INVESTIGATING THE MOLECULAR MECHANISMS OF DIFFERENTIATION FAILURE IN NF2 MUTANT NEUROEPITHELIAL STEM CELLS - ABSTRACT My long-term career goal is to develop more effective treatments for brain and spinal cord tumors in children and young adults with cancer predisposition syndromes. Spinal ependymoma (SP-EPN) is a molecularly distinct tumor that arises predominantly in the upper spinal cord. Most SP-EPN have biallelic genetic perturbations affecting the NF2 gene, and patients with NF2-associated schwannomatosis are predisposed to SP-EPN development. There is currently no effective medical therapy for SP-EPN. Surgical resection of late-stage symptomatic lesions is associated with high morbidity and mortality. There is a critical need to identify the molecular mechanisms underpinning pre-neoplastic lesions to halt progression in NF2 patients at an early stage. In our preliminary studies, we leveraged a unique spinal neuroepithelial stem (NES) cell model to show failure of differentiation at the radial-glia cell stage in NF2 knockout (NF2-/-) NES cells and the formation of pre- neoplastic growths in vitro and in vivo. Further, although most of the SP-EPN tumor display ependymal cell signatures, we identify a rare population within the tumor with radial-glia stem cell markers. The specific objectives of this study are: (i) to identify the cellular hierarchy in NF2-/- NES cell derived preneoplastic cells and patient-derived SP-EPN; (ii) to define the dysregulated signaling pathways in NF2-/- NES cells that fail to differentiate and (iii) to identify activated kinase targets that may rescue this differentiation failure. We hypothesize that derailed radial glia cells in the developing spinal cord are the source of SP-EPN formation and that targeting the aberrant signaling pathways in these cells will be important to prevent growth. To test this hypothesis, we will perform single cell RNA sequencing (scRNA seq) of NF2-/- NES cells during in vitro differentiation and xenografts (Aim 1). We will compare the molecular signatures of cells in pre-neoplastic growths with that of patient samples to validate the target pre-neoplastic cells in SP-EPN development. We will perform bulkRNA seq and multiplexed kinase inhibitor bead affinity chromatography and mass spectrometry (MiB/MS) to define the activated kinome profile of these pre-neoplastic cells compared to patient SP-EPN (Aim 2a) and the key signaling pathways that are disrupted in these cells. In Aim 2b we will test candidate kinase inhibitors to test in our NF2-/- NES cell model to determine if the failure of differentiation can be rescued pharmacologically. We expect to identify the cellular identity of NF2-/- preneoplastic cells that fail to differentiate, and the dysregulated signaling pathways that may be governing these cells. We aim to identify novel, rational therapeutic targets to stop the progression of tumor formation in children with NF2. To achieve research independence, I have identified three key areas in which I need mentorship and additional experience: (1) advanced cell engineering; (2) single cell analytics; (3) kinome profiling for drug discovery. I will accomplish this work under the mentorship of Dr Wade Clapp, a world expert in neurofibromatosis. Ultimately, I want to become an RO1 funded independent investigator directing a research program in brain and spinal cord tumorigenesis.
