Project Summary Copy gains or amplification of drug resistant oncogenes within the tumor contribute to genetic heterogeneity, therapeutic resistance and poor patient outcome. For example, chromosome 1q21-q25 and associated drug resistant oncogenes are gained in aggressive, drug resistant tumors. Additional studies have highlighted that additional amplifications are emerging after therapeutic treatment (e.g., DHFR and KRAS); while other oncogenic amplifications are transiently lost upon chemotherapy (i.e., EGFR). To date, little knowledge exists about how these regions are undergoing amplification, removal or how genetic heterogeneity occurs in cancer. Therefore, understanding how specific gains emerge or are lost will profoundly impact our understanding of tumorigenesis, chemotherapeutic response and drug resistance in cancer. Recently, the histone demethylase KDM4A was shown to be overexpressed in multiple cancers and directly generate transient site-specific copy gains (TSSGs) associated with drug resistance by promoting heterochromatin displacement and rereplication. TSSGs occurred during S phase and were lost upon late S/early G2 phase of cell cycle. This novel work challenged the paradigm that copy gains and amplifications of oncogenes and genomic loci occur randomly in tumors. We hypothesize that KDM4A as well as other chromatin factors are modulating the epigenome at specific locations throughout the genome and altering the susceptibility to TSSGs; while, specific cellular pathways regulate the removal of TSSGs. We have a set of unique tools to interrogate how chromatin regulators and associated states are directly modulating copy gains of oncogenic associated genes. The aims outlined in this proposal will uncover fundamental answers about new regulators of copy gain (aim 1), molecular features enhancing or suppressing the propensity of regions to undergo amplification (aim 2) and identify pathways and cellular processes driving copy gain removal (aim 3). In aim 1, we will couple a computational approach we developed with an unbiased genetic screen of chromatin factors in non- transformed cells so we can determine whether genetic perturbation of chromatin factors will promote copy alterations. In aim 2, we will use a novel sequencing strategy (rerep-seq) to characterize regions undergoing rereplication and establish the epigenomic features affiliated with these regions. In aim 3, we will use CRISPR- Display and live cell imaging to resolve TSSG removal kinetics and the nuclear/cellular localization. We will also use pharmacological and genetic screens to further resolve pathway(s) involved in removing TSSGs. This grant will uncover fundamental properties driving copy gains, elucidate pathways modulating copy gains and their fates, which in turn, identifies novel biomarkers and therapeutic targets in drug resistance tumors.