Kelly, Brendan, 2014-2015

Brendan Kelly
Brendan
Kelly

"Functional Analysis of the H3.3-K27M Mutation in Pediatric Glioma-Derived IPS Cells"

Host Mentor: Marius Wernig
Stanford University

A heterozygous missense mutation in the H3F3A gene, which encodes a lysine-27-to-methionine substitution in the replication-independent histone H3.3, has been implicated as the main tumor driver in a subset of pediatric high-grade gliomas. This mutant histone variant, H3.3-K27M, alters normal histone marks and promotes a globally hypomethylated DNA landscape. Recent evidence has led to speculation that this mutation promotes gliomagenesis, exclusively, in oligodendrocyte progenitor cells (OPCs) to form diffuse intrinsic pontine gliomas (DIPGs). Thus, the purpose of this study is to determine the epigenetic and oncogenic properties associated with this mutation in OPCs, in order to determine if it is the main tumor driver, in this particular cell of origin. To accomplish this, three patient-derived DIPG cell lines, which exhibit the H3.3-K27M mutation, will be reprogrammed into induced pluripotent stem cells (iPSCs), in order to reverse epigenetic configurations associated with the tumors. Once this is accomplished, specifically, the mutant allele will be corrected with with a floxed-wildtype version of H3F3A attached to an inducible mutant version, via the CRISPR/Cas9 system. These iPSCs will then be differentiated into cells of the neural lineage. This confers the ability to compare, with a great degree of accuracy, the phenotypic and epigenetic differences, and the degree to which this mutation is the main promoter of oncogenic properties in cells with and without this mutation. Therefore, through directed differentiation we intend to compare iPSC derived: OPCs, neural stem cells (NSCs), and astrocyte committed progenitors (ACPs), with and without the H3.3-K27M mutation, to determine whether this mutation is oncogenic, particularly, in OPCs.