MDB-17. THE PTBP2 SPLICING FACTOR IS ESSENTIAL IN GROUP 3/4 MEDULLOBLASTOMA

Abstract Medulloblastoma (MB) is a common malignant pediatric brain tumor with significant heterogeneity among its four molecular subgroups WNT, SHH, Group 3 and 4. Group 3/4 MB are aggressive with a relatively poor prognosis indicating a critical need for novel therapeutic targets. Since the transcriptomic profiling of MB subgroups has not resulted in the expected identification of therapeutic potential, we focused on a proteomic approach. Building upon our previously published quantitative proteomic study, we selected 157 proteins significantly over-expressed in Group 3/4 MB. By using targeted loss-of-function CRISPR-Cas9 screen in four MB cell lines, we identified 17 essential genes shared by at least 2 of the cell lines. The PTBP2 splicing factor was essential to the survival of all cell lines. PTBP2 is an RNA binding protein involved in splicing regulation playing an important role during neuronal maturation. We hypothesize that persistent expression of PTBP2 results in a differentiation arrest and preservation of proliferative potential. To evaluate the role of PTBP2 in group 3/4 MB biology, we used single target CRISPR-Cas9 to stably knock-out PTBP2 expression in D556 and MB002 MB cells. We performed bulk RNA-seq and quantitative proteomics of these KO clones along with CLIP-seq to map PTBP2 targeted transcripts. We also performed phenotype assays in KO clones. RNA-seq in PTBP2 KO MB cells revealed 2,213 differentially expressed genes predominantly involved in neuronal differentiation and antigen presentation. These genes were cross-referenced with quantitative proteomics and eCLIP data. Phenotype assays of PTBP2 KO cells revealed reduced proliferation and migration compared to controls. To study the effect of PTBP2 on MB differentiation, we stimulated MB002 PTBP2 KO cells with FBS and observed increased surface attachment and differentiated morphology compared to controls. This suggests that persistent PTBP2 expression inhibits differentiation in MB cells with an attendant increase of proliferation activity.