Single-cell genomics is essential to chart tumor ecosystems. Although single-cell RNA-Seq (scRNA-Seq) profiles RNA from cells dissociated from fresh tumors, single-nucleus RNA-Seq (snRNA-Seq) is ...needed to profile frozen or hard-to-dissociate tumors. Each requires customization to different tissue and tumor types, posing a barrier to adoption. Here, we have developed a systematic toolbox for profiling fresh and frozen clinical tumor samples using scRNA-Seq and snRNA-Seq, respectively. We analyzed 216,490 cells and nuclei from 40 samples across 23 specimens spanning eight tumor types of varying tissue and sample characteristics. We evaluated protocols by cell and nucleus quality, recovery rate and cellular composition. scRNA-Seq and snRNA-Seq from matched samples recovered the same cell types, but at different proportions. Our work provides guidance for studies in a broad range of tumors, including criteria for testing and selecting methods from the toolbox for other tumors, thus paving the way for charting tumor atlases.
Proteins are essential agents of biological processes. To date, large-scale profiling of cell line collections including the Cancer Cell Line Encyclopedia (CCLE) has focused primarily on genetic ...information whereas deep interrogation of the proteome has remained out of reach. Here, we expand the CCLE through quantitative profiling of thousands of proteins by mass spectrometry across 375 cell lines from diverse lineages to reveal information undiscovered by DNA and RNA methods. We observe unexpected correlations within and between pathways that are largely absent from RNA. An analysis of microsatellite instable (MSI) cell lines reveals the dysregulation of specific protein complexes associated with surveillance of mutation and translation. These and other protein complexes were associated with sensitivity to knockdown of several different genes. These data in conjunction with the wider CCLE are a broad resource to explore cellular behavior and facilitate cancer research.
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•Quantified the proteomes of 375 cell lines from diverse lineages in the CCLE•Correlated expression of proteins across many pathways•Downregulation of multiple protein complexes in microsatellite instability•Protein complexes associated with sensitivity to gene knockdown and mutation
Quantitative proteomes of 375 cancer cell lines for the Cancer Cell Line Encyclopedia (CCLE) reveal correlated protein expression between multiple pathways and complexes as well as associations between protein complexes and genetic features including microsatellite instability, individual gene mutations, and sensitivity to gene knockdowns.
To explore the distinct genotypic and phenotypic states of melanoma tumors, we applied single-cell RNA sequencing (RNA-seq) to 4645 single cells isolated from 19 patients, profiling malignant, ...immune, stromal, and endothelial cells. Malignant cells within the same tumor displayed transcriptional heterogeneity associated with the cell cycle, spatial context, and a drug-resistance program. In particular, all tumors harbored malignant cells from two distinct transcriptional cell states, such that tumors characterized by high levels of the MITF transcription factor also contained cells with low MITF and elevated levels of the AXL kinase. Single-cell analyses suggested distinct tumor microenvironmental patterns, including cell-to-cell interactions. Analysis of tumor-infiltrating T cells revealed exhaustion programs, their connection to T cell activation and clonal expansion, and their variability across patients. Overall, we begin to unravel the cellular ecosystem of tumors and how single-cell genomics offers insights with implications for both targeted and immune therapies.
Methods for highly multiplexed RNA imaging are limited in spatial resolution and thus in their ability to localize transcripts to nanoscale and subcellular compartments. We adapt expansion ...microscopy, which physically expands biological specimens, for long-read untargeted and targeted in situ RNA sequencing. We applied untargeted expansion sequencing (ExSeq) to the mouse brain, which yielded the readout of thousands of genes, including splice variants. Targeted ExSeq yielded nanoscale-resolution maps of RNAs throughout dendrites and spines in the neurons of the mouse hippocampus, revealing patterns across multiple cell types, layer-specific cell types across the mouse visual cortex, and the organization and position-dependent states of tumor and immune cells in a human metastatic breast cancer biopsy. Thus, ExSeq enables highly multiplexed mapping of RNAs from nanoscale to system scale.
Reliable methods to quantify dynamic signaling changes across diverse pathways are needed to better understand the effects of disease and drug treatment in cells and tissues but are presently ...lacking. Here, we present SigPath, a targeted mass spectrometry (MS) assay that measures 284 phosphosites in 200 phosphoproteins of biological interest. SigPath probes a broad swath of signaling biology with high throughput and quantitative precision. We applied the assay to investigate changes in phospho‐signaling in drug‐treated cancer cell lines, breast cancer preclinical models, and human medulloblastoma tumors. In addition to validating previous findings, SigPath detected and quantified a large number of differentially regulated phosphosites newly associated with disease models and human tumors at baseline or with drug perturbation. Our results highlight the potential of SigPath to monitor phosphoproteomic signaling events and to nominate mechanistic hypotheses regarding oncogenesis, response, and resistance to therapy.
SYNOPSIS
SigPath is a targeted, quantitative mass spectrometry assay that measures 284 phosphosites spanning 200 phosphoproteins with high throughput and quantitative precision across a broad swath of signaling biology of known interest.
The value of the assay is demonstrated by application to drug‐treated cancer cell lines, breast cancer preclinical models and human medulloblastoma tumors.
Large numbers of differentially regulated phosphosites newly associated with disease models and human tumors are identified.
The results highlight the potential use of SigPath to nominate mechanistic hypotheses regarding oncogenesis, response and resistance to therapy.
SigPath is a targeted, quantitative mass spectrometry assay that measures 284 phosphosites spanning 200 phosphoproteins with high throughput and quantitative precision across a broad swath of signaling biology of known interest.
Pathway analysis of PTM data sets is typically performed at a gene-centric level because of the lack of appropriately curated PTM signature databases. We have developed a PTM signatures database ...(PTMsigDB) providing curated phosphorylation signatures of kinases, perturbations and signaling pathways to enable site-specific PTM signature enrichment analysis (PTM-SEA). Application of PTM-SEA to phosphoproteomes of several cell lines perturbed with growth factors, cell cycle inhibitors, or a specific PI3K inhibitor demonstrated the potential of our site centric approach to study dysregulated pathways in cancers.
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Highlights
•Database of PTM site-specific phosphorylation signatures of kinases, perturbations and signaling pathways (PTMsigDB).•PTM signature enrichment analysis (PTM-SEA) outperformed gene-centric analysis in detection of EGF induced phospho signaling events.•PI3K perturbation signatures were readily detected in PI3Ka inhibited human breast cancer cells.•PTMsigDB and PTM-SEA can be freely accessed at https://github.com/broadinstitute/ssGSEA2.0.
Signaling pathways are orchestrated by post-translational modifications (PTMs) such as phosphorylation. However, pathway analysis of PTM data sets generated by mass spectrometry (MS)-based proteomics is typically performed at a gene-centric level because of the lack of appropriately curated PTM signature databases and bioinformatic tools that leverage PTM site-specific information. Here we present the first version of PTMsigDB, a database of modification site-specific signatures of perturbations, kinase activities and signaling pathways curated from more than 2,500 publications. We adapted the widely used single sample Gene Set Enrichment Analysis approach to utilize PTMsigDB, enabling PTMSignature Enrichment Analysis (PTM-SEA) of quantitative MS data. We used a well-characterized data set of epidermal growth factor (EGF)-perturbed cancer cells to evaluate our approach and demonstrated better representation of signaling events compared with gene-centric methods. We then applied PTM-SEA to analyze the phosphoproteomes of cancer cells treated with cell-cycle inhibitors and detected mechanism-of-action specific signatures of cell cycle kinases. We also applied our methods to analyze the phosphoproteomes of PI3K-inhibited human breast cancer cells and detected signatures of compounds inhibiting PI3K as well as targets downstream of PI3K (AKT, MAPK/ERK) covering a substantial fraction of the PI3K pathway. PTMsigDB and PTM-SEA can be freely accessed at https://github.com/broadinstitute/ssGSEA2.0.
Background
There is limited knowledge of the genetic alterations in acral melanoma metastases at different anatomic sites. Here, we characterized the genetic abnormalities of metastases in a ...51‐year‐old man with stage IIIC heel melanoma who developed concomitant brain and cutaneous metastases in spite of multiple treatment modalities.
Methods
Melanoma cells were isolated following palliative resection of the patient's cortical tumor and biopsy of cutaneous thigh metastasis. Mutational analysis using polymerase chain reaction amplification and BLAST, as well as exome sequencing (160 Mb coverage) was performed on the tumors, cell lines generated thereof and normal lymph nodes.
Results
All specimens had neuroblastoma RAS viral oncogene homolog Q61K mutations. There was a 40‐fold higher somatic mutation frequency in the brain metastasis compared to the cutaneous metastasis. The former showed truncations of DNA mismatch repair genes (MLH1 and MSH2), and non‐canonical BRAF (v‐raf murine sarcoma viral oncogene homolog B1), PIK3CA and NF‐1 mutations not observed in the extracranial lesion. Genomic profiling of each cell line was concordant with the respective original tumor tissue.
Conclusions
We present the mutational differences between brain and cutaneous acral melanoma metastases in a patient with concomitant lesions. Further genetic and functional studies are needed to understand the biology of metastatic disease appearing at different sites.
An Oncogenic Role for ETV1 in Melanoma JANE-VALBUENA, Judit; WIDLUND, Hans R; HAHN, William C ...
Cancer research (Chicago, Ill.),
03/2010, Letnik:
70, Številka:
5
Journal Article
Recenzirano
Odprti dostop
Copy gains involving chromosome 7p represent one of the most common genomic alterations found in melanomas, suggesting the presence of "driver" cancer genes. We identified several tumor samples that ...harbored focal amplifications situated at the peak of common chromosome 7p gains, in which the minimal common overlapping region spanned the ETV1 oncogene. Fluorescence in situ hybridization analysis revealed copy gains spanning the ETV1 locus in >40% of cases, with ETV1 amplification (>6 copies/cell) present in 13% of primary and 18% of metastatic melanomas. Melanoma cell lines, including those with ETV1 amplification, exhibited dependency on ETV1 expression for proliferation and anchorage-independent growth. Moreover, overexpression of ETV1 in combination with oncogenic NRAS(G12D) transformed primary melanocytes and promoted tumor formation in mice. ETV1 overexpression elevated microphthalmia-associated transcription factor expression in immortalized melanocytes, which was necessary for ETV1-dependent oncogenicity. These observations implicate deregulated ETV1 in melanoma genesis and suggest a pivotal lineage dependency mediated by oncogenic ETS transcription factors in this malignancy.
Novel treatment strategies are needed for older patients with acute myeloid leukemia (AML). This randomized phase 2 trial compared the efficacy and safety of 20 mg/m2 of IV decitabine on days 1 to 10 ...alone (arm A) with those of 1.3 mg/m2 of subcutaneous bortezomib (arm B) on days 1, 4, 8, and 11 for up to 4 10-day cycles followed by monthly 5-day cycles. Previously untreated AML patients age ≥60 years (excluding those with FLT3 mutations and favorable-risk cytogenetics) without restrictions in performance status (PS) or organ function were eligible. Median age was 72.4 years (range, 60.5-92.3 years); 31 patients (19%) had baseline PS ≥2, 35 (22%) had an antecedent hematological disorder, 58 had (39%) adverse cytogenetics, and 7 (5%) and 23 (14%) had abnormal cardiac or renal function. There were no statistically significant differences in overall survival (OS) or responses between the 2 treatment arms. The overall response rate (complete remission + complete remission with incomplete blood count recovery) was 39% (n = 64), with median OS of 9.3 months. Nineteen responders (31%) underwent allogeneic stem cell transplantation. The most common adverse event was febrile neutropenia, and there were no unexpected toxicities. Adding bortezomib to decitabine did not improve outcomes, but responses were better than those in previous trials using 5-day decitabine cycles. This trial was registered at www.clinicaltrials.gov as #NCT01420926.
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Combined treatment with dabrafenib and trametinib (CombiDT) achieves clinical responses in only about 15% of patients with BRAF inhibitor (BRAFi)-refractory metastatic melanoma in contrast to the ...higher response rate observed in BRAFi-naïve patients. Identifying correlates of response and mechanisms of resistance in this population will facilitate clinical management and rational therapeutic development.
To determine correlates of benefit from CombiDT therapy in patients with BRAFi-refractory metastatic melanoma.
Single-center, single-arm, open-label phase 2 trial of CombiDT treatment in patients with BRAF V600 metastatic melanoma resistant to BRAFi monotherapy conducted between September 2012 and October 2014 at the University of Texas MD Anderson Cancer Center. Key eligibility criteria for participants included BRAF V600 metastatic melanoma, prior BRAFi monotherapy, measurable disease (RECIST 1.1), and tumor accessible for biopsy.
Patients were treated with dabrafenib (150 mg, twice daily) and trametinib (2 mg/d) continuously until disease progression or intolerance. All participants underwent a mandatory baseline biopsy, and optional biopsy specimens were obtained on treatment and at disease progression. Whole-exome sequencing, reverse transcription polymerase chain reaction analysis for BRAF splicing, RNA sequencing, and immunohistochemical analysis were performed on tumor samples, and blood was analyzed for levels of circulating BRAF V600.
The primary end point was overall response rate (ORR). Progression-free survival (PFS) and overall survival (OS) were secondary clinical end points.
A total of 28 patients were screened, and 23 enrolled. Among evaluable patients, the confirmed ORR was 10%; disease control rate (DCR) was 45%, and median PFS was 13 weeks. Clinical benefit was associated with duration of prior BRAFi therapy greater than 6 months (DCR, 73% vs 11% for ≤6 months; P = .02) and decrease in circulating BRAF V600 at day 8 of cycle 1 (DCR, 75% vs 18% for no decrease; P = .02) but not with pretreatment mitogen-activated protein kinase (MAPK) pathway mutations or activation. Biopsy specimens obtained during treatment demonstrated that CombiDT therapy failed to achieve significant MAPK pathway inhibition or immune infiltration in most patients.
The baseline presence of MAPK pathway alterations was not associated with benefit from CombiDT in patients with BRAFi-refractory metastatic melanoma. Failure to inhibit the MAPK pathway provides a likely explanation for the limited clinical benefit of CombiDT in this setting. Circulating BRAF V600 is a promising early biomarker of clinical response.
clinicaltrials.gov Identifier: NCT01619774.
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