Viviane Tabar - US grants

[unreadable] DESCRIPTION (provided by applicant): Brain irradiation is a powerful tool in the treatment of cancers including primary and metastatic many tumors, and even leukemias and lymphomas. However its use is restricted by the poor tolerance of the nervous tissue and the development of serious side effects among long-term survivors. These include a decline in IQ and cognitive function. This phenomenon is very frequent and irreversible. Its pathogenesis is not fully elucidated but it involves extensive demyelination occurring in a delayed fashion (months to years). Based on preliminary data obtained in the rat, we hypothesize that radiation exhausts the pool of dividing oligodendrocyte progenitors (OPC) and therefore the brain's ability to replace the myelinating cells. We have developed significant experience in the neural differentiation of human ES (hES) cells and we propose the transplantation of hES-derived neural precursors as an effective strategy for repair of radiation-induced brain damage. [unreadable] [unreadable] We will study the temporal and topographic pattern of cell loss following whole brain irradiation in the rat at five timepoints over the course of one year. The rats will receive BrdU prior to sacrifice and the brains will be processed for BrdU, NG2 (an OPC marker) and other neural markers. We will then graft irradiated rats with two different populations of neural precursors obtained from hES cells: a multipotential neural stem cell-like population, and an oligodendroglial progenitor population. In our preliminary data we have successfully derived such cells from hES cells and established stable eGFP-expressing hES cells via lentiviral gene transfer for reliable in vitro and in vivo identification. Grafted animals will be examined at 6 weeks after transplantation to assess cell survival, migration, and ability to replace the OPC niche, and at 6-8 months after transplantation to evaluate the ability of hES cells to contribute to remyelination of the injured brain. This work addresses a therapeutically important problem in brain cancer treatment and will provide a wealth of information on the behavior of grafted human ES cells within the context of an injured brain. If successful, we plan to initiate future studies addressing the functional impact of hES-derived neural precursor grafts on radiation-induced cognitive decline. This grant will only use the INIH-approved cell line: HI. NIH code WA01. [unreadable] [unreadable]

[unreadable] DESCRIPTION (provided by applicant): The goal of this project is to investigate the potential of human embryonic stem (hES) cell derived oligoprogenitors as restorative therapy for radiation-induced damage in the brain at the structural and functional levels. The project is based on a substantial body of preliminary data including high yield derivation of oligoprogenitors from human ES cells (NIH approved lines WA-01 and WA-09), survival and differentiation of hES derived progenitors in rat brains, and a quantitative study of the deleterious and irreversible effects of radiation to the adult rat brain. Radiation damage to the brain is a significant clinical problem in cancer management, affecting patients with primary as well as metastatic brain tumors. The effects of radiation to the brain generally occur late (>6-12 months after exposure) and lead to irreversible deterioration in cognitive function. The pathogenesis of late radiation damage is complex, but the pathological outcome is characterized by demyelination and subsequent cerebral atrophy. The radiation model remains essentially unexplored as a possible target for cell replacement despite significant literature pertaining to histopathological changes and the terrible toll on quality of life of the cancer patient. We demonstrate that irradiation results in exhaustion of the cycling oligodendrocyte progenitor pool, with subsequent inability of the brain to replace mature myelinating cells. We have recently developed protocols for the highly efficient differentiation of hES cells into oligoprogenitors. Here we propose to address their potential for repairing radiation damage to the CNS. We will purify the oligoprogenitor populations and perform pilot studies in irradiated rats in an effort to optimize cell numbers, migration, survival and favorable timing of graft placement post irradiation. Once these parameters are elucidated, we will proceed with long term oligoprogenitor or sham grafts in irradiated rats and perform careful behavioral assessments of learning, memory and cognitive tasks, as well electrical studies on brain slices to study conduction across the corpus callosum. We hope to demonstrate the role of hES derived oligoprogenitors in cytoarchitectural restoration of myelination and tissue and behavioral function post radiation. This proposal will use exclusively NIH approved hES lines: WA-01 and WA-09. [unreadable] [unreadable] [unreadable] [unreadable]

? DESCRIPTION (provided by applicant): This proposal aims at designing a cell-therapy based approach to hypopituitarism , with special focus on pituitary dysfunction due to radiation injury. The pituitary gland is a master endocrine organ that orchestrates the function of multiple glands in the organism, under direct control by the hypothalamus. Hypopituitarism is a very prevalent problem following radiation treatment to the brain. It is dose-related, progressive and irreversible; it also contributes to poor quality of life and other substantial problems among cancer survivors. Hypopituitarism is also an important permanent consequence of infection, traumatic brain injury or congenital conditions. We have developed a protocol for the specification of pituitary cells from human ES cells and further differentiation into hormone producing cells. We have also previously optimized a brain radiation model in the rat, using a clinically relevant fractionated radiation course. The animals are well studied for growth, weight and behavioral parameters and we have some preliminary evidence of pituitary dysfunction. Here we propose to optimize the human ES and iPS differentiation protocol to allow the enrichment into specific pituitary cell subtypes. Validation of cell phenotypes is obtained through gene expression and marker profiles, measurements of hormone secretions, as well as in vivo function. Human ES derived pituitary cells are grafted into hypophysectomized nude rats and basal hormone levels are measured in the serum at different time points. Grafts will be placed subcutaneously or orthotopically into the hypothalamus / pituitary interface. We will also graft the cells into the irradiated rats with evidence of pituitary damage. Analysis will focus on revovery of hormone levels in the plasma, as well as evidence of integration of the graft into the hypothalamic - pituitary axis, using stimulation tests. Furthermore, functional analysis will be attempted by asking whetehjr female rats with cell grafts can recover their ability to reproduce and lactate.The proposal addresses a novel problem that has received little attention to date. As we succeed in achieving long term remission among cancer patients, questions of poor quality of life among survivors have acquired a more pressing nature. This project will provide much needed insight into the feasibility of cell based therapy to ameliorate these consequences of brain radiation. It will also undertake the derivation of specific pituitary cell subtypes from humn ES or iPS cells, an unreported achievement in human cells to date.

Abstract / Project Summary DIPGs are rapidly lethal brain tumors of childhood. The majority of patients die within a year or two of diagnosis, as there is no effective treatment for this devastating disease. Their critical location in the brainstem has long prohibited access to tumor tissue. Recently, sequencing studies revealed novel histone mutations consisting of single amino acid substitutions in the tail of the histone H3 variants. A pattern of exclusive co-occurrence of histone mutations with specific signaling pathway alterations as well as age of onset and brain region alterations emerged. Taking advantage of these data and of the strict developmental window of the tumors, our team has built what is effectively the first genetically-engineered human ES cell-based model of a tumor. The model yielded tumors that recapitulated the genetic, transcriptomal, epigenetic and histological features of the disease, thus providing valuable tools for the study of the disease. Genome wide studies suggested that the histone mutation led to a resetting of the developmental status of the cells to an earlier more primitive stem cell state. Importantly, our modeling system served as an effective platform for drug screens, leading to the identification of a novel protein ?protein interaction network as a key component of the proliferative and growth machinery of these tumors, centered on the protein menin. Menin is a unique protein with multiple partners; it is oncogenic in the context of the MLL rearranged leukemias. Our data show that silencing menin or inhibiting its MLL interaction with a menin inhibitor leads to a decrease in proliferation and increased cell death, a completely novel finding never reported in glioma, that raises the promise of a therapeutic strategy. The proposal aims to expand our hES modeling platform by building new models that are representative of the genetic diversity of the somatic mutations described in brainstem gliomas. We will also study the events downstream of the histone mutations, with emphasis on the molecular basis of the oncogenic role of menin. The ultimate goal is to develop a therapeutic strategy for DIPGs, taking advantage of newly synthesized menin inhibitors. Our studies should also contribute to developing hES cells into a more widely applicable platform for cancer modeling capitalizing on their many advantages, including access to an unlimited supply of stage appropriate human cells for mechanistic or therapeutic studies, the ability to study tumor biology from the step of initiation to tumor maintenance, and the ease of implementing sophisticated genetic tools.

PROJECT SUMMARY/ABSTRACT Malignant gliomas are primary brain cancers which can be prognostically stratified on the basis of a single mutation in the gene for isocitrate dehydrogenase (IDH). While gliomas are highly lethal cancers, the IDH mutation confers a significantly better prognosis, compared to IDH wild type gliomas, for unclear reasons. The IDH mutation results in a neomorphic gain of function which produces high levels of the oncometabolite R-2- hydroxyglutarate (2HG) and global genomic hypermethylation. IDH mutant gliomas are characterized by an immune quiescent tumor microenvironment, with significant infiltration with glioma associated macrophages and microglia (GAMs), and high levels of 2HG detected in the microenvironment. The central hypothesis is that 2HG promotes immune quiescence in IDH mutant glioma by specifying GAM function through activation of glioma-associated lineage determining transcription factors, leading to downregulation of antigen presentation capacity of GAMs, and inhibition of peripheral monocyte differentiation. The overarching goal of this study is to investigate how 2HG affects the immune function of GAMs, and how this in turn gives rise to an immune quiescent phenotype. The objective of this proposal is to determine the molecular basis for GAM specification and to develop immune functional assays and novel cellular platforms to interrogate the relationship between 2HG and GAM function in IDH mutant gliomas. The interest of the proposed work lies in providing greater understanding of the biology of the glioma tumor microenvironment in order to inform emerging immunotherapies, most of which have failed in glioma. In addition, IDH inhibitors have entered the clinical trial phase in gliomas, and it is critical to understand whether inhibiting 2HG could have an unintended consequences by reversing immune quiescence and leading to a pro-inflammatory tumor accelerating phenotype.