Nair, Asha

AbstractArboviruses target bone forming osteoblasts and perturb bone remodeling via paracrine factors. We previously reported that Zika virus (ZIKV) infection of early‐stage human mesenchymal stromal cells (MSCs) inhibited the osteogenic lineage commitment of MSCs. To understand the physiological interplay between bone development and ZIKV pathogenesis, we employed a primary in vitro model to examine the biological responses of MSCs to ZIKV infection at different stages of osteogenesis. Precommitted MSCs were infected at the late stage of osteogenic stimulation (Day 7) with ZIKV (multiplicity of infection of 5). We observe that MSCs infected at the late stage of differentiation are highly susceptible to ZIKV infection similar to previous observations with early stage infected MSCs (Day 0). However, in contrast to ZIKV infection at the early stage of differentiation, infection at a later stage significantly elevates the key osteogenic markers and calcium content. Comparative RNA sequencing (RNA‐seq) of early and late stage infected MSCs reveals that ZIKV infection alters the mRNA transcriptome during osteogenic induction of MSCs (1251 genes). ZIKV infection provokes a robust antiviral response at both stages of osteogenic differentiation as reflected by the upregulation of interferon responsive genes (n > 140). ZIKV infection enhances the expression of immune‐related genes in early stage MSCs while increasing cell cycle genes in late stage MSCs. Remarkably, ZIKA infection in early stage MSCs also activates lipid metabolism‐related pathways. In conclusion, ZIKV infection has differentiation stage‐dependent effects on MSCs and this mechanistic understanding may permit the development of new therapeutic or preventative measures for bone‐related effects of ZIKV infection.

Abstract Gallbladder cancer (GBC) is one of the deadliest cancers, with a 5-year-survival-rate of less than 5 percent for late-stage disease. The response rate to chemotherapy among GBC patients is generally poor. Recent research has attempted to identify diagnostic, prognostic, and predictive biomarkers, however, currently, no biomarkers can accurately diagnose GBC and predict patients’ prognosis. Integrative analysis of molecular and clinical characterization has not been fully established, and minimal improvement has been made to the survival of these patients, in part due to the heterogeneity of GBC. Machine learning techniques have been proven to empower analysis of big data in oncology, allowing for improvement in the generation of biomarkers to predict patient outcomes. Using machine learning, we can utilize high-throughput RNA sequencing with clinicopathologic data to develop a predictive tool for GBC prognosis. Current predictive models for GBC outcomes often utilize clinical data only, with the highest C-statistic reported being 0.71. C-statistic values over 0.7 generally indicate good models, however 0.8 is the threshold for strong predictive models. We aim to build a superior algorithm to predict overall survival in GBC patients with advanced disease, using machine learning approaches to prioritize biomarkers for GBC prognosis. We have identified over 80 fresh frozen GBC tissue samples from Mayo Clinic Rochester, Dongsan Medical Center in Daegu, Korea, University of the Witwatersrand, in Johannesburg, South Africa, Lithuanian University of Health Science in Vilnius, Lithuania, and University of Calgary in Calgary, Canada, from patients enrolled between 2012 and 2021. We will perform next-generation RNA sequencing on these tissue samples. The patients’ clinical, pathologic and survival data will be abstracted from the medical record uniformly across sites. Feature engineering and dimensionality reduction will be performed. Then random forests, support vector machines, and gradient boosting machines will be applied to train the data. Variable importance will prioritize multi-omic markers. Standard 5-fold cross validation will be used to assess performance of each ML algorithm. If overall survival can be better predicted with the addition patients’ transcriptional sequencing data compared to using clinical profiles alone, we can gain a greater understanding of key biomarkers driving the tumor phenotype. Citation Format: Linsey Jackson, Loretta Allotey, Valles Kenneth, Gavin Oliver, Asha Nair, Daniel O'Brien, Rondell Graham, Mitesh Borad, Arjun Athreya, Lewis Roberts. Prognostic biomarkers for gallbladder cancer: A machine learning approach [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 1944.

This study compares gene expression in mucosa of the terminal ileum, right colon, and left colon in patients with diarrhea- or constipation-predominant irritable bowel syndrome (IBS) and contrasts expression between these two disease entities and also between each entity and mucosa from healthy controls. The study shows there is differential expression of genes related to immune, transmitter, nociceptive, ion channel, and transporter functions, as well as reduced serine protease inhibition, in patients with IBS.

Skeletal muscle insulin resistance contributes to the pathogenesis of type 2 diabetes mellitus. Epidemiological studies have demonstrated that vitamin D deficiency is associated with the presence of type 2 diabetes in humans. To probe the role of the vitamin D receptor (VDR), an intracellular and nuclear protein that mediates the bioactivity of the active vitamin D ligand, 1,25‐dihydroxyvitamin D, in skeletal muscle mediated glucose disposal, we deleted the skeletal muscle VDR in adult mice. We assessed glucose disposal, insulin concentrations and sensitivity, and skeletal muscle gene expression in Vdr knockout and wild‐type Vdr mice. Following the induction of a skeletal muscle‐specific Cre recombinase with tamoxifen in adult male Vdr fl/fl mice, efficient deletion of the Vdr was demonstrated. In skeletal muscle‐specific Vdr knockout mice, basal glucose concentrations were similar to those in wild‐type Vdr mice. In Vdr knockout mice, blood glucose concentrations decreased more rapidly following the administration of intraperitoneal glucose than in control mice expressing wild‐type Vdr (Figure 1A and Figure 1B). The AUC for glucose concentrations in Vdr fl/fl .actin iCre mice treated with tamoxifen was reduced by 20% (p=0.015) compared to the AUC of blood glucose concentrations prior to tamoxifen treatment. Pre‐and post‐treatment AUC’s for blood glucose in Vdr fl/fl .actin iCre mice treated with vehicle and in Vdr fl/fl mice treated with tamoxifen were unchanged (p=0.97,0.23). Plasma insulin concentrations were identical in knockout and wild‐type Vdr mice following a bolus of glucose, demonstrating that deletion of the Vdr in skeletal muscle did not influence the release of insulin in response to glucose (one‐way ANOVA, p=0.45). Insulin sensitivity was similar in Vdr knockout and wild‐type Vdr mice. Gene expression analysis of skeletal muscle mRNA derived from Vdr knockout mice and wild‐type mice demonstrated an increase in the expression of genes associated with oxidative phosphorylation, fatty acid oxidation and adipogenesis pathways that could potentially alter glucose utilization. We conclude that selective deletion of the Vdr in the skeletal muscle of adult mice results in more efficient glucose disposal than in wild‐type Vdr mice; basal and glucose‐stimulated insulin serum concentrations are identical in knock‐out and wild type VDR mice with no changes in insulin sensitivity. The data suggest that modulation of the vitamin‐D endocrine system is unlikely to improve glucose utilization.

Abstract Polymorphous low-grade neuroepithelial tumor of the young (PLNTY) is a recently described epileptogenic tumor characterized by oligodendroglioma-like components, aberrant CD34 expression, and frequent mitogen-activated protein kinase (MAPK) pathway activation. We molecularly profiled 13 cases with diagnostic histopathological features of PLNTY (10 female; median age, 16 years; range, 5–52). Patients frequently presented with seizures (9 of 12 with available history) and temporal lobe tumors (9 of 13). MAPK pathway activating alterations were identified in all 13 cases. Fusions were present in the 7 youngest patients: FGFR2-CTNNA3 (n = 2), FGFR2-KIAA1598 (FGFR2-SHTN1) (n = 1), FGFR2-INA (n = 1), FGFR2-MPRIP (n = 1), QKI-NTRK2 (n = 1), and KIAA1549-BRAF (n = 1). BRAF V600E mutation was present in 6 patients (17 years or older). Two fusion-positive cases additionally harbored TP53/RB1 abnormalities suggesting biallelic inactivation. Copy number changes predominantly involving whole chromosomes were observed in all 10 evaluated cases, with losses of chromosome 10q occurring with FGFR2-KIAA1598 (SHTN1)/CTNNA3 fusions. The KIAA1549-BRAF and QKI-NTRK2 fusions were associated respectively with a 7q34 deletion and 9q21 duplication. This study shows that despite its name, PLNTY also occurs in older adults, who frequently show BRAF V600E mutation. It also expands the spectrum of the MAPK pathway activating alterations associated with PLNTY and demonstrates recurrent chromosomal copy number changes consistent with chromosomal instability.

Abstract Patients with glioblastoma (GBM) have a median survival of 15 months despite aggressive treatment. Immunotherapies such as dendritic cell (DC) vaccines have modest clinical efficacy in small clinical trials. Treatment-related pseudo-progression confounds outcome assessment by MRI, particularly in patients receiving immunotherapy. Thus, there is a need for additional non-invasive methods to monitor treatment response. Extracellular vesicles (EVs), especially plasma exosomes, contain tumor-specific microRNA (miRNA) cargo that could serve as a liquid biopsy to distinguish true progression from treatment-related pseudo-progression. Plasma exosomes were isolated by serial density gradient ultracentrifugation from 20 newly diagnosed GBM patients enrolled in a clinical trial of allogeneic tumor lysate-pulsed autologous DC vaccination. Short non-coding RNA sequencing and bioinformatics analysis was performed for each patient at three time points (TP): pre-vaccine (TP1), post-initial vaccine (TP2), and at end of treatment (TP3). miRNA expression analysis revealed a total of 14 upregulated and 12 downregulated miRNAs across time points (p-value < 0.05, |logFC| >1), few of which have been previously reported to be differentially expressed in GBM. Interestingly, patients’ miRNA profile expression differed more at the beginning of treatment (e.g. TP1-vs-TP2) and at subsequent time points (e.g. TP2-vs-TP3). Ingenuity Pathway Analysis is in progress to identify pathways associated with immunotherapy treatment response in malignant gliomas. In conclusion, miRNA sequencing from GBM patients’ plasma exosomes enrolled in our DC clinical trial shows marked differential miRNA expression between time points. These results suggest that as patients progress through treatment, consistent differences in plasma exosomal miRNA expression profile can be identified that could be utilized as predictors of treatment response. Thus, plasma EVs may serve as a robust platform to monitor treatment outcome.

Abstract Long non-coding RNAs (lncRNA) are key regulatory RNAs that do not code for protein. Recently lncRNAs have been recognized as key regulators of gene expression and chromatin organization. Although, deregulation of lncRNAs such as HOTAIR, MALAT1 and H19 has been reported in solid cancers, role of lncRNAs in lymphoma or other hematologic malignancies is not known. The aim of this study is to identify lymphoma-associated lncRNAs and elucidate their role in disease pathogenesis. Next generation RNA-sequencing and validation studies were carried out on mantle cell lymphoma (MCL) patient samples along with normal controls. As a result, hundreds of lncRNAs were found overexpressed in the MCL samples as compared with normal B cells, including RP11-24J23.2 (50 fold), RP11-12A2.3 (36 fold) and DLEU7-AS1 (19 fold). Quantitative RT-PCR (QRT-PCR) confirmed higher expression of RP11-24J23.2, RP11-12A2.3, and DLEU7-AS1 in MCL patient samples as compared to normal B cells. To investigate the biological significance of lncRNAs in MCL cells, we overexpressed RP11-24J23.2, RP11-12A2.3, and DLEU7-AS1 lncRNAs and analyzed effects on cell proliferation and apoptosis. To achieve overexpression lncRNAs, RP11-24J23.2, RP11-12A2.3, and DLEU7-AS1 were cloned individually in expression pcDNA3.1 generating constructs: pcDNA3RP11-24J23.2, pcDNA3RP11-12A2.3 and pcDNA3DLEU7-AS1and then transfected into MCL cell lines along with pcDNA3.1. QRT-PCR revealed effective overexpression (~100 fold increase over baseline) for each transfected lncRNAs construct in Granta and Mino cells. Analysis of proliferation and survival showed that overexpressing RP11-24J23.2, RP11-12A2.3 or DLEU7-AS1 promotes cell proliferation and survival in these cells as compared to the vector controls. Most of the lncRNAs found so far have been linked to the transcriptional regulation of the target genes through their association with chromatin remodeling factors such as Polycomb Repressive Complex 2 (PRC2). To identify the functional relevance of RP11-24J23.2, RP11-12A2.3, and DLEU7-AS1 lncRNAs, we examined the binding of the lymphoma specific lncRNAs with PRC2 complex, by performing RNA immunoprecipitation (RNA-IP) using specific antibodies to EZH2, EED and SUZ12. The RNA-IP results demonstrate high affinity binding of all three lncRNAs with SUZ12 and EED but a weak binding with EZH2. Furthermore, RNA-IP using antibody to histone 3-lysine 27 trimethylation (H3K27me3), a suppressive histone mark associated with PRC2 complex, also showed efficient binding with RP11-24J23.2, RP11-12A2.3, and DLEU7-AS1. These results suggest that RP11-24J23.2, RP11-12A2.3, and DLEU7-AS1 lncRNAs can directly associated with PRC2 complex at chromatin. To understand the mechanism of increased cell growth after overexpression of lncRNAs, RP11-24J23.2, RP11-12A2.3 or DLEU7-AS1, we analyzed expression level of important genes signature of MCL pathogenesis that include cyclin D1, cyclin D3, c-Myc, P14, and SOX11, a member of the group C SOX (SRY-related HMG-box) transcription factor family. Our results demonstrate that overexpression of lncRNAs RP11-24J23.2, RP11-12A2.3 or DLEU7-AS1 enhanced the levels of cyclin D1 (>2 fold) mRNA without any impact on cyclin D3 or c-Myc transcript levels. In contrast overexpression of RP11-24J23.2, RP11-12A2.3 or DLEU7-AS1 lncRNAs decreased SOX11 (0.08, 0.1 and 0.06 respectively) and P14 (0.06, 0.7 and 0.40 respectively) mRNA expression in Mino cells. To gain understanding of the mechanism involved in the suppression of SOX11 by the lncRNAs, we performed Chromatin immunoprecipitation assays with EZH2, EED and SUZ12 antibodies on Mino cells transfected with pcDNA3RP11-24J23.2, pcDNA3RP11-12A2.3 and pcDNA3DLEU7-AS1 or vector control. The data showed that overexpression of RP11-12A2.3 and DLEU7-AS1 significantly increased the binding of EED and to a lesser extent of EZH2 to SOX11 promoter while the binding of SUZ12 to SOX11 promoter was not changed as compared to vector alone. In conclusion, our results imply that lncRNAs are overexpressed in the MCL. Moreover, lymphoma associated lncRNAs (RP11-24J23.2, RP11-12A2.3 and DLEU7-AS1) regulate SOX11 expression via PRC2 complex contributing towards growth of MCL cells. Specific nature of lncRNAs found in lymphoma samples suggests that lncRNAs can be used as potential biomarkers for mantle cell lymphoma. Disclosures No relevant conflicts of interest to declare.

AbstractMAPK pathway activation has been recurrently observed in desmoplastic infantile ganglioglioma/astrocytoma (DIG/DIA) with reported disproportionally low mutation allele frequencies relative to the apparent high tumor content, suggesting that MAPK pathway alterations may be subclonal. We sought to expand the number of molecularly profiled cases and investigate if tumor cell composition could account for the observed low mutation allele frequencies. Molecular (targeted neuro-oncology next-generation sequencing/RNA sequencing and OncoScan microarray) and immunohistochemical (CD68-PGM1/CD163/CD14/CD11c/lysozyme/CD3/CD20/CD34/PD-L1) studies were performed in 7 DIG. Activating MAPK pathway alterations were identified in 4 (57%) cases: 3 had a BRAF mutation (V600E/V600D/V600_W604delinsDQTDG, at 8%–27% variant allele frequency) and 1 showed a TPM3-NTRK1 fusion. Copy number changes were infrequent and nonrecurrent. All tumors had at least 30% of cells morphologically and immunophenotypically consistent with microglial/macrophage lineage. Two subtotally resected tumors regrew; 1 was re-excised and received adjuvant treatment (chemotherapy/targeted therapy), with clinical response to targeted therapy only. Even with residual tumor, all patients are alive (median follow-up, 83 months; 19–139). This study further supports DIG as another MAPK pathway-driven neuroepithelial tumor, thus expanding potential treatment options for tumors not amenable to surgical cure, and suggests that DIG is a microglia/macrophage-rich neuroepithelial tumor with frequent low driver mutation allele frequencies.

AbstractPrimary aneurysmal bone cyst (ABC) is a benign multiloculated cystic lesion of bone that is defined cytogenetically by USP6 gene rearrangements. Rearrangements involving USP6 are promoter swaps, usually generated by fusion of the noncoding upstream exons of different partner genes with exon 1 or 2 of USP6, thus leading to transcriptional upregulation of full‐length USP6 coding sequence. Testing for USP6 rearrangements is used diagnostically to distinguish it from secondary ABC and other giant cell‐rich primary bone tumors. In this report, we present a case of a 16‐year‐old male with a primary ABC of the left distal femur. USP6 break apart fluorescence in situ hybridization was positive for a rearrangement and conventional chromosome analysis identified a reciprocal X;17 translocation. In order to identify the putative USP6 fusion partner, we performed RNA sequencing and uncovered a novel USP9X‐USP6 promoter swap fusion. This result was confirmed by reverse transcription‐polymerase chain reaction (RT‐PCR) and by mate pair sequencing thus showing the utility of these alternative methodologies in identifying novel fusion candidates. Ubiquitin‐specific protease 9X (USP9X), like USP6, encodes a highly conserved substrate‐specific deubiquitylating enzyme. USP9X is highly expressed in a number of tissue types and acts as both an oncogene and tumor suppressor in several human cancers. We conclude that oncogenic activation of USP6 via USP9X promoter exchange represents a novel driver of primary ABC formation.