Publications by authors named "Anatoly Urisman"

37 Publications

Therapy-Induced Evolution of Human Lung Cancer Revealed by Single-Cell RNA Sequencing.

Cell 2020 Sep 20;182(5):1232-1251.e22. Epub 2020 Aug 20.

Department of Medicine, University of California, San Francisco, San Francisco, CA 94158, USA; Helen Diller Family Comprehensive Cancer Center, University of California, San Francisco, San Francisco, CA 94158, USA; Department of Cellular and Molecular Pharmacology, University of California, San Francisco, San Francisco, CA 94158, USA. Electronic address:

Lung cancer, the leading cause of cancer mortality, exhibits heterogeneity that enables adaptability, limits therapeutic success, and remains incompletely understood. Single-cell RNA sequencing (scRNA-seq) of metastatic lung cancer was performed using 49 clinical biopsies obtained from 30 patients before and during targeted therapy. Over 20,000 cancer and tumor microenvironment (TME) single-cell profiles exposed a rich and dynamic tumor ecosystem. scRNA-seq of cancer cells illuminated targetable oncogenes beyond those detected clinically. Cancer cells surviving therapy as residual disease (RD) expressed an alveolar-regenerative cell signature suggesting a therapy-induced primitive cell-state transition, whereas those present at on-therapy progressive disease (PD) upregulated kynurenine, plasminogen, and gap-junction pathways. Active T-lymphocytes and decreased macrophages were present at RD and immunosuppressive cell states characterized PD. Biological features revealed by scRNA-seq were biomarkers of clinical outcomes in independent cohorts. This study highlights how therapy-induced adaptation of the multi-cellular ecosystem of metastatic cancer shapes clinical outcomes.
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http://dx.doi.org/10.1016/j.cell.2020.07.017DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7484178PMC
September 2020

Complement activation on endothelium initiates antibody-mediated acute lung injury.

J Clin Invest 2020 11;130(11):5909-5923

Department of Medicine, UCSF, San Francisco, California, USA.

Antibodies targeting human leukocyte antigen (HLA)/major histocompatibility complex (MHC) proteins limit successful transplantation and transfusion, and their presence in blood products can cause lethal transfusion-related acute lung injury (TRALI). It is unclear which cell types are bound by these anti-leukocyte antibodies to initiate an immunologic cascade resulting in lung injury. We therefore conditionally removed MHC class I (MHC I) from likely cellular targets in antibody-mediated lung injury. Only the removal of endothelial MHC I reduced lung injury and mortality, related mechanistically to absent endothelial complement fixation and lung platelet retention. Restoration of endothelial MHC I rendered MHC I-deficient mice susceptible to lung injury. Neutrophil responses, including neutrophil extracellular trap (NET) release, were intact in endothelial MHC I-deficient mice, whereas complement depletion reduced both lung injury and NETs. Human pulmonary endothelial cells showed high HLA class I expression, and posttransfusion complement activation was increased in clinical TRALI. These results indicate that the critical source of antigen for anti-leukocyte antibodies is in fact the endothelium, which reframes our understanding of TRALI as a rapid-onset vasculitis. Inhibition of complement activation may have multiple beneficial effects of reducing endothelial injury, platelet retention, and NET release in conditions where antibodies trigger these pathogenic responses.
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http://dx.doi.org/10.1172/JCI138136DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7598054PMC
November 2020

Structural Insights into the SPRED1-Neurofibromin-KRAS Complex and Disruption of SPRED1-Neurofibromin Interaction by Oncogenic EGFR.

Cell Rep 2020 07;32(3):107909

NCI RAS Initiative, Cancer Research Technology Program, Frederick National Laboratory for Cancer Research, Leidos Biomedical Research, Inc., Frederick, MD 21701, USA. Electronic address:

Sprouty-related, EVH1 domain-containing (SPRED) proteins negatively regulate RAS/mitogen-activated protein kinase (MAPK) signaling following growth factor stimulation. This inhibition of RAS is thought to occur primarily through SPRED1 binding and recruitment of neurofibromin, a RasGAP, to the plasma membrane. Here, we report the structure of neurofibromin (GTPase-activating protein [GAP]-related domain) complexed with SPRED1 (EVH1 domain) and KRAS. The structure provides insight into how the membrane targeting of neurofibromin by SPRED1 allows simultaneous interaction with activated KRAS. SPRED1 and NF1 loss-of-function mutations occur across multiple cancer types and developmental diseases. Analysis of the neurofibromin-SPRED1 interface provides a rationale for mutations observed in Legius syndrome and suggests why SPRED1 can bind to neurofibromin but no other RasGAPs. We show that oncogenic EGFR(L858R) signaling leads to the phosphorylation of SPRED1 on serine 105, disrupting the SPRED1-neurofibromin complex. The structural, biochemical, and biological results provide new mechanistic insights about how SPRED1 interacts with neurofibromin and regulates active KRAS levels in normal and pathologic conditions.
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http://dx.doi.org/10.1016/j.celrep.2020.107909DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7437355PMC
July 2020

Small Airway Disease: A Step Closer to Etiology-Based Classification of Bronchiolitis.

Surg Pathol Clin 2020 Mar 30;13(1):189-196. Epub 2019 Nov 30.

Department of Pathology, University of California San Francisco, 505 Parnassus Avenue, Box 0102, San Francisco, CA 94143, USA.

Three major histologic patterns of bronchiolitis: obliterative bronchiolitis, follicular bronchiolitis, and diffuse panbronchiolitis, are reviewed in detail. These distinct patterns of primary bronchiolar injury provide a useful starting point for formulating a differential diagnosis and considering possible causes. In support of the aim toward a cause-based classification system of small airway disease, a simple diagnostic algorithm is provided for further subclassification of the above 3 bronchiolitis patterns according to the major associated etiologic subgroups.
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http://dx.doi.org/10.1016/j.path.2019.10.004DOI Listing
March 2020

Inhibition of cyclin-dependent kinase 7 down-regulates yes-associated protein expression in mesothelioma cells.

J Cell Mol Med 2020 01 21;24(1):1087-1098. Epub 2019 Nov 21.

Department of Surgery, Thoracic Oncology Laboratory, Comprehensive Cancer Center, University of California, San Francisco, CA, USA.

Cyclin-dependent kinase 7 (CDK7) is a protein kinase that plays a major role in transcription initiation. Yes-associated protein (YAP) is a main effector of the Hippo/YAP signalling pathway. Here, we investigated the role of CDK7 on YAP regulation in human malignant pleural mesothelioma (MPM). We found that in microarray samples of human MPM tissue, immunohistochemistry staining showed correlation between the expression level of CDK7 and YAP (n = 70, r = .513). In MPM cells, CDK7 expression level was significantly correlated with GTIIC reporter activity (r = .886, P = .019). Inhibition of CDK7 by siRNA decreased the YAP protein level and the GTIIC reporter activity in the MPM cell lines 211H, H290 and H2052. Degradation of the YAP protein was accelerated after CDK7 knockdown in 211H, H290 and H2052 cells. Inhibition of CDK7 reduced tumour cell migration and invasion, as well as tumorsphere formation ability. Restoration of the CDK7 gene rescued the YAP protein level and GTIIC reporter activity after siRNA knockdown in 211H and H2052 cells. Finally, we performed a co-immunoprecipitation analysis using an anti-YAP antibody and captured the CDK7 protein in 211H cells. Our results suggest that CDK7 inhibition reduces the YAP protein level by promoting its degradation and suppresses the migration and invasion of MPM cells. Cyclin-dependent kinase 7 may be a promising therapeutic target for MPM.
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http://dx.doi.org/10.1111/jcmm.14841DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6933402PMC
January 2020

Adaptive Resistance to Dual BRAF/MEK Inhibition in BRAF-Driven Tumors through Autocrine FGFR Pathway Activation.

Clin Cancer Res 2019 12 12;25(23):7202-7217. Epub 2019 Sep 12.

Helen Diller Comprehensive Cancer Center, University of California, San Francisco, San Francisco, California.

Purpose: Combined MAPK pathway inhibition using dual BRAF and MEK inhibitors has prolonged the duration of clinical response in patients with BRAF-driven tumors compared with either agent alone. However, resistance frequently arises.

Experimental Design: We generated cell lines resistant to dual BRAF/MEK inhibition and utilized a pharmacologic synthetic lethal approach to identify a novel, adaptive resistance mechanism mediated through the fibroblast growth factor receptor (FGFR) pathway.

Results: In response to drug treatment, transcriptional upregulation of FGF1 results in autocrine activation of FGFR, which potentiates extracellular signal-regulated kinases (ERK) activation. FGFR inhibition overcomes resistance to dual BRAF/MEK inhibitors in both cell lines and patient-derived xenograft (PDX) models. Abrogation of this bypass mechanism in the first-line setting enhances tumor killing and prevents the emergence of drug-resistant cells. Moreover, clinical data implicate serum FGF1 levels in disease prognosis.

Conclusions: Taken together, these results describe a new, adaptive resistance mechanism that is more commonly observed in the context of dual BRAF/MEK blockade as opposed to single-agent treatment and reveal the potential clinical utility of FGFR-targeting agents in combination with BRAF and MEK inhibitors as a promising strategy to forestall resistance in a subset of BRAF-driven cancers.
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http://dx.doi.org/10.1158/1078-0432.CCR-18-2779DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6891193PMC
December 2019

Cucurbitacin E inhibits the Yes‑associated protein signaling pathway and suppresses brain metastasis of human non‑small cell lung cancer in a murine model.

Oncol Rep 2019 Aug 20;42(2):697-707. Epub 2019 Jun 20.

Department of Surgery, Helen Diller Family Comprehensive Cancer Center, University of California, San Francisco, San Francisco, CA 94115, USA.

Human non‑small cell lung cancer (NSCLC) is associated with an extremely poor prognosis especially for the 40% of patients who develop brain metastasis, and few treatment strategies exist. Cucurbitacin E (CuE), an oxygenated tetracyclic triterpenoid isolated from plants particularly of the family Cucurbitaceae, has shown anti‑tumorigenic properties in several types of cancer, yet the mechanism remains unclear. Yes‑associated protein (YAP), a main mediator of the Hippo signaling pathway, promotes tumorigenesis, drug resistance and metastasis in human NSCLC. The present study was designed to ascertain whether CuE inhibits YAP and its downstream gene expression in the human NSCLC cell lines H2030‑BrM3 (K‑rasG12C mutation) and PC9‑BrM3 (EGFRΔexon19 mutation), which have high potential for brain metastasis. The efficacy of CuE in suppressing brain metastasis of H2030‑BrM3 cells in a murine model was also investigated. It was found that after CuE treatment in H2030‑BrM3 and PC9‑BrM3 cells, YAP protein expression was decreased, and YAP signaling GTIIC reporter activity and expression of the downstream genes CTGF and CYR61 were significantly (P<0.01) decreased. CuE treatment also reduced the migration and invasion abilities of the H2030‑BrM3 and PC9‑BrM3 cells. Finally, our in vivo study showed that CuE treatment (0.2 mg/kg) suppressed H2030‑BrM3 cell brain metastasis and that mice treated with CuE survived longer than the control mice treated with 10% DMSO (P=0.02). The present study is the first to demonstrate that CuE treatment inhibits YAP and the signaling downstream gene expression in human NSCLC in vitro, and suppresses brain metastasis of NSCLC in a murine model. More studies to verify the promising efficacy of CuE in inhibiting brain metastasis of NSCLC and various other cancers may be warranted.
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http://dx.doi.org/10.3892/or.2019.7207DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6610039PMC
August 2019

RIT1 oncoproteins escape LZTR1-mediated proteolysis.

Science 2019 03;363(6432):1226-1230

Helen Diller Family Comprehensive Cancer Center, University of California San Francisco, San Francisco, CA, USA.

RIT1 oncoproteins have emerged as an etiologic factor in Noonan syndrome and cancer. Despite the resemblance of RIT1 to other members of the Ras small guanosine triphosphatases (GTPases), mutations affecting RIT1 are not found in the classic hotspots but rather in a region near the switch II domain of the protein. We used an isogenic germline knock-in mouse model to study the effects of RIT1 mutation at the organismal level, which resulted in a phenotype resembling Noonan syndrome. By mass spectrometry, we detected a RIT1 interactor, leucine zipper-like transcription regulator 1 (LZTR1), that acts as an adaptor for protein degradation. Pathogenic mutations affecting either RIT1 or LZTR1 resulted in incomplete degradation of RIT1. This led to RIT1 accumulation and dysregulated growth factor signaling responses. Our results highlight a mechanism of pathogenesis that relies on impaired protein degradation of the Ras GTPase RIT1.
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http://dx.doi.org/10.1126/science.aav1444DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6986682PMC
March 2019

Chemically reprogramming the phospho-transfer reaction to crosslink protein kinases to their substrates.

Protein Sci 2019 03 31;28(3):654-662. Epub 2019 Jan 31.

Department of Cellular and Molecular Pharmacology, University of California San Francisco, San Francisco, California.

The proteomic mapping of enzyme-substrate interactions is challenged by their transient nature. A method to capture interacting protein kinases in complexes with a single substrate of interest would provide a new tool for mapping kinase signaling networks. Here, we describe a nucleotide-based substrate analog capable of reprogramming the wild-type phosphoryl-transfer reaction to produce a kinase-acrylamide-based thioether crosslink to mutant substrates with a cysteine nucleophile substituted at the native phosphorylation site. A previously reported ATP-based methacrylate crosslinker (ATP-MA) was capable of mediating kinase crosslinking to short peptides but not protein substrates. Exploration of structural variants of ATP-MA to enable crosslinking of protein substrates to kinases led to the discovery that an ADP-based methacrylate (ADP-MA) crosslinker was superior to the ATP scaffold at crosslinking in vitro. The improved efficiency of ADP-MA over ATP-MA is due to reduced inhibition of the second step of the kinase-substrate crosslinking reaction by the product of the first step of the reaction. The new probe, ADP-MA, demonstrated enhanced in vitro crosslinking between the Src tyrosine kinase and its substrate Cortactin in a phosphorylation site-specific manner. The kinase-substrate crosslinking reaction can be carried out in a complex mammalian cell lysate setting, although the low abundance of endogenous kinases remains a significant challenge for efficient capture.
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http://dx.doi.org/10.1002/pro.3570DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6371225PMC
March 2019

Pathologic Complete Response to Neoadjuvant Crizotinib in a Lung Adenocarcinoma Patient With a MET Exon 14 Skipping Mutation.

Clin Lung Cancer 2019 03 20;20(2):e137-e141. Epub 2018 Nov 20.

Department of Medicine, University of California, San Francisco, San Francisco, CA; Hellen Diller Family Comprehensive Cancer Center, University of California, San Francisco, San Francisco, CA. Electronic address:

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http://dx.doi.org/10.1016/j.cllc.2018.11.003DOI Listing
March 2019

Macrophages promote epithelial proliferation following infectious and non-infectious lung injury through a Trefoil factor 2-dependent mechanism.

Mucosal Immunol 2019 01 18;12(1):64-76. Epub 2018 Oct 18.

Department of Pathobiology, University of Pennsylvania School of Veterinary Medicine, Philadelphia, PA, 19104, USA.

Coordinated efforts between macrophages and epithelia are considered essential for wound healing, but the macrophage-derived molecules responsible for repair are poorly defined. This work demonstrates that lung macrophages rely upon Trefoil factor 2 to promote epithelial proliferation following damage caused by sterile wounding, Nippostrongylus brasiliensis or Bleomycin sulfate. Unexpectedly, the presence of T, B, or ILC populations was not essential for macrophage-driven repair. Instead, conditional deletion of TFF2 in myeloid-restricted CD11c TFF2 mice exacerbated lung pathology and reduced the proliferative expansion of CD45 EpCAM pro-SPC alveolar type 2 cells. TFF2 deficient macrophages had reduced expression of the Wnt genes Wnt4 and Wnt16 and reconstitution of hookworm-infected CD11c TFF2 mice with rWnt4 and rWnt16 restored the proliferative defect in lung epithelia post-injury. These data reveal a previously unrecognized mechanism wherein lung myeloid phagocytes utilize a TFF2/Wnt axis as a mechanism that drives epithelial proliferation following lung injury.
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http://dx.doi.org/10.1038/s41385-018-0096-2DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6301101PMC
January 2019

A 36-Year-Old Man With Renal Failure, Fever, and Hypocomplementemia.

Arthritis Care Res (Hoboken) 2019 04;71(4):449-455

University of California, San Francisco.

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http://dx.doi.org/10.1002/acr.23770DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6478622PMC
April 2019

Axonal G3BP1 stress granule protein limits axonal mRNA translation and nerve regeneration.

Nat Commun 2018 08 22;9(1):3358. Epub 2018 Aug 22.

Department of Biological Sciences, University of South Carolina, Columbia, 29208, SC, USA.

Critical functions of intra-axonally synthesized proteins are thought to depend on regulated recruitment of mRNA from storage depots in axons. Here we show that axotomy of mammalian neurons induces translation of stored axonal mRNAs via regulation of the stress granule protein G3BP1, to support regeneration of peripheral nerves. G3BP1 aggregates within peripheral nerve axons in stress granule-like structures that decrease during regeneration, with a commensurate increase in phosphorylated G3BP1. Colocalization of G3BP1 with axonal mRNAs is also correlated with the growth state of the neuron. Disrupting G3BP functions by overexpressing a dominant-negative protein activates intra-axonal mRNA translation, increases axon growth in cultured neurons, disassembles axonal stress granule-like structures, and accelerates rat nerve regeneration in vivo.
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http://dx.doi.org/10.1038/s41467-018-05647-xDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6105716PMC
August 2018

Surgical Management of Pulmonary Mucormycosis in Third-Trimester Pregnancy.

Thorac Cardiovasc Surg Rep 2018 Jan 28;7(1):e27-e29. Epub 2018 Jun 28.

Department of Surgery, University of California, San Francisco, San Francisco, California, United States.

 Pulmonary mucormycosis is a rare fungal infection that carries a high mortality. Given the rarity of this disease, its management has not been well established.  We report a 36-year-old female presenting with right middle and lower lobe pulmonary mucormycosis during the third trimester of pregnancy. Diagnosis was established using chest computed tomography followed by bronchoalveolar lavage and lung biopsy. Prompt initiation of amphotericin B and right middle and lower lobe lobectomy resulted in maternal survival and fetal viability.  This favorable outcome is attributed to extensive communication between treatment teams in addition to comprehensive surgical planning.
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http://dx.doi.org/10.1055/s-0038-1660806DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6023714PMC
January 2018

Inhibition of yes-associated protein suppresses brain metastasis of human lung adenocarcinoma in a murine model.

J Cell Mol Med 2018 06 24;22(6):3073-3085. Epub 2018 Mar 24.

Department of Surgery, Helen Diller Family Comprehensive Cancer Center, University of California, San Francisco, CA, USA.

Yes-associated protein (YAP) is a main mediator of the Hippo pathway and promotes cancer development and progression in human lung cancer. We sought to determine whether inhibition of YAP suppresses metastasis of human lung adenocarcinoma in a murine model. We found that metastatic NSCLC cell lines H2030-BrM3(K-ras mutation) and PC9-BrM3 (EGFR mutation) had a significantly decreased p-YAP(S127)/YAP ratio compared to parental H2030 (K-ras mutation) and PC9 (EGFR mutation) cells (P < .05). H2030-BrM3 cells had significantly increased YAP mRNA and expression of Hippo downstream genes CTGF and CYR61 compared to parental H2030 cells (P < .05). Inhibition of YAP by short hairpin RNA (shRNA) and small interfering RNA (siRNA) significantly decreased mRNA expression in downstream genes CTGF and CYR61 in H2030-BrM3 cells (P < .05). In addition, inhibiting YAP by YAP shRNA significantly decreased migration and invasion abilities of H2030-BrM3 cells (P < .05). We are first to show that mice inoculated with YAP shRNA-transfected H2030-BrM3 cells had significantly decreased metastatic tumour burden and survived longer than control mice (P < .05). Collectively, our results suggest that YAP plays an important role in promoting lung adenocarcinoma brain metastasis and that direct inhibition of YAP by shRNA suppresses H2030-BrM3 cell brain metastasis in a murine model.
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http://dx.doi.org/10.1111/jcmm.13582DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5980132PMC
June 2018

Locally translated mTOR controls axonal local translation in nerve injury.

Science 2018 03;359(6382):1416-1421

Department of Biomolecular Sciences, Weizmann Institute of Science, Rehovot 76100, Israel.

How is protein synthesis initiated locally in neurons? We found that mTOR (mechanistic target of rapamycin) was activated and then up-regulated in injured axons, owing to local translation of mTOR messenger RNA (mRNA). This mRNA was transported into axons by the cell size-regulating RNA-binding protein nucleolin. Furthermore, mTOR controlled local translation in injured axons. This included regulation of its own translation and that of retrograde injury signaling molecules such as importin β1 and STAT3 (signal transducer and activator of transcription 3). Deletion of the mTOR 3' untranslated region (3'UTR) in mice reduced mTOR in axons and decreased local translation after nerve injury. Both pharmacological inhibition of mTOR in axons and deletion of the mTOR 3'UTR decreased proprioceptive neuronal survival after nerve injury. Thus, mRNA localization enables spatiotemporal control of mTOR pathways regulating local translation and long-range intracellular signaling.
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http://dx.doi.org/10.1126/science.aan1053DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6501578PMC
March 2018

Revealing nascent proteomics in signaling pathways and cell differentiation.

Proc Natl Acad Sci U S A 2018 03 21;115(10):2353-2358. Epub 2018 Feb 21.

Helen Diller Family Comprehensive Cancer Center, University of California, San Francisco, CA 94158;

Regulation of gene expression at the level of protein synthesis is a crucial element in driving how the genetic landscape is expressed. However, we are still limited in technologies that can quantitatively capture the immediate proteomic changes that allow cells to respond to specific stimuli. Here, we present a method to capture and identify nascent proteomes in situ across different cell types without disturbing normal growth conditions, using O-propargyl-puromycin (OPP). Cell-permeable OPP rapidly labels nascent elongating polypeptides, which are subsequently conjugated to biotin-azide, using click chemistry, and captured with streptavidin beads, followed by digestion and analysis, using liquid chromatography-tandem mass spectrometry. Our technique of OPP-mediated identification (OPP-ID) allows detection of widespread proteomic changes within a short 2-hour pulse of OPP. We illustrate our technique by recapitulating alterations of proteomic networks induced by a potent mammalian target of rapamycin inhibitor, MLN128. In addition, by employing OPP-ID, we identify more than 2,100 proteins and uncover distinct protein networks underlying early erythroid progenitor and differentiation states not amenable to alternative approaches such as amino acid analog labeling. We present OPP-ID as a method to quantitatively identify nascent proteomes across an array of biological contexts while preserving the subtleties directing signaling in the native cellular environment.
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http://dx.doi.org/10.1073/pnas.1707514115DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5877968PMC
March 2018

Checkpoint inhibitor is active against large cell neuroendocrine carcinoma with high tumor mutation burden.

J Immunother Cancer 2017 09 19;5(1):75. Epub 2017 Sep 19.

Department of Surgery, University of California, San Francisco, USA.

Background: Large cell neuroendocrine tumor (LCNEC) of the lung is a rare and aggressive tumor similar to small cell lung cancer (SCLC). Thus, it is often treated similarly to SCLC in the front-line setting with a platinum doublet. However, treatment for patients beyond the first line remains undefined.

Case Presentation: We report the case of a patient with stage IB LCNEC (PD-L1 negative but positive for PD-L1 amplification and tumor mutation burden high) who progressed after adjuvant chemotherapy after surgery and subsequent therapy with an antibody drug conjugate targeting a neuroendocrine-specific cell surface marker but achieved a significant and durable response with pembrolizumab, a humanized IgG4 monoclonal anti-PD-1 antibody.

Conclusions: Immunotherapy with checkpoint inhibitors is an effective treatment option for patients with metastatic LCNEC, even if PD-L1 expression is negative.
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http://dx.doi.org/10.1186/s40425-017-0281-yDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5604145PMC
September 2017

A Case of Metastatic Atypical Neuroendocrine Tumor with Translocation and Diffuse Brain Metastases.

Oncologist 2017 07 15;22(7):768-773. Epub 2017 May 15.

Department of Medicine, University of California, San Francisco, San Francisco, California, USA.

A challenge in precision medicine requires identification of actionable driver mutations. Critical to such effort is the deployment of sensitive and well-validated assays for mutation detection. Although identification of such alterations within the tumor tissue remains the gold standard, many advanced non-small cell lung cancer cases have only limited tissue samples, derived from small biopsies or fine-needle aspirates, available for testing. More recently, noninvasive methods using either circulating tumor cells or tumor DNA (ctDNA) have become an alternative method for identifying molecular biomarkers and screening patients eligible for targeted therapies. In this article, we present a case of a 52-year-old never-smoking male who presented with widely metastatic atypical neuroendocrine tumor to the bones and the brain. Molecular genotyping using DNA harvested from a bone metastasis was unsuccessful due to limited material. Subsequent ctDNA analysis revealed an translocation. The clinical significance of the mutation in this particular cancer type and therapeutic strategies are discussed.

Key Points: To our knowledge, this index case represents the first reported translocation identified in an atypical carcinoid tumor.Liquid biopsy such as circulating tumor DNA is a feasible alternative platform for identifying sensitizing genomic alterations.Second-generation inhibitors represent a new paradigm for treating -positive patients with brain metastases.
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http://dx.doi.org/10.1634/theoncologist.2017-0054DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5507651PMC
July 2017

Pathologic Findings and Prognosis in a Large Prospective Cohort of Chronic Hypersensitivity Pneumonitis.

Chest 2017 09 20;152(3):502-509. Epub 2017 Feb 20.

Department of Medicine, University of California, San Francisco, San Francisco, CA. Electronic address:

Background: The ability of specific histopathologic features to predict mortality or lung transplantation in patients with chronic hypersensitivity pneumonitis (HP) is unknown.

Methods: Patients with chronic HP diagnosed by surgical lung biopsy were identified from an ongoing longitudinal cohort. The surgical lung biopsy slides were evaluated prospectively by an experienced thoracic pathologist using a standardized checklist to differentiate the major pathologic patterns and score the presence of specific histopathologic features. Cox proportional hazard analysis was used to identify independent predictors of transplant-free survival, and Kaplan-Meier analysis was used to visualize outcomes.

Results: One hundred nineteen patients were identified. Patients with a fibrotic nonspecific interstitial pneumonia (f-NSIP) pattern, bronchiolocentric fibrosis (BF) pattern, or usual interstitial pneumonia (UIP) pattern had significantly worse transplant-free survival than did those with a cellular NSIP (c-NSIP) pattern or peribronchiolar inflammation with poorly formed granulomas (PI-PFG) pattern. No survival difference among patients with an f-NSIP pattern, a BF pattern, or a UIP pattern was found. Fibroblastic foci were identified in a subset of biopsy samples from all pathologic patterns. Peribronchiolar fibrosis was noted in all UIP cases. Independent predictors of time to death or transplantation included the presence of fibroblast foci or dense collagen fibrosis.

Conclusions: Histopathologic patterns of c-NSIP and PI-PFG had a better transplant-free survival than did patterns of UIP, f-NSIP, and BF. The presence of fibroblast foci or dense collagen fibrosis correlated with progression to death or lung transplantation. Identification of fibroblast foci on biopsy samples, regardless of the underlying histopathologic pattern, may be a clinically useful predictor of survival in patients with HP.
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http://dx.doi.org/10.1016/j.chest.2017.02.011DOI Listing
September 2017

The use of pretest probability increases the value of high-resolution CT in diagnosing usual interstitial pneumonia.

Thorax 2017 May 12;72(5):424-429. Epub 2017 Jan 12.

Department of Medicine, University of California San Francisco, San Francisco, California, USA.

Background: Recent studies have suggested that non-definitive patterns on high-resolution CT (HRCT) scan provide sufficient diagnostic specificity to forgo surgical lung biopsy in the diagnosis of idiopathic pulmonary fibrosis (IPF). The objective of this study was to determine test characteristics of non-definitive HRCT patterns for identifying histopathological usual interstitial pneumonia (UIP).

Methods: Patients with biopsy-proven interstitial lung disease (ILD) and non-definitive HRCT scans were identified from two academic ILD centres. Test characteristics for HRCT patterns as predictors of UIP on surgical lung biopsy were derived and validated in independent cohorts.

Results: In the derivation cohort, 64/385 (17%) had possible UIP pattern on HRCT; 321/385 (83%) had inconsistent with UIP pattern. 113/385 (29%) patients had histopathological UIP pattern in the derivation cohort. Possible UIP pattern had a specificity of 91.2% (95% CI 87.2% to 94.3%) and a positive predictive value (PPV) of 62.5% (95% CI 49.5% to 74.3%) for UIP pattern on surgical lung biopsy. The addition of age, sex and total traction bronchiectasis score improved the PPV. Inconsistent with UIP pattern demonstrated poor PPV (22.7%, 95% CI 18.3% to 27.7%). HRCT pattern specificity was nearly identical in the validation cohort (92.7%, 95% CI 82.4% to 98.0%). The substantially higher prevalence of UIP pattern in the validation cohort improved the PPV of HRCT patterns.

Conclusions: A possible UIP pattern on HRCT has high specificity for UIP on surgical lung biopsy, but PPV is highly dependent on underlying prevalence. Adding clinical and radiographic features to possible UIP pattern on HRCT may provide sufficient probability of histopathological UIP across prevalence ranges to change clinical decision-making.
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http://dx.doi.org/10.1136/thoraxjnl-2016-209671DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5555580PMC
May 2017

An Optimized Chromatographic Strategy for Multiplexing In Parallel Reaction Monitoring Mass Spectrometry: Insights from Quantitation of Activated Kinases.

Mol Cell Proteomics 2017 02 11;16(2):265-277. Epub 2016 Dec 11.

§Department of Pharmaceutical Chemistry, University of California San Francisco, San Francisco, California.

Reliable quantitation of protein abundances in defined sets of cellular proteins is critical to numerous biological applications. Traditional immunodetection-based methods are limited by the quality and availability of specific antibodies, especially for site-specific post-translational modifications. Targeted proteomic methods, including the recently developed parallel reaction monitoring (PRM) mass spectrometry, have enabled accurate quantitative measurements of up to a few hundred specific target peptides. However, the degree of practical multiplexing in label-free PRM workflows remains a significant limitation for the technique. Here we present a strategy for significantly increasing multiplexing in label-free PRM that takes advantage of the superior separation characteristics and retention time stability of meter-scale monolithic silica-C18 column-based chromatography. We show the utility of the approach in quantifying kinase abundances downstream of previously developed active kinase enrichment methodology based on multidrug inhibitor beads. We examine kinase activation dynamics in response to three different MAP kinase inhibitors in colorectal carcinoma cells and demonstrate reliable quantitation of over 800 target peptides from over 150 kinases in a single label-free PRM run. The kinase activity profiles obtained from these analyses reveal compensatory activation of TGF-β family receptors as a response to MAPK blockade. The gains achieved using this label-free PRM multiplexing strategy will benefit a wide array of biological applications.
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http://dx.doi.org/10.1074/mcp.M116.058172DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5294213PMC
February 2017

Quantitative Proteomics Reveals Fundamental Regulatory Differences in Oncogenic HRAS and Isocitrate Dehydrogenase (IDH1) Driven Astrocytoma.

Mol Cell Proteomics 2017 01 10;16(1):39-56. Epub 2016 Nov 10.

From the ‡Department of Pharmaceutical Chemistry, University of California, San Francisco, 94158-2517 California;

Glioblastoma multiformes (GBMs) are high-grade astrocytomas and the most common brain malignancies. Primary GBMs are often associated with disturbed RAS signaling, and expression of oncogenic HRAS results in a malignant phenotype in glioma cell lines. Secondary GBMs arise from lower-grade astrocytomas, have slower progression than primary tumors, and contain IDH1 mutations in over 70% of cases. Despite significant amount of accumulating genomic and transcriptomic data, the fundamental mechanistic differences of gliomagenesis in these two types of high-grade astrocytoma remain poorly understood. Only a few studies have attempted to investigate the proteome, phosphorylation signaling, and epigenetic regulation in astrocytoma. In the present study, we applied quantitative phosphoproteomics to identify the main signaling differences between oncogenic HRAS and mutant IDH1-driven glioma cells as models of primary and secondary GBM, respectively. Our analysis confirms the driving roles of the MAPK and PI3K/mTOR signaling pathways in HRAS driven cells and additionally uncovers dysregulation of other signaling pathways. Although a subset of the signaling changes mediated by HRAS could be reversed by a MEK inhibitor, dual inhibition of MEK and PI3K resulted in more complete reversal of the phosphorylation patterns produced by HRAS expression. In contrast, cells expressing mutant IDH1 did not show significant activation of MAPK or PI3K/mTOR pathways. Instead, global downregulation of protein expression was observed. Targeted proteomic analysis of histone modifications identified significant histone methylation, acetylation, and butyrylation changes in the mutant IDH1 expressing cells, consistent with a global transcriptional repressive state. Our findings offer novel mechanistic insight linking mutant IDH1 associated inhibition of histone demethylases with specific histone modification changes to produce global transcriptional repression in secondary glioblastoma. Our proteomic datasets are available for download and provide a comprehensive catalogue of alterations in protein abundance, phosphorylation, and histone modifications in oncogenic HRAS and IDH1 driven astrocytoma cells beyond the transcriptomic level.
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http://dx.doi.org/10.1074/mcp.M116.063883DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5217781PMC
January 2017

Activation of HIPK2 Promotes ER Stress-Mediated Neurodegeneration in Amyotrophic Lateral Sclerosis.

Neuron 2016 07 16;91(1):41-55. Epub 2016 Jun 16.

Department of Pathology, University of California, San Francisco, San Francisco, CA 94158, USA; Department of Neurology, University of California, San Francisco, San Francisco, CA 94158, USA; Neuroscience Graduate Program, University of California, San Francisco, San Francisco, CA 94158, USA; Pathology Service 113B, VA Medical Center, San Francisco, CA 94121, USA. Electronic address:

Persistent accumulation of misfolded proteins causes endoplasmic reticulum (ER) stress, a prominent feature in many neurodegenerative diseases including amyotrophic lateral sclerosis (ALS). Here we report the identification of homeodomain interacting protein kinase 2 (HIPK2) as the essential link that promotes ER-stress-induced cell death via the IRE1α-ASK1-JNK pathway. ER stress, induced by tunicamycin or SOD1(G93A), activates HIPK2 by phosphorylating highly conserved serine and threonine residues (S359/T360) within the activation loop of the HIPK2 kinase domain. In SOD1(G93A) mice, loss of HIPK2 delays disease onset, reduces cell death in spinal motor neurons, mitigates glial pathology, and improves survival. Remarkably, HIPK2 activation positively correlates with TDP-43 proteinopathy in NEFH-tTA/tetO-hTDP-43ΔNLS mice, sporadic ALS and C9ORF72 ALS, and blocking HIPK2 kinase activity protects motor neurons from TDP-43 cytotoxicity. These results reveal a previously unrecognized role of HIPK2 activation in ER-stress-mediated neurodegeneration and its potential role as a biomarker and therapeutic target for ALS. VIDEO ABSTRACT.
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http://dx.doi.org/10.1016/j.neuron.2016.05.021DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4938715PMC
July 2016

Enhanced MET Translation and Signaling Sustains K-Ras-Driven Proliferation under Anchorage-Independent Growth Conditions.

Cancer Res 2015 Jul 14;75(14):2851-62. Epub 2015 May 14.

Helen Diller Family Comprehensive Cancer Center, University of California San Francisco, San Francisco, California.

Oncogenic K-Ras mutation occurs frequently in several types of cancers, including pancreatic and lung cancers. Tumors with K-Ras mutation are resistant to chemotherapeutic drugs as well as molecular targeting agents. Although numerous approaches are ongoing to find effective ways to treat these tumors, there are still no effective therapies for K-Ras mutant cancer patients. Here we report that K-Ras mutant cancers are more dependent on K-Ras in anchorage-independent culture conditions than in monolayer culture conditions. In seeking to determine mechanisms that contribute to the K-Ras dependency in anchorage-independent culture conditions, we discovered the involvement of Met in K-Ras-dependent, anchorage-independent cell growth. The Met signaling pathway is enhanced and plays an indispensable role in anchorage-independent growth even in cells in which Met is not amplified. Indeed, Met expression is elevated under anchorage-independent growth conditions and is regulated by K-Ras in a MAPK/ERK kinase (MEK)-dependent manner. Remarkably, in spite of a global downregulation of mRNA translation during anchorage-independent growth, we find that Met mRNA translation is specifically enhanced under these conditions. Importantly, ectopic expression of an active Met mutant rescues K-Ras ablation-derived growth suppression, indicating that K-Ras-mediated Met expression drives "K-Ras addiction" in anchorage-independent conditions. Our results indicate that enhanced Met expression and signaling is essential for anchorage-independent growth of K-Ras mutant cancer cells and suggests that pharmacological inhibitors of Met could be effective for K-Ras mutant tumor patients.
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http://dx.doi.org/10.1158/0008-5472.CAN-14-1623DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4506276PMC
July 2015

Pulmonary pathology in connective tissue disease.

Semin Respir Crit Care Med 2014 Apr 25;35(2):201-12. Epub 2014 Mar 25.

Department of Pathology, University of California San Francisco, San Francisco, California.

A significant proportion of patients with autoimmune connective tissue disease (CTD) show lung involvement that results in clinical interstitial lung disease (ILD). Surgical lung biopsy is helpful for diagnosis of CTD-ILD in many cases. In this review, we discuss the histologic manifestations of different types of CTD-ILD, focusing on patterns of disease and their differential diagnoses. Acquired autoimmune connective tissue diseases will be covered in this review, while lung involvement in vasculitides, heritable connective tissue disorders, and drug-induced CTD-like conditions will not be discussed.
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http://dx.doi.org/10.1055/s-0034-1371543DOI Listing
April 2014

Global cellular response to chemotherapy-induced apoptosis.

Elife 2013 Oct 29;2:e01236. Epub 2013 Oct 29.

Department of Pharmaceutical Chemistry , University of California, San Francisco , San Francisco , United States ; Department of Laboratory Medicine , University of California, San Francisco , San Francisco , United States.

How cancer cells globally struggle with a chemotherapeutic insult before succumbing to apoptosis is largely unknown. Here we use an integrated systems-level examination of transcription, translation, and proteolysis to understand these events central to cancer treatment. As a model we study myeloma cells exposed to the proteasome inhibitor bortezomib, a first-line therapy. Despite robust transcriptional changes, unbiased quantitative proteomics detects production of only a few critical anti-apoptotic proteins against a background of general translation inhibition. Simultaneous ribosome profiling further reveals potential translational regulation of stress response genes. Once the apoptotic machinery is engaged, degradation by caspases is largely independent of upstream bortezomib effects. Moreover, previously uncharacterized non-caspase proteolytic events also participate in cellular deconstruction. Our systems-level data also support co-targeting the anti-apoptotic regulator HSF1 to promote cell death by bortezomib. This integrated approach offers unique, in-depth insight into apoptotic dynamics that may prove important to preclinical evaluation of any anti-cancer compound. DOI:http://dx.doi.org/10.7554/eLife.01236.001.
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http://dx.doi.org/10.7554/eLife.01236DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3808542PMC
October 2013

Association of large-airway lymphocytic bronchitis with bronchiolitis obliterans syndrome.

Am J Respir Crit Care Med 2013 Feb 13;187(4):417-23. Epub 2012 Dec 13.

Department of Medicine, University of California at San Francisco, San Francisco, CA 94121, USA.

Rationale: Lung transplantation offers great promise for otherwise terminal lung diseases, but the development of bronchiolitis obliterans syndrome (BOS) continues to limit survival. Although acute rejection and lymphocytic bronchiolitis have been identified as risk factors for the development of BOS, it is unclear whether large-airway lymphocytic inflammation conveys the same risk.

Objectives: We evaluated lymphocytic bronchitis on endobronchial biopsies as a risk factor for BOS and mortality.

Methods: Endobronchial biopsies were collected and graded during surveillance after lung transplantation. We assessed samples with negative cultures collected in the first 90 days from 298 subjects and compared large-airway lymphocytic bronchitis assessed by a 0-2 "E-score" and with standard A and BR pathology scores for acute rejection and small-airway lymphocytic bronchiolitis, respectively.

Measurements And Main Results: We found surprisingly little association between large- and small-airway lymphocytic inflammation scores from a given bronchoscopy. Endobronchial lymphocytic bronchitis was more prevalent in subjects in BOS stage 0p and BOS stages 1-3 at the time of biopsy. Within 90 days after transplantation, increasing maximum E-score was associated with greater risk of BOS (adjusted hazard ratio, 1.76; 95% confidence interval, 1.11-2.78; P = 0.02) and in this analysis 90-day maximum E-scores were the only score type predictive of BOS (P < 0.01).

Conclusions: These results support a multicenter study to evaluate endoscopic biopsies for the identification of patients at increased risk for BOS. The association of endobronchial lymphocytic inflammation and BOS may have mechanistic implications.
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http://dx.doi.org/10.1164/rccm.201206-1025OCDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3603592PMC
February 2013

In-depth investigation of archival and prospectively collected samples reveals no evidence for XMRV infection in prostate cancer.

PLoS One 2012 18;7(9):e44954. Epub 2012 Sep 18.

Department of Laboratory Medicine, University of San Francisco, San Francisco, California, United States of America.

XMRV, or xenotropic murine leukemia virus (MLV)-related virus, is a novel gammaretrovirus originally identified in studies that analyzed tissue from prostate cancer patients in 2006 and blood from patients with chronic fatigue syndrome (CFS) in 2009. However, a large number of subsequent studies failed to confirm a link between XMRV infection and CFS or prostate cancer. On the contrary, recent evidence indicates that XMRV is a contaminant originating from the recombination of two mouse endogenous retroviruses during passaging of a prostate tumor xenograft (CWR22) in mice, generating laboratory-derived cell lines that are XMRV-infected. To confirm or refute an association between XMRV and prostate cancer, we analyzed prostate cancer tissues and plasma from a prospectively collected cohort of 39 patients as well as archival RNA and prostate tissue from the original 2006 study. Despite comprehensive microarray, PCR, FISH, and serological testing, XMRV was not detected in any of the newly collected samples or in archival tissue, although archival RNA remained XMRV-positive. Notably, archival VP62 prostate tissue, from which the prototype XMRV strain was derived, tested negative for XMRV on re-analysis. Analysis of viral genomic and human mitochondrial sequences revealed that all previously characterized XMRV strains are identical and that the archival RNA had been contaminated by an XMRV-infected laboratory cell line. These findings reveal no association between XMRV and prostate cancer, and underscore the conclusion that XMRV is not a naturally acquired human infection.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0044954PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3445615PMC
March 2013

The diagnosis and management of intradiaphragmatic extralobar pulmonary sequestrations: a report of 4 cases.

J Pediatr Surg 2012 Aug;47(8):1501-5

Department of Surgery, University of California, San Francisco, CA 94143-0570, USA.

Background/purpose: Intradiaphragmatic extralobar pulmonary sequestrations (IDEPSs) are a rare subset of bronchopulmonary sequestrations (BPS). We report the largest series of patients with IDEPS and describe the diagnostic and operative challenges associated with this condition.

Methods: We retrospectively reviewed our experience with fetal and pediatric BPS from 1995 to 2010 to identify patients with IDEPS.

Results: We identified 27 patients with BPS and 4 patients in whom the masses were within the diaphragm. In 1 patient, the prenatal ultrasound correctly identified the mass as being within the diaphragm itself, whereas the remaining cases were thought to be intraabdominal or had discordant preoperative imaging findings. The diagnosis of an IDEPS proved challenging to make prospectively using prenatal ultrasound, computed tomography, or magnetic resonance imaging. All patients underwent attempted resection. Two cases required a combined laparoscopic and thoracoscopic approach to accurately localize the mass. The postoperative recovery of these patients was uneventful.

Conclusions: We present the largest reported experience of IDEPS. Because preoperative imaging studies cannot always determine whether a sequestration is intraabdominal, intrathoracic, or intradiaphragmatic, operative planning may pose a challenge. However, the use of minimally invasive approaches can allow exploration of both the thoracic and abdominal cavities with low morbidity.
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http://dx.doi.org/10.1016/j.jpedsurg.2011.11.066DOI Listing
August 2012