Title: Isocitrate Dehydrogenase-1 R132H Mutation in Oligodendrocyte Progenitors and Proneural Glioma
Description: The identification of the isocitrate dehydrogenase-1 mutation (IDH-1 R132H) in human gliomas has been an important prognostic tool for diagnosing tumor subtype and determining prognosis value in patient care. However; it has not influenced current treatment practices because its role in glioma development is still not fully understood. IDH-1 is a cytoplasmic enzyme that normally functions to mediate the conversion of isocitrate to α-ketogluterate (α-KG). Extensive characterization of the IDH-1 R132H mutation demonstrated that it primarily results in defects associated with (1) decreased efficiency of normal IDH-1 enzymatic activity, (2) elevated production of R-2-hydroxygluterate (R-2HG), and (3) altered genome-wide DNA hypermethylation. Interestingly, the somatic point mutation is exclusively seen in the Proneural glioma subtype (PN), which has phenotypic similarities to oligodendrocyte progenitor cells (OPCs). This suggests that these tumors arise from OPCs or progenitors that acquire an OPC-like phenotype.

Thus far, the tumorigenic potential of IDH-1 R132H has not been extensively investigated in current in vivo models of GBM. Beyond its altered enzymatic affinity, the mechanism which facilitates OPC transformation is still not understood. In order to investigate the effects of the IDH-1 R132H mutation during PN gliomagenesis, we built upon previous work by our research group to design a novel in vivo experimental murine glioma system. While IDH-1 R132H mutations have been implicated as the earliest event in PN glioma formation the tumorigenic potential of this metabolic mutation on OPCs of the subcortical white matter has thus far not been tested. In this dissertation I describe my experimental approach and findings for specifically addressing two aims for understanding the relationship of the IDH-1 R132H mutation, OPCs and gliomagenesis: (1) determine whether the expression of the IDH-1 R132H mutant at tumor induction can effect glioma initiation and progression during gliomagenesis in a manner that is phenotypically and molecularly distinct from IDH-1 WT gliomagenesis; and (2) determine how expression of the IDH-1 R132H mutation and 2-HG are affecting OPC lineage development and whether this is facilitating these glial progenitors to acquire a phenotype that resembles cellular transformation.

I found that delivery of the IDH-1 R132H mutations to OPCs in the adult murine subcortical white matter was by itself not sufficient to form tumors; however, when expressed in the context of TP53 tumor suppressor deletions and PDGF, tumors formed and expressed both a histological and molecular signature resembling PN human gliomas. These murine gliomas were molecularly and metabolically distinct from IDH-1 wildtype murine gliomas, in that they showed elevated 2-HG production and evidence of a transcriptional and translational signature that was consistent with significantly increased protein synthesis activity. These particular results provided two major innovations in the study of gliomagenesis: (1) the first murine model of PN IDH-1 R132H mutant gliomagenesis initiated from the delivery of genetic alterations to OPCs, and (2) the isolation and stable propagation of tumor cells isolated from these gliomas that retain expression of the metabolic mutations. More importantly, my findings provide the first account of the IDH-1 R132H mutations driving a cumulative increase in global translation.

Alternatively, when I directly tested the effects of the IDH-1 R132H mutation in culture OPCs in the absence of additional genetic alterations, I found that the mutation has a role in impairing differentiation by retaining expression of immature lineage markers (PDGFR and NG2) and keeping OPCs highly proliferative when stimulated to differentiate. This is further supported by the inability of OPCs that expressed the IDH-1 R132H mutation to increase mRNA levels of myelin proteins during differentiation. Directly treating OPCs with 2-HG during differentiation recapitulated a cellular phenotype associated with reduced expression of a mature oligodendroctye marker (O1) and the retention of an immature profile (PDGFR+/O1-). However, while expression of IDH-1 R132H mutation had no effect on cell viability, 2-HG had a dose-dependent effect in decreasing cell viability both in conditions of OPC proliferation and differentiation. Analysis of translation activity in OPCs in response to IDH-1 R132H or R-2HG treatment revealed that both conditions had very distinct patterns in the cellular distribution of global protein synthesis.

These experiments provide a preliminary account of how the IDH-1 R132H mutation is negatively influencing the ability for OPCs to undergo normal lineage development and informs on impaired cell cycle exit playing a role in facilitating the acquisition of a transformed cellular phenotype. The capacity for OPCs to undergo cellular transformation into fully-formed PN IDH-1 R132H mutant gliomas has not been previously investigated. In this study, I engineered an experimental approach for targeting this progenitor population. I was able to address this gap in current understanding by providing comprehensive analysis and characterization of a murine model of the IDH-1 R132H mutation. By investigating the effects of IDH-1 R132H expression in non-transformed OPCs and PN gliomagenesis, I was able to identify a process of cellular adaption where the presence of the mutation throughout gliomagenesis defines a cellular context that is distinct from the setting of normal OPCs. This process involves global alterations in translation activity.

Furthermore, I believe that the mechanism by which this mutation drives tumor formation may be relevant to the mechanisms that are unique to the development and lineage progression of OPCs. Through the use of in vivo murine models, in vitro glial progenitor systems and an integrated molecular approach for identifying metabolic and phenotypic alterations, I was able to provide an account of my graduate research work dedicated to characterizing the effects of IDH-1 R132H in a mouse model of PN glioma and the effect of IDH1R132H on non-neoplastic OPCs as a novel approach to exploring its effects at early stages of gliomagenesis in the process of cellular transformation.