger mammalian genomes. Two major challenges include assembling and delivering long DNA sequences. Here we describe a pipeline for de novo DNA assembly and delivery that enables functional evaluation of mammalian genes on the length scale of 100 kb. The DNA assembly step is supported by an integrated robotic workcell. We assembled the 101 kb human HPRT1 gene in yeast, delivered it to mouse embryonic stem cells, and showed expression of the human protein from its full-length gene. This pipeline provides a framework for producing systematic, designer variants of any mammalian gene locus for functional evaluation in cells
ORIGINAL:0014531
ISSN: n/a
CID: 4336392

Profound Tissue Specificity in Proliferation Control Underlies Cancer Drivers and Aneuploidy Patterns

Sack, Laura Magill; Davoli, Teresa; Li, Mamie Z; Li, Yuyang; Xu, Qikai; Naxerova, Kamila; Wooten, Eric C; Bernardi, Ronald J; Martin, Timothy D; Chen, Ting; Leng, Yumei; Liang, Anthony C; Scorsone, Kathleen A; Westbrook, Thomas F; Wong, Kwok-Kin; Elledge, Stephen J
Genomics has provided a detailed structural description of the cancer genome. Identifying oncogenic drivers that work primarily through dosage changes is a current challenge. Unrestrained proliferation is a critical hallmark of cancer. We constructed modular, barcoded libraries of human open reading frames (ORFs) and performed screens for proliferation regulators in multiple cell types. Approximately 10% of genes regulate proliferation, with most performing in an unexpectedly highly tissue-specific manner. Proliferation drivers in a given cell type showed specific enrichment in somatic copy number changes (SCNAs) from cognate tumors and helped predict aneuploidy patterns in those tumors, implying that tissue-type-specific genetic network architectures underlie SCNA and driver selection in different cancers. In vivo screening confirmed these results. We report a substantial contribution to the catalog of SCNA-associated cancer drivers, identifying 147 amplified and 107 deleted genes as potential drivers, and derive insights about the genetic network architecture of aneuploidy in tumors.
PMID: 29576454
ISSN: 1097-4172
CID: 3011212

Tumor aneuploidy correlates with markers of immune evasion and with reduced response to immunotherapy

Davoli, Teresa; Uno, Hajime; Wooten, Eric C; Elledge, Stephen J
Immunotherapies based on immune checkpoint blockade are highly effective in a subset of patients. An ongoing challenge is the identification of biomarkers that predict which patients will benefit from these therapies. Aneuploidy, also known as somatic copy number alterations (SCNAs), is widespread in cancer and is posited to drive tumorigenesis. Analyzing 12 human cancer types, we find that, for most, highly aneuploid tumors show reduced expression of markers of cytotoxic infiltrating immune cells, especially CD8+ T cells, and increased expression of cell proliferation markers. Different types of SCNAs predict the proliferation and immune signatures, implying distinct underlying mechanisms. Using published data from two clinical trials of immune checkpoint blockade therapy for metastatic melanoma, we found that tumor aneuploidy inversely correlates with patient survival. Together with other tumor characteristics such as tumor mutational load, aneuploidy may thus help identify patients most likely to respond to immunotherapy.
PMCID:5592794
PMID: 28104840
ISSN: 1095-9203
CID: 2667872

A genetic interaction analysis identifies cancer drivers that modify EGFR dependency

Liao, Sida; Davoli, Teresa; Leng, Yumei; Li, Mamie Z; Xu, Qikai; Elledge, Stephen J
A large number of cancer drivers have been identified through tumor sequencing efforts, but how they interact and the degree to which they can substitute for each other have not been systematically explored. To comprehensively investigate how cancer drivers genetically interact, we searched for modifiers of epidermal growth factor receptor (EGFR) dependency by performing CRISPR, shRNA, and expression screens in a non-small cell lung cancer (NSCLC) model. We elucidated a broad spectrum of tumor suppressor genes (TSGs) and oncogenes (OGs) that can genetically modify proliferation and survival of cancer cells when EGFR signaling is altered. These include genes already known to mediate EGFR inhibitor resistance as well as many TSGs not previously connected to EGFR and whose biological functions in tumorigenesis are not well understood. We show that mutation of PBRM1, a subunit of the SWI/SNF complex, attenuates the effects of EGFR inhibition in part by sustaining AKT signaling. We also show that mutation of Capicua (CIC), a transcriptional repressor, suppresses the effects of EGFR inhibition by partially restoring the EGFR-promoted gene expression program, including the sustained expression of Ets transcription factors such as ETV1 Together, our data provide strong support for the hypothesis that many cancer drivers can substitute for each other in certain contexts and broaden our understanding of EGFR regulation.
PMCID:5322732
PMID: 28167502
ISSN: 1549-5477
CID: 2667862

Functional genomics reveals that tumors with activating phosphoinositide 3-kinase mutations are dependent on accelerated protein turnover

Davoli, Teresa; Mengwasser, Kristen E; Duan, Jingjing; Chen, Ting; Christensen, Camilla; Wooten, Eric C; Anselmo, Anthony N; Li, Mamie Z; Wong, Kwok-Kin; Kahle, Kristopher T; Elledge, Stephen J
Activating mutations in the phosphoinositide 3-kinase (PI3K) signaling pathway are frequently identified in cancer. To identify pathways that support PI3K oncogenesis, we performed a genome-wide RNAi screen in isogenic cell lines harboring wild-type or mutant PIK3CA to search for PI3K synthetic-lethal (SL) genes. A combined analysis of these results with a meta-analysis of two other large-scale RNAi screening data sets in PI3K mutant cancer cell lines converged on ribosomal protein translation and proteasomal protein degradation as critical nononcogene dependencies for PI3K-driven tumors. Genetic or pharmacologic inhibition of either pathway alone, but not together, selectively killed PI3K mutant tumor cells in an mTOR-dependent manner. The expression of ribosomal and proteasomal components was significantly up-regulated in primary human colorectal tumors harboring PI3K pathway activation. Importantly, a PI3K SL gene signature containing the top hits of the SL genes identified in our meta-analysis robustly predicted overall patient survival in colorectal cancer, especially among patients with tumors with an activated PI3K pathway. These results suggest that disruption of protein turnover homeostasis via ribosome or proteasome inhibition may be a novel treatment strategy for PI3K mutant human tumors.
PMCID:5238728
PMID: 28087713
ISSN: 1549-5477
CID: 2523682

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