Dipg-33. Combining Clonal Tracking With Single Cell Rna Sequencing Reveals Temporal Shifts in Cell States Associated With Trametinib Resistance in Mapk Altered Diffuse Midline Glioma

BACKGROUND H3K27-altered diffuse midline glioma (DMG) is an aggressive and incurable paediatric high-grade glioma with median overall survival of 9-12 months. A distinct subset of DMG harbour alterations in the MAPK pathway, and we have previously shown that DMG-MAPK may exhibit sensitivity to MEK inhibitors but develop resistance under long-term exposure to drug. Elucidating these resistance mechanisms could spearhead better treatment strategies for these patients. METHODS To study clonal evolution under trametinib, we used CloneSifter lineage tracking technology to uniquely barcode 300,000 DMG-MAPK cells which were then treated with a GI80 dose of trametinib for 11 weeks. We extracted DNA to quantify barcode enrichment at endpoint across six technical replicate experiments and leveraged CloneSifter’s compatibility with single cell RNA sequencing (scRNA-seq) to couple clonal identity with transcriptional states in ~90,000 trametinib treated cells at varying timepoints. RESULTS DNA sequencing revealed 0.5% (1500/300,000) of the initial barcodes were enriched across trametinib replicates and were also detectable by scRNA-seq. Conducting cNMF analysis on the single cell dataset showed populations of trametinib treated cells associated with each other in a temporal manner, whereby early and late-stage resistant populations were defined by neuronal and stem cell like markers, respectively. A time-dependent enrichment of CloneSifter barcodes was also observed, suggesting dynamic expansion of clones over the treatment window. Notably, week 11 trametinib treated cells showed higher expression of resistance driver genes related to EMT, stemness and p38/MAPK upregulation. CONCLUSIONS Utilizing the sequence-specific reporter construct built in to the CloneSifter system allows for retrieval of resistant clones marked by enriched barcodes under treatment prior to drug exposure, enabling further characterization of monoclonal populations of trametinib-resistant cells. In summary, we have employed novel tools to study resistance development in DMG-MAPK that could be exploited to devise rational treatment strategies for these patients in an evolutionary-guided manner.