DDDR-39. Untangling MEK inhibitor resistance in DMG-MAPK using coupled lineage tracing and single cell RNA sequencing

MAPK pathway alterations occur in 10-20% of diffuse midline glioma, H3K27-altered (DMG-H3K27), resulting in a proposed novel subtype of the disease with distinct clinical features. In a previous co-clinical trial platform, we showed an increased sensitivity to MEK inhibitors in DMG-MAPK patient-derived models, though resistance occurred in vitro, in vivo and clinically. To understand the nature of these resistance mechanisms, we used sophisticated coupling of retrievable high-complexity barcoding and single cell RNA sequencing in DMG-MAPK cells. Lineage tracing conducted in several technical replicate experiments showed a subset of clones is consistently enriched across replicates when treated with a GI90 concentration of trametinib for 11 weeks. Interrogation of clonal dynamics using mathematical modelling showed barcodes commonly enriched across replicates exhibit significantly higher clonal fitness compared to replicate-specific enriched barcodes, suggesting a deterministic pattern of resistance. Serial single cell RNA sequencing analysis at weeks 5, 8 and 11 post-treatment initiation showed some resistant H3.3K27M/BRAF-mutant cells adopted astrocyte-like and neural progenitor-like metaprograms in which barcodes were commonly enriched across two cell states, suggesting a subset of clones circumvent trametinib treatment by phenotypic switching; simultaneously, a late-emerging resistant population of cells activate a distinct mesenchymal program. Furthermore, we determine the previously identified MAP2K1 (MEK1) mutations to arise specifically from a small population of radial glia-like stem cells that appear acutely predisposed to the acquisition of these mutations. Together, our data suggest two clonally and mechanistically distinct resistance mechanisms can be active in DMG-MAPK in response to MEK inhibition, involving both transcriptional plasticity and a progenitor-like state primed for the acquisition of genetic determinants which circumvent drug response. With trametinib-resistant cells being highly sensitive to selective SRC inhibition, evolutionary-guided combinatorial treatment schedules may provide for strategies to significantly lengthen time taken for resistance development.