Immunotherapy for Pediatric Brain and Spine Tumors: Current State and Future Directions.

BACKGROUND: Brain tumors are the most common solid tumors and the leading cause of cancer-related deaths in children. Incidence in the USA has been on the rise for the last 2 decades. While therapeutic advances in diagnosis and treatment have improved survival and quality of life in many children, prognosis remains poor and current treatments have significant long-term sequelae. SUMMARY: There is a substantial need for the development of new therapeutic approaches, and since the introduction of immunotherapy by immune checkpoint inhibitors, there has been an exponential increase in clinical trials to adopt these and other immunotherapy approaches in children with brain tumors. In this review, we summarize the current immunotherapy landscape for various pediatric brain tumor types including choroid plexus tumors, embryonal tumors (medulloblastoma, AT/RT, PNETs), ependymoma, germ cell tumors, gliomas, glioneuronal and neuronal tumors, and mesenchymal tumors. We discuss the latest clinical trials and noteworthy preclinical studies to treat these pediatric brain tumors using checkpoint inhibitors, cellular therapies (CAR-T, NK, T cell), oncolytic virotherapy, radioimmunotherapy, tumor vaccines, immunomodulators, and other targeted therapies. KEY MESSAGES: The current landscape for immunotherapy in pediatric brain tumors is still emerging, but results in certain tumors have been promising. In the age of targeted therapy, genetic tumor profiling, and many ongoing clinical trials, immunotherapy will likely become an increasingly effective tool in the neuro-oncologist armamentarium.

Distinct subcellular changes in proteasome activity and linkage-specific protein polyubiquitination in the amygdala during the consolidation and reconsolidation of a fear memory.

Numerous studies have supported a critical role for the ubiquitin-proteasome system (UPS) in the memory consolidation and reconsolidation processes. The protein targets and functional role of ubiquitin-proteasome activity can vary widely across cellular compartments, however, it is unknown how UPS activity changes within the nuclear, cytoplasmic, and synaptic regions in response to learning or memory retrieval. Additionally, while previous studies have focused on degradation-specific protein polyubiquitination, it is unknown how learning alters other polyubiquitin tags that are not targeted by the proteasome. Using cellular fractionation protocols in combination with linkage-specific polyubiquitin antibodies, we examined subcellular changes in ubiquitin-proteasome activity in the amygdala during memory consolidation and reconsolidation. Following memory acquisition, overall protein ubiquitination and proteasome activity simultaneously increased in the nucleus and decreased in the synaptic and cytoplasmic regions. The nuclear increases were associated with upregulation of degradation-specific (K48) and degradation-independent (K63, M1) polyubiquitin tags, suggesting multiple functions for ubiquitin signaling within this region. Interestingly, retrieval induced a very different pattern of ubiquitin-proteasome activity in the amygdala, consisting of increases in overall protein ubiquitination and proteasome activity and K48-, K63-, and M1-polyubiquitin tags in the synaptic, but not nuclear or cytoplasmic regions. Collectively, learning and memory retrieval dynamically and differentially alter degradation-dependent and degradation-independent ubiquitin-proteasome activity across different cellular compartments, suggesting that the UPS may serve unique functions during memory consolidation and reconsolidation.

Quantifying Subcellular Ubiquitin-proteasome Activity in the Rodent Brain.

The ubiquitin-proteasome system is a key regulator of protein degradation and a variety of other cellular processes in eukaryotes. In the brain, increases in ubiquitin-proteasome activity are critical for synaptic plasticity and memory formation and aberrant changes in this system are associated with a variety of neurological, neurodegenerative and psychiatric disorders. One of the issues in studying ubiquitin-proteasome functioning in the brain is that it is present in all cellular compartments, in which the protein targets, functional role and mechanisms of regulation can vary widely. As a result, the ability to directly compare brain ubiquitin protein targeting and proteasome catalytic activity in different subcellular compartments within the same animal is critical for fully understanding how the UPS contributes to synaptic plasticity, memory and disease. The method described here allows collection of nuclear, cytoplasmic and crude synaptic fractions from the same rodent (rat) brain, followed by simultaneous quantification of proteasome catalytic activity (indirectly, providing activity of the proteasome core only) and linkage-specific ubiquitin protein tagging. Thus, the method can be used to directly compare subcellular changes in ubiquitin-proteasome activity in different brain regions in the same animal during synaptic plasticity, memory formation and different disease states. This method can also be used to assess the subcellular distribution and function of other proteins within the same animal.

Males and Females Differ in the Subcellular and Brain Region Dependent Regulation of Proteasome Activity by CaMKII and Protein Kinase A.

The ubiquitin-proteasome system (UPS) controls the degradation of ~90% of short-lived proteins in cells and is involved in activity- and learning-dependent synaptic plasticity in the brain. Calcium/calmodulin dependent protein kinase II (CaMKII) and Protein Kinase A (PKA) can regulate activity of the proteasome. However, there have been a number of conflicting reports regarding under what conditions CaMKII and PKA regulate proteasome activity in the brain. Furthermore, this work has been done exclusively in males, leaving questions about whether these kinases also regulate the proteasome in females. Here, using subcellular fractionation protocols in combination with in vitro pharmacology and proteasome activity assays, we investigated the conditions under which CaMKII and PKA regulate proteasome activity in the brains of male and female rats. In males, nuclear proteasome chymotrypsin activity was regulated by PKA in the amygdala but CaMKII in the hippocampus. Conversely, in females CaMKII regulated nuclear chymotrypsin activity in the amygdala, but not hippocampus. Additionally, in males CaMKII and PKA regulated proteasome trypsin activity in the cytoplasm of hippocampal, but not amygdala cells, while in females both CaMKII and PKA could regulate this activity in the nucleus of cells in both regions. Proteasome peptidylglutamyl activity was regulated by CaMKII and PKA activity in the nuclei of amygdala and hippocampus cells in males. However, in females PKA regulated nuclear peptidylglutamyl activity in the amygdala, but not hippocampus. Collectively, these results suggest that CaMKII- and PKA-dependent regulation of proteasome activity in the brain varies significantly across subcellular compartments and between males and females.

The Ubiquitin-Proteasome System and Memory: Moving Beyond Protein Degradation.

Cellular models of memory formation have focused on the need for protein synthesis. Recently, evidence has emerged that protein degradation mediated by the ubiquitin-proteasome system (UPS) is also important for this process. This has led to revised cellular models of memory formation that focus on a balance between protein degradation and synthesis. However, protein degradation is only one function of the UPS. Studies using single-celled organisms have shown that non-proteolytic ubiquitin-proteasome signaling is involved in histone modifications and DNA methylation, suggesting that ubiquitin and the proteasome can regulate chromatin remodeling independent of protein degradation. Despite this evidence, the idea that the UPS is more than a protein degradation pathway has not been examined in the context of memory formation. In this article, we summarize recent findings implicating protein degradation in memory formation and discuss various ways in which both ubiquitin signaling and the proteasome could act independently to regulate epigenetic-mediated transcriptional processes necessary for learning-dependent synaptic plasticity. We conclude by proposing comprehensive models of how non-proteolytic functions of the UPS could work in concert to control epigenetic regulation of the cellular memory consolidation process, which will serve as a framework for future studies examining the role of the UPS in memory formation.