Ratan Bhardwaj

Progressive Movement Disorder in Brothers Carrying a GNAO1 Mutation Responsive to Deep Brain Stimulation

GNAO1, located on chromosome 16q12.2, encodes for 1 of the heterotrimeric guanine binding proteins subunits (G proteins), specifically Gαo, which has been implicated as having an important role in brain function. GNAO1 mutations have been shown to impart oncogene properties as well as cause epileptic encephalopathy. The authors report 2 cases of brothers with a severe movement disorder and hypotonia without epilepsy who have been confirmed by whole exome sequencing to have a novel mutation in GNAO1. Their movement disorder improved significantly with deep brain stimulation.

Wnt pathway in atypical teratoid rhabdoid tumors.

BACKGROUND Atypical teratoid rhabdoid tumor (ATRT) is an aggressive pediatric brain tumor with limited therapeutic options. The hypothesis for this study was that the Wnt pathway triggered by the Wnt5B ligand plays an important role in ATRT biology. To address this hypothesis, the role of WNT5B and other Wnt pathway genes was analyzed in ATRT tissues and ATRT primary cell lines. METHODS Transcriptome-sequencing analyses were performed using nanoString platforms, immunohistochemistry, Western blotting, quantitative reverse transcriptase PCR, immunoprecipitation, short interference RNA studies, cell viability studies, and drug dose response (DDR) assays. RESULTS Our transcriptome-sequencing results of Wnt pathway genes from ATRT tissues and cell lines indicated that the WNT5B gene is significantly upregulated in ATRT samples compared with nontumor brain samples. These results also indicated a differential expression of both canonical and noncanonical Wnt genes. Imunoprecipitation studies indicated that Wnt5B binds to Frizzled1 and Ryk receptors. Inhibition of WNT5B by short interference RNA decreased the expression of FRIZZLED1 and RYK. Cell viability studies a indicated significant decrease in cell viability by inhibiting Frizzled1 receptor. DDR assays showed promising results with some inhibitors. CONCLUSIONS These promising therapeutic options will be studied further before starting a translational clinical trial. The success of these options will improve care for these patients.

Detection of an atypical teratoid rhabdoid brain tumor gene deletion in circulating blood using next-generation sequencing.

Circulating biomarkers such as somatic chromosome mutations are novel diagnostic tools to detect cancer noninvasively. We describe focal deletions found in a patient with atypical teratoid rhabdoid tumor, a highly aggressive early childhood pediatric tumor. First, we used magnetic resonance imaging (MRI) and histopathology to study the tumor anatomy. Next, we used whole genome sequencing (Next Gen Sequencing) and Bioinformatics interrogation to discover the presence of 3 focal deletions in tumor tissue and 2 of these 3 focal deletions in patient’s blood also. About 20% of the blood DNA sequencing reads matched the tumor DNA reads at the SMARCB1 gene locus. Circulating, tumor-specific DNA aberrations are a promising biomarker for atypical teratoid rhabdoid tumor patients. The high percentage of tumor DNA detected in blood indicates that either circulating brain tumor cells lyse in the blood or that contents of brain tumor cells traverse a possibly compromised blood-brain barrier in this patient.

Abstract 3923: A role for fibroblast growth factor receptors in growth of atypical teratoid rhabdoid tumor cells and as potential therapeutic targets

Malignant rhabdoid tumors are characterized by the loss of the SWI/SNF complex subunit SMARCB1. Atypical Teratoid Rhabdoid Tumor (ATRT) is a rare form of pediatric MRT that occurs in the CNS. Current treatment modalities for this tumor type continue to be surgical resection, chemotherapy and radiotherapy, but overall survival is still relatively low for younger children with this disease. We initially tested the sensitivity of three ATRT cell lines to 48 chemotherapeutic agents and selective inhibitors. Our results indicated that two cell lines CHLA-04-ATRT and CHLA-05-ATRT were sensitive to FGFR inhibition. Further analysis of a panel of five ATRT cell lines indicated that the cell lines CHLA-04-ATRT, CHLA-05-ATRT, CHLA-266 and BT-12 were sensitive to the FGFR inhibitors PD173074, BGJ398 and AZD4547. Interestingly, the ATRT cell line CHLA-06-ATRT did not show sensitivity to FGFR inhibition. Analysis of FGFR protein expression by immunoblot analysis indicated that CHLA-04-ATRT and CHLA-266 cells expressed both FGFR1 and FGFR2, while the cell lines CHLA-05-ATRT and BT-12 expressed only FGFR1. The CHLA-06-ATRT cell line did not show expression of any FGFRs. We further demonstrated functional FGFR activity in the ATRT cell lines by treating growth factor-deprived ATRT cells with FGF-1 or FGF-2, both of which led to dose dependent cell growth only in the cell lines expressing FGFR1 or FGFR2. We are currently investigating the expression of FGFR isoforms in ATRT patient samples. Taken together, our results demonstrate a potential role for FGFR1 and FGFR2 in the survival and proliferation of ATRT cells and indicate that FGFRs could serve as potential targets for therapy in ATRT. Citation Format: David O. Azorsa, Oliver B. Pepper, Madhavi Chakravadhanula, David W. Lee, Justin J. Montoya, Victor Ozols, Ratan Bhardwaj, Robert J. Arceci. A role for fibroblast growth factor receptors in growth of atypical teratoid rhabdoid tumor cells and as potential therapeutic targets. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 3923. doi:10.1158/1538-7445.AM2015-3923

Abstract 5430: Frizzled-1 receptor, a potential therapeutic target for atypical teratoid rhabdoid tumor

Atypical Teratoid Rhabdoid Tumors (ATRTs) are aggressive tumor types in the field of pediatric neuro-oncology that need new therapeutic strategies since they are associated with significantly worse overall prognosis than other pediatric tumors. A characteristic feature of ATRTs is usually an aberration of SMARCB1/INI1/hSNF5 gene. One of the important developmental pathways for cellular development is the Wnt pathway. Wnt proteins are a family of 19 secreted glycoproteins and act as ligands that bind transmembrane receptors such as the Frizzled receptors. There are 10 known Frizzled receptors, and the binding of a specific Wnt ligand to a specific Frizzled receptor plays a crucial role in the regulation of diverse cellular processes. Perturbation of the activities of Wnt ligands can result in defects in embryonic development and diseased states such as cancer. In our preliminary transcriptome analyses results, Wnt5b and Frizzled-1 receptor (Fz-1) were significantly upregulated in 20 ATRT samples and 3 ATRT cell lines. The binding of Wnt5b with Fz-1 was further confirmed by immunoprecipitation and Western blotting analyses. Treatment of ATRT cells with Fz-1 specific siRNA did not indicate a significant decrease in cell viability. Similarly treatment of ATRT cells with a Fz-1 specific monoclonal antibody (Fz-1Mab) also did not indicate significant decrease in cell viability. However, in this study treatment of cells with a combination of Fz-1 specific siRNA and Fz-1Mab indicated a significant decrease in cell viability. A dose response analysis was performed over time to test various doses of Fz-1Mab with constant Fz-1 siRNA concentrations; a cell titre glo assay was then performed to assess cell viability. The physiologically relevant Fz-1Mab dose was then used to assess the effects of using this combination treatment on Fz-1 and Wnt5b protein levels. A decrease in Fz-1 and Wnt5b protein levels when Fz-1 receptor is blocked by siRNA and Mab indicates that Wnt5b bound to Fz-1 receptor plays an important role in ATRT disease etiology and could be a therapeutic target. Citation Format: Madhavi Chakravadhanula, Victor Ozols, Chris Hampton, Ratan Bhardwaj. Frizzled-1 receptor, a potential therapeutic target for atypical teratoid rhabdoid tumor. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 5430. doi:10.1158/1538-7445.AM2014-5430

Abstract 1564: Partial immunovesiculectomy by Th-1 dendritic cell vaccination: Implications for immunotherapy of solid tumors

The efficient and specific manner by which the mammalian immune system identifies and eradicates target antigen has stood as a testament to both its power and potential to similarly eradicate neoplastic self. In spite of this undeniable potential, the recalcitrance of the immune system to directed manipulation has been formidable, and the manner by which self-directed cellular (Th-1) immunity might be reproducibly promulgated has not yet been elucidated. Recent basic advances in the understanding of dendritic cell (DC) maturation, Th-1 polarization, T-cell homing, and plasmacytoid DC biology might allow promulgation of self-directed cellular immunity provided that all important aspects of such promulgation have been identified and are properly implemented. To investigate this hypothesis, seminal vesicle (SV), was chosen as a model organ system in the wild type mouse. In this system, SV serves as a proxy tumor from an immunological perspective: antigenically distinguishable from other organ systems yet comprised entirely of self tissue antigens and stringently protected from immunological recognition by mechanisms of central and peripheral tolerance. Equivalent class I and class II antigenic environments were provided to spleen-derived DC by electroporation with SV mRNA and incubation with SV lysate. DC were matured with a full complement of inflammatory cytokines. DC were applied i.p. in the vicinity of the SV so as to have access to the specific lymphatics that drain the area. Plasmacytoid recruitment and IFN-γ secretion was induced by local i.p. administration of particulate imiquimod. Two months post-vaccination, SV and other organs were harvested and examined for pathological changes by HE 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 1564. doi:1538-7445.AM2012-1564

Abstract LB-132: The potential role of the multienzyme aminoacyl-tRNA synthetase complex in Th-1 immunity: implications for cancer immunotherapy

Proceedings: AACR 103rd Annual Meeting 2012‐‐ Mar 31‐Apr 4, 2012; Chicago, IL Recent advances in dendritic cell (DC)-based cancer vaccines have highlighted the potential utility of arming the immune system to target neoplastic self. A strong, yet controlled, TH1 response has typically been the goal of DC-based protocols, as it is cell-based adaptive immunity that possesses the ability to most-efficiently destroy the nascent tumor or occult micrometastases. Nonetheless, many DC-intrinsic factors have impeded the development of a vaccine strong enough to impart meaningful clinical results, and there remains a need for further study of the parameters required for maximal, targeted DC activity. Previously, we have shown that contemporaneous loading of DC with overlapping MHC class I and MHC class II peptide antigens leads to an enhanced TH1 response when compared to canonical DC stimulation as assayed by release of IL-12, sCTLA-4, proliferation of CD8+ CD25+, INF-γ release, and specific killing of target both in vitro and in vivo. These and other data have lead to the hypothesis that DC possess a sensor mechanism capable of discerning amino acid sequence similarities between peptides bound by MHC class I and class II and that tRNA synthetases may serve as critical components of the putative recognition machinery due to their inherent ability to recognize and distinguish between individual amino acid side chains. Here we report that the aminoacyl-tRNA synthetase complex (MACS), a large molecular complex comprised of at least nine aminoacyl-tRNA synthetases, possess interesting characteristics that might link it to this phenomenon. Components of the MACS complex, including the p43 structural subunit, were found in DC exosomes, the contents of which mimic those of the late endosome, a known cross-presentation compartment. MARS (methionyl-tRNA synthetase), a component of the MACS, as well as non-MACS components VARS (valyl-tRNA synthetase) and GARS (glycyl-tRNA synthetase) were found within exosomes as well. UVC cross-linking of exosomal extracts resulted in the incorporation of individual tRNA-synthetase isoforms into a very large complex of indeterminate size. p43 was differentially released from the MACS in response to the loading of DC only with matched class I (GFP mRNA) and class II (recombinant GFP protein) determinants. siRNA knockdown of MACS structural component p38 was able to ablate the ability of doubly-loaded DC to enhance the generation of CD8+CD25+ cells as well as the ability of these cells to secrete IFN-γ. Further, siRNA knockdown of p38 abrogated the release of p43 from DC loaded with matched class I and II determinants. With an enhanced understanding of these regulatory phenomena, the development of small molecule TH1 agonists and/or immunotherapeutic protocols with enhanced clinical efficacy become realistic and achievable goals. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr LB-132. doi:1538-7445.AM2012-LB-132