Using an integrated approach to characterize the pancreatic tissue and isolated islets from a 33-year-old with 17 years of type 1 diabetes (T1D), we found that donor islets contained β cells without insulitis and lacked glucose-stimulated insulin secretion despite a normal insulin response to cAMP-evoked stimulation. With these unexpected findings for T1D, we sequenced the donor DNA and found a pathogenic heterozygous variant in the gene encoding hepatocyte nuclear factor-1α (HNF1A). In one of the first studies of human pancreatic islets with a disease-causing HNF1A variant associated with the most common form of monogenic diabetes, we found that HNF1A dysfunction leads to insulin-insufficient diabetes reminiscent of T1D by impacting the regulatory processes critical for glucose-stimulated insulin secretion and suggest a rationale for a therapeutic alternative to current treatment.
Abstract Background: Preclinical studies have shown the combination of imetelstat (GRN163L), an inhibitor of telomerase, and T results in synergistic growth inhibition and restoration of T sensitivity in T-resistant cells (Clin Can Res 12(10):3184–92, 2006). Here we translate those findings to the clinic with the first-in-man phase I trial of imetelstat+ T in patients (pts) with T-refractory HER2+ metastatic disease. Methods: T (6 mg/kg q3 wk) was administered with increasing doses of imetelstat (240/300/375 mg/m2 q3 wk) using a standard 3+3 dose escalation design. Maximum Tolerated Dose (MTD) was based on toxicity observed during cycle 1. Responding or stable pts continued treatment until progression. Tumor biopsy and bone marrow aspirate for biologic correlates were obtained at baseline and prior to cycle 2. Limited pharmacokinetics (PK) for T and imetelstat were included. Results: Ten pts were enrolled; median age was 54 (28–64). Patients were extensively pre-treated with the number of prior regimens ranging from 3 to >12. Prior cytotoxic therapies included anthracyclines (n = 9), taxanes (n = 9), vinorelbine (n = 8), capecitabine (n = 8), gemcitabine (n = 6), ixabepilone (n = 4), and eribulin (n = 4). Prior anti-HER2 targeted therapies included trastuzumab (n = 10), capecitabine (n = 8), T-DM1 (n = 6), and pertuzumab (n = 1). Therapy was well tolerated; no treatment related grade 4 toxicities were observed. Myelosuppression was limited to one pt with Grade 2 anemia and one each with Grade 2/3 thrombocytopenia. MTD was not reached. There were no objective responses; 2 pts. in cohort 3 had SD. Serial tumor biopsies and bone marrow aspirates have been analyzed for 7 pts (cohort 1: 001–004; cohort 2: 005–007; cohort 3: analysis ongoing). Tumor hTERT (p = ns) and phosphorylated HER2 (p = 0.03) decreased after treatment in nearly all pts. Bone marrow S-phase did not change consistently with treatment. Conclusion: The combination of trastuzumab + imetelstat is well tolerated and results in decreases in HER2 phosphorylation similar to that seen in preclinical models. Additional biologic correlates and PK analyses are ongoing. Further study of this combination in less heavily pre-treated patients is planned. Citation Information: Cancer Res 2012;72(24 Suppl):Abstract nr P5-18-13.
Abstract Neurofibromatosis type 1 (NF1) is a genetic disorder with a range of clinical manifestations such as widespread growth of benign tumours called neurofibromas, pain, learning disorders, bone deformities, vascular abnormalities and even malignant tumours. With the establishment of the Children’s Tumour Foundation biobank, neurofibroma samples can now be collected directly from patients to be analysed by the larger scientific community. This work describes a pilot study to characterize one class of neurofibroma, cutaneous neurofibromas, by molecularly profiling of ~40 cutaneous neurofibromas collected from 11 individual patients. Data collected from each tumour includes (1) SNP Arrays, (2) Whole genome sequencing (WGS) and (3) RNA-Sequencing. These data are now freely available for further analysis at http://www.synapse.org/cutaneousNF .
Abstract Background: Neurofibromatosis type I (NF1) is a genetic disorder that disrupts neurological tissue growth and can lead to a diverse set of symptoms including systematic growth of benign tumors, learning disorders and bone deformities. It is a rare disease occurring in only 1 in 3,000 people worldwide. While the disease has been linked to loss of function in the NF1 gene - a known tumor suppressor - there is a high degree of phenotypic diversity in the NF1 patient population, making it difficult to identify the underlying cause of the disease and treat it effectively. In this work we seek to improve overall knowledge of dermal NF1 through global molecular characterization of the disease. Methods: We have collected four dermal neurofibromas and peripheral blood from each of 11 NF1 patients. We analyzed each sample using (1) Whole genome sequencing (WGS) on the Illumina HiSeq X platform, (2) Illumina OMNI2.5 Arrays (3) RNA-Sequencing on an Illumina HiSeq v4 machine and (4) iTRAQ-labeled proteomics. WGS data for both tumor and blood samples from each patient were used to identify patient-specific germ-line mutations as well as tumor-specific somatic mutations in each sample. Single nucleotide polymorphisms identified by the OMNI Arrays were used to identify copy number alterations in both blood and tumor samples. RNA-Seq data and proteomics data were mapped to transcripts and proteins respectively. Results: Preliminary analysis of this data illustrates a diverse genomic landscape of NF1. Hierarchical clustering of copy number alterations largely show samples clustering by tissue, suggesting that most copy number alterations are somatic and not shared across the germline. However, there are two patients that show germline copy number alterations, including one patient with loss in the NF1 region. WGS analysis suggests similar diversity with each patient possessing a distinct combination of germline and somatic mutations of NF1 and other cancer-related genes. Cluster analysis of the RNA-Seq data shows no patient-specific clusters, suggesting that that each tumor executes a unique transcriptional program. Conclusion: This work represents a first-ever attempt to profile the diversity of dermal neurofibromatosis at a molecular level. Preliminary analysis of the data underscores the complexity of this disease and explains, in part, previous difficulty in identifying effective treatments. Ongoing work includes expanding the analysis to include more patient samples and other types of NF1-derived tumors. As an orphan disease, NF1 has been poorly characterized compared to more common cancers. To rectify this, the Children's Tumor Foundation and Sage Bionetworks are collaborating to make NF1 data available to the public to accelerate research and the drug discovery pipeline. We expect that this data will be a resource for other NF1 researchers to assist in the study of this disease at the molecular level. All data and preliminary results are publicly available at http://www.synapse.org/dermalNF Citation Format: Sara JC Gosline, Pamela Knight, Thomas Yu, Nripesh Prasad, Angela Jones, Shristi Shrestha, Braden Boone, Shawn E. Levy, Andrew J. Link, Allison C. Galassie, Hubert Weinberg, Stephen Friend, Salvatore La Rosa, Justin Guinney, Annette Bakker. The molecular landscape of dermal neurofibromatosis. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 772.
It is estimated that ∼1% of the world's population has intellectual disability, with males affected more often than females. OGT is an X-linked gene encoding for the enzyme O-GlcNAc transferase (OGT), which carries out the reversible addition of N-acetylglucosamine (GlcNAc) to Ser/Thr residues of its intracellular substrates. Three missense mutations in the tetratricopeptide (TPR) repeats of OGT have recently been reported to cause X-linked intellectual disability (XLID). Here, we report the discovery of two additional novel missense mutations (c.775 G>A, p.A259T, and c.1016 A>G, p.E339G) in the TPR domain of OGT that segregate with XLID in affected families. Characterization of all five of these XLID missense variants of OGT demonstrates modest declines in thermodynamic stability and/or activities of the variants. We engineered each of the mutations into a male human embryonic stem cell line using CRISPR/Cas9. Investigation of the global O-GlcNAc profile as well as OGT and O-GlcNAc hydrolase levels by Western blotting showed no gross changes in steady-state levels in the engineered lines. However, analyses of the differential transcriptomes of the OGT variant–expressing stem cells revealed shared deregulation of genes involved in cell fate determination and liver X receptor/retinoid X receptor signaling, which has been implicated in neuronal development. Thus, here we reveal two additional mutations encoding residues in the TPR regions of OGT that appear causal for XLID and provide evidence that the relatively stable and active TPR variants may share a common, unelucidated mechanism of altering gene expression profiles in human embryonic stem cells. It is estimated that ∼1% of the world's population has intellectual disability, with males affected more often than females. OGT is an X-linked gene encoding for the enzyme O-GlcNAc transferase (OGT), which carries out the reversible addition of N-acetylglucosamine (GlcNAc) to Ser/Thr residues of its intracellular substrates. Three missense mutations in the tetratricopeptide (TPR) repeats of OGT have recently been reported to cause X-linked intellectual disability (XLID). Here, we report the discovery of two additional novel missense mutations (c.775 G>A, p.A259T, and c.1016 A>G, p.E339G) in the TPR domain of OGT that segregate with XLID in affected families. Characterization of all five of these XLID missense variants of OGT demonstrates modest declines in thermodynamic stability and/or activities of the variants. We engineered each of the mutations into a male human embryonic stem cell line using CRISPR/Cas9. Investigation of the global O-GlcNAc profile as well as OGT and O-GlcNAc hydrolase levels by Western blotting showed no gross changes in steady-state levels in the engineered lines. However, analyses of the differential transcriptomes of the OGT variant–expressing stem cells revealed shared deregulation of genes involved in cell fate determination and liver X receptor/retinoid X receptor signaling, which has been implicated in neuronal development. Thus, here we reveal two additional mutations encoding residues in the TPR regions of OGT that appear causal for XLID and provide evidence that the relatively stable and active TPR variants may share a common, unelucidated mechanism of altering gene expression profiles in human embryonic stem cells.
Getting higher yields of monoclonal antibody (MAb) is a problem in Hybridoma Technology which has two major bottlenecks: a) poor yield of hybridized cells, b) low cellular productivity of MAb in culture. There are three ways of obtaining high MAb yield in vitro a) Large scale culture, b) high density culture and c) enhancing individual cellular productivity in culture. Currently, the focus is on the correct synergistic combination of fortified nutrient media, bioreactor design and mode of operation. Maximization of cell culture longevity, maintenance of high specific antibody secretion rates, nutrient supplementation, waste product minimization and control of environmental conditions are important parameters for improvement of large scale production of MAb. Though, MAb yields have improved rapidly over the decade, there is a growing concern for the decrease in quality of MAb secreted. Further research is therefore necessary to take full advantage of MAb as a potential diagnostic agent for in vivo therapy.
Single cell RNA-sequencing (scRNA-seq) approaches are continuously improving at the point of data collection and analysis to simultaneously profile all cell types within islet microorgans to better understand heterogeneity and phenotype-function relationships and how these change with aging and disease. To better understand the advantages and limitations of scRNA-seq, we used the ChromiumTMplatform to profile human islets with a robust insulin and glucagon secretion by perifusion (5 nondiabetic donors, ages 14-66 years) and compared these results to scRNA-seq analysis of purified α and β cells. In context of the same donor (n=2), purified α and β cells (using NTPDase3 marker) showed high correlation (r=0.99) with the Seurat-identified α and β subsets originating from dispersed whole islets. Based on the analysis of a very large pool of single cells (79,881 cells; 2316 genes/cell), the Seurat-guided analysis identified cell types that segregated into eight different clusters (17% β, 66% α, 4% δ, 4% ductal, 4% mesenchymal, 2% acinar, 2% endothelial, and 1% immune). Interstingly, in this large single cell context, we did not identify the four β cell subsets previously reported on the basis of alternative cell surface markers, but found a β cell cluster with highly expressed ER stress-associated genes and an α cell cluster with higher cell cycle regulation marker expression. We concurrently investigated expression of functionally important MAFA and MAFB genes, which are expressed only in a fraction of β cells by histological analysis, and found that PERK-mediated UPR transcripts were enriched in the MAFB-expressing β cells, while the MAFA-expressing β cells differentially expressed genes associated with β cell function. Overall, our study indicates high fidelity of α and β cell clustering in dispersed and sorted islet cells and points to a different pattern of β cell heterogeneity when β cells are purified using NTPDase3 expression compared to other cell surface markers. Disclosure S. Shrestha: None. D.C. Saunders: None. J.T. Walker: None. R. Haliyur: None. G. Poffenberger: None. R. Aramandla: None. A. Jones: None. N. Prasad: None. S.E. Levy: None. A.C. Powers: None. M. Brissova: None.
Vaccine development for influenza A/H5N1 is an important public health priority, but H5N1 vaccines are less immunogenic than seasonal influenza vaccines. Adjuvant System 03 (AS03) markedly enhances immune responses to H5N1 vaccine antigens, but the underlying molecular mechanisms are incompletely understood.We compared the safety (primary endpoint), immunogenicity (secondary), gene expression (tertiary) and cytokine responses (exploratory) between AS03-adjuvanted and unadjuvanted inactivated split-virus H5N1 influenza vaccines. In a double-blinded clinical trial, we randomized twenty adults aged 18-49 to receive two doses of either AS03-adjuvanted (n = 10) or unadjuvanted (n = 10) H5N1 vaccine 28 days apart. We used a systems biology approach to characterize and correlate changes in serum cytokines, antibody titers, and gene expression levels in six immune cell types at 1, 3, 7, and 28 days after the first vaccination.Both vaccines were well-tolerated. Nine of 10 subjects in the adjuvanted group and 0/10 in the unadjuvanted group exhibited seroprotection (hemagglutination inhibition antibody titer > 1:40) at day 56. Within 24 hours of AS03-adjuvanted vaccination, increased serum levels of IL-6 and IP-10 were noted. Interferon signaling and antigen processing and presentation-related gene responses were induced in dendritic cells, monocytes, and neutrophils. Upregulation of MHC class II antigen presentation-related genes was seen in neutrophils. Three days after AS03-adjuvanted vaccine, upregulation of genes involved in cell cycle and division was detected in NK cells and correlated with serum levels of IP-10. Early upregulation of interferon signaling-related genes was also found to predict seroprotection 56 days after first vaccination.Using this cell-based systems approach, novel mechanisms of action for AS03-adjuvanted pandemic influenza vaccination were observed.ClinicalTrials.gov NCT01573312.
Abstract Clinical and pathologic heterogeneity in type 1 diabetes is increasingly being recognized. Findings in the islets and pancreas of a 22-year-old male with 8 years of type 1 diabetes were discordant with expected results and clinical history (islet autoantibodies negative, hemoglobin A1c 11.9%) and led to comprehensive investigation to define the functional, molecular, genetic, and architectural features of the islets and pancreas to understand the cause of the donor’s diabetes. Examination of the donor’s pancreatic tissue found substantial but reduced β-cell mass with some islets devoid of β cells (29.3% of 311 islets) while other islets had many β cells. Surprisingly, isolated islets from the donor pancreas had substantial insulin secretion, which is uncommon for type 1 diabetes of this duration. Targeted and whole-genome sequencing and analysis did not uncover monogenic causes of diabetes but did identify high-risk human leukocyte antigen haplotypes and a genetic risk score suggestive of type 1 diabetes. Further review of pancreatic tissue found islet inflammation and some previously described α-cell molecular features seen in type 1 diabetes. By integrating analysis of isolated islets, histological evaluation of the pancreas, and genetic information, we concluded that the donor’s clinical insulin deficiency was most likely the result autoimmune-mediated β-cell loss but that the constellation of findings was not typical for type 1 diabetes. This report highlights the pathologic and functional heterogeneity that can be present in type 1 diabetes.
