Publications by authors named "Benjamin G Neel"

190 Publications

SHP2 drives inflammation-triggered insulin resistance by reshaping tissue macrophage populations.

Sci Transl Med 2021 Apr 28;13(591). Epub 2021 Apr 28.

INSERM UMR-S 1166, Sorbonne Université, Hôpital de la Pitié-Salpêtrière, Paris F-75013, France.

Insulin resistance is a key event in type 2 diabetes onset and a major comorbidity of obesity. It results from a combination of fat excess-triggered defects, including lipotoxicity and metaflammation, but the causal mechanisms remain difficult to identify. Here, we report that hyperactivation of the tyrosine phosphatase SHP2 found in Noonan syndrome (NS) led to an unsuspected insulin resistance profile uncoupled from altered lipid management (for example, obesity or ectopic lipid deposits) in both patients and mice. Functional exploration of an NS mouse model revealed this insulin resistance phenotype correlated with constitutive inflammation of tissues involved in the regulation of glucose metabolism. Bone marrow transplantation and macrophage depletion improved glucose homeostasis and decreased metaflammation in the mice, highlighting a key role of macrophages. In-depth analysis of bone marrow-derived macrophages in vitro and liver macrophages showed that hyperactive SHP2 promoted a proinflammatory phenotype, modified resident macrophage homeostasis, and triggered monocyte infiltration. Consistent with a role of SHP2 in promoting inflammation-driven insulin resistance, pharmaceutical SHP2 inhibition in obese diabetic mice improved insulin sensitivity even better than conventional antidiabetic molecules by specifically reducing metaflammation and alleviating macrophage activation. Together, these results reveal that SHP2 hyperactivation leads to inflammation-triggered metabolic impairments and highlight the therapeutical potential of SHP2 inhibition to ameliorate insulin resistance.
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http://dx.doi.org/10.1126/scitranslmed.abe2587DOI Listing
April 2021

Genetically Defined Syngeneic Mouse Models of Ovarian Cancer as Tools for the Discovery of Combination Immunotherapy.

Cancer Discov 2021 Feb 6;11(2):384-407. Epub 2020 Nov 6.

Whitehead Institute for Biomedical Research, Cambridge, Massachusetts.

Despite advances in immuno-oncology, the relationship between tumor genotypes and response to immunotherapy remains poorly understood, particularly in high-grade serous tubo-ovarian carcinomas (HGSC). We developed a series of mouse models that carry genotypes of human HGSCs and grow in syngeneic immunocompetent hosts to address this gap. We transformed murine-fallopian tube epithelial cells to phenocopy homologous recombination-deficient tumors through a combined loss of and overexpression of and , which was contrasted with an identical model carrying wild-type . For homologous recombination-proficient tumors, we constructed genotypes combining loss of and overexpression of , and driven by or or overexpression. These lines form tumors recapitulating human disease, including genotype-driven responses to treatment, and enabled us to identify follistatin as a driver of resistance to checkpoint inhibitors. These data provide proof of concept that our models can identify new immunotherapy targets in HGSC. SIGNIFICANCE: We engineered a panel of murine fallopian tube epithelial cells bearing mutations typical of HGSC and capable of forming tumors in syngeneic immunocompetent hosts. These models recapitulate tumor microenvironments and drug responses characteristic of human disease. In a -overexpressing model, immune-checkpoint resistance was driven by follistatin..
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http://dx.doi.org/10.1158/2159-8290.CD-20-0818DOI Listing
February 2021

Genetically Defined, Syngeneic Organoid Platform for Developing Combination Therapies for Ovarian Cancer.

Cancer Discov 2021 Feb 6;11(2):362-383. Epub 2020 Nov 6.

Laura and Isaac Perlmutter Cancer Center, NYU Grossman School of Medicine, NYU Langone Health, New York, New York.

The paucity of genetically informed, immunocompetent tumor models impedes evaluation of conventional, targeted, and immune therapies. By engineering mouse fallopian tube epithelial organoids using lentiviral gene transduction and/or CRISPR/Cas9 mutagenesis, we generated multiple high-grade serous tubo-ovarian cancer (HGSC) models exhibiting mutational combinations seen in patients with HGSC. Detailed analysis of homologous recombination (HR)-proficient ( ), HR-deficient ( ), and unclassified ( ) organoids revealed differences in properties (proliferation, differentiation, and "secretome"), copy-number aberrations, and tumorigenicity. Tumorigenic organoids had variable sensitivity to HGSC chemotherapeutics, and evoked distinct immune microenvironments that could be modulated by neutralizing organoid-produced chemokines/cytokines. These findings enabled development of a chemotherapy/immunotherapy regimen that yielded durable, T cell-dependent responses in HGSC; in contrast, tumors failed to respond. Mouse and human HGSC models showed genotype-dependent similarities in chemosensitivity, secretome, and immune microenvironment. Genotype-informed, syngeneic organoid models could provide a platform for the rapid evaluation of tumor biology and therapeutics. SIGNIFICANCE: The lack of genetically informed, diverse, immunocompetent models poses a major barrier to therapeutic development for many malignancies. Using engineered fallopian tube organoids to study the cell-autonomous and cell-nonautonomous effects of specific combinations of mutations found in HGSC, we suggest an effective combination treatment for the currently intractable -amplified subgroup..
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http://dx.doi.org/10.1158/2159-8290.CD-20-0455DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7858239PMC
February 2021

Hyperactive CDK2 Activity in Basal-like Breast Cancer Imposes a Genome Integrity Liability that Can Be Exploited by Targeting DNA Polymerase ε.

Mol Cell 2020 11 4;80(4):682-698.e7. Epub 2020 Nov 4.

Department of Pathology, New York University School of Medicine, New York, NY 10016, USA; Laura & Isaac Perlmutter Cancer Center, New York University School of Medicine, New York, NY 10016, USA. Electronic address:

Knowledge of fundamental differences between breast cancer subtypes has driven therapeutic advances; however, basal-like breast cancer (BLBC) remains clinically intractable. Because BLBC exhibits alterations in DNA repair enzymes and cell-cycle checkpoints, elucidation of factors enabling the genomic instability present in this subtype has the potential to reveal novel anti-cancer strategies. Here, we demonstrate that BLBC is especially sensitive to suppression of iron-sulfur cluster (ISC) biosynthesis and identify DNA polymerase epsilon (POLE) as an ISC-containing protein that underlies this phenotype. In BLBC cells, POLE suppression leads to replication fork stalling, DNA damage, and a senescence-like state or cell death. In contrast, luminal breast cancer and non-transformed mammary cells maintain viability upon POLE suppression but become dependent upon an ATR/CHK1/CDC25A/CDK2 DNA damage response axis. We find that CDK1/2 targets exhibit hyperphosphorylation selectively in BLBC tumors, indicating that CDK2 hyperactivity is a genome integrity vulnerability exploitable by targeting POLE.
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http://dx.doi.org/10.1016/j.molcel.2020.10.016DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7687292PMC
November 2020

Quantitative phosphoproteomic analysis reveals involvement of PD-1 in multiple T cell functions.

J Biol Chem 2020 12 19;295(52):18036-18050. Epub 2020 Oct 19.

Columbia Center for Translational Immunology, Columbia University Medical Center, New York, USA; Division of Rheumatology, Columbia University Medical Center, New York, New York, USA. Electronic address:

Programmed cell death protein 1 (PD-1) is a critical inhibitory receptor that limits excessive T cell responses. Cancer cells have evolved to evade these immunoregulatory mechanisms by upregulating PD-1 ligands and preventing T cell-mediated anti-tumor responses. Consequently, therapeutic blockade of PD-1 enhances T cell-mediated anti-tumor immunity, but many patients do not respond and a significant proportion develop inflammatory toxicities. To improve anti-cancer therapy, it is critical to reveal the mechanisms by which PD-1 regulates T cell responses. We performed global quantitative phosphoproteomic interrogation of PD-1 signaling in T cells. By complementing our analysis with functional validation assays, we show that PD-1 targets tyrosine phosphosites that mediate proximal T cell receptor signaling, cytoskeletal organization, and immune synapse formation. PD-1 ligation also led to differential phosphorylation of serine and threonine sites within proteins regulating T cell activation, gene expression, and protein translation. predictions revealed that kinase/substrate relationships engaged downstream of PD-1 ligation. These insights uncover the phosphoproteomic landscape of PD-1-triggered pathways and reveal novel PD-1 substrates that modulate diverse T cell functions and may serve as future therapeutic targets. These data are a useful resource in the design of future PD-1-targeting therapeutic approaches.
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http://dx.doi.org/10.1074/jbc.RA120.014745DOI Listing
December 2020

Raymond L. Erikson (1936-2020).

Cell 2020 05 28;181(5):961-963. Epub 2020 May 28.

Department of Pharmacology, Weill Cornell Medicine, New York, NY 10021, USA. Electronic address:

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http://dx.doi.org/10.1016/j.cell.2020.04.051DOI Listing
May 2020

SHP2 inhibition diminishes KRASG12C cycling and promotes tumor microenvironment remodeling.

J Exp Med 2021 Jan;218(1)

Laura and Isaac Perlmutter Cancer Center, New York University School of Medicine, NYU Langone Health, New York, NY.

KRAS is the most frequently mutated human oncogene, and KRAS inhibition has been a longtime goal. Recently, inhibitors were developed that bind KRASG12C-GDP and react with Cys-12 (G12C-Is). Using new affinity reagents to monitor KRASG12C activation and inhibitor engagement, we found that an SHP2 inhibitor (SHP2-I) increases KRAS-GDP occupancy, enhancing G12C-I efficacy. The SHP2-I abrogated RTK feedback signaling and adaptive resistance to G12C-Is in vitro, in xenografts, and in syngeneic KRASG12C-mutant pancreatic ductal adenocarcinoma (PDAC) and non-small cell lung cancer (NSCLC). SHP2-I/G12C-I combination evoked favorable but tumor site-specific changes in the immune microenvironment, decreasing myeloid suppressor cells, increasing CD8+ T cells, and sensitizing tumors to PD-1 blockade. Experiments using cells expressing inhibitor-resistant SHP2 showed that SHP2 inhibition in PDAC cells is required for PDAC regression and remodeling of the immune microenvironment but revealed direct inhibitory effects on tumor angiogenesis and vascularity. Our results demonstrate that SHP2-I/G12C-I combinations confer a substantial survival benefit in PDAC and NSCLC and identify additional potential combination strategies.
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http://dx.doi.org/10.1084/jem.20201414DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7549316PMC
January 2021

U.S. Biomedical Research Needs More Immigrant Scientists, Not Fewer!

Cancer Cell 2020 09;38(3):308

Department of Pathology and Department of Medicine, Laura and Isaac Perlmutter Cancer Center, NYU Grossman School of Medicine, New York, NY, USA. Electronic address:

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http://dx.doi.org/10.1016/j.ccell.2020.08.010DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7489325PMC
September 2020

Distinct fibroblast functional states drive clinical outcomes in ovarian cancer and are regulated by TCF21.

J Exp Med 2020 08;217(8)

Department of Medical Biophysics, University of Toronto, Toronto, Ontario, Canada.

Recent studies indicate that cancer-associated fibroblasts (CAFs) are phenotypically and functionally heterogeneous. However, little is known about CAF subtypes, the roles they play in cancer progression, and molecular mediators of the CAF "state." Here, we identify a novel cell surface pan-CAF marker, CD49e, and demonstrate that two distinct CAF states, distinguished by expression of fibroblast activation protein (FAP), coexist within the CD49e+ CAF compartment in high-grade serous ovarian cancers. We show for the first time that CAF state influences patient outcomes and that this is mediated by the ability of FAP-high, but not FAP-low, CAFs to aggressively promote proliferation, invasion and therapy resistance of cancer cells. Overexpression of the FAP-low-specific transcription factor TCF21 in FAP-high CAFs decreases their ability to promote invasion, chemoresistance, and in vivo tumor growth, indicating that it acts as a master regulator of the CAF state. Understanding CAF states in more detail could lead to better patient stratification and novel therapeutic strategies.
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http://dx.doi.org/10.1084/jem.20191094DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7398174PMC
August 2020

Piecing Together a Broken Tumor Suppressor Phosphatase for Cancer Therapy.

Cell 2020 04;181(3):514-517

Perlmutter Cancer Center, NYU School of Medicine, NY Langone Health, New York, NY 10016, USA. Electronic address:

Members of the PP2A family of serine/threonine phosphatases are important human tumor suppressor genes. Unlike most tumor suppressors, they are rarely mutated/deleted, but rather are impaired by "inhibitor proteins." Two papers in this issue of Cell show how some phenothiazine derivatives reactivate specific PP2A isozymes with potential benefit in cancer and other diseases.
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http://dx.doi.org/10.1016/j.cell.2020.04.005DOI Listing
April 2020

The Protein Tyrosine Phosphatase Receptor Delta Regulates Developmental Neurogenesis.

Cell Rep 2020 01;30(1):215-228.e5

Program in Neurosciences and Mental Health, Hospital for Sick Children, Toronto M5G 1X8, ON, Canada; Center for Integrative Biology, Facultad de Ciencias, Universidad Mayor, Santiago 8580745, Chile. Electronic address:

PTPRD is a receptor protein tyrosine phosphatase that is genetically associated with neurodevelopmental disorders. Here, we asked whether Ptprd mutations cause aberrant neural development by perturbing neurogenesis in the murine cortex. We show that loss of Ptprd causes increases in neurogenic transit-amplifying intermediate progenitor cells and cortical neurons and perturbations in neuronal localization. These effects are intrinsic to neural precursor cells since acute Ptprd knockdown causes similar perturbations. PTPRD mediates these effects by dephosphorylating receptor tyrosine kinases, including TrkB and PDGFRβ, and loss of Ptprd causes the hyperactivation of TrkB and PDGFRβ and their downstream MEK-ERK signaling pathway in neural precursor cells. Moreover, inhibition of aberrant TrkB or MEK activation rescues the increased neurogenesis caused by knockdown or homozygous loss of Ptprd. These results suggest that PTPRD regulates receptor tyrosine kinases to ensure appropriate numbers of intermediate progenitor cells and neurons, suggesting a mechanism for its genetic association with neurodevelopmental disorders.
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http://dx.doi.org/10.1016/j.celrep.2019.11.033DOI Listing
January 2020

The sixth international RASopathies symposium: Precision medicine-From promise to practice.

Am J Med Genet A 2020 03 11;182(3):597-606. Epub 2019 Dec 11.

Cincinnati Children's Hospital Medical Center and University of Cincinnati, School of Medicine, Cincinnati, Ohio.

The RASopathies are a group of genetic disorders that result from germline pathogenic variants affecting RAS-mitogen activated protein kinase (MAPK) pathway genes. RASopathies share RAS/MAPK pathway dysregulation and share phenotypic manifestations affecting numerous organ systems, causing lifelong and at times life-limiting medical complications. RASopathies may benefit from precision medicine approaches. For this reason, the Sixth International RASopathies Symposium focused on exploring precision medicine. This meeting brought together basic science researchers, clinicians, clinician scientists, patient advocates, and representatives from pharmaceutical companies and the National Institutes of Health. Novel RASopathy genes, variants, and animal models were discussed in the context of medication trials and drug development. Attempts to define and measure meaningful endpoints for treatment trials were discussed, as was drug availability to patients after trial completion.
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http://dx.doi.org/10.1002/ajmg.a.61434DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7021559PMC
March 2020

Corrigendum to: Off-target inhibition by active site-targeting SHP2 inhibitors.

FEBS Open Bio 2019 Dec;9(12):2174

Laura and Isaac Perlmutter Cancer Center, NYU Langone Health, New York, NY, USA.

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http://dx.doi.org/10.1002/2211-5463.12754DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6886290PMC
December 2019

Both fallopian tube and ovarian surface epithelium are cells-of-origin for high-grade serous ovarian carcinoma.

Nat Commun 2019 11 26;10(1):5367. Epub 2019 Nov 26.

Laura and Isaac Perlmutter Cancer Center, NYU Langone Health, New York, NY, 10016, USA.

The cell-of-origin of high grade serous ovarian carcinoma (HGSOC) remains controversial, with fallopian tube epithelium (FTE) and ovarian surface epithelium (OSE) both considered candidates. Here, by using genetically engineered mouse models and organoids, we assessed the tumor-forming properties of FTE and OSE harboring the same oncogenic abnormalities. Combined RB family inactivation and Tp53 mutation in Pax8 + FTE caused Serous Tubal Intraepithelial Carcinoma (STIC), which metastasized rapidly to the ovarian surface. These events were recapitulated by orthotopic injection of mutant FTE organoids. Engineering the same genetic lesions into Lgr5 + OSE or OSE-derived organoids also caused metastatic HGSOC, although with longer latency and lower penetrance. FTE- and OSE-derived tumors had distinct transcriptomes, and comparative transcriptomics and genomics suggest that human HGSOC arises from both cell types. Finally, FTE- and OSE-derived organoids exhibited differential chemosensitivity. Our results comport with a dualistic origin for HGSOC and suggest that the cell-of-origin might influence therapeutic response.
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http://dx.doi.org/10.1038/s41467-019-13116-2DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6879755PMC
November 2019

Catalytic dysregulation of SHP2 leading to Noonan syndromes affects platelet signaling and functions.

Blood 2019 12;134(25):2304-2317

Institut des Maladies Métaboliques et Cardiovasculaires, INSERM U1048 and Université Paul Sabatier, Toulouse, France.

Src homology 2 domain-containing phosphatase 2 (SHP2), encoded by the PTPN11 gene, is a ubiquitous protein tyrosine phosphatase that is a critical regulator of signal transduction. Germ line mutations in the PTPN11 gene responsible for catalytic gain or loss of function of SHP2 cause 2 disorders with multiple organ defects: Noonan syndrome (NS) and NS with multiple lentigines (NSML), respectively. Bleeding anomalies have been frequently reported in NS, but causes remain unclear. This study investigates platelet activation in patients with NS and NSML and in 2 mouse models carrying PTPN11 mutations responsible for these 2 syndromes. Platelets from NS mice and patients displayed a significant reduction in aggregation induced by low concentrations of GPVI and CLEC-2 agonists and a decrease in thrombus growth on a collagen surface under arterial shear stress. This was associated with deficiencies in GPVI and αIIbβ3 integrin signaling, platelet secretion, and thromboxane A2 generation. Similarly, arterial thrombus formation was significantly reduced in response to a local carotid injury in NS mice, associated with a significant increase in tail bleeding time. In contrast, NSML mouse platelets exhibited increased platelet activation after GPVI and CLEC-2 stimulation and enhanced platelet thrombotic phenotype on collagen matrix under shear stress. Blood samples from NSML patients also showed a shear stress-dependent elevation of platelet responses on collagen matrix. This study brings new insights into the understanding of SHP2 function in platelets, points to new thrombopathies linked to platelet signaling defects, and provides important information for the medical care of patients with NS in situations involving risk of bleeding.
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http://dx.doi.org/10.1182/blood.2019001543DOI Listing
December 2019

Activated Thiol Sepharose-based proteomic approach to quantify reversible protein oxidation.

FASEB J 2019 11 26;33(11):12336-12347. Epub 2019 Aug 26.

Department of Medical Biophysics, University of Toronto, Toronto, Ontario, Canada.

Reactive oxygen species (ROS) can act as second messengers in various signaling pathways, and abnormal oxidation contributes to multiple diseases, including cancer. Detecting and quantifying protein oxidation is crucial for a detailed understanding of reduction-oxidation reaction (redox) signaling. We developed an Activated Thiol Sepharose-based proteomic (ATSP) approach to quantify reversible protein oxidation. ATSP can enrich HO-sensitive thiol peptides, which are more likely to contain reactive cysteines involved in redox signaling. We applied our approach to analyze hereditary leiomyomatosis and renal cell carcinoma (HLRCC), a type of kidney cancer that harbors fumarate hydratase (FH)-inactivating mutations and has elevated ROS levels. Multiple proteins were oxidized in FH-deficient cells, including many metabolic proteins such as the pyruvate kinase M2 isoform (PKM2). Treatment of HLRCC cells with dimethyl fumarate or PKM2 activators altered PKM2 oxidation levels. Finally, we found that ATSP could detect Src homology region 2 domain-containing phosphatase-2 and PKM2 oxidation in cells stimulated with platelet-derived growth factor. This newly developed redox proteomics workflow can detect reversible oxidation of reactive cysteines and can be employed to analyze multiple physiologic and pathologic conditions.-Xu, Y., Andrade, J., Ueberheide, B., Neel, B. G. Activated Thiol Sepharose-based proteomic approach to quantify reversible protein oxidation.
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http://dx.doi.org/10.1096/fj.201900693RDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6902679PMC
November 2019

Migration rather than proliferation transcriptomic signatures are strongly associated with breast cancer patient survival.

Sci Rep 2019 07 29;9(1):10989. Epub 2019 Jul 29.

Center for Bioinformatics and Computational Biology, University of Maryland, College Park, Maryland, 20742, USA.

The efficacy of prospective cancer treatments is routinely estimated by in vitro cell-line proliferation screens. However, it is unclear whether tumor aggressiveness and patient survival are influenced more by the proliferative or the migratory properties of cancer cells. To address this question, we experimentally measured proliferation and migration phenotypes across more than 40 breast cancer cell-lines. Based on the latter, we built and validated individual predictors of breast cancer proliferation and migration levels from the cells' transcriptomics. We then apply these predictors to estimate the proliferation and migration levels of more than 1000 TCGA breast cancer tumors. Reassuringly, both estimates increase with tumor's aggressiveness, as qualified by its stage, grade, and subtype. However, predicted tumor migration levels are significantly more strongly associated with patient survival than the proliferation levels. We confirmed these findings by conducting siRNA knock-down experiments on the highly migratory MDA-MB-231 cell lines and deriving gene knock-down based proliferation and migration signatures. We show that cytoskeletal drugs might be more beneficial in patients with high predicted migration levels. Taken together, these results testify to the importance of migration levels in determining patient survival.
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http://dx.doi.org/10.1038/s41598-019-47440-wDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6662662PMC
July 2019

Phase I study of local radiation and tremelimumab in patients with inoperable locally recurrent or metastatic breast cancer.

Oncotarget 2019 Apr 26;10(31):2947-2958. Epub 2019 Apr 26.

Department of Medical Oncology, Princess Margaret Cancer Centre, University Health Network, University of Toronto, Toronto, Canada.

Immunotherapy has shown modest activity in metastatic breast cancer (MBC). In this phase I dose escalation study, we assessed safety of tremelimumab, a humanized anti-CTLA4 monoclonal antibody, at starting dose 3 mg/kg, on the third day of palliative radiotherapy (2000cGy in 5 daily fractions) in patients with MBC. Primary objective was to determine the maximum tolerated dose (MTD) of tremelimumab combined with RT. Secondary objective was to assess response. Among 6 patients enrolled between July 2010 and October 2011, 5 had hormone receptor-positive MBC, 1 had triple negative MBC. Median age was 45 years. Common toxicities included lymphopenia (83%), fatigue (50%) and rash (33%). One dose-limiting toxicity occurred at 6 mg/kg, however the trial closed before MTD could be determined. One patient discontinued treatment due to a pathological fracture. Best response was stable disease (SD), 1 patient had SD for >6 months. Median follow up was 27.0 months. Median OS was 50.8 months, with 1 patient surviving >8 years. Peripheral blood mononuclear cell (PBMC) profiles showed increasing proliferating (Ki67+) Treg cells 1 week post treatment in 5 patients. Overall, tremelimumab at 3 mg/kg combined with RT appears to be a tolerable treatment strategy. Further studies are needed to optimize this combination approach.
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http://dx.doi.org/10.18632/oncotarget.26893DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6508206PMC
April 2019

The Noonan Syndrome-linked Raf1L613V mutation drives increased glial number in the mouse cortex and enhanced learning.

PLoS Genet 2019 04 24;15(4):e1008108. Epub 2019 Apr 24.

School of Life Sciences, Arizona State University, Tempe, Arizona, United States of America.

RASopathies are a family of related syndromes caused by mutations in regulators of the RAS/Extracellular Regulated Kinase 1/2 (ERK1/2) signaling cascade that often result in neurological deficits. RASopathy mutations in upstream regulatory components, such as NF1, PTPN11/SHP2, and RAS have been well-characterized, but mutation-specific differences in the pathogenesis of nervous system abnormalities remain poorly understood, especially those involving mutations downstream of RAS. Here, we assessed cellular and behavioral phenotypes in mice expressing a Raf1L613V gain-of-function mutation associated with the RASopathy, Noonan Syndrome. We report that Raf1L613V/wt mutants do not exhibit a significantly altered number of excitatory or inhibitory neurons in the cortex. However, we observed a significant increase in the number of specific glial subtypes in the forebrain. The density of GFAP+ astrocytes was significantly increased in the adult Raf1L613V/wt cortex and hippocampus relative to controls. OLIG2+ oligodendrocyte progenitor cells were also increased in number in mutant cortices, but we detected no significant change in myelination. Behavioral analyses revealed no significant changes in voluntary locomotor activity, anxiety-like behavior, or sociability. Surprisingly, Raf1L613V/wt mice performed better than controls in select aspects of the water radial-arm maze, Morris water maze, and cued fear conditioning tasks. Overall, these data show that increased astrocyte and oligodendrocyte progenitor cell (OPC) density in the cortex coincides with enhanced cognition in Raf1L613V/wt mutants and further highlight the distinct effects of RASopathy mutations on nervous system development and function.
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http://dx.doi.org/10.1371/journal.pgen.1008108DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6502435PMC
April 2019

An organoid platform for ovarian cancer captures intra- and interpatient heterogeneity.

Nat Med 2019 05 22;25(5):838-849. Epub 2019 Apr 22.

Center for Molecular Medicine, University Medical Center Utrecht, Utrecht University, Utrecht, the Netherlands.

Ovarian cancer (OC) is a heterogeneous disease usually diagnosed at a late stage. Experimental in vitro models that faithfully capture the hallmarks and tumor heterogeneity of OC are limited and hard to establish. We present a protocol that enables efficient derivation and long-term expansion of OC organoids. Utilizing this protocol, we have established 56 organoid lines from 32 patients, representing all main subtypes of OC. OC organoids recapitulate histological and genomic features of the pertinent lesion from which they were derived, illustrating intra- and interpatient heterogeneity, and can be genetically modified. We show that OC organoids can be used for drug-screening assays and capture different tumor subtype responses to the gold standard platinum-based chemotherapy, including acquisition of chemoresistance in recurrent disease. Finally, OC organoids can be xenografted, enabling in vivo drug-sensitivity assays. Taken together, this demonstrates their potential application for research and personalized medicine.
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http://dx.doi.org/10.1038/s41591-019-0422-6DOI Listing
May 2019

N-Glycoproteomics of Patient-Derived Xenografts: A Strategy to Discover Tumor-Associated Proteins in High-Grade Serous Ovarian Cancer.

Cell Syst 2019 04 10;8(4):345-351.e4. Epub 2019 Apr 10.

University of Toronto, Department of Medical Biophysics, Toronto, ON M5G 1L7, Canada; Princess Margaret Cancer Centre, University Health Network, Toronto, ON M5G 2M9, Canada. Electronic address:

High-grade serous ovarian carcinoma (HGSC) is the most common and lethal subtype of gynecologic malignancy in women. The current standard of treatment combines cytoreductive surgery and chemotherapy. Despite the efficacy of initial treatment, most patients develop cancer recurrence, and 70% of patients die within 5 years of initial diagnosis. CA125 is the current FDA-approved biomarker used in the clinic to monitor response to treatment and recurrence, but its impact on patient survival is limited. New strategies for the discovery of HGSC biomarkers are urgently needed. Here, we describe a proteomics strategy to detect tumor-associated proteins in serum of HGSC patient-derived xenograft models. We demonstrate proof-of-concept applicability using two independent, longitudinal serum cohorts from HGSC patients.
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http://dx.doi.org/10.1016/j.cels.2019.03.011DOI Listing
April 2019

The transgenic mouse: a new model to delineate platelet and leukocyte functions.

Blood 2019 01 14;133(4):331-343. Epub 2018 Nov 14.

Institute of Cardiovascular Sciences, College of Medical and Dental Sciences, University of Birmingham, Birmingham, United Kingdom.

Conditional knockout (KO) mouse models are invaluable for elucidating the physiological roles of platelets. The () transgenic mouse is the current model of choice for generating megakaryocyte/platelet-specific KO mice. Platelets and leukocytes work closely together in a wide range of disease settings, yet the specific contribution of platelets to these processes remains unclear. This is partially a result of the transgene being expressed in a variety of leukocyte populations. To overcome this issue, we developed a transgenic mouse strain in which expression is driven by the endogenous locus. By crossing and mice to the dual-fluorescence reporter mouse and performing a head-to-head comparison, we demonstrate more stringent megakaryocyte lineage-specific expression of the transgene. Broader tissue expression was observed with the transgene, leading to recombination in many hematopoietic lineages, including monocytes, macrophages, granulocytes, and dendritic and B and T cells. Direct comparison of phenotypes of , or conditional KO mice generated using either the or strains revealed similar platelet phenotypes. However, additional inflammatory and immunological anomalies were observed in -generated KO mice as a result of nonspecific deletion in other hematopoietic lineages. By excluding leukocyte contributions to phenotypes, the mouse will advance our understanding of the role of platelets in inflammation and other pathophysiological processes in which platelet-leukocyte interactions are involved.
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http://dx.doi.org/10.1182/blood-2018-09-877787DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6484457PMC
January 2019

Pathologic Oxidation of PTPN12 Underlies ABL1 Phosphorylation in Hereditary Leiomyomatosis and Renal Cell Carcinoma.

Cancer Res 2018 12 8;78(23):6539-6548. Epub 2018 Oct 8.

Department of Medical Biophysics, University of Toronto, Toronto, Ontario, Canada.

: Hereditary leiomyomatosis and renal cell carcinoma (HLRCC) is an inherited cancer syndrome associated with a highly aggressive form of type 2 papillary renal cell carcinoma (PRCC). Germline inactivating alterations in () cause HLRCC and result in elevated levels of reactive oxygen species (ROS). Recent work indicates that PRCC cells have increased activation of ABL1, which promotes tumor growth, but how ABL1 is activated remains unclear. Given that oxidation can regulate protein-tyrosine phosphatase (PTP) catalytic activity, inactivation of an ABL-directed PTP by ROS might account for ABL1 activation in this malignancy. Our group previously developed "q-oxPTPome," a method that globally monitors the oxidation of classical PTPs. In this study, we present a refined q-oxPTPome, increasing its sensitivity by >10×. Applying q-oxPTPome to FH-deficient cell models showed that multiple PTPs were either highly oxidized (including PTPN12) or overexpressed. Highly oxidized PTPs were those with relatively high sensitivity to exogenous HO. Most PTP oxidation in FH-deficient cells was reversible, although nearly 40% of PTPN13 was irreversibly oxidized to the sulfonic acid state. Using substrate-trapping mutants, we mapped PTPs to their putative substrates and found that only PTPN12 could target ABL1. Furthermore, knockdown experiments identified PTPN12 as the major ABL1 phosphatase, and overexpression of PTPN12 inhibited ABL1 phosphorylation and HLRCC cell growth. These results show that ROS-induced oxidation of PTPN12 accounts for ABL1 phosphorylation in HLRCC-associated PRCC, revealing a novel mechanism for inactivating a tumor suppressor gene product and establishing a direct link between pathologic PTP oxidation and neoplastic disease. SIGNIFICANCE: This work identifies a novel mechanism of activation of the oncogenic kinase ABL1 via ROS-induced, oxidation-mediated inactivation of cognate protein tyrosine phosphatases.
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http://dx.doi.org/10.1158/0008-5472.CAN-18-0901DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6279512PMC
December 2018

Off-target inhibition by active site-targeting SHP2 inhibitors.

FEBS Open Bio 2018 Sep 1;8(9):1405-1411. Epub 2018 Aug 1.

Laura and Isaac Perlmutter Cancer Center NYU Langone Health New York NY USA.

Due to the involvement of SHP2 (SH2 domain-containing protein-tyrosine phosphatase) in human disease, including Noonan syndrome and cancer, several inhibitors targeting SHP2 have been developed. Here, we report that the commonly used SHP2 inhibitor NSC-87877 does not exhibit robust inhibitory effects on growth factor-dependent MAPK (mitogen-activated protein kinase) pathway activation and that the recently developed active site-targeting SHP2 inhibitors IIB-08, 11a-1, and GS-493 show off-target effects on ligand-evoked activation/trans-phosphorylation of the PDGFRβ (platelet-derived growth factor receptor β). GS-493 also inhibits purified human PDGFRβ and SRC , whereas PDGFRβ inhibition by IIB-08 and 11a-1 occurs only in the cellular context. Our results argue for extreme caution in inferring specific functions for SHP2 based on studies using these inhibitors.
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http://dx.doi.org/10.1002/2211-5463.12493DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6120237PMC
September 2018

SHP2 Inhibition Prevents Adaptive Resistance to MEK Inhibitors in Multiple Cancer Models.

Cancer Discov 2018 10 25;8(10):1237-1249. Epub 2018 Jul 25.

Laura and Isaac Perlmutter Cancer Center, New York University School of Medicine, NYU Langone Health, New York, New York.

Adaptive resistance to MEK inhibitors (MEKi) typically occurs via induction of genes for different receptor tyrosine kinases (RTK) and/or their ligands, even in tumors of the same histotype, making combination strategies challenging. SHP2 () is required for RAS/ERK pathway activation by most RTKs and might provide a common resistance node. We found that combining the SHP2 inhibitor SHP099 with a MEKi inhibited the proliferation of multiple cancer cell lines knockdown/MEKi treatment had similar effects, whereas expressing SHP099 binding-defective mutants conferred resistance, demonstrating that SHP099 is on-target. SHP099/trametinib was highly efficacious in xenograft and/or genetically engineered models of -mutant pancreas, lung, and ovarian cancers and in wild-type RAS-expressing triple-negative breast cancer. SHP099 inhibited activation of KRAS mutants with residual GTPase activity, impeded SOS/RAS/MEK/ERK1/2 reactivation in response to MEKi, and blocked ERK1/2-dependent transcriptional programs. We conclude that SHP099/MEKi combinations could have therapeutic utility in multiple malignancies. MEK inhibitors show limited efficacy as single agents, in part because of the rapid development of adaptive resistance. We find that SHP2/MEK inhibitor combinations prevent adaptive resistance in multiple cancer models expressing mutant and wild-type KRAS. .
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http://dx.doi.org/10.1158/2159-8290.CD-18-0444DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6170706PMC
October 2018

Vitamin C in Stem Cell Reprogramming and Cancer.

Trends Cell Biol 2018 09 30;28(9):698-708. Epub 2018 Apr 30.

Department of Pathology, New York University (NYU) School of Medicine, New York, NY 10016, USA; Laura and Isaac Perlmutter Cancer Center, NYU School of Medicine, New York, NY 10016, USA. Electronic address:

Vitamin C is an essential dietary requirement for humans. In addition to its known role as an antioxidant, vitamin C is a cofactor for Fe- and α-ketoglutarate-dependent dioxygenases (Fe/α-KGDDs) which comprise a large number of diverse enzymes, including collagen prolyl hydroxylases and epigenetic regulators of histone and DNA methylation. Vitamin C can modulate embryonic stem cell (ESC) function, enhance reprogramming of fibroblasts to induced pluripotent stem cells (iPSCs), and hinder the aberrant self-renewal of hematopoietic stem cells (HSCs) through its ability to enhance the activity of either Jumonji C (JmjC) domain-containing histone demethylases or ten-eleven translocation (TET) DNA hydroxylases. Given that epigenetic dysregulation is a known driver of malignancy, vitamin C may play a novel role as an epigenetic anticancer agent.
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http://dx.doi.org/10.1016/j.tcb.2018.04.001DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6102081PMC
September 2018

Noonan syndrome-causing SHP2 mutants impair ERK-dependent chondrocyte differentiation during endochondral bone growth.

Hum Mol Genet 2018 07;27(13):2276-2289

INSERM UMR 1043, Centre of Pathophysiology of Toulouse Purpan (CPTP), University of Toulouse Paul Sabatier, Toulouse, France.

Growth retardation is a constant feature of Noonan syndrome (NS) but its physiopathology remains poorly understood. We previously reported that hyperactive NS-causing SHP2 mutants impair the systemic production of insulin-like growth factor 1 (IGF1) through hyperactivation of the RAS/extracellular signal-regulated kinases (ERK) signalling pathway. Besides endocrine defects, a direct effect of these mutants on growth plate has not been explored, although recent studies have revealed an important physiological role for SHP2 in endochondral bone growth. We demonstrated that growth plate length was reduced in NS mice, mostly due to a shortening of the hypertrophic zone and to a lesser extent of the proliferating zone. These histological features were correlated with decreased expression of early chondrocyte differentiation markers, and with reduced alkaline phosphatase staining and activity, in NS murine primary chondrocytes. Although IGF1 treatment improved growth of NS mice, it did not fully reverse growth plate abnormalities, notably the decreased hypertrophic zone. In contrast, we documented a role of RAS/ERK hyperactivation at the growth plate level since 1) NS-causing SHP2 mutants enhance RAS/ERK activation in chondrocytes in vivo (NS mice) and in vitro (ATDC5 cells) and 2) inhibition of RAS/ERK hyperactivation by U0126 treatment alleviated growth plate abnormalities and enhanced chondrocyte differentiation. Similar effects were obtained by chronic treatment of NS mice with statins. In conclusion, we demonstrated that hyperactive NS-causing SHP2 mutants impair chondrocyte differentiation during endochondral bone growth through a local hyperactivation of the RAS/ERK signalling pathway, and that statin treatment may be a possible therapeutic approach in NS.
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http://dx.doi.org/10.1093/hmg/ddy133DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6005060PMC
July 2018

SHP2 regulates skeletal cell fate by modifying SOX9 expression and transcriptional activity.

Bone Res 2018 6;6:12. Epub 2018 Apr 6.

1Department of Orthopaedics, Brown University Alpert Medical School and Rhode Island Hospital, Providence, RI 02903 USA.

Chondrocytes and osteoblasts differentiate from a common mesenchymal precursor, the osteochondroprogenitor (OCP), and help build the vertebrate skeleton. The signaling pathways that control lineage commitment for OCPs are incompletely understood. We asked whether the ubiquitously expressed protein-tyrosine phosphatase SHP2 (encoded by ) affects skeletal lineage commitment by conditionally deleting in mouse limb and head mesenchyme using "Cre-loxP"-mediated gene excision. SHP2-deficient mice have increased cartilage mass and deficient ossification, suggesting that SHP2-deficient OCPs become chondrocytes and not osteoblasts. Consistent with these observations, the expression of the master chondrogenic transcription factor SOX9 and its target genes , and were increased in SHP2-deficient chondrocytes, as revealed by gene expression arrays, qRT-PCR, in situ hybridization, and immunostaining. Mechanistic studies demonstrate that SHP2 regulates OCP fate determination via the phosphorylation and SUMOylation of SOX9, mediated at least in part via the PKA signaling pathway. Our data indicate that SHP2 is critical for skeletal cell lineage differentiation and could thus be a pharmacologic target for bone and cartilage regeneration.
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http://dx.doi.org/10.1038/s41413-018-0013-zDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5886981PMC
April 2018

Gain-of-function mutations in the gene encoding the tyrosine phosphatase SHP2 induce hydrocephalus in a catalytically dependent manner.

Sci Signal 2018 03 20;11(522). Epub 2018 Mar 20.

Aflac Cancer and Blood Disorders Center, Department of Pediatrics, Children's Healthcare of Atlanta, Emory University School of Medicine, Atlanta, GA 30322, USA.

Catalytically activating mutations in , which encodes the protein tyrosine phosphatase SHP2, cause 50% of Noonan syndrome (NS) cases, whereas inactivating mutations in are responsible for nearly all cases of the similar, but distinct, developmental disorder Noonan syndrome with multiple lentigines (NSML; formerly called LEOPARD syndrome). However, both types of disease mutations are gain-of-function mutations because they cause SHP2 to constitutively adopt an open conformation. We found that the catalytic activity of SHP2 was required for the pathogenic effects of gain-of-function, disease-associated mutations on the development of hydrocephalus in the mouse. Targeted pan-neuronal knockin of a allele encoding the active SHP2 E76K mutant resulted in hydrocephalus due to aberrant development of ependymal cells and their cilia. These pathogenic effects of the E76K mutation were suppressed by the additional mutation C459S, which abolished the catalytic activity of SHP2. Moreover, ependymal cells in NSML mice bearing the inactive SHP2 mutant Y279C were also unaffected. Mechanistically, the SHP2 E76K mutant induced developmental defects in ependymal cells by enhancing dephosphorylation and inhibition of the transcription activator STAT3. Whereas STAT3 activity was reduced in cells, the activities of the kinases ERK and AKT were enhanced, and neural cell-specific knockout mice also manifested developmental defects in ependymal cells and cilia. These genetic and biochemical data demonstrate a catalytic-dependent role of SHP2 gain-of-function disease mutants in the pathogenesis of hydrocephalus.
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http://dx.doi.org/10.1126/scisignal.aao1591DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5915342PMC
March 2018

A Genomically Characterized Collection of High-Grade Serous Ovarian Cancer Xenografts for Preclinical Testing.

Am J Pathol 2018 05 16;188(5):1120-1131. Epub 2018 Feb 16.

Princess Margaret Cancer Centre, University Health Network, Toronto, Ontario, Canada; Department of Medical Biophysics, University of Toronto, Toronto, Ontario, Canada; Laura and Isaac Perlmutter Cancer Center, New York University Langone Medical Center, New York, New York. Electronic address:

High-grade serous ovarian cancer (HGSC) is the leading cause of morbidity and mortality from gynecologic malignant tumors. Overall survival remains low because of the nearly ubiquitous emergence of platinum resistance and the paucity of effective next-line treatments. Current cell culture-based models show limited similarity to HGSC and are therefore unreliable predictive models for preclinical evaluation of investigational drugs. This deficiency could help explain the low overall rate of successful drug development and the decades of largely unchanged approaches to HGSC treatment. We used gene expression, copy number variation, and exome sequencing analyses to credential HGSC patient-derived xenografts (PDXs) as effective preclinical models that recapitulate the features of human HGSC. Mice bearing PDXs were also treated with standard-of-care carboplatin therapy. PDXs showed similar sensitivity to carboplatin as the patient's tumor at the time of sampling. PDXs also recapitulated the diversity of genomic alterations (copy number variation and mutation profiles) previously described in large data sets that profiled HGSC. Furthermore, mRNA profiling showed that the PDXs represent all HGSC subtypes with the exception of the immunoreactive group. Credentialing of PDX models of HGSC should aid progress in HGSC research by providing improved preclinical models of HGSC that can be used to test novel targets and more accurately evaluate their likelihood of success.
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http://dx.doi.org/10.1016/j.ajpath.2018.01.019DOI Listing
May 2018