Abstract 1225: Novel cellular barcoding to dissect melanoma heterogeneity and drug resistance

Abstract Melanoma is the most common cause of death in skin cancer. While the development of targeted therapies (MAPK pathway inhibitors) led to a substantial improvement in overall survival of patients with melanoma harboring the BRAFV600E mutation, approximately 40% of treated patients acquire resistance and experience tumor recurrence after one year. Melanoma drug resistance is mediated by drug-tolerant persister (DTP) cell subpopulations present in tumors. DTP cells are either intrinsically rare sub-populations pre-existing prior to therapies, or adaptive sub-populations that acquire genetic and epigenetic alterations during treatments. Our lab has established six PDX models that responded to the treatment of BRAF/MEK inhibitor combination then relapsed in six to 40 weeks. We combined cell lineage barcoding and single-cell transcriptomics to trace the emergence of DTP cells in melanoma recurrence. The recent development of cellular barcoding facilitates the labelling of tens to millions of cancer clones and enables the identification of the complex mechanisms associated with clonal fate in response to therapy and in different microenvironments. We have constructed a library of approximately 15 million barcodes into a lentiviral vector. Melanoma cells were infected with the barcode library to ensure each cell carried a unique barcode. Labelled melanoma cells were injected into NSG mice, which were then treated with BRAF/MEK inhibitors, and they developed drug resistance with prolonged treatments. Treated tumors were harvested at different timepoints until relapse. The barcodes are localized close to the polyA tail of the expressed mRNA, the 3’ end sequence of which could be retrieved simultaneously through single-cell RNA sequencing (scRNAseq). Thus, we can trace the clonal information from the relapsed tumor back to earlier timepoints such as the treatment naive tumor and minimal residual disease (MRD) stage. Our data showed that the diversity of barcodes in MRD stage had no significance compared to treatment naive tumor, while the diversity of barcodes in the relapsed tumor decreased around 10-fold from the MRD stage. The most dominant sub-populations in MRD arose from minor sub-populations in the treatment naive tumor, while most of the dominant sub-populations in the relapsed tumor were daughter cells from minor sub-populations in MRD stage. The top three sub-populations dominated 25% of the relapsed tumor. The three dominant drug-resistant sub-populations shared a similar phenotype shown by scRNAseq clusters. The data indicate that the heterogeneity of melanoma is maintained in the early treatment stage but is lost in acquired resistance. And the lineages of DTP cells change continuously under prolonged BRAF/MEKi drug pressure. Citation Format: Haiyin Li, Yeqing Chen, Jessica Kaster, Nazifa Promi, Meenhard Herlyn. Novel cellular barcoding to dissect melanoma heterogeneity and drug resistance [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 1225.