Final Report Summary - CROSSTALK IN GBMS (GENOMIC AND TRANSCRIPTIONAL ANALYSIS OF GLIOBLASTOMA MICROENVIRONMENTAL CELLULAR SUBSETS USING ANTIBODY MICROARRAYS) Dr. Gini successfully performed her research project entitled “Genomic and Transcriptional Analysis of Glioblastoma Microenvironmental Cellular Subsets Using Antibody Microarrays” during the three years of her fellowship. She started her research in the Mischel Lab at UCLA (Los Angeles, U.S.A.) on October 1st 2011 under the mentorship of Professor Paul Mischel. In August 2012, Professor Mischel was recruited to the Ludwig Institute for Cancer Research, San Diego Branch, on the UCSD campus. Dr. Gini moved with the Mischel Lab on August 1st 2012 to continue her project. Then, Dr. Gini efficiently transferred her acquired knowledge and professional skills to the Bonetti Lab, in the Department of Neurological Sciences and Movement at the University of Verona (Italy), during the third phase of the fellowship. As demonstrated below, Dr. Gini has shown outstanding achievements, confirmed by her relevant scientific publications, published or in preparation in high impact factor journals. Specifically, the project objectives of the three years of Dr. Gini’s fellowship were the following.Aims of the outgoing phase:1- Application of nano- and microfluidic technologies for cancer research.2- Genomic, transcriptomic and proteomic profiling of single cells.3- Functional analysis of the genomic, transcriptomic and proteomic finding.Aims of the return phase:1- To introduce innovative diagnostic nanotechnology for GBM analysis in the applicant’s European country, in Bonetti Lab, at the University of Verona, Italy.2- To bridge a new set of collaborations between the Mischel and Bonetti laboratories, paving the way towards joint implementation of these tools for molecular analysis of patients enrolled in GBM trials.3- To gain additional insights in GBM etiology.Outgoing phase:1- Application of nano- and microfluidic technologies for cancer research. DEAL (DNA Encoded Antibody Library) sorting of different cell phenotypes from heterogeneous patient’s tumor samples: DEAL sorting and microfluidic strategies were applied to extensively characterize Epidermal Growth Factor Receptor (EGFR) wild type and EGFRvIII positive cells from heterogeneous, patient derived GBM samples, helping the design of more efficient protocol of treatments for EGFRvIII positive GBMs. EGFR hyperactivated GBMs represent approximately 50% of the GBM patients (Gini B, Clinical Cancer Research, 2013; Nathanson DA, Gini B et al., Science, 2014). Single Cell Barcode Chip Analysis (SCBC): Dr. Gini is closely collaborating with the Heath Lab (Caltech, Pasadena, CA) mediating the translation of the SCBC approach in the clinic. A manuscript applying this technology to guide combinatorial targeted therapy for GBM patients has been submitted. This novel single cell phospho-proteome approach aims at personalizing cancer treatments (Gini B, Shin YS, Wei W et al., submitted).2- Genomic, transcriptional and proteomic profiling of single cells. Patient derived GBM neurospheres were characterized at the genomic, transcriptomic and proteomic levels in bulk assays using respectively SNPs array, gene expression analysis and biochemical tests. Single cell technologies were used to profile the GBM genome, transcriptome and proteome by respectively SNPs array, single cell RNA sequencing and single cell proteomic. Additionally, Fluorescence In Situ Hybridization (FISH) and molecular biology strategies were matched in the evaluation of the cancer genome. Significant insights were achieved from these combined measurements and their relevance in GBM was confirmed by functional tests. 3- Functional analysis. Functional screening validated the most relevant finding of the previously mentioned single cell tests and bulk assays. Specifically, Dr. Gini importantly contributed to the clarification of the role of tumor heterogeneity and pathway cross talk in EGFR and mTOR kinase inhibitor resistance and characterized overcoming approaches (Nathanson DA, Gini B et al., Science, 2014; Gini, et al., Clinical Cancer Research, 2013; Gini B and Mischel PS, 2014, Cancer Discovery; Gini B, Shin YS, Wei W et al., submitted).Return phase:1- To introduce innovative diagnostic nanotechnology for GBM analysis in the applicant’s European country, in Bonetti Lab, at the University of Verona, Italy.Dr. Gini matured the professional skills to successfully mediate the adoption of the DEAL (DNA Encoded Antibody Library) based Single Cell Barcode Chip (SCBC) platform analysis for the treatment protocol design of GBM patients at the University of Verona, through close collaboration with the Mischel Lab (Gini B, Shin YS, Wei W et al., submitted). 2- To bridge a new set of collaborations between the Mischel and Bonetti laboratories, paving the way towards joint implementation of these tools for molecular analysis of patients enrolled in GBM trials.The SCBC was already successfully applied to a set of freshly resected GBM biopsies from the surgery room as timely fashion tool for planning more effective combinatorial treatments. The incoming application of this technology to a broader number of GBM patients through joint clinical trials opens up the possibility to stratify GBM patients and to adopt tailored treatment protocols for different patient subgroups. 3- To gain additional insights in GBM etiology.Two major microenvironmental factors contributing to GBM etiology and to its treatment refractory phenotype were characterized: tumor hypoxia and pro-inflammatory immunitary lineages (Gini et al., 2014, in preparation). This analysis allowed to further elucidate the mechanisms through which cancers adapt to targeted therapies and to identify new druggable targets. Main results:The SCBC analysis of primary and recurrent GBM biopsies, from the surgery room, confirmed the alterations identified at diagnosis and pointed to more specific combinatorial treatments. EGFR triggered samples were tested for sensitivity to EGFR kinase inhibition or inhibition of the downstream effector mechanistic Target of Rapamycin (mTOR). Two major compensatory pathways were identified after an in vitro treatment of just 60 hours, and they were relying on the Extracellular-Signal-Regulated kinase (ERK) signaling and on the Eukaryotic Translation Initiation factor 4-E Binding Protein 1 (4-E-BP1) signaling. Simultaneous inhibition of both, main triggering pathways and their redundant branches, more effectively inhibited tumor cell proliferation and induced cell death (Gini B, Shin YS, Wei W et al., submitted). The new combinatory treatments identified will guide the design of incoming clinical trials.Since EGFRvIII addicted GBMs showed to become resistant to kinase inhibition through down regulation of the episomal level of EGFRvIII (Nathanson DA, Gini B, et al., 2014, Science; Gini B and Mischel PS, 2014, Cancer Discovery), we hypothesized that a drug holiday would rescue the oncogene copies and the sensitivity to the drug. Indeed, patient derived, primary GBM neurospheres re-sensitized to targeted therapy affecting the EGFR after a drug holiday, during which tumor cells reconstituted the sensitive genetic background. This insight will be translated in new treatment regimens and doses for GBM patients. Microenvironmental factors contributing to GBMs progression and resistance to treatment were investigated. In these analyses, hypoxia and pro-inflammatory cell lineages were considered. Tumor hypoxia resulted correlated with resistance to mTORki in GBM. In this condition, the Nuclear Factor kappa-light-chain-enhancer of activated B cells (NF-kB) pathway was significantly up-regulated, as well as the Phosphatidylinositol 3-Kinase (PI3K)/Protein Kinase B (PKB) pathway. Combinatorial treatment of mTORki and NF-kB inhibitors are currently under investigation as potential synergistic therapies in GBMs cells. Of note, the onset of the resistance phenotype resulted Hypoxia-Inducible Factor-1-alpha (HIF-1-alpha) independent (Gini et al., 2014, in preparation). The stromal modulation of the GBM phenotype was assessed analyzing the Tumor-Associated Macrophages (TAMs). M1 macrophages are pro-inflammatory and tumor suppressive whereas M2 macrophages are anti-inflammatory and tumor promoting. Using M1 conditioned medium, GBM U251 cells growth was significantly inhibited with induction of apoptosis. Preliminary IHC analysis on low grade gliomas for M2 markers confirmed a reduced time to progression in patients with high scored IHC stain. Specifically, the relevant results achieved by Dr. Gini with her fellowship are subject of the following papers, submitted or recently published in high impact factor journals:- Nathanson DA, Gini B, Mottahedeh J, Visnyei K, Koga T, Gomez G, Eskin A, Hwang K, Wang J, Masui K, Paucar A, Yang H, Ohashi M, Zhu S, Wykosky J, Reed R, Nelson SF, Cloughesy TF, James CD, Rao PN, Kornblum HI, Heath JR, Cavenee WK, Furnari FB, Mischel PS. 2014. Targeted Therapy Resistance Mediated by Dynamic Regulation of Extrachromosomal Mutant EGFR DNA. Science, 343, 6166:72-76. This research has been highlighted in Nature Medicine, 20, 28 (2014) and Science Signaling, 7, 307, ec5 (2014).- Gini B, Mischel PS. 2014. Greater than the sum of its parts: Single nucleus sequencing identifies convergent evolution of independent EGFR mutants in GBM. Cancer Discovery; 4(8): 876-8.- Gini B, Shin YS, Matsutani T, Masui K, Yang H, Gu Y, Herrmann K, Hwang K, Bonetti B, Chopra R, James CD, Cavenee WK, Cloughesy TF, Mischel PS, Heath JR and Wei W. 2014. Single cell proteomic network analysis defines effective targeted combination therapy in glioblastoma; submitted.- Gini B, Yang H, Bonetti B, Cavenee WK, Mischel PS. 2014. Hypoxia regulation of sensitivity to the mechanistic Target Of Rapamycin kinase inhibition (mTORki) in Glioblastoma Multiforme; in preparation.- Gini B, Zanca C, Guo D, Matsutani T, Masui K, Ikegami S, Yang H, Nathanson D, Villa GR, Shackelford D, Zhu S, Tanaka K, Babic I, Akhavan D, Lin K, Assuncao A, Gu Y, Bonetti B, Mortensen DS, Xu S, Raymon HK, Cavenee WK, Furnari FB, James CD, Kroemer G, Heath JR, Hege K, Chopra R, Cloughesy TF, Mischel PS. 2013. The mTOR kinase inhibitors, CC214-1 and CC214-2, preferentially block the growth of EGFRvIII-activated glioblastomas. Clinical Cancer Research. 19(20):5722-32.- Gini B, Masui K, Wykosky J, Zanca C, Mischel PS, Furnari F, Cavenee W. 2013. A Tale of Two Approaches: Complementary Mechanisms of Cytotoxic and Targeted Therapy Resistance May Inform Next Generation Cancer Treatments. Carcinogenesis. 34(4):725-38. - Iwanami A, Gini B, Zanca C, Matsutani T, Assuncao A, Nael A, Dang J, Yang H, Zhu S, Kohyama J, Kitabayashi I, Cavenee WK, Cloughesy TF, Furnari FB, Nakamura M, Toyama Y, Okano H, Mischel PS. 2013. PML mediates glioblastoma resistance to mammalian target of rapamycin (mTOR)-targeted therapies. PNAS. 110(11): 4339-44.- Masui K, Tanaka K, Akhavan D, Babic I, Gini B, Matsutani T, Iwanami A, Zhu S, Yang H, Yong WH, Cloughesy TF, Cavenee WK, Mellinghoff IK, Paul S. Mischel. 2013. mTORComplex 2 controls FOXO acetylation to promote aggressive glioblastoma growth through metabolic reprogramming. Cell Metabolism. 5;18(5):726-39.- Babic I, Anderson ES, Tanaka K, Guo D, Li B, Zhu S, Gu Y, Villa G, Akhavan D, Nathanson D, Gini B, Mareninov S, Li R, Espindola C, Kurdistani SK, Eskin A, Nelson SF, Yong WH, Cavenee WK, Cloughesy TF, Christofk HR, Black DL, Mischel PS. 2013. Oncogene-induced alternative splicing of Max promotes glycolytic tumor growth in brain cancer. Cell Metabolism. 17(6): 1000-1008.- Chaumeil M, Gini B, Yang H, Iwanami A, Subramanian S, Ozawa T, Pieper RO, Mischel PS, James CD, Mitchel SB, Ronen SM. 2012. Longitudinal evaluation of MPIO-labeled cell biodistribution in glioblastoma using high resolution and contrast enhanced MR imaging at 14.1 Tesla. Neuro-Oncology. 14(8):1050-61- Tanaka K, Babic I, Nathanson D, Akhavan D, Guo D, Gini B, Dang J, Zhu S, Yang H, de Jesus J, Amzajerdi AN, Zhang Y, Dibble CC, Dan H, Rinkenbaugh A, Yong WH, Vinters HV, Gera JF, Cavenee WK, Cloughesy TF, Manning BD, Baldwin AS, Mischel PS. 2011. Oncogenic EGFR signaling activates an mTORC2-NF-κB pathway that promotes chemotherapy resistance. Cancer Discov. Nov 1;1(6):524-538.- Guo D, Reinitz F, Youssef M, Hong C, Nathanson D, Akhavan D, Kuga D, Amzajerdi AN, Soto H, Zhu S, Babic I, Tanaka K, Dang J, Iwanami A, Gini B, Dejesus J, Lisiero DD, Huang TT, Prins RM, Wen PY, Robins HI, Prados MD, Deangelis LM, Mellinghoff IK, Mehta MP, James CD, Chakravarti A, Cloughesy TF, Tontonoz P, Mischel PS. 2011. An LXR agonist promotes GBM cell death through inhibition of an EGFR/AKT/SREBP-1/LDLR-dependent pathway. Cancer Discov. 1(5):442-456.Dr. Gini project has strong socio-economic impact with regard to the protocols of treatment of cancer patients. Specifically, the single cell technology tests on freshly resected GBMs allowed to gain significant achievements in the treatment design for GBMs patients, with the potential to reduce the socio-economic burden dependent on the management of patient diagnosed with cancer. Of note, this methodology, while optimized in GBMs, can be applied for the analysis of other forms of cancer. Contacts: Professor Bruno Bonetti, bruno.bonetti@univr.it; Professor Paul Mischel, pmischel@ucsd.edu; Dr. Beatrice Gini, ginibea@hotmail.com
