Targeted profiling of human extrachromosomal DNA by CRISPR-CATCH

Extrachromosomal DNA (ecDNA) is a common mode of oncogene amplification but is challenging to analyze. Here, we adapt CRISPR-CATCH, in vitro CRISPR-Cas9 treatment and pulsed field gel electrophoresis of agarose-entrapped genomic DNA, previously developed for bacterial chromosome segments, to isolate megabase-sized human ecDNAs. We demonstrate strong enrichment of ecDNA molecules containing EGFR, FGFR2 and MYC from human cancer cells and NRAS ecDNA from human metastatic melanoma with acquired therapeutic resistance. Targeted enrichment of ecDNA versus chromosomal DNA enabled phasing of genetic variants, identified the presence of an EGFRvIII mutation exclusively on ecDNAs and supported an excision model of ecDNA genesis in a glioblastoma model. CRISPR-CATCH followed by nanopore sequencing enabled single-molecule ecDNA methylation profiling and revealed hypomethylation of the EGFR promoter on ecDNAs. We distinguished heterogeneous ecDNA species within the same sample by size and sequence with base-pair resolution and discovered functionally specialized ecDNAs that amplify select enhancers or oncogene-coding sequences

Durable Suppression of Acquired MEK Inhibitor Resistance in Cancer by Sequestering MEK from ERK and Promoting Antitumor T-cell Immunity.

MAPK targeting in cancer often fails due to MAPK reactivation. MEK inhibitor (MEKi) monotherapy provides limited clinical benefits but may serve as a foundation for combination therapies. Here, we showed that combining a type II RAF inhibitor (RAFi) with an allosteric MEKi durably prevents and overcomes acquired resistance among cancers with KRAS, NRAS, NF1, BRAF <sup>non-V600</sup> , and BRAF <sup>V600</sup> mutations. Tumor cell-intrinsically, type II RAFi plus MEKi sequester MEK in RAF complexes, reduce MEK/MEK dimerization, and uncouple MEK from ERK in acquired-resistant tumor subpopulations. Immunologically, this combination expands memory and activated/exhausted CD8 <sup>+</sup> T cells, and durable tumor regression elicited by this combination requires CD8 <sup>+</sup> T cells, which can be reinvigorated by anti-PD-L1 therapy. Whereas MEKi reduces dominant intratumoral T-cell clones, type II RAFi cotreatment reverses this effect and promotes T-cell clonotypic expansion. These findings rationalize the clinical development of type II RAFi plus MEKi and their further combination with PD-1/L1-targeted therapy. SIGNIFICANCE: Type I RAFi + MEKi are indicated only in certain BRAF <sup>V600MUT</sup> cancers. In contrast, type II RAFi + MEKi are durably active against acquired MEKi resistance across broad cancer indications, which reveals exquisite MAPK addiction. Allosteric modulation of MAPK protein/protein interactions and temporal preservation of intratumoral CD8 <sup>+</sup> T cells are mechanisms that may be further exploited.This article is highlighted in the In This Issue feature, p. 521

Integration of Electrochemistry with Ultra-Performance Liquid Chromatography/Mass Spectrometry

This study presents the development of ultra-performance liquid chromatography (UPLC) mass spectrometry (MS) combined with electrochemistry (EC) for the first time and its application for the structural analysis of proteins/peptides that contain disulfide bonds. In our approach, a protein/peptide mixture sample undergoes a fast UPLC separation and subsequent electrochemical reduction in an electrochemical flow cell followed by online MS and tandem mass spectrometry (MS/MS) analyses. The electrochemical cell is coupled to the mass spectrometer using our recently developed desorption electrospray ionization (DESI) interface. Using this UPLC/EC/DESI-MS method, peptides that contain disulfide bonds can be differentiated from those without disulfide bonds, as the former are electroactive and reducible. MS/MS analysis of the disulfide-reduced peptide ions provides increased information on the sequence and disulfide-linkage pattern. In a reactive DESI- MS detection experiment in which a supercharging reagent was used to dope the DESI spray solvent, increased charging was obtained for the UPLC-separated proteins. Strikingly, upon online electrolytic reduction, supercharged proteins (e.g., α-lactalbumin) showed even higher charging, which will be useful in top- down protein structure MS analysis as increased charges are known to promote protein ion dissociation. Also, the separation speed and sensitivity are enhanced by approximately 1(~)2 orders of magnitude by using UPLC for the liquid chromatography (LC)/EC/MS platform, in comparison to the previously used high- performance liquid chromatography (HPLC). This UPLC/EC/DESI-MS method combines the power of fast UPLC separation, fast electrochemical conversion, and online MS structural analysis for a potentially valuable tool for proteomics research and bioanalysis