Temporal chromatin accessibility changes define transcriptional states essential for osteosarcoma metastasis

The metastasis-invasion cascade describes the series of steps required for a cancer cell to successfully spread from its primary tumor and ultimately grow within a secondary organ. Despite metastasis being a dynamic, multistep process, most omics studies to date have focused on comparing primary tumors to the metastatic deposits that define end-stage disease. This static approach means we lack information about the genomic and epigenomic changes that occur during the majority of tumor progression. One particularly understudied phase of tumor progression is metastatic colonization, during which cells must adapt to the new microenvironment of the secondary organ. Through temporal profiling of chromatin accessibility and gene expression in vivo, we identify dynamic changes in the epigenome that occur as osteosarcoma tumors form and grow within the lung microenvironment. Furthermore, we show through paired in vivo and in vitro CRISPR drop-out screens and pharmacological validation that the upstream transcription factors represent a class of metastasis-specific dependency genes. While current models depict lung colonization as a discrete step within the metastatic cascade, our study shows it is a defined trajectory through multiple epigenetic states, revealing new therapeutic opportunities undetectable with standard approaches.

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Field-specific reporting Sequencing data generated for this manuscript can be found at the Gene Expression Omnibus (GEO) under the accession number GSE215765.The MNNG-HOS JQ1 RNA-seq data that was previously published is also available on GEO under the accession number GSE74230.DepMap data are available at https://depmap.org/portal/download/custom/. Human genome reference hg19 was used for sequencing alignment in this manuscript.
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Replicate experiments were performed as described in the methods.In vivo experiments were performed in triplicate, ex vivo experiments in triplicate, and in vitro experiments in quadruplicate or greater.All experiments showed similar results.
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anti-KLF4 was purchased from R&D systems (AF3640) and used at 0.5 ug/mL anti-cyclophilin B was purchased from abcam (ab16045) and used at 1:10,000 dilution anti-H3K27ac was purchased from abcam (ab4729) and used at 8 ug per ChIP-seq All secondary antibodies were used at a 1:10,000 dilution.Anti-goat and was purchased from Thermo Scientific, product # 31433, lot # RA2143996).Anti-rabbit was purchased from Thermo Scientific, product # 31460, lot # QG221919).
Antibody validation data are available on supplier websites.
was used for data collection.
anti-KLF4 was validated by western blot in HT-29 human colon adenocarcinoma cell line, SW480 human colorectal adenocarcinoma cell line, and HCT-116 human colorectal carcinoma cell line (https://www.rndsystems.com/products/human-klf4-antibody_af3640)anti-cyclophilin B was validated by western blot in Wild-type HAP1 whole cell lysate, PPIB (Cyclophilin B) knockout HAP1 whole cell lysate, Jurkat whole cell lysate, and U87-MG whole cell lysate.Ab16045 was shown to specifically react with PPIB in wild-type HAP1 cells as signal was lost in PPIB knockout cells.(https://www.abcam.com/products/primary-antibodies/cyclophilin-b-antibody-ab16045.html)MG63.3-GFP and 143b-HOS-GFP cell lines are originally from the lab of Dr. Chand Khanna.MG63.3-Cas9i-GFP was derived from MG63.3-GFP through lentiviral transduction, as described previously (doi: 10.1172/JCI127718) Please select the one below that is the best fit for your research.If you are not sure, read the appropriate sections before making your selection.
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