Plasticity And Vulnerability Associated With Extrachromosomal And Intrachromosomal Braf Amplifications.

Abstract Cancer cells display two modes of focal amplifications (FA): extrachromosomal double minutes (DMs/ecDNAs) and intrachromosomal homogenously staining regions (HSRs). Understanding the plasticity of these two modes is critical for preventing targeted therapy resistance. We developed a combined BRAF plus MEK inhibitor resistance melanoma model that bears high BRAF amplifications through both ecDNA and HSR modes, and investigated FA dynamics in the context of drug resistance plasticity. Cells harboring FAs displayed mode switching between ecDNAs and HSRs, from both de novo genetic changes and selection of pre-existing subpopulations. We found that copy number plasticity is not exclusive to ecDNAs. Single cell-derived clones with HSRs also exhibit BRAF copy number and corresponding HSR length plasticity that allows them to respond to dose reduction and recover from drug addiction. In addition, upon kinase inhibitor escalation, we observed reproducible selection for cells with BRAF kinase domain duplications residing on ecDNAs. Whole genome sequencing of sublines with different FA formats allowed fine reconstructions of BRAF amplicon and revealed conserved structures during the transitions due to drug dose manipulations. The amplification boundaries in our cell line model were found consistent with those in clinically observed cases of combined BRAF/MAPK inhibitor resistance. RNA-seq and CRISPR screening performed on parental, ecDNA and HSR sublines revealed distinct biology and vulnerabilities associated with each kind of FA. Vulnerabilities for genes involved in ecDNA maintenance in DNA repair pathways identified in our model are consistent with previous reports, such as a role for DNA-dependent protein kinase (DNA-PK/PRKDC). Our screen reveals many new targets. In sum, the karyotypic plasticity of FAs allows cancer cells to respond to drug dose challenges through a myriad of mechanisms, including increases or decreases in DMs, shortening of HSRs, and acquisition of secondary resistance mechanisms. The cellular vulnerabilities identified in our study provide roadmaps for therapeutically targeting each kind of FA mode. Citation Format: Kai Song, Jenna Minami, Trevor Ridgley, Arthur Huang, Rachana Jayaraman, William Crosson, Jesus Salazar, Eli Pazol, Senaratne Niroshi, Rao Nagesh, Kim Paraiso, Thomas Graeber. Plasticity and vulnerability associated with extrachromosomal and intrachromosomal BRAF amplifications [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 2036.