Publications by authors named "Steven A Carr"

263 Publications

Phenotype, specificity and avidity of antitumour CD8 T cells in melanoma.

Nature 2021 Jul 21. Epub 2021 Jul 21.

Department of Medical Oncology, Dana-Farber Cancer Institute, Boston, MA, USA.

Interactions between T cell receptors (TCRs) and their cognate tumour antigens are central to antitumour immune responses; however, the relationship between phenotypic characteristics and TCR properties is not well elucidated. Here we show, by linking the antigenic specificity of TCRs and the cellular phenotype of melanoma-infiltrating lymphocytes at single-cell resolution, that tumour specificity shapes the expression state of intratumoural CD8 T cells. Non-tumour-reactive T cells were enriched for viral specificities and exhibited a non-exhausted memory phenotype, whereas melanoma-reactive lymphocytes predominantly displayed an exhausted state that encompassed diverse levels of differentiation but rarely acquired memory properties. These exhausted phenotypes were observed both among clonotypes specific for public overexpressed melanoma antigens (shared across different tumours) or personal neoantigens (specific for each tumour). The recognition of such tumour antigens was provided by TCRs with avidities inversely related to the abundance of cognate targets in melanoma cells and proportional to the binding affinity of peptide-human leukocyte antigen (HLA) complexes. The persistence of TCR clonotypes in peripheral blood was negatively affected by the level of intratumoural exhaustion, and increased in patients with a poor response to immune checkpoint blockade, consistent with chronic stimulation mediated by residual tumour antigens. By revealing how the quality and quantity of tumour antigens drive the features of T cell responses within the tumour microenvironment, we gain insights into the properties of the anti-melanoma TCR repertoire.
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http://dx.doi.org/10.1038/s41586-021-03704-yDOI Listing
July 2021

Structure of PDE3A-SLFN12 complex reveals requirements for activation of SLFN12 RNase.

Nat Commun 2021 07 16;12(1):4375. Epub 2021 Jul 16.

Cancer Program, Broad Institute of MIT and Harvard, Cambridge, MA, USA.

DNMDP and related compounds, or velcrins, induce complex formation between the phosphodiesterase PDE3A and the SLFN12 protein, leading to a cytotoxic response in cancer cells that express elevated levels of both proteins. The mechanisms by which velcrins induce complex formation, and how the PDE3A-SLFN12 complex causes cancer cell death, are not fully understood. Here, we show that PDE3A and SLFN12 form a heterotetramer stabilized by binding of DNMDP. Interactions between the C-terminal alpha helix of SLFN12 and residues near the active site of PDE3A are required for complex formation, and are further stabilized by interactions between SLFN12 and DNMDP. Moreover, we demonstrate that SLFN12 is an RNase, that PDE3A binding increases SLFN12 RNase activity, and that SLFN12 RNase activity is required for DNMDP response. This new mechanistic understanding will facilitate development of velcrin compounds into new cancer therapies.
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http://dx.doi.org/10.1038/s41467-021-24495-wDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8285493PMC
July 2021

Profiling SARS-CoV-2 HLA-I peptidome reveals T cell epitopes from out-of-frame ORFs.

Cell 2021 07 3;184(15):3962-3980.e17. Epub 2021 Jun 3.

Harvard Medical School, Boston, MA 02115, USA; Center for Virology and Vaccine Research, Beth Israel Deaconess Medical Center, Boston, MA, USA; Ragon Institute of MGH, MIT and Harvard, Cambridge, MA, USA; Massachusetts Consortium on Pathogen Readiness, Boston, MA, USA.

T cell-mediated immunity plays an important role in controlling SARS-CoV-2 infection, but the repertoire of naturally processed and presented viral epitopes on class I human leukocyte antigen (HLA-I) remains uncharacterized. Here, we report the first HLA-I immunopeptidome of SARS-CoV-2 in two cell lines at different times post infection using mass spectrometry. We found HLA-I peptides derived not only from canonical open reading frames (ORFs) but also from internal out-of-frame ORFs in spike and nucleocapsid not captured by current vaccines. Some peptides from out-of-frame ORFs elicited T cell responses in a humanized mouse model and individuals with COVID-19 that exceeded responses to canonical peptides, including some of the strongest epitopes reported to date. Whole-proteome analysis of infected cells revealed that early expressed viral proteins contribute more to HLA-I presentation and immunogenicity. These biological insights, as well as the discovery of out-of-frame ORF epitopes, will facilitate selection of peptides for immune monitoring and vaccine development.
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http://dx.doi.org/10.1016/j.cell.2021.05.046DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8173604PMC
July 2021

MS-Based HLA-II Peptidomics Combined With Multiomics Will Aid the Development of Future Immunotherapies.

Mol Cell Proteomics 2021 Jun 17;20:100116. Epub 2021 Jun 17.

Broad Institute of MIT and Harvard, Cambridge, Massachusetts, USA. Electronic address:

Immunotherapies have emerged to treat diseases by selectively modulating a patient's immune response. Although the roles of T and B cells in adaptive immunity have been well studied, it remains difficult to select targets for immunotherapeutic strategies. Because human leukocyte antigen class II (HLA-II) peptides activate CD4+ T cells and regulate B cell activation, proliferation, and differentiation, these peptide antigens represent a class of potential immunotherapy targets and biomarkers. To better understand the molecular basis of how HLA-II antigen presentation is involved in disease progression and treatment, systematic HLA-II peptidomics combined with multiomic analyses of diverse cell types in healthy and diseased states is required. For this reason, MS-based innovations that facilitate investigations into the interplay between disease pathologies and the presentation of HLA-II peptides to CD4+ T cells will aid in the development of patient-focused immunotherapies.
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http://dx.doi.org/10.1016/j.mcpro.2021.100116DOI Listing
June 2021

STAG2 loss rewires oncogenic and developmental programs to promote metastasis in Ewing sarcoma.

Cancer Cell 2021 Jun;39(6):827-844.e10

Dana-Farber/Boston Children's Cancer and Blood Disorders Center, Boston, MA, USA; Broad Institute of MIT and Harvard, Cambridge, MA, USA. Electronic address:

The core cohesin subunit STAG2 is recurrently mutated in Ewing sarcoma but its biological role is less clear. Here, we demonstrate that cohesin complexes containing STAG2 occupy enhancer and polycomb repressive complex (PRC2)-marked regulatory regions. Genetic suppression of STAG2 leads to a compensatory increase in cohesin-STAG1 complexes, but not in enhancer-rich regions, and results in reprogramming of cis-chromatin interactions. Strikingly, in STAG2 knockout cells the oncogenic genetic program driven by the fusion transcription factor EWS/FLI1 was highly perturbed, in part due to altered enhancer-promoter contacts. Moreover, loss of STAG2 also disrupted PRC2-mediated regulation of gene expression. Combined, these transcriptional changes converged to modulate EWS/FLI1, migratory, and neurodevelopmental programs. Finally, consistent with clinical observations, functional studies revealed that loss of STAG2 enhances the metastatic potential of Ewing sarcoma xenografts. Our findings demonstrate that STAG2 mutations can alter chromatin architecture and transcriptional programs to promote an aggressive cancer phenotype.
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http://dx.doi.org/10.1016/j.ccell.2021.05.007DOI Listing
June 2021

Deep learning boosts sensitivity of mass spectrometry-based immunopeptidomics.

Nat Commun 2021 06 7;12(1):3346. Epub 2021 Jun 7.

Chair of Proteomics and Bioanalytics, Technical University of Munich (TUM), Freising, Germany.

Characterizing the human leukocyte antigen (HLA) bound ligandome by mass spectrometry (MS) holds great promise for developing vaccines and drugs for immune-oncology. Still, the identification of non-tryptic peptides presents substantial computational challenges. To address these, we synthesized and analyzed >300,000 peptides by multi-modal LC-MS/MS within the ProteomeTools project representing HLA class I & II ligands and products of the proteases AspN and LysN. The resulting data enabled training of a single model using the deep learning framework Prosit, allowing the accurate prediction of fragment ion spectra for tryptic and non-tryptic peptides. Applying Prosit demonstrates that the identification of HLA peptides can be improved up to 7-fold, that 87% of the proposed proteasomally spliced HLA peptides may be incorrect and that dozens of additional immunogenic neo-epitopes can be identified from patient tumors in published data. Together, the provided peptides, spectra and computational tools substantially expand the analytical depth of immunopeptidomics workflows.
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http://dx.doi.org/10.1038/s41467-021-23713-9DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8184761PMC
June 2021

Identification of a Novel Regulator of Clostridioides difficile Cortex Formation.

mSphere 2021 Jun 28;6(3):e0021121. Epub 2021 May 28.

Department of Molecular Biology and Microbiology, Tufts University School of Medicine, Boston, Massachusetts, USA.

Clostridioides difficile is a leading cause of health care-associated infections worldwide. These infections are transmitted by C. difficile's metabolically dormant, aerotolerant spore form. Functional spore formation depends on the assembly of two protective layers, a thick layer of modified peptidoglycan known as the cortex layer and a multilayered proteinaceous meshwork known as the coat. We previously identified two spore morphogenetic proteins, SpoIVA and SipL, that are essential for recruiting coat proteins to the developing forespore and making functional spores. While SpoIVA and SipL directly interact, the identities of the proteins they recruit to the forespore remained unknown. Here, we used mass spectrometry-based affinity proteomics to identify proteins that interact with the SpoIVA-SipL complex. These analyses identified the family-specific, sporulation-induced bitopic membrane protein CD3457 (renamed SpoVQ) as a protein that interacts with SipL and SpoIVA. Loss of SpoVQ decreased heat-resistant spore formation by ∼5-fold and reduced cortex thickness ∼2-fold; the thinner cortex layer of Δ spores correlated with higher levels of spontaneous germination (i.e., in the absence of germinant). Notably, loss of SpoVQ in either or mutants prevented cortex synthesis altogether and greatly impaired the localization of a SipL-mCherry fusion protein around the forespore. Thus, SpoVQ is a novel regulator of C. difficile cortex synthesis that appears to link cortex and coat formation. The identification of SpoVQ as a spore morphogenetic protein further highlights how family-specific mechanisms control spore formation in C. difficile. The Centers for Disease Control has designated Clostridioides difficile as an urgent threat because of its intrinsic antibiotic resistance. C. difficile persists in the presence of antibiotics in part because it makes metabolically dormant spores. While recent work has shown that preventing the formation of infectious spores can reduce C. difficile disease recurrence, more selective antisporulation therapies are needed. The identification of spore morphogenetic factors specific to C. difficile would facilitate the development of such therapies. In this study, we identified SpoVQ (CD3457) as a spore morphogenetic protein specific to the family that regulates the formation of C. difficile's protective spore cortex layer. SpoVQ acts in concert with the known spore coat morphogenetic factors, SpoIVA and SipL, to link formation of the protective coat and cortex layers. These data reveal a novel pathway that could be targeted to prevent the formation of infectious C. difficile spores.
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http://dx.doi.org/10.1128/mSphere.00211-21DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8265636PMC
June 2021

Human plasma proteomic profiles indicative of cardiorespiratory fitness.

Nat Metab 2021 Jun 27;3(6):786-797. Epub 2021 May 27.

Division of Cardiovascular Medicine, Beth Israel Deaconess Medical Center, Boston, MA, USA.

Maximal oxygen uptake (VOmax) is a direct measure of human cardiorespiratory fitness and is associated with health. However, the molecular determinants of interindividual differences in baseline (intrinsic) VOmax, and of increases of VOmax in response to exercise training (ΔVOmax), are largely unknown. Here, we measure ~5,000 plasma proteins using an affinity-based platform in over 650 sedentary adults before and after a 20-week endurance-exercise intervention and identify 147 proteins and 102 proteins whose plasma levels are associated with baseline VOmax and ΔVOmax, respectively. Addition of a protein biomarker score derived from these proteins to a score based on clinical traits improves the prediction of an individual's ΔVOmax. We validate findings in a separate exercise cohort, further link 21 proteins to incident all-cause mortality in a community-based cohort and reproduce the specificity of ~75% of our key findings using antibody-based assays. Taken together, our data shed light on biological pathways relevant to cardiorespiratory fitness and highlight the potential additive value of protein biomarkers in identifying exercise responsiveness in humans.
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http://dx.doi.org/10.1038/s42255-021-00400-zDOI Listing
June 2021

Avadomide induces degradation of ZMYM2 fusion oncoproteins in hematologic malignancies.

Blood Cancer Discov 2021 May 10;2(3):250-265. Epub 2021 Mar 10.

Broad Institute of MIT and Harvard, Cambridge, Massachusetts, USA.

Thalidomide analogs exert their therapeutic effects by binding to the CRL4 E3 ubiquitin ligase, promoting ubiquitination and subsequent proteasomal degradation of specific protein substrates. Drug-induced degradation of IKZF1 and IKZF3 in B-cell malignancies demonstrates the clinical utility of targeting disease-relevant transcription factors for degradation. Here, we found that avadomide (CC-122) induces CRBN-dependent ubiquitination and proteasomal degradation of ZMYM2 (ZNF198), a transcription factor involved in balanced chromosomal rearrangements with and in aggressive forms of hematologic malignancies. The minimal drug-responsive element of ZMYM2 is a zinc-chelating MYM domain and is contained in the N-terminal portion of ZMYM2 that is universally included in the derived fusion proteins. We demonstrate that avadomide has the ability to induce proteasomal degradation of ZMYM2-FGFR1 and ZMYM2-FLT3 chimeric oncoproteins, both and . Our findings suggest that patients with hematologic malignancies harboring these fusion proteins may benefit from avadomide treatment.
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http://dx.doi.org/10.1158/2643-3230.bcd-20-0105DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8133393PMC
May 2021

Cell-specific transcriptional control of mitochondrial metabolism by TIF1γ drives erythropoiesis.

Science 2021 05;372(6543):716-721

Department of Stem Cell and Regenerative Biology, Harvard University, Cambridge, MA 01238, USA.

Transcription and metabolism both influence cell function, but dedicated transcriptional control of metabolic pathways that regulate cell fate has rarely been defined. We discovered, using a chemical suppressor screen, that inhibition of the pyrimidine biosynthesis enzyme dihydroorotate dehydrogenase (DHODH) rescues erythroid differentiation in bloodless zebrafish mutant embryos defective for transcriptional intermediary factor 1 gamma (). This rescue depends on the functional link of DHODH to mitochondrial respiration. The transcription elongation factor TIF1γ directly controls coenzyme Q (CoQ) synthesis gene expression. Upon loss, CoQ levels are reduced, and a high succinate/α-ketoglutarate ratio leads to increased histone methylation. A CoQ analog rescues 's bloodless phenotype. These results demonstrate that mitochondrial metabolism is a key output of a lineage transcription factor that drives cell fate decisions in the early blood lineage.
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http://dx.doi.org/10.1126/science.aaz2740DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8177078PMC
May 2021

Epigenetic silencing by SETDB1 suppresses tumour intrinsic immunogenicity.

Nature 2021 Jul 5;595(7866):309-314. Epub 2021 May 5.

Broad Institute of MIT and Harvard, Cambridge, MA, USA.

Epigenetic dysregulation is a defining feature of tumorigenesis that is implicated in immune escape. Here, to identify factors that modulate the immune sensitivity of cancer cells, we performed in vivo CRISPR-Cas9 screens targeting 936 chromatin regulators in mouse tumour models treated with immune checkpoint blockade. We identified the H3K9 methyltransferase SETDB1 and other members of the HUSH and KAP1 complexes as mediators of immune escape. We also found that amplification of SETDB1 (1q21.3) in human tumours is associated with immune exclusion and resistance to immune checkpoint blockade. SETDB1 represses broad domains, primarily within the open genome compartment. These domains are enriched for transposable elements (TEs) and immune clusters associated with segmental duplication events, a central mechanism of genome evolution. SETDB1 loss derepresses latent TE-derived regulatory elements, immunostimulatory genes, and TE-encoded retroviral antigens in these regions, and triggers TE-specific cytotoxic T cell responses in vivo. Our study establishes SETDB1 as an epigenetic checkpoint that suppresses tumour-intrinsic immunogenicity, and thus represents a candidate target for immunotherapy.
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http://dx.doi.org/10.1038/s41586-021-03520-4DOI Listing
July 2021

Proteomics of protein trafficking by in vivo tissue-specific labeling.

Nat Commun 2021 04 22;12(1):2382. Epub 2021 Apr 22.

Department of Genetics, Blavatnik Institute, Harvard Medical School, Boston, MA, USA.

Conventional approaches to identify secreted factors that regulate homeostasis are limited in their abilities to identify the tissues/cells of origin and destination. We established a platform to identify secreted protein trafficking between organs using an engineered biotin ligase (BirA*G3) that biotinylates, promiscuously, proteins in a subcellular compartment of one tissue. Subsequently, biotinylated proteins are affinity-enriched and identified from distal organs using quantitative mass spectrometry. Applying this approach in Drosophila, we identify 51 muscle-secreted proteins from heads and 269 fat body-secreted proteins from legs/muscles, including CG2145 (human ortholog ENDOU) that binds directly to muscles and promotes activity. In addition, in mice, we identify 291 serum proteins secreted from conditional BirA*G3 embryo stem cell-derived teratomas, including low-abundance proteins with hormonal properties. Our findings indicate that the communication network of secreted proteins is vast. This approach has broad potential across different model systems to identify cell-specific secretomes and mediators of interorgan communication in health or disease.
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http://dx.doi.org/10.1038/s41467-021-22599-xDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8062696PMC
April 2021

Suppression of pancreatic ductal adenocarcinoma growth and metastasis by fibrillar collagens produced selectively by tumor cells.

Nat Commun 2021 04 20;12(1):2328. Epub 2021 Apr 20.

Koch Institute for Integrative Cancer Research, Massachusetts Institute of Technology, Cambridge, MA, USA.

Pancreatic ductal adenocarcinoma (PDAC) has a collagen-rich dense extracellular matrix (ECM) that promotes malignancy of cancer cells and presents a barrier for drug delivery. Data analysis of our published mass spectrometry (MS)-based studies on enriched ECM from samples of progressive PDAC stages reveal that the C-terminal prodomains of fibrillar collagens are partially uncleaved in PDAC ECM, suggesting reduced procollagen C-proteinase activity. We further show that the enzyme responsible for procollagen C-proteinase activity, bone morphogenetic protein1 (BMP1), selectively suppresses tumor growth and metastasis in cells expressing high levels of COL1A1. Although BMP1, as a secreted proteinase, promotes fibrillar collagen deposition from both cancer cells and stromal cells, only cancer-cell-derived procollagen cleavage and deposition suppresses tumor malignancy. These studies reveal a role for cancer-cell-derived fibrillar collagen in selectively restraining tumor growth and suggest stratification of patients based on their tumor epithelial collagen I expression when considering treatments related to perturbation of fibrillar collagens.
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http://dx.doi.org/10.1038/s41467-021-22490-9DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8058088PMC
April 2021

Targeting acute myeloid leukemia dependency on VCP-mediated DNA repair through a selective second-generation small-molecule inhibitor.

Sci Transl Med 2021 03;13(587)

INSERM U1186, Gustave-Roussy Cancer Center, Université Paris-Saclay, 94805 Villejuif, France.

The development and survival of cancer cells require adaptive mechanisms to stress. Such adaptations can confer intrinsic vulnerabilities, enabling the selective targeting of cancer cells. Through a pooled in vivo short hairpin RNA (shRNA) screen, we identified the adenosine triphosphatase associated with diverse cellular activities (AAA-ATPase) valosin-containing protein (VCP) as a top stress-related vulnerability in acute myeloid leukemia (AML). We established that AML was the most responsive disease to chemical inhibition of VCP across a panel of 16 cancer types. The sensitivity to VCP inhibition of human AML cell lines, primary patient samples, and syngeneic and xenograft mouse models of AML was validated using -directed shRNAs, overexpression of a dominant-negative VCP mutant, and chemical inhibition. By combining mass spectrometry-based analysis of the VCP interactome and phospho-signaling studies, we determined that VCP is important for ataxia telangiectasia mutated (ATM) kinase activation and subsequent DNA repair through homologous recombination in AML. A second-generation VCP inhibitor, CB-5339, was then developed and characterized. Efficacy and safety of CB-5339 were validated in multiple AML models, including syngeneic and patient-derived xenograft murine models. We further demonstrated that combining DNA-damaging agents, such as anthracyclines, with CB-5339 treatment synergizes to impair leukemic growth in an MLL-AF9-driven AML murine model. These studies support the clinical testing of CB-5339 as a single agent or in combination with standard-of-care DNA-damaging chemotherapy for the treatment of AML.
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http://dx.doi.org/10.1126/scitranslmed.abg1168DOI Listing
March 2021

Blockade of IL-22 signaling reverses erythroid dysfunction in stress-induced anemias.

Nat Immunol 2021 04 22;22(4):520-529. Epub 2021 Mar 22.

Department of Cancer Immunology and Virology, Dana-Farber Cancer Institute, Boston, MA, USA.

Patients with myelodysplastic syndromes (MDSs) display severe anemia but the mechanisms underlying this phenotype are incompletely understood. Right open-reading-frame kinase 2 (RIOK2) encodes a protein kinase located at 5q15, a region frequently lost in patients with MDS del(5q). Here we show that hematopoietic cell-specific haploinsufficient deletion of Riok2 (Riok2Vav1) led to reduced erythroid precursor frequency leading to anemia. Proteomic analysis of Riok2Vav1 erythroid precursors suggested immune system activation, and transcriptomic analysis revealed an increase in p53-dependent interleukin (IL)-22 in Riok2Vav1 CD4 T cells (T22). Further, we discovered that the IL-22 receptor, IL-22RA1, was unexpectedly present on erythroid precursors. Blockade of IL-22 signaling alleviated anemia not only in Riok2Vav1 mice but also in wild-type mice. Serum concentrations of IL-22 were increased in the subset of patients with del(5q) MDS as well as patients with anemia secondary to chronic kidney disease. This work reveals a possible therapeutic opportunity for reversing many stress-induced anemias by targeting IL-22 signaling.
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http://dx.doi.org/10.1038/s41590-021-00895-4DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8026551PMC
April 2021

Proteomic profiling reveals biomarkers and pathways in type 2 diabetes risk.

JCI Insight 2021 Mar 8;6(5). Epub 2021 Mar 8.

Cardiovascular Institute.

Recent advances in proteomic technologies have made high-throughput profiling of low-abundance proteins in large epidemiological cohorts increasingly feasible. We investigated whether aptamer-based proteomic profiling could identify biomarkers associated with future development of type 2 diabetes (T2DM) beyond known risk factors. We identified dozens of markers with highly significant associations with future T2DM across 2 large longitudinal cohorts (n = 2839) followed for up to 16 years. We leveraged proteomic, metabolomic, genetic, and clinical data from humans to nominate 1 specific candidate to test for potential causal relationships in model systems. Our studies identified functional effects of aminoacylase 1 (ACY1), a top protein association with future T2DM risk, on amino acid metabolism and insulin homeostasis in vitro and in vivo. Furthermore, a loss-of-function variant associated with circulating levels of the biomarker WAP, Kazal, immunoglobulin, Kunitz, and NTR domain-containing protein 2 (WFIKKN2) was, in turn, associated with fasting glucose, hemoglobin A1c, and HOMA-IR measurements in humans. In addition to identifying potentially novel disease markers and pathways in T2DM, we provide publicly available data to be leveraged for insights about gene function and disease pathogenesis in the context of human metabolism.
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http://dx.doi.org/10.1172/jci.insight.144392DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8021115PMC
March 2021

Biomarkers to distinguish bacterial from viral pediatric clinical pneumonia in a malaria endemic setting.

Clin Infect Dis 2021 Feb 3. Epub 2021 Feb 3.

Broad Institute of MIT and Harvard, Cambridge, MA.

Background: Differential etiologies of pediatric acute febrile respiratory illness pose challenges for all populations globally but especially in malaria-endemic settings because the pathogens responsible overlap in clinical presentation and frequently occur together. Rapid identification of bacterial pneumonia with high quality diagnostic tools would enable appropriate, point of care antibiotic treatment. Current diagnostics are insufficient, and the discovery and development of new tools is needed. We report a unique biomarker signature identified in blood samples to accomplish this.

Methods: Blood samples from 195 pediatric Mozambican patients with clinical pneumonia were analyzed with an aptamer-based, high dynamic range, quantitative assay (~1200 proteins). We identified new biomarkers using a training set of samples from patients with established bacterial, viral, or malarial pneumonia. Proteins with significantly variable abundance across etiologies (FDR<0.01) formed the basis for predictive diagnostic models derived from machine learning techniques (Random Forest, Elastic Net). Validation on a dedicated test set of samples was performed.

Results: Significantly different abundances between bacterial and viral infections (219 proteins) and bacterial infections and mixed (viral and malaria) infections (151 proteins) were found. Predictive models achieved >90% sensitivity and >80% specificity, regardless of number of pathogen classes. Bacterial pneumonia was strongly associated with neutrophil markers, in particular degranulation including HP, LCN2, LTF, MPO, MMP8, PGLYRP1, RETN, SERPINA1, S100A9, and SLPI.

Conclusion: Blood protein signatures highly associated with neutrophil biology reliably differentiated bacterial pneumonia from other causes. With appropriate technology, these markers could provide the basis for a rapid diagnostic for field-based triage for antibiotic treatment of pediatric pneumonia.
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http://dx.doi.org/10.1093/cid/ciaa1843DOI Listing
February 2021

Dual functions of SPOP and ERG dictate androgen therapy responses in prostate cancer.

Nat Commun 2021 02 2;12(1):734. Epub 2021 Feb 2.

Department of Biomedical Research, University of Bern, 3008, Bern, Switzerland.

Driver genes with a mutually exclusive mutation pattern across tumor genomes are thought to have overlapping roles in tumorigenesis. In contrast, we show here that mutually exclusive prostate cancer driver alterations involving the ERG transcription factor and the ubiquitin ligase adaptor SPOP are synthetic sick. At the molecular level, the incompatible cancer pathways are driven by opposing functions in SPOP. ERG upregulates wild type SPOP to dampen androgen receptor (AR) signaling and sustain ERG activity through degradation of the bromodomain histone reader ZMYND11. Conversely, SPOP-mutant tumors stabilize ZMYND11 to repress ERG-function and enable oncogenic androgen receptor signaling. This dichotomy regulates the response to therapeutic interventions in the AR pathway. While mutant SPOP renders tumor cells susceptible to androgen deprivation therapies, ERG promotes sensitivity to high-dose androgen therapy and pharmacological inhibition of wild type SPOP. More generally, these results define a distinct class of antagonistic cancer drivers and a blueprint toward their therapeutic exploitation.
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http://dx.doi.org/10.1038/s41467-020-20820-xDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7854732PMC
February 2021

CBL mutations drive PI3K/AKT signaling via increased interaction with LYN and PIK3R1.

Blood 2021 Apr;137(16):2209-2220

Department of Medical Oncology, Dana-Farber Cancer Institute, Boston, MA.

Casitas B-lineage lymphoma (CBL) encodes an E3 ubiquitin ligase and signaling adaptor that regulates receptor and nonreceptor tyrosine kinases. Recurrent CBL mutations occur in myeloid neoplasms, including 10% to 20% of chronic myelomonocytic leukemia (CMML) cases, and selectively disrupt the protein's E3 ubiquitin ligase activity. CBL mutations have been associated with poor prognosis, but the oncogenic mechanisms and therapeutic implications of CBL mutations remain incompletely understood. We combined functional assays and global mass spectrometry to define the phosphoproteome, CBL interactome, and mechanism of signaling activation in a panel of cell lines expressing an allelic series of CBL mutations. Our analyses revealed that increased LYN activation and interaction with mutant CBL are key drivers of enhanced CBL phosphorylation, phosphoinositide-3-kinase regulatory subunit 1 (PIK3R1) recruitment, and downstream phosphatidylinositol 3-kinase (PI3K)/AKT signaling in CBL-mutant cells. Signaling adaptor domains of CBL, including the tyrosine kinase-binding domain, proline-rich region, and C-terminal phosphotyrosine sites, were all required for the oncogenic function of CBL mutants. Genetic ablation or dasatinib-mediated inhibition of LYN reduced CBL phosphorylation, CBL-PIK3R1 interaction, and PI3K/AKT signaling. Furthermore, we demonstrated in vitro and in vivo antiproliferative efficacy of dasatinib in CBL-mutant cell lines and primary CMML. Overall, these mechanistic insights into the molecular function of CBL mutations provide rationale to explore the therapeutic potential of LYN inhibition in CBL-mutant myeloid malignancies.
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http://dx.doi.org/10.1182/blood.2020006528DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8063090PMC
April 2021

Proteogenomic insights into the biology and treatment of HPV-negative head and neck squamous cell carcinoma.

Cancer Cell 2021 Mar 7;39(3):361-379.e16. Epub 2021 Jan 7.

Tisch Cancer Institute, Icahn School of Medicine at Mount Sinai, New York, NY 10029, USA; Department of Population Health Science and Policy, Icahn School of Medicine at Mount Sinai, New York, NY 10029, USA.

We present a proteogenomic study of 108 human papilloma virus (HPV)-negative head and neck squamous cell carcinomas (HNSCCs). Proteomic analysis systematically catalogs HNSCC-associated proteins and phosphosites, prioritizes copy number drivers, and highlights an oncogenic role for RNA processing genes. Proteomic investigation of mutual exclusivity between FAT1 truncating mutations and 11q13.3 amplifications reveals dysregulated actin dynamics as a common functional consequence. Phosphoproteomics characterizes two modes of EGFR activation, suggesting a new strategy to stratify HNSCCs based on EGFR ligand abundance for effective treatment with inhibitory EGFR monoclonal antibodies. Widespread deletion of immune modulatory genes accounts for low immune infiltration in immune-cold tumors, whereas concordant upregulation of multiple immune checkpoint proteins may underlie resistance to anti-programmed cell death protein 1 monotherapy in immune-hot tumors. Multi-omic analysis identifies three molecular subtypes with high potential for treatment with CDK inhibitors, anti-EGFR antibody therapy, and immunotherapy, respectively. Altogether, proteogenomics provides a systematic framework to inform HNSCC biology and treatment.
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http://dx.doi.org/10.1016/j.ccell.2020.12.007DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7946781PMC
March 2021

Regulation of purine metabolism connects KCTD13 to a metabolic disorder with autistic features.

iScience 2021 Jan 11;24(1):101935. Epub 2020 Dec 11.

Stanley Center for Psychiatric Research, Broad Institute of MIT and Harvard, Cambridge, MA 02142, USA.

Genetic variation of the 16p11.2 deletion locus containing the gene and of is linked with autism. This genetic connection suggested that substrates of a CUL3-KCTD13 ubiquitin ligase may be involved in disease pathogenesis. Comparison of mutant ( ) and wild-type neuronal ubiquitylomes identified adenylosuccinate synthetase (ADSS), an enzyme that catalyzes the first step in adenosine monophosphate (AMP) synthesis, as a KCTD13 ligase substrate. In neurons, there were increased levels of succinyl-adenosine (S-Ado), a metabolite downstream of ADSS. Notably, S-Ado levels are elevated in adenylosuccinate lyase deficiency, a metabolic disorder with autism and epilepsy phenotypes. The increased S-Ado levels in neurons were decreased by treatment with an ADSS inhibitor. Lastly, functional analysis of human variants suggests that variation may alter ubiquitination of ADSS. These data suggest that succinyl-AMP metabolites accumulate in neurons, and this observation may have implications for our understanding of 16p11.2 deletion syndrome.
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http://dx.doi.org/10.1016/j.isci.2020.101935DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7773955PMC
January 2021

The conserved translation factor LepA is required for optimal synthesis of a porin family in .

J Bacteriol 2020 Dec 23. Epub 2020 Dec 23.

Department of Immunology and Infectious Disease, Harvard TH Chan School of Public Health, Boston, Massachusetts, 02115, United States

The recalcitrance of mycobacteria to antibiotic therapy is in part due to its ability to build proteins into a multi-layer cell wall. Proper synthesis of both cell wall constituents and associated proteins is crucial to maintaining cell integrity, and intimately tied to antibiotic susceptibility. How mycobacteria properly synthesize the membrane-associated proteome, however, remains poorly understood. Recently, we found that loss of in (Msm) altered tolerance to rifampin, a drug that targets a non-ribosomal cellular process. LepA is a ribosome-associated GTPase found in bacteria, mitochondria, and chloroplasts, yet its physiological contribution to cellular processes is not clear. To uncover the determinants of LepA-mediated drug tolerance, we characterized the whole-cell proteomes and transcriptomes of a deletion mutant relative to strains with We find that LepA is important for the steady-state abundance of a number of membrane-associated proteins, including an outer membrane porin, MspA, which is integral to nutrient uptake and drug susceptibility. Loss of LepA leads to a decreased amount of porin in the membrane which leads to the drug tolerance phenotype of the mutant. In mycobacteria, the translation factor LepA modulates mycobacterial membrane homeostasis, which in turn affects antibiotic tolerance.The mycobacterial cell wall is a promising target for new antibiotics due to the abundance of important membrane-associated proteins. Defining mechanisms of synthesis of the membrane proteome will be critical to uncovering and validating drug targets. We found that LepA, a universally conserved translation factor, controls the synthesis of a number of major membrane proteins in LepA primarily controls synthesis of the major porin MspA. Loss of LepA results in decreased permeability through the loss of this porin, including permeability to antibiotics like rifampin and vancomycin. In mycobacteria, regulation from the ribosome is critical for the maintenance of membrane homeostasis and, importantly, antibiotic susceptibility.
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http://dx.doi.org/10.1128/JB.00604-20DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8095456PMC
December 2020

Cohesin mutations alter DNA damage repair and chromatin structure and create therapeutic vulnerabilities in MDS/AML.

JCI Insight 2021 02 8;6(3). Epub 2021 Feb 8.

Department of Medical Oncology, Dana-Farber Cancer Institute, Boston, Massachusetts, USA.

The cohesin complex plays an essential role in chromosome maintenance and transcriptional regulation. Recurrent somatic mutations in the cohesin complex are frequent genetic drivers in cancer, including myelodysplastic syndromes (MDS) and acute myeloid leukemia (AML). Here, using genetic dependency screens of stromal antigen 2-mutant (STAG2-mutant) AML, we identified DNA damage repair and replication as genetic dependencies in cohesin-mutant cells. We demonstrated increased levels of DNA damage and sensitivity of cohesin-mutant cells to poly(ADP-ribose) polymerase (PARP) inhibition. We developed a mouse model of MDS in which Stag2 mutations arose as clonal secondary lesions in the background of clonal hematopoiesis driven by tet methylcytosine dioxygenase 2 (Tet2) mutations and demonstrated selective depletion of cohesin-mutant cells with PARP inhibition in vivo. Finally, we demonstrated a shift from STAG2- to STAG1-containing cohesin complexes in cohesin-mutant cells, which was associated with longer DNA loop extrusion, more intermixing of chromatin compartments, and increased interaction with PARP and replication protein A complex. Our findings inform the biology and therapeutic opportunities for cohesin-mutant malignancies.
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http://dx.doi.org/10.1172/jci.insight.142149DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7934867PMC
February 2021

The SARS-CoV-2 RNA-protein interactome in infected human cells.

Nat Microbiol 2021 03 21;6(3):339-353. Epub 2020 Dec 21.

Helmholtz Institute for RNA-based Infection Research, Helmholtz-Center for Infection Research, Würzburg, Germany.

Characterizing the interactions that SARS-CoV-2 viral RNAs make with host cell proteins during infection can improve our understanding of viral RNA functions and the host innate immune response. Using RNA antisense purification and mass spectrometry, we identified up to 104 human proteins that directly and specifically bind to SARS-CoV-2 RNAs in infected human cells. We integrated the SARS-CoV-2 RNA interactome with changes in proteome abundance induced by viral infection and linked interactome proteins to cellular pathways relevant to SARS-CoV-2 infections. We demonstrated by genetic perturbation that cellular nucleic acid-binding protein (CNBP) and La-related protein 1 (LARP1), two of the most strongly enriched viral RNA binders, restrict SARS-CoV-2 replication in infected cells and provide a global map of their direct RNA contact sites. Pharmacological inhibition of three other RNA interactome members, PPIA, ATP1A1, and the ARP2/3 complex, reduced viral replication in two human cell lines. The identification of host dependency factors and defence strategies as presented in this work will improve the design of targeted therapeutics against SARS-CoV-2.
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http://dx.doi.org/10.1038/s41564-020-00846-zDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7906908PMC
March 2021

Proteogenomic Landscape of Breast Cancer Tumorigenesis and Targeted Therapy.

Cell 2020 11 18;183(5):1436-1456.e31. Epub 2020 Nov 18.

Office of Cancer Clinical Proteomics Research, National Cancer Institute, Bethesda, MD 20892, USA.

The integration of mass spectrometry-based proteomics with next-generation DNA and RNA sequencing profiles tumors more comprehensively. Here this "proteogenomics" approach was applied to 122 treatment-naive primary breast cancers accrued to preserve post-translational modifications, including protein phosphorylation and acetylation. Proteogenomics challenged standard breast cancer diagnoses, provided detailed analysis of the ERBB2 amplicon, defined tumor subsets that could benefit from immune checkpoint therapy, and allowed more accurate assessment of Rb status for prediction of CDK4/6 inhibitor responsiveness. Phosphoproteomics profiles uncovered novel associations between tumor suppressor loss and targetable kinases. Acetylproteome analysis highlighted acetylation on key nuclear proteins involved in the DNA damage response and revealed cross-talk between cytoplasmic and mitochondrial acetylation and metabolism. Our results underscore the potential of proteogenomics for clinical investigation of breast cancer through more accurate annotation of targetable pathways and biological features of this remarkably heterogeneous malignancy.
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http://dx.doi.org/10.1016/j.cell.2020.10.036DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8077737PMC
November 2020

Avant-garde: an automated data-driven DIA data curation tool.

Nat Methods 2020 12 16;17(12):1237-1244. Epub 2020 Nov 16.

Broad Institute of MIT and Harvard, Cambridge, MA, USA.

Several challenges remain in data-independent acquisition (DIA) data analysis, such as to confidently identify peptides, define integration boundaries, remove interferences, and control false discovery rates. In practice, a visual inspection of the signals is still required, which is impractical with large datasets. We present Avant-garde as a tool to refine DIA (and parallel reaction monitoring) data. Avant-garde uses a novel data-driven scoring strategy: signals are refined by learning from the dataset itself, using all measurements in all samples to achieve the best optimization. We evaluate the performance of Avant-garde using benchmark DIA datasets and show that it can determine the quantitative suitability of a peptide peak, and reach the same levels of selectivity, accuracy, and reproducibility as manual validation. Avant-garde is complementary to existing DIA analysis engines and aims to establish a strong foundation for subsequent analysis of quantitative mass spectrometry data.
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http://dx.doi.org/10.1038/s41592-020-00986-4DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7723322PMC
December 2020

Proximity labeling in mammalian cells with TurboID and split-TurboID.

Nat Protoc 2020 12 2;15(12):3971-3999. Epub 2020 Nov 2.

Department of Genetics, Stanford University, Stanford, CA, USA.

This protocol describes the use of TurboID and split-TurboID in proximity labeling applications for mapping protein-protein interactions and subcellular proteomes in live mammalian cells. TurboID is an engineered biotin ligase that uses ATP to convert biotin into biotin-AMP, a reactive intermediate that covalently labels proximal proteins. Optimized using directed evolution, TurboID has substantially higher activity than previously described biotin ligase-related proximity labeling methods, such as BioID, enabling higher temporal resolution and broader application in vivo. Split-TurboID consists of two inactive fragments of TurboID that can be reconstituted through protein-protein interactions or organelle-organelle interactions, which can facilitate greater targeting specificity than full-length enzymes alone. Proteins biotinylated by TurboID or split-TurboID are then enriched with streptavidin beads and identified by mass spectrometry. Here, we describe fusion construct design and characterization (variable timing), proteomic sample preparation (5-7 d), mass spectrometric data acquisition (2 d), and proteomic data analysis (1 week).
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http://dx.doi.org/10.1038/s41596-020-0399-0DOI Listing
December 2020

Neurogranin, Encoded by the Schizophrenia Risk Gene NRGN, Bidirectionally Modulates Synaptic Plasticity via Calmodulin-Dependent Regulation of the Neuronal Phosphoproteome.

Biol Psychiatry 2021 02 29;89(3):256-269. Epub 2020 Jul 29.

Picower Institute for Learning and Memory, Massachusetts Institute of Technology, Cambridge, Massachusetts; Department of Brain and Cognitive Sciences, Massachusetts Institute of Technology, Cambridge, Massachusetts. Electronic address:

Background: Neurogranin (Ng), encoded by the schizophrenia risk gene NRGN, is a calmodulin-binding protein enriched in the postsynaptic compartments, and its expression is reduced in the postmortem brains of patients with schizophrenia. Experience-dependent translation of Ng is critical for encoding contextual memory, and Ng regulates developmental plasticity in the primary visual cortex during the critical period. However, the overall impact of Ng on the neuronal signaling that regulates synaptic plasticity is unknown.

Methods: Altered Ng expression was achieved via virus-mediated gene manipulation in mice. The effect on long-term potentiation (LTP) was accessed using spike timing-dependent plasticity protocols. Quantitative phosphoproteomics analyses led to discoveries in significant phosphorylated targets. An identified candidate was examined with high-throughput planar patch clamp and was validated with pharmacological manipulation.

Results: Ng bidirectionally modulated LTP in the hippocampus. Decreasing Ng levels significantly affected the phosphorylation pattern of postsynaptic density proteins, including glutamate receptors, GTPases, kinases, RNA binding proteins, selective ion channels, and ionic transporters, some of which highlighted clusters of schizophrenia- and autism-related genes. Hypophosphorylation of NMDA receptor subunit Grin2A, one significant phosphorylated target, resulted in accelerated decay of NMDA receptor currents. Blocking protein phosphatase PP2B activity rescued the accelerated NMDA receptor current decay and the impairment of LTP mediated by Ng knockdown, implicating the requirement of synaptic PP2B activity for the deficits.

Conclusions: Altered Ng levels affect the phosphorylation landscape of neuronal proteins. PP2B activity is required for mediating the deficit in synaptic plasticity caused by decreasing Ng levels, revealing a novel mechanistic link of a schizophrenia risk gene to cognitive deficits.
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http://dx.doi.org/10.1016/j.biopsych.2020.07.014DOI Listing
February 2021
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