Leveraging Medulloblastoma Clonal Dynamics to Overcome Treatment Resistance

Abstract Purpose: Medulloblastoma (MB) is a common pediatric brain tumor with distinct molecular subgroups, of which, Group 3 MB is associated with increased recurrence, metastatic potential and poor patient outcomes. Small molecule inhibitors targeting BMI1 have been shown to be efficacious against several types of malignant tumors, including pediatric MB. While our previously published in vivo study provides a promising proof-of-concept for the therapeutic targeting of BMI1 in Group 3 MB with small molecule inhibitor, it is not sufficient to eradicate the tumour. Experimental design: In this study, following preclinical validation of BMI1 inhibitor PTC-596, DNA barcoding technology was leveraged to profile in vivo clonal dynamics of Group 3 MB in response to the established chemoradiotherapy regimen alone and in combination with PTC-596. Following demonstration of a small number of treatment-refractory clones we sought to identify potential druggable molecular vulnerabilities by utilizing phosphoproteomic profiling and genome-wide CRISPR screening. Results: By comparing the changes in phosphorylation pattern of key signaling kinases post PTC-596 treatment with the list of sensitizer genes from in vitro genome-wide CRISPR/Cas9 screen and to the essential genes in human neural stem cells (hNSCs), we identified several context-specific regulators of mTOR, AKT and PLK1 pathways. Subsequently, targeting the PI3K pathway with Enzastaurin was shown to be most meanable to synergistic targeting alongside BMI1 inhibition. Conclusion: This work provides the foundation for clinical validation of small-molecule inhibitors synergistic with PTC-596 to improve the durability of remissions and extend survival of patients with treatment-refractory Group 3 MB.