Publications by authors named "Reena Shakya"

31 Publications

Intein-mediated cytoplasmic reconstitution of a split toxin enables selective cell ablation in mixed populations and tumor xenografts.

Proc Natl Acad Sci U S A 2020 09 24;117(36):22090-22100. Epub 2020 Aug 24.

Department of Chemistry and Biochemistry, The Ohio State University, Columbus, OH 43210;

The application of proteinaceous toxins for cell ablation is limited by their high on- and off-target toxicity, severe side effects, and a narrow therapeutic window. The selectivity of targeting can be improved by intein-based toxin reconstitution from two dysfunctional fragments provided their cytoplasmic delivery via independent, selective pathways. While the reconstitution of proteins from genetically encoded elements has been explored, exploiting cell-surface receptors for boosting selectivity has not been attained. We designed a robust splitting algorithm and achieved reliable cytoplasmic reconstitution of functional diphtheria toxin from engineered intein-flanked fragments upon receptor-mediated delivery of one of them to the cells expressing the counterpart. Retargeting the delivery machinery toward different receptors overexpressed in cancer cells enables selective ablation of specific subpopulations in mixed cell cultures. In a mouse model, the transmembrane delivery of a split-toxin construct potently inhibits the growth of xenograft tumors expressing the split counterpart. Receptor-mediated delivery of engineered split proteins provides a platform for precise therapeutic and experimental ablation of tumors or desired cell populations while also greatly expanding the applicability of the intein-based protein transsplicing.
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http://dx.doi.org/10.1073/pnas.2006603117DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7486740PMC
September 2020

Ablation of the Brca1-Palb2 Interaction Phenocopies Fanconi Anemia in Mice.

Cancer Res 2020 10 30;80(19):4172-4184. Epub 2020 Jul 30.

Department of Cancer Biology and Genetics, The Ohio State University Comprehensive Cancer Center, Columbus, Ohio.

Heterozygous mutations in the gene predispose women to breast and ovarian cancer, while biallelic BRCA1 mutations are a cause of Fanconi anemia (FA), a rare genetic disorder characterized by developmental abnormalities, early-onset bone marrow failure, increased risk of cancers, and hypersensitivity to DNA-crosslinking agents. BRCA1 is critical for homologous recombination of DNA double-strand breaks (DSB). Through its coiled-coil domain, BRCA1 interacts with an essential partner, PALB2, recruiting BRCA2 and RAD51 to sites of DNA damage. Missense mutations within the coiled-coil domain of BRCA1 (e.g., L1407P) that affect the interaction with PALB2 have been reported in familial breast cancer. We hypothesized that if PALB2 regulates or mediates BRCA1 tumor suppressor function, ablation of the BRCA1-PALB2 interaction may also elicit genomic instability and tumor susceptibility. We generated mice defective for the Brca1-Palb2 interaction (Brca1 L1363P in mice) and established MEF cells from these mice. MEF exhibited hypersensitivity to DNA-damaging agents and failed to recruit Rad51 to DSB. mice were viable but exhibited various FA symptoms including growth retardation, hyperpigmentation, skeletal abnormalities, and male/female infertility. Furthermore, all mice exhibited macrocytosis and died due to bone marrow failure or lymphoblastic lymphoma/leukemia with activating Notch1 mutations. These phenotypes closely recapitulate clinical features observed in patients with FA. Collectively, this model effectively demonstrates the significance of the BRCA1-PALB2 interaction in genome integrity and provides an FA model to investigate hematopoietic stem cells for mechanisms underlying progressive failure of hematopoiesis and associated development of leukemia/lymphoma, and other FA phenotypes. SIGNIFICANCE: A new Brca1 mouse model for Fanconi anemia (FA) complementation group S provides a system in which to study phenotypes observed in human FA patients including bone marrow failure..
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http://dx.doi.org/10.1158/0008-5472.CAN-20-0486DOI Listing
October 2020

CD200 promotes immunosuppression in the pancreatic tumor microenvironment.

J Immunother Cancer 2020 06 23;8(1). Epub 2020 Jun 23.

The James Comprehensive Cancer Center, The Ohio State University, Columbus, Ohio, United States

Background: A significant challenge to overcome in pancreatic ductal adenocarcinoma (PDAC) is the profound systemic immunosuppression that renders this disease non-responsive to immunotherapy. Our supporting data provide evidence that CD200, a regulator of myeloid cell activity, is expressed in the PDAC microenvironment. Additionally, myeloid-derived suppressor cells (MDSC) isolated from patients with PDAC express elevated levels of the CD200 receptor (CD200R). Thus, we hypothesize that CD200 expression in the PDAC microenvironment limits responses to immunotherapy by promoting expansion and activity of MDSC.

Methods: Immunofluorescent staining was used to determine expression of CD200 in murine and human PDAC tissue. Flow cytometry was utilized to test for CD200R expression by immune populations in patient blood samples. In vivo antibody blocking of CD200 was conducted in subcutaneous MT-5 tumor-bearing mice and in a genetically engineered PDAC model (KPC-Brca2 mice). Peripheral blood mononuclear cells (PBMC) from patients with PDAC were analyzed by single-cell RNA sequencing. MDSC expansion assays were completed using healthy donor PBMC stimulated with IL-6/GM-CSF in the presence of recombinant CD200 protein.

Results: We found expression of CD200 by human pancreatic cell lines (BxPC3, MiaPaca2, and PANC-1) as well as on primary epithelial pancreatic tumor cells and smooth muscle actin+ stromal cells. CD200R expression was found to be elevated on CD11b+CD33+HLA-DR MDSC immune populations from patients with PDAC (p=0.0106). Higher expression levels of CD200R were observed in CD15+ MDSC compared with CD14+ MDSC (p<0.001). In vivo studies demonstrated that CD200 antibody blockade limited tumor progression in MT-5 subcutaneous tumor-bearing and in KPC-Brca2 mice (p<0.05). The percentage of intratumoral MDSC was significantly reduced in anti-CD200 treated mice compared with controls. Additionally, in vivo blockade of CD200 can also significantly enhance the efficacy of PD-1 checkpoint antibodies compared with single antibody therapies (p<0.05). Single-cell RNA sequencing of PBMC from patients revealed that CD200R+ MDSC expressed genes involved in cytokine signaling and MDSC expansion. Further, in vitro cytokine-driven expansion and the suppressive activity of human MDSC was enhanced when cocultured with recombinant CD200 protein.

Conclusions: These results indicate that CD200 expression in the PDAC microenvironment may regulate MDSC expansion and that targeting CD200 may enhance activity of checkpoint immunotherapy.
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http://dx.doi.org/10.1136/jitc-2019-000189DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7312341PMC
June 2020

Modeling Brain Metastases Through Intracranial Injection and Magnetic Resonance Imaging.

J Vis Exp 2020 06 7(160). Epub 2020 Jun 7.

The Comprehensive Cancer Center, The Ohio State University; Department of Radiation Oncology, The Ohio State University;

Metastatic spread of cancer is an unfortunate consequence of disease progression, aggressive cancer subtypes, and/or late diagnosis. Brain metastases are particularly devastating, difficult to treat, and confer a poor prognosis. While the precise incidence of brain metastases in the United States remains hard to estimate, it is likely to increase as extracranial therapies continue to become more efficacious in treating cancer. Thus, it is necessary to identify and develop novel therapeutic approaches to treat metastasis at this site. To this end, intracranial injection of cancer cells has become a well-established method in which to model brain metastasis. Previously, the inability to directly measure tumor growth has been a technical hindrance to this model; however, increasing availability and quality of small animal imaging modalities, such as magnetic resonance imaging (MRI), are vastly improving the ability to monitor tumor growth over time and infer changes within the brain during the experimental period. Herein, intracranial injection of murine mammary tumor cells into immunocompetent mice followed by MRI is demonstrated. The presented injection approach utilizes isoflurane anesthesia and a stereotactic setup with a digitally controlled, automated drill and needle injection to enhance precision, and reduce technical error. MRI is measured over time using a 9.4 Tesla instrument in The Ohio State University James Comprehensive Cancer Center Small Animal Imaging Shared Resource. Tumor volume measurements are demonstrated at each time point through use of ImageJ. Overall, this intracranial injection approach allows for precise injection, day-to-day monitoring, and accurate tumor volume measurements, which combined greatly enhance the utility of this model system to test novel hypotheses on the drivers of brain metastases.
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http://dx.doi.org/10.3791/61272DOI Listing
June 2020

Murine models for familial pancreatic cancer: Histopathology, latency and drug sensitivity among cancers of Palb2, Brca1 and Brca2 mutant mouse strains.

PLoS One 2019 26;14(12):e0226714. Epub 2019 Dec 26.

Department of Cancer Biology and Genetics, The Ohio State University Comprehensive Cancer Center, Columbus, Ohio, United States of America.

Alterations of the PALB2 tumor suppressor gene have been identified in familial breast, ovarian and pancreatic cancer cases. PALB2 cooperates with BRCA1/2 proteins through physical interaction in initiation of homologous recombination, in maintenance of genome integrity following DNA double-strand breaks. To determine if the role of PALB2 as a linker between BRCA1 and BRCA2 is critical for BRCA1/2-mediated tumor suppression, we generated Palb2 mouse pancreatic cancer models and compared tumor latencies, phenotypes and drug responses with previously generated Brca1/2 pancreatic cancer models. For development of Palb2 pancreatic cancer, we crossed conditional Palb2 null mouse with mice carrying the KrasG12D; p53R270H; Pdx1-Cre (KPC) constructs, and these animals were observed for pancreatic tumor development. Individual deletion of Palb2, Brca1 or Brca2 genes in pancreas per se using Pdx1-Cre was insufficient to cause tumors, but it reduced pancreata size. Concurrent expression of mutant KrasG12D and p53R270H, with tumor suppressor inactivated strains in Palb2-KPC, Brca1-KPC or Brca2-KPC, accelerated pancreatic ductal adenocarcinoma (PDAC) development. Moreover, most Brca1-KPC and some Palb2-KPC animals developed mucinous cystic neoplasms with PDAC, while Brca2-KPC and KPC animals did not. 26% of Palb2-KPC mice developed MCNs in pancreata, which resemble closely the Brca1 deficient tumors. However, the remaining 74% of Palb2-KPC animals developed PDACs without any cysts like Brca2 deficient tumors. In addition, the number of ADM lesions and immune cells infiltrations (CD3+ and F/480+) were significantly increased in Brca1-KPC tumors, but not in Brca2-KPC tumors. Interestingly, the level of ADM lesions and infiltration of CD3+ or F/480+ cells in Palb2-KPC tumors were intermediate between Brca1-KPC and Brca2-KPC tumors. As expected, disruption of Palb2 and Brca1/2 sensitized tumor cells to DNA damaging agents in vitro and in vivo. Altogether, Palb2-KPC PDAC exhibited features observed in both Brca1-KPC and Brca2-KPC tumors, which could be due to its role, as a linker between Brca1 and Brca2.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0226714PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6932818PMC
April 2020

Inhibition of histone deacetylase 2 reduces MDM2 expression and reduces tumor growth in dedifferentiated liposarcoma.

Oncotarget 2019 Oct 1;10(55):5671-5679. Epub 2019 Oct 1.

Department of Biomedical Informatics, The Ohio State University, Columbus, Ohio, USA.

Dedifferentiated liposarcoma (DDLPS) is a highly morbid mesenchymal tumor characterized and driven by genomic amplification of the gene. Direct inhibition of MDM2 has shown promise pre-clinically, but has yet to be validated in clinical trials. Early studies have demonstrated that pan-histone deacetylase (HDAC) inhibition may have anti-MDM2 effects. Here we present , , and mouse xenograft studies that suggest that specifically targeting HDAC2 reduces MDM2 expression and has anti-tumor affects in DDLPS. Two independent datasets, The Cancer Genome Atlas (TCGA; = 58) and the Memorial Sloan-Kettering Cancer Center Dataset (MSKCC; = 63), were used to identify the co-expression between class I and , and their clinical impact. 2 was highly co-expressed with (TCGA: Spearman's coefficient = 0.29, = 0.03; MSKCC: Spearman's coefficient = 0.57, < 0.001). As both a continuous and dichotomous predictor, elevated expression was associated with worsened disease-free survival in the TCGA (Continuous: Hazard-ratio (HR) 1.7; 95% Confidence Interval (95%CI) 0.97-2.9; = 0.06; Dichotomous: HR 7.1, 95%CI 2.5-19.8, < 0.001) and distant recurrence-free survival in the MSKCC (Continuous: HR 2.2; 95%CI 1.1-4.8; = 0.04; Dichotomous: HR 2.8, 95%CI 1.2-6.4, = 0.02). , treatment of DDLPS cell lines with the HDAC inhibitors MI-192 (HDAC2/3 inhibitor) or romidepsin (HDAC1/2 inhibitor) reduced expression and induced apoptosis. In a murine DDLPS xenograft model, romidepsin reduced tumor growth and lowered tumor MDM2 expression. RNA-sequencing of romidepsin treated mouse tumors demonstrated markers of TP53 reactivation. Taken together, our data supports the hypothesis that targeting HDAC2 may represent a potential strategy to modulate MDM2 expression in DDLPS.
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http://dx.doi.org/10.18632/oncotarget.27144DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6779286PMC
October 2019

Modeling Human Cancer-induced Cachexia.

Cell Rep 2019 08;28(6):1612-1622.e4

Arthur G. James Comprehensive Cancer Center Cancer Cachexia Program, The Ohio State University, Columbus, OH 43210, USA; Department of Pediatrics, Medical University of South Carolina, Charleston, SC 29425, USA; Hollings Cancer Center, Medical University of South Carolina, Charleston, SC 29425, USA. Electronic address:

Cachexia is a wasting syndrome characterized by pronounced skeletal muscle loss. In cancer, cachexia is associated with increased morbidity and mortality and decreased treatment tolerance. Although advances have been made in understanding the mechanisms of cachexia, translating these advances to the clinic has been challenging. One reason for this shortcoming may be the current animal models, which fail to fully recapitulate the etiology of human cancer-induced tissue wasting. Because pancreatic ductal adenocarcinoma (PDA) presents with a high incidence of cachexia, we engineered a mouse model of PDA that we named KPP. KPP mice, similar to PDA patients, progressively lose skeletal and adipose mass as a consequence of their tumors. In addition, KPP muscles exhibit a similar gene ontology as cachectic patients. We envision that the KPP model will be a useful resource for advancing our mechanistic understanding and ability to treat cancer cachexia.
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http://dx.doi.org/10.1016/j.celrep.2019.07.016DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6733019PMC
August 2019

Degree of Amplification Affects Clinical Outcomes in Dedifferentiated Liposarcoma.

Oncologist 2019 07 24;24(7):989-996. Epub 2019 Apr 24.

Division of Medical Oncology, Department of Internal Medicine, The Ohio State University, Columbus, Ohio, USA

Background: Dedifferentiated liposarcomas (DDLPS) are mesenchymal tumors associated with universally poor response to treatment. Genomic amplification of murine double minute 2 () is used as a diagnostic biomarker; however, no established biomarkers exist to guide DDLPS treatment. In the largest study of its kind, we report that the extent of amplification, not simply the presence of amplification, may be biologically important to the actions of DDLPS.

Patients And Methods: The distribution of amplification in DDLPS was assessed using data from a commercial sequencing laboratory ( = 642) and The Cancer Genome Atlas ( = 57). Data from two retrospective clinical trials ( = 15, = 16) and one prospective clinical trial ( = 25) were used to test 's utility as a clinical biomarker. in vitro and in vivo assessments were conducted in DDLPS cell lines.

Results: Genomic amplification follows a highly reproducible log-normal distribution. In patients with DDLPS treated with complete tumor resection, elevated was associated with shortened time to recurrence as measured by genomic amplification ( = .003) and mRNA expression ( = .04). In patients requiring systemic therapy, higher amplification was associated with reduced overall survival ( = .04). Doxorubicin treatment of DDLPS cells in vitro demonstrated variable sensitivity based on baseline levels, and doxorubicin treatment elevated MDM2 expression. In vivo, treatment with doxorubicin followed by an MDM2 inhibitor improved doxorubicin sensitivity.

Conclusion: amplification levels in DDLPS follow a reproducible distribution and are associated with clinical outcomes and drug sensitivity. These results suggest that a prospective study of as a predictive biomarker in DDLPS is warranted.

Implications For Practice: No validated biomarkers exist for treatment selection in dedifferentiated liposarcoma (DDLPS). Although murine double minute 2 () is currently used for diagnosis, the clinical relevance of amplification has yet to be fully assessed. This study found that amplification follows a predictable distribution in DDLPS and correlates with clinical and biological outcomes. These data suggests that amplification may be a useful biomarker in DDLPS.
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http://dx.doi.org/10.1634/theoncologist.2019-0047DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6656442PMC
July 2019

Synergy from gene expression and network mining (SynGeNet) method predicts synergistic drug combinations for diverse melanoma genomic subtypes.

NPJ Syst Biol Appl 2019 26;5. Epub 2019 Feb 26.

Institute for Informatics, Washington University in St. Louis, St. Louis, MO, USA.

Systems biology perspectives are crucial for understanding the pathophysiology of complex diseases, and therefore hold great promise for the discovery of novel treatment strategies. Drug combinations have been shown to improve durability and reduce resistance to available first-line therapies in a variety of cancers; however, traditional drug discovery approaches are prohibitively cost and labor-intensive to evaluate large-scale matrices of potential drug combinations. Computational methods are needed to efficiently model complex interactions of drug target pathways and identify mechanisms underlying drug combination synergy. In this study, we employ a computational approach, SynGeNet (Synergy from Gene expression and Network mining), which integrates transcriptomics-based connectivity mapping and network centrality analysis to analyze disease networks and predict drug combinations. As an exemplar of a disease in which combination therapies demonstrate efficacy in genomic-specific contexts, we investigate malignant melanoma. We employed SynGeNet to generate drug combination predictions for each of the four major genomic subtypes of melanoma (BRAF, NRAS, NF1, and triple wild type) using publicly available gene expression and mutation data. We validated synergistic drug combinations predicted by our method across all genomic subtypes using results from a high-throughput drug screening study across. Finally, we prospectively validated the drug combination for -mutant melanoma that was top ranked by our approach, vemurafenib (BRAF inhibitor) + tretinoin (retinoic acid receptor agonist), using both in vitro and in vivo models of -mutant melanoma and RNA-sequencing analysis of drug-treated melanoma cells to validate the predicted mechanisms. Our approach is applicable to a wide range of disease domains, and, importantly, can model disease-relevant protein subnetworks in precision medicine contexts.
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http://dx.doi.org/10.1038/s41540-019-0085-4DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6391384PMC
April 2020

Loss of TXNIP enhances peritoneal metastasis and can be abrogated by dual TORC1/2 inhibition.

Oncotarget 2018 Nov 2;9(86):35676-35686. Epub 2018 Nov 2.

Department of Biomedical Informatics, Division of Bioinformatics, The Ohio State University, Columbus, OH 43210, USA.

Peritoneal metastasis (PM) is a debilitating consequence of multiple cancers. As cancer cells lose tonic signaling related to attachment dependence, critical morphologic shifts result in alteration of the transcriptome. Identifying key genes associated with this transformation may lead to targeted therapies for this devastating complication. TC71, CHLA9, PANC1, YOU and HEYA8 cell lines were grown as tumor spheroids in polyHEMA coated plates. Temporal profiling of transcriptomic alterations over 72 hrs was used to develop a comprehensive PM model. We identified transcriptomic outliers using Gaussian mixtures model clustering to identify drivers of spheroid formation. Outliers were validated in The Cancer Genome Atlas (TCGA) and an ovarian tissue microarray (TMA) and by modulation in ovarian cancer models and in peritoneal xenograft models. Outlier analysis of PM genes identified the gene TXNIP and the TORC signaling as central to PM. Ovarian cancer spheroids isolated from patient ascites had significantly higher TXNIP than their attached counterparts ( = 0.047). TXNIP levels predicted progression-free (log-rank = 0.026) survival in stage 1/2 ovarian cancer and overall survival (log rank = 0.047) in stage 3/4 ovarian cancer. , TXNIP silencing was associated with increased mTOR signaling and enhanced spheroid development which could be overcome by TAK228, a TORC1/2 inhibitor. Similarly, peritoneal xenograft models of carcinomatosis were prevented by TAK228. PM is driven by TXNIP-associated TORC1/2 signaling. This work provides the first evidence that TORC1/2 inhibition may prevent PM.
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http://dx.doi.org/10.18632/oncotarget.26281DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6235015PMC
November 2018

MAX Mutations in Endometrial Cancer: Clinicopathologic Associations and Recurrent MAX p.His28Arg Functional Characterization.

J Natl Cancer Inst 2018 05;110(5):517-526

James Comprehensive Cancer Center.

Background: Genomic studies have revealed that multiple genes are mutated at varying frequency in endometrial cancer (EC); however, the relevance of many of these mutations is poorly understood. An EC-specific recurrent mutation in the MAX transcription factor p.His28Arg was recently discovered. We sought to assess the functional consequences of this hotspot mutation and determine its association with cancer-relevant phenotypes.

Methods: MAX was sequenced in 509 endometrioid ECs, and associations between mutation status and clinicopathologic features were assessed. EC cell lines stably expressing MAXH28R were established and used for functional experiments. DNA binding was examined using electrophoretic mobility shift assays and chromatin immunoprecipitation. Transcriptional profiling was performed with microarrays. Murine flank (six to 11 mice per group) and intraperitoneal tumor models were used for in vivo studies. Vascularity of xenografts was assessed by MECA-32 immunohistochemistry. The paracrine pro-angiogenic nature of MAXH28R-expressing EC cells was tested using microfluidic HUVEC sprouting assays and VEGFA enzyme-linked immunosorbent assays. All statistical tests were two-sided.

Results: Twenty-two of 509 tumors harbored mutations in MAX, including 12 tumors with the p.His28Arg mutation. Patients with a MAX mutation had statistically significantly reduced recurrence-free survival (hazard ratio = 4.00, 95% confidence interval = 1.15 to 13.91, P = .03). MAXH28R increased affinity for canonical E-box sequences, and MAXH28R-expressing EC cells dramatically altered transcriptional profiles. MAXH28R-derived xenografts statistically significantly increased vascular area compared with MAXWT and empty vector tumors (P = .003 and P = .008, respectively). MAXH28R-expressing EC cells secreted nearly double the levels of VEGFA compared with MAXWT cells (P = .03, .005, and .005 at 24, 48, and 72 hours, respectively), and conditioned media from MAXH28R cells increased sprouting when applied to HUVECs.

Conclusion: These data highlight the importance of MAX mutations in EC and point to increased vascularity as one mechanism contributing to clinical aggressiveness of EC.
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http://dx.doi.org/10.1093/jnci/djx238DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6279289PMC
May 2018

Sex as a biological variable in response to temozolomide.

Neuro Oncol 2017 06;19(6):873-874

Department of Neurological Surgery, Dardinger Laboratory for Neuro-oncology and Neurosciences, The Ohio State University Wexner Medical Center, Columbus, Ohio, USA.

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http://dx.doi.org/10.1093/neuonc/nox040DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5464457PMC
June 2017

The Exportin-1 Inhibitor Selinexor Exerts Superior Antitumor Activity when Combined with T-Cell Checkpoint Inhibitors.

Mol Cancer Ther 2017 03 1;16(3):417-427. Epub 2017 Feb 1.

Department of Hematology and Medical Oncology, Winship Cancer Institute of Emory University, Atlanta, Georgia.

Selinexor, a selective inhibitor of nuclear export (SINE) compound targeting exportin-1, has previously been shown to inhibit melanoma cell growth We hypothesized that combining selinexor with antibodies that block or disrupt T-cell checkpoint molecule signaling would exert superior antimelanoma activity. , selinexor increased and gene expression in leukocytes and induced gene expression in human melanoma cell lines. Mice bearing syngeneic B16F10 melanoma tumors demonstrated a significant reduction in tumor growth rate in response to the combination of selinexor and anti-PD-1 or anti-PD-L1 antibodies ( < 0.05). Similar results were obtained in B16F10-bearing mice treated with selinexor combined with anti-CTLA4 antibody. Immunophenotypic analysis of splenocytes by flow cytometry revealed that selinexor alone or in combination with anti-PD-L1 antibody significantly increased the frequency of both natural killer cells ( ≤ 0.050) and CD4 T cells with a Th1 phenotype ( ≤ 0.050). Further experiments indicated that the antitumor effect of selinexor in combination with anti-PD-1 therapy persisted under an alternative dosing schedule but was lost when selinexor was administered daily. These data indicate that the efficacy of selinexor against melanoma may be enhanced by disrupting immune checkpoint activity. .
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http://dx.doi.org/10.1158/1535-7163.MCT-16-0498DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5407496PMC
March 2017

Stromal Senescence By Prolonged CDK4/6 Inhibition Potentiates Tumor Growth.

Mol Cancer Res 2017 03 30;15(3):237-249. Epub 2016 Dec 30.

Department of Molecular Genetics, The Ohio State University, Columbus, Ohio.

Senescent cells within the tumor microenvironment (TME) adopt a proinflammatory, senescence-associated secretory phenotype (SASP) that promotes cancer initiation, progression, and therapeutic resistance. Here, exposure to palbociclib (PD-0332991), a CDK4/6 inhibitor, induces senescence and a robust SASP in normal fibroblasts. Senescence caused by prolonged CDK4/6 inhibition is DNA damage-independent and associated with Mdm2 downregulation, whereas the SASP elicited by these cells is largely reliant upon NF-κB activation. Based upon these observations, it was hypothesized that the exposure of nontransformed stromal cells to PD-0332991 would promote tumor growth. Ongoing clinical trials of CDK4/6 inhibitors in melanoma prompted a validation of this hypothesis using a suite of genetically defined melanoma cells (i.e., mutant, mutant, and wild-type). When cultured in the presence of CDK4/6i-induced senescent fibroblasts, melanoma cell lines exhibited genotype-dependent proliferative responses. However, , PD-0332991-treated fibroblasts enhanced the growth of all melanoma lines tested and promoted the recruitment of Gr-1-positive immune cells. These data indicate that prolonged CDK4/6 inhibitor treatment causes normal fibroblasts to enter senescence and adopt a robust SASP. Such senescent cells suppress the antitumor immune response and promote melanoma growth in immunocompetent, models. The ability of prolonged CDK4/6 inhibitor treatment to induce cellular senescence and a robust SASP in primary cells may hinder therapeutic efficacy and promote long-term, gerontogenic consequences that should be considered in clinical trials aiming to treat melanoma and other cancer types. .
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http://dx.doi.org/10.1158/1541-7786.MCR-16-0319DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5334447PMC
March 2017

IL-6 and PD-L1 antibody blockade combination therapy reduces tumour progression in murine models of pancreatic cancer.

Gut 2018 02 21;67(2):320-332. Epub 2016 Oct 21.

Department of Hematology and Medical Oncology, Winship Cancer Institute of Emory University, Atlanta, Georgia, USA.

Objective: Limited efficacy of immune checkpoint inhibitors in pancreatic ductal adenocarcinoma (PDAC) has prompted investigation into combination therapy. We hypothesised that interleukin 6 (IL-6) blockade would modulate immunological features of PDAC and enhance the efficacy of anti-programmed death-1-ligand 1 (PD-L1) checkpoint inhibitor therapy.

Design: Transcription profiles and IL-6 secretion from primary patient-derived pancreatic stellate cells (PSCs) were analyzed via Nanostring and immunohistochemistry, respectively. In vivo efficacy and mechanistic studies were conducted with antibodies (Abs) targeting IL-6, PD-L1, CD4 or CD8 in subcutaneous or orthotopic models using Panc02, MT5 or KPC-luc cell lines; and the aggressive, genetically engineered PDAC model (Kras, Trp53, Pdx1-cre, Brca2 (KPC-Brca2 mice)). Systemic and local changes in immunophenotype were measured by flow cytometry or immunohistochemical analysis.

Results: PSCs (n=12) demonstrated prominent IL-6 expression, which was localised to stroma of tumours. Combined IL-6 and PD-L1 blockade elicited efficacy in mice bearing subcutaneous MT5 (p<0.02) and Panc02 tumours (p=0.046), which was accompanied by increased intratumoural effector T lymphocytes (CD62LCD44). CD8-depleting but not CD4-depleting Abs abrogated the efficacy of combined IL-6 and PD-L1 blockade in mice bearing Panc02 tumours (p=0.0016). This treatment combination also elicited significant antitumour activity in mice bearing orthotopic KPC-luc tumours and limited tumour progression in KPC-Brca2 mice (p<0.001). Histological analysis revealed increased T-cell infiltration and reduced α-smooth muscle actin cells in tumours from multiple models. Finally, IL-6 and PD-L1 blockade increased overall survival in KPC-Brca2 mice compared with isotype controls (p=0.0012).

Conclusions: These preclinical results indicate that targeted inhibition of IL-6 may enhance the efficacy of anti-PD-L1 in PDAC.
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http://dx.doi.org/10.1136/gutjnl-2016-311585DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5406266PMC
February 2018

Stromal ETS2 Regulates Chemokine Production and Immune Cell Recruitment during Acinar-to-Ductal Metaplasia.

Neoplasia 2016 09;18(9):541-52

Comprehensive Cancer Center, The Ohio State University, Columbus, OH 43210, USA; Department of Cancer Biology & Genetics, The Ohio State University, Columbus, OH 43210, USA. Electronic address:

Preclinical studies have suggested that the pancreatic tumor microenvironment both inhibits and promotes tumor development and growth. Here we establish the role of stromal fibroblasts during acinar-to-ductal metaplasia (ADM), an initiating event in pancreatic cancer formation. The transcription factor V-Ets avian erythroblastosis virus E26 oncogene homolog 2 (ETS2) was elevated in smooth muscle actin-positive fibroblasts in the stroma of pancreatic ductal adenocarcinoma (PDAC) patient tissue samples relative to normal pancreatic controls. LSL-Kras(G12D/+); LSL-Trp53(R172H/+); Pdx-1-Cre (KPC) mice showed that ETS2 expression initially increased in fibroblasts during ADM and remained elevated through progression to PDAC. Conditional ablation of Ets-2 in pancreatic fibroblasts in a Kras(G12D)-driven mouse ADM model decreased the amount of ADM events. ADMs from fibroblast Ets-2-deleted animals had reduced epithelial cell proliferation and increased apoptosis. Surprisingly, fibroblast Ets-2 deletion significantly altered immune cell infiltration into the stroma, with an increased CD8+ T-cell population, and decreased presence of regulatory T cells (Tregs), myeloid-derived suppressor cells, and mature macrophages. The mechanism involved ETS2-dependent chemokine ligand production in fibroblasts. ETS2 directly bound to regulatory sequences for Ccl3, Ccl4, Cxcl4, Cxcl5, and Cxcl10, a group of chemokines that act as potent mediators of immune cell recruitment. These results suggest an unappreciated role for ETS2 in fibroblasts in establishing an immune-suppressive microenvironment in response to oncogenic Kras(G12D) signaling during the initial stages of tumor development.
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http://dx.doi.org/10.1016/j.neo.2016.07.006DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5031867PMC
September 2016

Genetic ablation of Smoothened in pancreatic fibroblasts increases acinar-ductal metaplasia.

Genes Dev 2016 09 15;30(17):1943-55. Epub 2016 Sep 15.

Comprehensive Cancer Center, The Ohio State University, Columbus, Ohio 43210, USA; Cancer Biology and Genetics Department, The Ohio State University, Columbus, Ohio 43210, USA;

The contribution of the microenvironment to pancreatic acinar-to-ductal metaplasia (ADM), a preneoplastic transition in oncogenic Kras-driven pancreatic cancer progression, is currently unclear. Here we show that disruption of paracrine Hedgehog signaling via genetic ablation of Smoothened (Smo) in stromal fibroblasts in a Kras(G12D) mouse model increased ADM. Smo-deleted fibroblasts had higher expression of transforming growth factor-α (Tgfa) mRNA and secreted higher levels of TGFα, leading to activation of EGFR signaling in acinar cells and increased ADM. The mechanism involved activation of AKT and noncanonical activation of the GLI family transcription factor GLI2. GLI2 was phosphorylated at Ser230 in an AKT-dependent fashion and directly regulated Tgfa expression in fibroblasts lacking Smo Additionally, Smo-deleted fibroblasts stimulated the growth of Kras(G12D)/Tp53(R172H) pancreatic tumor cells in vivo and in vitro. These results define a non-cell-autonomous mechanism modulating Kras(G12D)-driven ADM that is balanced by cross-talk between Hedgehog/SMO and AKT/GLI2 pathways in stromal fibroblasts.
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http://dx.doi.org/10.1101/gad.283499.116DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5066238PMC
September 2016

The DNA resection protein CtIP promotes mammary tumorigenesis.

Oncotarget 2016 May;7(22):32172-83

Institute for Cancer Genetics, Department of Pathology and Cell Biology, and Herbert Irving Comprehensive Cancer Center, Columbia University Medical Center, New York, NY 10032, USA.

Many DNA repair factors act to suppress tumor formation by preserving genomic stability. Similarly, the CtIP protein, which interacts with the BRCA1 tumor suppressor, is also thought to have tumor suppression activity. Through its role in DNA end resection, CtIP facilitates DNA double-strand break (DSB) repair by homologous recombination (DSBR-HR) and microhomology-mediated end joining (MMEJ). In addition, however, CtIP has also been implicated in the formation of aberrant chromosomal rearrangements in an MMEJ-dependent manner, an activity that could potentially promote tumor development by increasing genome instability. To clarify whether CtIP acts in vivo to suppress or promote tumorigenesis, we have examined its oncogenic potential in mouse models of human breast cancer. Surprisingly, mice heterozygous for a null Ctip allele did not display an increased susceptibility to tumor formation. Moreover, mammary-specific biallelic CtIP ablation did not elicit breast tumors in a manner reminiscent of BRCA1 loss. Instead, CtIP inactivation dramatically reduced the kinetics of mammary tumorigenesis in mice bearing mammary-specific lesions of the p53 gene. Thus, unlike other repair factors, CtIP is not a tumor suppressor, but has oncogenic properties that can promote tumorigenesis, consistent with its ability to facilitate MMEJ-dependent chromosomal instability. Consequently, inhibition of CtIP-mediated MMEJ may prove effective against tumor types, such as human breast cancer, that display MMEJ-dependent chromosomal rearrangements.
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http://dx.doi.org/10.18632/oncotarget.8605DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5078005PMC
May 2016

Single agent BMS-911543 Jak2 inhibitor has distinct inhibitory effects on STAT5 signaling in genetically engineered mice with pancreatic cancer.

Oncotarget 2015 Dec;6(42):44509-22

Division of Medical Oncology, Department of Internal Medicine, The Arthur G. James Cancer Hospital and Richard J. Solove Research Institute, The Ohio State University, Columbus, OH 43210, USA.

The Jak/STAT pathway is activated in human pancreatic ductal adenocarcinoma (PDAC) and cooperates with mutant Kras to drive initiation and progression of PDAC in murine models. We hypothesized that the small-molecule Jak2 inhibitor (BMS-911543) would elicit anti-tumor activity against PDAC and decrease immune suppressive features of the disease. We used an aggressive genetically engineered PDAC model with mutant KrasG12D, tp53R270H, and Brca1 alleles (KPC-Brca1 mice). Mice with confirmed tumor burden were treated orally with vehicle or 30 mg/kg BMS-911543 daily for 14 days. Histologic analysis of pancreata from treated mice revealed fewer foci of adenocarcinoma and significantly decreased Ki67+ cells versus controls. In vivo administration of BMS-911543 significantly reduced pSTAT5 and FoxP3 positive cells within the pancreas, but did not alter STAT3 phosphorylation. Continuous dosing of KPC-Brca1 mice with BMS-911543 resulted in a median survival of 108 days, as compared to a median survival of 87 days in vehicle treated animals, a 23% increase (p = 0.055). In vitro experiments demonstrated that PDAC cell lines were poorly sensitive to BMS-911543, requiring high micromolar concentrations to achieve targeted inhibition of Jak/STAT signaling. Similarly, BMS-911543 had little in vitro effect on the viability of both murine and human PDAC-derived stellate cell lines. However, BMS-911543 potently inhibited phosphorylation of pSTAT3 and pSTAT5 at low micromolar doses in human PBMC and reduced in vitro differentiation of Foxp3+ T regulatory cells. These results indicate that single agent Jak2i deserves further study in preclinical models of PDAC and has distinct inhibitory effects on STAT5 mediated signaling.
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http://dx.doi.org/10.18632/oncotarget.6332DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4792572PMC
December 2015

The transcriptional regulatory network of proneural glioma determines the genetic alterations selected during tumor progression.

Cancer Res 2014 Mar 3;74(5):1440-1451. Epub 2014 Jan 3.

Gabriele Bartoli Brain Tumor Laboratory, Department of Neurosurgery, Irving Research Cancer Center, Columbia University Medical Center, New York, NY.

Proneural glioblastoma is defined by an expression pattern resembling that of oligodendrocyte progenitor cells and carries a distinctive set of genetic alterations. Whether there is a functional relationship between the proneural phenotype and the associated genetic alterations is unknown. To evaluate this possible relationship, we performed a longitudinal molecular characterization of tumor progression in a mouse model of proneural glioma. In this setting, the tumors acquired remarkably consistent genetic deletions at late stages of progression, similar to those deleted in human proneural glioblastoma. Further investigations revealed that p53 is a master regulator of the transcriptional network underlying the proneural phenotype. This p53-centric transcriptional network and its associated phenotype were observed at both the early and late stages of progression, and preceded the proneural-specific deletions. Remarkably, deletion of p53 at the time of tumor initiation obviated the acquisition of later deletions, establishing a link between the proneural transcriptional network and the subtype-specific deletions selected during glioma progression.
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http://dx.doi.org/10.1158/0008-5472.CAN-13-2150DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3981545PMC
March 2014

High prevalence of BRCA1 and BRCA2 germline mutations with loss of heterozygosity in a series of resected pancreatic adenocarcinoma and other neoplastic lesions.

Clin Cancer Res 2013 Jul 8;19(13):3396-403. Epub 2013 May 8.

Henry D. Janowitz Division of Gastroenterology, Icahn School of Medicine at Mount Sinai, New York, NY 10029, USA.

Purpose: Pancreatic ductal adenocarcinoma (PDAC) is associated with the breast ovarian cancer syndrome (BRCA1/BRCA2) mutations. It is unknown if this association is causal.

Experimental Design: This is a single-site study of patients who underwent surgical pancreatic tumor resection and self-identified as Ashkenazi Jewish. DNA from normal pancreatic tissue was genotyped for the three Ashkenazi Jewish BRCA1/2 founder mutations BRCA1 185delAG, BRCA1 5382insC, and BRCA2 6174delT, and loss of heterozygosity (LOH) was determined by sequencing DNA from microdissected tumor. When additional tumor tissue was available, p53 immunohistochemistry (IHC) was conducted.

Results: Thirty-seven patients underwent surgery for PDAC, seven for intraductal papillary mucinous neoplasm (IPMN), and 19 for other diseases. A high prevalence of BRCA1/2 mutations was found in the surgical cohort (12/63; 19.0%; P < 0.001), PDAC cohort (8/37; 21.6%; P < 0.001), and IPMN cohort (2/7; 28.6%; P = .01) compared with published control mutation frequency. A high prevalence of BRCA1 185delAG (8.1%; P < 0.001) and BRCA2 6174delT (10.8%; P < 0.001) in Ashkenazi Jewish patients with PDAC was shown. BRCA1/2 LOH was found in 2 of 4 BRCA1-associated PDACs and 3 of 4 BRCA2-associated PDACs. Positive p53 IHC was found in 5 of 8 BRCA1/2 PDACs.

Conclusions: We show a high prevalence of BRCA1/2 mutations with LOH in an Ashkenazi Jewish cohort of surgically resected PDAC and neoplastic lesions, suggesting that these germline mutations are causal in selected individuals.
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http://dx.doi.org/10.1158/1078-0432.CCR-12-3020DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3959126PMC
July 2013

Hypomethylating therapy in an aggressive stroma-rich model of pancreatic carcinoma.

Cancer Res 2013 Jan 29;73(2):885-96. Epub 2012 Nov 29.

Institute for Cancer Genetics, Columbia University Medical Center, New York, New York 10032, USA.

Pancreatic ductal adenocarcinoma (PDAC) is a lethal malignancy that resists current treatments. To test epigenetic therapy against this cancer, we used the DNA demethylating drug 5-aza-2'-deoxycytidine (DAC) in an aggressive mouse model of stromal rich PDAC (KPC-Brca1 mice). In untreated tumors, we found globally decreased 5-methyl-cytosine (5-mC) in malignant epithelial cells and in cancer-associated myofibroblasts (CAF), along with increased amounts of 5-hydroxymethyl-cytosine (5-HmC) in CAFs, in progression from pancreatic intraepithelial neoplasia to PDAC. DAC further reduced DNA methylation and slowed PDAC progression, markedly extending survival in an early-treatment protocol and significantly though transiently inhibiting tumor growth when initiated later, without adverse side effects. Escaping tumors contained areas of sarcomatoid transformation with disappearance of CAFs. Mixing-allografting experiments and proliferation indices showed that DAC efficacy was due to inhibition of both the malignant epithelial cells and the CAFs. Expression profiling and immunohistochemistry highlighted DAC induction of STAT1 in the tumors, and DAC plus IFN-γ produced an additive antiproliferative effect on PDAC cells. DAC induced strong expression of the testis antigen deleted in azoospermia-like (DAZL) in CAFs. These data show that DAC is effective against PDAC in vivo and provide a rationale for future studies combining hypomethylating agents with cytokines and immunotherapy.
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http://dx.doi.org/10.1158/0008-5472.CAN-12-1880DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3548986PMC
January 2013

BRCA1 tumor suppression depends on BRCT phosphoprotein binding, but not its E3 ligase activity.

Science 2011 Oct;334(6055):525-8

Institute for Cancer Genetics, Columbia University, New York, NY 10032, USA.

Germline mutations of the breast cancer 1 (BRCA1) gene are a major cause of familial breast and ovarian cancer. The BRCA1 protein displays E3 ubiquitin ligase activity, and this enzymatic function is thought to be required for tumor suppression. To test this hypothesis, we generated mice that express an enzymatically defective Brca1. We found that this mutant Brca1 prevents tumor formation to the same degree as does wild-type Brca1 in three different genetically engineered mouse (GEM) models of cancer. In contrast, a mutation that ablates phosphoprotein recognition by the BRCA C terminus (BRCT) domains of BRCA1 elicits tumors in each of the three GEM models. Thus, BRCT phosphoprotein recognition, but not the E3 ligase activity, is required for BRCA1 tumor suppression.
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http://dx.doi.org/10.1126/science.1209909DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3904783PMC
October 2011

Ret-dependent cell rearrangements in the Wolffian duct epithelium initiate ureteric bud morphogenesis.

Dev Cell 2009 Aug;17(2):199-209

Department of Genetics and Development, Columbia University Medical Center, New York, NY 10032, USA.

While the genetic control of renal branching morphogenesis has been extensively described, the cellular basis of this process remains obscure. GDNF/RET signaling is required for ureter and kidney development, and cells lacking Ret are excluded from the tips of the branching ureteric bud in chimeric kidneys. Here, we find that this exclusion results from earlier Ret-dependent cell rearrangements in the caudal Wolffian duct, which generate a specialized epithelial domain that later emerges as the tip of the primary ureteric bud. By juxtaposing cells with elevated or reduced RET activity, we find that Wolffian duct cells compete, based on RET signaling levels, to contribute to this domain. At the same time, the caudal Wolffian duct transiently converts from a simple to a pseudostratified epithelium, a process that does not require Ret. Thus, both Ret-dependent cell movements and Ret-independent changes in the Wolffian duct epithelium contribute to ureteric bud formation.
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http://dx.doi.org/10.1016/j.devcel.2009.07.013DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2762206PMC
August 2009

E3 ligase activity of BRCA1 is not essential for mammalian cell viability or homology-directed repair of double-strand DNA breaks.

Proc Natl Acad Sci U S A 2008 Dec 16;105(52):20876-81. Epub 2008 Dec 16.

Institute for Cancer Genetics, Department of Pathology and Cell Biology, Columbia University, New York, NY 10032, USA.

Hereditary cases of breast and ovarian cancer are often attributed to germ-line mutations of the BRCA1 tumor suppressor gene. Although BRCA1 is involved in diverse cellular processes, its role in the maintenance of genomic integrity may be a key component of its tumor suppression activity. The protein encoded by BRCA1 interacts in vivo with the related BARD1 protein to form a heterodimeric complex that acts as a ubiquitin E3 ligase. Because the enzymatic activity of the BRCA1/BARD1 heterodimer is conserved over a broad phylogenetic range, it is thought to be critical for the central functions of BRCA1. To test this hypothesis, we have generated isogenic clones of embryonic stem cells that do or do not express an enzymatically proficient Brca1 polypeptide. Surprisingly, cells lacking the ubiquitin ligase activity of BRCA1 are viable and do not accumulate spontaneous cytogenetic rearrangements. Gene targeting efficiencies are modestly reduced in these cells, and chromosomal rearrangements arise at elevated rates in response to genotoxic stress. Nonetheless, cells lacking Brca1 enzymatic activity are not hypersensitive to the DNA cross-linking agent mitomycin C. They also form Rad51 focus in response to ionizing radiation and repair chromosome breaks by homologous recombination at wild-type levels. These results indicate that key aspects of BRCA1 function in genome maintenance, including its role in homology-directed repair of double-strand DNA breaks, do not depend on the E3 ligase activity of BRCA1.
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http://dx.doi.org/10.1073/pnas.0811203106DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2603436PMC
December 2008

The basal-like mammary carcinomas induced by Brca1 or Bard1 inactivation implicate the BRCA1/BARD1 heterodimer in tumor suppression.

Proc Natl Acad Sci U S A 2008 May 28;105(19):7040-5. Epub 2008 Apr 28.

Department of Pathology and Cell Biology, Institute for Cancer Genetics, Columbia University Medical Center, 1130 Saint Nicholas Avenue, New York, NY 10032, USA.

Women with germ-line mutations of the BRCA1 tumor suppressor gene are highly susceptible to breast and ovarian cancer. The protein product of BRCA1 is involved in a broad spectrum of biological processes and interacts with many diverse proteins. One of these, BARD1, associates with BRCA1 to form a heterodimeric complex that is enzymatically active as an ubiquitin E3 ligase. Although the BRCA1/BARD1 heterodimer has been implicated in several aspects of BRCA1 function, its role in tumor suppression has not been evaluated. To address this question, we generated mouse strains carrying conditional alleles of either Bard1 or Brca1 and used Cre recombination to inactivate these genes in mammary epithelial cells. Significantly, the conditional Bard1- and Brca1-mutant mice developed breast carcinomas that are indistinguishable from each other (and from those of double conditional Bard1/Brca1-mutant animals) with respect to their frequency, latency, histopathology, and cytogenetic features. Reminiscent of the basal-like breast carcinomas seen in human BRCA1 mutation carriers, these tumors are "triple negative" for estrogen and progesterone receptor expression and HER2/neu amplification. They also express basal cytokeratins CK5 and CK14, have an elevated frequency of p53 lesions, and display high levels of chromosomal instability. The remarkable similarities between the mammary carcinomas of Bard1-, Brca1-, and Bard1/Brca1-mutant mice indicate that the tumor suppressor activities of both genes are mediated through the BRCA1/BARD1 heterodimer.
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http://dx.doi.org/10.1073/pnas.0711032105DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2365565PMC
May 2008

Branching morphogenesis of the ureteric epithelium during kidney development is coordinated by the opposing functions of GDNF and Sprouty1.

Dev Biol 2006 Nov 25;299(2):466-77. Epub 2006 Aug 25.

Division of Hematology/Oncology, Mount Sinai School of Medicine, One Gustave L. Levy Place, New York, NY 10029, USA.

Branching of ureteric bud-derived epithelial tubes is a key morphogenetic process that shapes development of the kidney. Glial cell line-derived neurotrophic factor (GDNF) initiates ureteric bud formation and promotes subsequent branching morphogenesis. Exactly how GDNF coordinates branching morphogenesis is unclear. Here we show that the absence of the receptor tyrosine kinase antagonist Sprouty1 (Spry1) results in irregular branching morphogenesis characterized by both increased number and size of ureteric bud tips. Deletion of Spry1 specifically in the epithelium is associated with increased epithelial Wnt11 expression as well as increased mesenchymal Gdnf expression. We propose that Spry1 regulates a Gdnf/Ret/Wnt11-positive feedback loop that coordinates mesenchymal-epithelial dialogue during branching morphogenesis. Genetic experiments indicate that the positive (GDNF) and inhibitory (Sprouty1) signals have to be finely balanced throughout renal development to prevent hypoplasia or cystic hyperplasia. Epithelial cysts develop in Spry1-deficient kidneys that share several molecular characteristics with those observed in human disease, suggesting that Spry1 null mice may be useful animal models for cystic hyperplasia.
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http://dx.doi.org/10.1016/j.ydbio.2006.08.051DOI Listing
November 2006

GDNF/Ret signaling and the development of the kidney.

Bioessays 2006 Feb;28(2):117-27

Department of Genetics and Development, Columbia University Medical Center, New York 10032, USA.

Signaling by GDNF through the Ret receptor is required for normal growth of the ureteric bud during kidney development. However, the precise role of GDNF/Ret signaling in renal branching morphogenesis and the specific responses of ureteric bud cells to GDNF remain unclear. Recent studies have provided new insight into these issues. The localized expression of GDNF by the metanephric mesenchyme, together with several types of negative regulation, is important to elicit and correctly position the initial budding event from the Wolffian duct. GDNF also promotes the continued branching of the ureteric bud. However, it does not provide the positional information required to specify the pattern of ureteric bud growth and branching, as its site of synthesis can be drastically altered with minimal effects on kidney development. Cells that lack Ret are unable to contribute to the tip of the ureteric bud, apparently because GDNF-driven proliferation is required for the formation and growth of this specialized epithelial domain.
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http://dx.doi.org/10.1002/bies.20357DOI Listing
February 2006

The role of GDNF in patterning the excretory system.

Dev Biol 2005 Jul;283(1):70-84

Department of Genetics and Development, Columbia University Medical Center, New York, NY 10032, USA.

Mesenchymal-epithelial interactions are an important source of information for pattern formation during organogenesis. In the developing excretory system, one of the secreted mesenchymal factors thought to play a critical role in patterning the growth and branching of the epithelial ureteric bud is GDNF. We have tested the requirement for GDNF as a paracrine chemoattractive factor by altering its site of expression during excretory system development. Normally, GDNF is secreted by the metanephric mesenchyme and acts via receptors on the Wolffian duct and ureteric bud epithelium. Misexpression of GDNF in the Wolffian duct and ureteric buds resulted in formation of multiple, ectopic buds, which branched independently of the metanephric mesenchyme. This confirmed the ability of GDNF to induce ureter outgrowth and epithelial branching in vivo. However, in mutant mice lacking endogenous GDNF, kidney development was rescued to a substantial degree by GDNF supplied only by the Wolffian duct and ureteric bud. These results indicate that mesenchymal GDNF is not required as a chemoattractive factor to pattern the growth of the ureteric bud within the developing kidney, and that any positional information provided by the mesenchymal expression of GDNF may provide for renal branching morphogenesis is redundant with other signals.
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http://dx.doi.org/10.1016/j.ydbio.2005.04.008DOI Listing
July 2005

Sprouty1 is a critical regulator of GDNF/RET-mediated kidney induction.

Dev Cell 2005 Feb;8(2):229-39

Department of Medicine, Mount Sinai School of Medicine, One Gustave L. Levy Place, New York, New York, USA.

Intercellular signaling molecules and their receptors, whose expression must be tightly regulated in time and space, coordinate organogenesis. Regulators of intracellular signaling pathways provide an additional level of control. Here we report that loss of the receptor tyrosine kinase (RTK) antagonist, Sprouty1 (Spry1), causes defects in kidney development in mice. Spry1(-/-) embryos have supernumerary ureteric buds, resulting in the development of multiple ureters and multiplex kidneys. These defects are due to increased sensitivity of the Wolffian duct to GDNF/RET signaling, and reducing Gdnf gene dosage correspondingly rescues the Spry1 null phenotype. We conclude that the function of Spry1 is to modulate GDNF/RET signaling in the Wolffian duct, ensuring that kidney induction is restricted to a single site. These results demonstrate the importance of negative feedback regulation of RTK signaling during kidney induction and suggest that failures in feedback control may underlie some human congenital kidney malformations.
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http://dx.doi.org/10.1016/j.devcel.2004.12.004DOI Listing
February 2005