Studies in animal models have shown that traumatic brain injury (TBI) induces the rapid accumulation of many of the same key proteins that form pathologic aggregates in neurodegenerative diseases. Here, we examined whether this rapid process also occurs in humans after TBI. Brain tissue from 18 cases who died after TBI and from 6 control cases was examined using immunohistochemistry. Following TBI, widespread axonal injury was persistently identified by the accumulation of neurofilament protein and amyloid precursor protein (APP) in axonal bulbs and varicosities. Axonal APP was found to co-accumulate with its cleavage enzymes, beta-site APP cleaving enzyme (BACE), presenilin-1 (PS1) and their product, amyloid-beta (Abeta). In addition, extensive accumulation of alpha-synuclein (alpha-syn) was found in swollen axons and tau protein was found to accumulate in both axons and neuronal cell bodies. These data show rapid axonal accumulation of proteins implicated in neurodegenerative diseases including Alzheimer's disease and the synucleinopathies. The cause of axonal pathology can be attributed to disruption of axons due to trauma, or as a secondary effect of raised intracranial pressure or hypoxia. Such axonal pathology in humans may provide a unique environment whereby co-accumulation of APP, BACE, and PS1 leads to intra-axonal production of Abeta as well as accumulation of alpha-syn and tau. This process may have important implications for survivors of TBI who have been shown to be at greater risk of developing neurodegenerative diseases.

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The majority of oncogenic drivers are intracellular proteins, thus constraining their immunotherapeutic targeting to mutated peptides (neoantigens) presented by individual human leukocyte antigen (HLA) allotypes1. However, most cancers have a modest mutational burden that is insufficient to generate responses using neoantigen-based therapies2,3. Neuroblastoma is a paediatric cancer that harbours few mutations and is instead driven by epigenetically deregulated transcriptional networks4. Here we show that the neuroblastoma immunopeptidome is enriched with peptides derived from proteins that are essential for tumourigenesis and focus on targeting the unmutated peptide QYNPIRTTF, discovered on HLA-A*24:02, which is derived from the neuroblastoma dependency gene and master transcriptional regulator PHOX2B. To target QYNPIRTTF, we developed peptide-centric chimeric antigen receptors (CARs) using a counter-panning strategy with predicted potentially cross-reactive peptides. We further hypothesized that peptide-centric CARs could recognize peptides on additional HLA allotypes when presented in a similar manner. Informed by computational modelling, we showed that PHOX2B peptide-centric CARs also recognize QYNPIRTTF presented by HLA-A*23:01 and the highly divergent HLA-B*14:02. Finally, we demonstrated potent and specific killing of neuroblastoma cells expressing these HLAs in vitro and complete tumour regression in mice. These data suggest that peptide-centric CARs have the potential to vastly expand the pool of immunotherapeutic targets to include non-immunogenic intracellular oncoproteins and widen the population of patients who would benefit from such therapy by breaking conventional HLA restriction.

mnd2 mice die prematurely as a result of neurodegeneration 30-40 days after birth due to loss of the enzymatic activity of the mitochondrial quality control protease HtrA2/Omi. Here, we show that transgenic expression of human HtrA2/Omi in the central nervous system of mnd2 mice rescues them from neurodegeneration and prevents their premature death. Interestingly, adult transgenic mnd2 mice develop accelerated aging phenotypes, such as premature weight loss, hair loss, reduced fertility, curvature of the spine, heart enlargement, increased autophagy, and death by 12-17 months of age. These mice also have elevated levels of clonally expanded mitochondrial DNA (mtDNA) deletions in their tissues. Our results provide direct genetic evidence linking mitochondrial protein quality control to mtDNA deletions and aging in mammals. Cell Death and Differentiation (2013) 20, 259-269; doi:10.1038/cdd.2012 published online 14 September 2012 Mitochondria are dynamic organelles primarily involved in the production of adenosine triphosphate (ATP) through oxidative phosphorylation. They also play important roles in diverse cellular processes such as cell death, autophagy and innate immunity. 1 As a consequence of oxidative phosphorylation, mitochondria produce reactive oxygen species (ROS), which damages mitochondrial proteins, lipids and nucleic acids, because of their proximity to the source of ROS production. Accumulation over time of mutations and deletions in mitochondrial DNA (mtDNA) together with increased protein misfolding, as a result of ROS damage, leads to ageassociated decline in mitochondrial function, which is believed to be responsible for organismal aging and age-associated diseases. [2][3][4] To maintain optimal mitochondrial function over time, a number of quality control mechanisms exist that monitor and regulate all aspects of mitochondrial physiology. Damaged and unfolded mitochondrial proteins are removed by mitochondrial quality control proteases, which recognize these proteins and degrade them. 5,6 The ATP-dependent AAA (ATPase-Associated with diverse cellular Activities) proteases, are among the best-characterized proteases implicated in mitochondrial protein quality control. 7 The AAA proteases, ClpXP and Lon, located in the matrix are involved in quality control of matrix proteins. Although no specific mitochondrial substrate has been identified for the ClpXP protease, in vitro studies showed that Lon protease preferentially targets oxidatively damaged matrix aconitase. 8 Two additional AAA proteases, Paraplegin (encoded by the SPG7 gene) and YME1L, are associated with the inner membrane with their catalytic sites facing the matrix and intermembrane space, respectively. 7 These proteases are believed to be primarily involved in the degradation of damaged and unfolded membrane proteins of the electron transport chain. In addition, Paraplegin has been shown to process the mitochondrial ribosomal protein MrpL32, 9 suggesting that it might also function in mitochondrial ribosome assembly. Loss-of-functi...

Accumulation of aggregated α-synuclein into Lewy bodies is thought to contribute to the onset and progression of dopaminergic neuron degeneration in Parkinson's disease (PD) and related disorders. Although protein aggregation is associated with perturbation of proteostasis, how α-synuclein aggregation affects the brain proteome and signaling remains uncertain. In a mouse model of α-synuclein aggregation, 6% of 6215 proteins and 1.6% of 8183 phosphopeptides changed in abundance, indicating conservation of proteostasis and phosphorylation signaling. The proteomic analysis confirmed changes in abundance of proteins that regulate dopamine synthesis and transport, synaptic activity and integrity, and unearthed changes in mRNA binding, processing and protein translation. Phosphorylation signaling changes centered on axonal and synaptic cytoskeletal organization and structural integrity. Proteostatic responses included a significant increase in the levels of Lmp7, a component of the immunoproteasome. Increased Lmp7 levels and activity were also quantified in postmortem human brains with PD and dementia with Lewy bodies. Functionally, the immunoproteasome degrades α-synuclein aggregates and generates potentially antigenic peptides. Expression and activity of the immunoproteasome may represent testable targets to induce adaptive responses that maintain proteome integrity and modulate immune responses in protein aggregation disorders.

Background: Neuroblastoma (NB) is an embryonal tumor of the sympathetic nervous system that accounts for 12% of childhood cancer deaths. While the introduction of GD2 immunotherapy provides an improvement in time to progression, the therapy is toxic and impact on overall survival is minimal, supporting an urgent need for novel immunotherapies. To date, the cell surface landscape (surfaceome) of NB remains undefined, hindering the identification of immunotherapeutic targets. Methods: To identify NB surfaceome proteins, we performed plasma membrane protein extraction using sucrose gradient ultracentrifugation coupled to mass spectrometry (nLC-MS/MS) in NB cell lines (n=12) and patient derived xenografts (PDX; n=10). These data were integrated with existing RNA-sequencing (NB=153; Normal=7859) and H3K27ac chromatin immunoprecipitation (ChIP)-sequencing data (from overlapping NB cell lines) to evaluate extracellular proteins differentially expressed in NB compared to normal tissues. Candidate targets were validated by immunohistochemistry on NB tumor and normal tissue microarrays (TMAs), flow cytometry and immunofluorescence. In-vitro functional studies were performed following genetic manipulation of candidate targets to assess cell proliferation, differentiation and viability. Finally, we tested ADCT-701 (a DLK1-directed antibody drug conjugate [ADC] with a pyrrolobenzodiazepine [PBD] warhead) in eight PDX models (study ongoing, total of 12 models initiated) with varying levels of DLK1 expression. At enrollment, two mice were each treated with a single dose of saline or 1mg/kg of B12-PL1601 (non-targeting PBD-conjugated ADC) or 1mg/kg ADCT-701 and mice were evaluated for 100 days or until tumor reached 2.0cm3. Results: We yielded on average 66% (range:60-68%) membrane protein enrichment with high reproducibility between biological replicates (80%; range:78-84%) and identified 4826 unique membrane proteins. Our approach confirmed known cell surface proteins in development as immunotherapeutic targets in NB (ALK, GPC2, NCAM1, DLL3 and CD276). Here, we prioritized DLK1 for further evaluation due to it being the only candidate with expression directly associated with a super enhancer element (P=6.09X10-5). RNA-sequencing and tissue microarray analysis of NB and normal tissues showed DLK1 to be overexpressed in a large subset of high-risk NB with minimal expression in normal tissues, excepting adrenal medulla and pituitary. Flow cytometry and immunofluorescence confirmed cell surface expression of DLK1 in a panel of NB cell lines. Genetic depletion of DLK1 using shRNA resulted in neurite outgrowth (P=7.26X10-5) and terminal differentiation. Full proteome analysis of DLK1 knockdown and control cell lines using MS showed regulation of proteins that control outgrowth of neurites (P=3.37X10-3) and development of neurons (P=3.76X10-3). To date, ADCT-701 treatment resulted in maintained complete response (N=2), complete response (N=3) and stable disease (N=1) in models with high DLK1 expression, while those with low/no expression showed disease progression (N=2). Conclusion: DLK1 is an epigenetically regulated immunotherapeutic target in neuroblastoma. ADCT-701 shows potent activity in preclinical models of NB and should be prioritized for clinical development. Citation Format: Amber K. Weiner, Alexander B. Radaoui, Matthew Tsang, Dan Martinez, Simone Sidoli, Karina L. Conkrite, Alberto Delaidelli, Jo Lynne Rokita, Maria V. Lane, Zalman Vaksman, Komal S. Rathi, Pichai Raman, Jennifer Pogoriler, Tricia Bhatti, Bruce Pawel, Beverly Teicher, Stephen W. Erickson, Poul Sorensen, Yael P. Mosse, Kateryna Krytska, Francesca Zammarchi, Patrick H. van Berkel, Malcolm A. Smith, Benjamin A. Garcia, John M. Maris, Sharon J. Diskin. A multi-omic surfaceome study identifies DLK1 as an epigenetically regulated protein and immunotherapeutic target in neuroblastoma [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference on Molecular Targets and Cancer Therapeutics; 2019 Oct 26-30; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2019;18(12 Suppl):Abstract nr LB-B04. doi:10.1158/1535-7163.TARG-19-LB-B04

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Background: Children with high-risk neuroblastoma have a poor prognosis despite intensive multimodal chemoradiotherapy. While monoclonal antibodies targeting the disialoganglioside GD2 improve outcomes in neuroblastoma, this therapy is associated with significant “on target-off tumor” toxicities. Thus, a major challenge remains in identifying novel cell surface molecules that meet the stringent criteria for modern immunotherapeutics, including unique tumor expression compared to normal childhood tissues, and preferably that these cell surface molecules be required for tumor sustenance and thus may be less susceptible to immune escape mechanisms. Methods: Differentially expressed genes that represent putative cell surface immunotherapeutic targets were initially identified by comparing high-risk neuroblastoma RNA sequencing data (N=126 primary tumors) to paired normal tissue data (GTEx; N=25 unique normal tissues; N=1-313 replicate samples/tissue type). For further prioritization, gene sets were filtered by in silico cell surface prediction and by absolute RNA expression, and then by assessing primary tumor DNA copy number via SNP genotyping data (N=177). For prioritized genes, protein expression and cellular localization was confirmed by Western blot, immunohistiochemistry (IHC), immunofluroescence (IF) and membrane extraction techniques in neuroblastoma cell lines and primary tumors. For functional characterization, neuroblastoma cell lines (N=12) were subjected to both gain and loss of function studies for each candidate gene. Results: The initial transcriptome-based discovery effort identified 649 significantly differentially expressed genes (log-fold change tumor vs. normal >1 for each tissue; adjusted p<0.05), 86 (13%) of which were predicted to be potential cell surface molecules. Through our analytic pipeline, we prioritized the extracellular glycosylphosphatidylinositol (GPI) anchored, signaling co-receptor Glypican-2 (GPC2) for validation given robust differential RNA expression (log-fold change tumor vs. normal tissue = 2.1-8.2; p<3 x 10-10), high-level absolute RNA expression (median FPKM=57; 85% of tumors with FPKM >25) and consistent DNA copy number gain (31% of primary neuroblastomas; N=177) associated with significantly higher GPC2 expression (p<0.0001). Immunoblot analysis confirmed ubiquitous GPC2 protein expression (N=8 high-risk neuroblastomas and 23 cell lines) and membrane extraction, IF, and IHC confirmed dense plasma membrane associated GPC2 protein expression in neuroblastoma cell lines. IHC analysis of primary neuroblastoma tumors (N=165) compared to a parallel array of pediatric normal tissues (N=41) further confirmed GPC2 protein expression to be membrane associated and tumor specific with very limited normal tissue expression. Lentiviral mediated RNAi induced GPC2 depletion in a panel of 12 neuroblastoma cell lines resulted in significant apoptosis and growth inhibition both in transient CellTiter-Glo and Caspase-Glo assays (20-87% decreased growth and 1.5-18.4-fold increased caspase 3/7 level vs. control) and with longer term real-time monitoring of cell growth (RT-CES). GPC2 overexpression resulted in significantly increased cellular proliferation (2.7-fold growth increase vs. control, p<0.0001). Finally, GPC2 was also found to be significantly differentially overexpressed in other embryonal cancers, most notably medulloblastoma. Conclusions: GPC2 is a candidate cell surface immunotherapeutic target and putative oncogene in high-risk neuroblastoma. More globally, these data show that genome-wide transcriptome analysis integrated with genomic and functional validation can identify differentially expressed cell surface oncogenes that may be attractive immunotherapeutic targets. Development of a GPC2 directed chimeric antigen receptor is ongoing, and progress will be reported. This abstract is also presented as Poster A13. Citation Format: Kristopher R. Bosse, Pichai Raman, Robyn T. Sussman, Michael Randall, Dan Martinez, Zhongyu Zhu, Bruce Pawel, Tricia Bhatti, Javed Khan, Dimiter S. Dimitrov, Crystal Mackall, John M. Maris. GPC2 is a candidate immunotherapeutic target and putative oncogene in high-risk neuroblastoma and other pediatric cancers. [abstract]. In: Proceedings of the AACR Special Conference on Advances in Pediatric Cancer Research: From Mechanisms and Models to Treatment and Survivorship; 2015 Nov 9-12; Fort Lauderdale, FL. Philadelphia (PA): AACR; Cancer Res 2016;76(5 Suppl):Abstract nr PR02.