Post-translational regulation of cerebellar development and its implications in pediatric brain cancer

Tumors often result from temporal and spatial dysregulation of developmental signaling pathways. A striking example is medulloblastoma, the most common malignant pediatric brain tumor. During normal cerebellar development, granule neuron precursors (GNPs) proliferate extensively in response to hedgehog (Hh) signaling before differentiating into granule neurons. Sustained Hh signaling in GNPs, due either to reduced function of pathway components that negatively regulate Hh signal transduction or to heightened function of activating Hh signal transducers, results in continued maintenance and division of precursor cells beyond the normal proliferative stage of development, and eventually to formation of a medulloblastoma. To better understand the molecular mechanisms driving proliferation in GNPs and medulloblastoma, I characterized changes in transcript, protein and protein phosphorylation at the beginning of the proliferative stage of GNP development (P1), at peak proliferation (P7) and in the first moments of differentiation (P14). The proliferative period of GNP development had few changes in transcript (<5% of all transcripts changed greater than 2-fold, of which 92% decreased), or protein abundance (<4% of all quantified proteins changed greater than 2-fold, 50% decreased). In contrast, GNP differentiation was accompanied by a moderate increase in transcription (<5% changed 2-fold, 86% of which increased) and widespread protein turn-over (31% of the entire proteome changed 2-fold or more, equally up and down). Notably, phosphorylation was highest at P7 (4.9% of the phosphorylation sites increased >2-fold between P1 and P7) but differentiation was accompanied by widespread dephosphorylation (21.4% of the phosphoproteome decreased > 2-fold). The medulloblastoma-like, peak proliferative stage of GNP development was characterized by high levels of phosphorylation of a specific set of proteins which become dephosphorylated upon differentiation. This suggests that increased activity of a particular kinase might played a role in proliferation of GNPs, and by inference, medulloblastoma. Protein kinase CK2 emerged as a driver of hundreds of phosphorylation events during the proliferative, medulloblastoma-like stage of GNP growth, including three of the eight genes commonly amplified in medulloblastoma. I found that CK2 is required for normal GNP proliferation due to its action on two late steps in Hh signal transduction: stabilizing the transcription factor Gli2 and promoting Gli2-regulated transcription. Treatment of highly aggressive human SHH MB cell lines with CK2 inhibitors resulted in a dose-dependent increase in cell death. Furthermore, treatment of medulloblastoma-bearing mice with CK2 inhibitors blocked growth of mouse medulloblastomas resistant to previous hedgehog inhibitors and resulted in longterm tumor regression. This work has now directly led to a phase 1/2 clinical trial investigating the use of the CK2 inhibitor CX-4945 for treatment of SHH medulloblastoma.

Keywords: Developmental Biology.
Advisor: Fuller Margaret T.
Chen James Kenneth 
Elias Joshua
Shatz Carla J.