MS CETSA deep functional proteomics uncovers DNA repair programs leading to gemcitabine resistance.

Mechanisms for resistance to cytotoxic cancer drugs are dependent on dynamic changes in the biochemistry of cellular pathways, information which is hard to obtain at the systems level. Here we use a deep functional proteomics implementation of the Cellular Thermal Shift Assay to reveal a range of induced biochemical responses to gemcitabine in resistant and sensitive diffuse large B cell lymphoma cell lines. Initial responses in both, gemcitabine resistant and sensitive cells, reflect known targeted effects by gemcitabine on ribonucleotide reductase and DNA damage responses. However, later responses diverge dramatically where sensitive cells show induction of characteristic CETSA signals for early apoptosis, while resistant cells reveal biochemical modulations reflecting transition through a distinct DNA-damage signaling state, including opening of cell cycle checkpoints and induction of translesion DNA synthesis programs, allowing bypass of damaged DNA-adducts. The results also show the induction of a protein ensemble, labeled the Auxiliary DNA Damage Repair, likely supporting DNA replication at damaged sites that can be attenuated in resistant cells by an ATR inhibitor, thus re-establishing gemcitabine sensitivity and demonstrating ATR as a key signaling node of this response.