Uncovering ATR signaling networks underlying chemotherapeutic synergies

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PROJECT SUMMARY/ABSTRACT The DNA damage sensor kinase ATR is essential for human cell viability and plays a crucial role in cell tolerance to DNA replication stress1–3. Cancer cells often feature elevated levels of replication stress due to oncogene action and are therefore susceptible to inhibitors of ATR, making ATR a prime target in anti-cancer therapies4–7. While ATR inhibitors have been shown to be particularly effective in combination with replication stress-inducing agents, the molecular mechanisms behind these drug synergies remain poorly understood, primarily due to the sheer complexity of the ATR signaling network5,8–11. To uncover the signaling events that underlie these therapeutic synergies, I will utilize mass spectrometry to perform both unbiased and targeted phosphoproteomic screens in cancer cells lines treated with different chemotherapeutic agents shown to synergize with ATR inhibitors. Pilot unbiased screens I conducted in HCT116 cancer cells have begun to identify and quantify ATR signaling in response to chemotherapies CPT and Olaparib, allowing each response to be barcoded for direct comparison. Comparing pilot barcodes revealed dramatic differences in ATR signaling induced by each drug, also raising questions regarding whether such ATR signaling varies further between cancers resistant to these chemotherapies8,12. Moreover, when investigating CPT-induced ATR signaling, we uncovered a set of non- canonical signaling events that are independent of DNA resection, a process required for the activation of canonical ATR signaling1,13. These non-canonical signaling substrates include proteins involved in the dissolution of R-loops: DNA:RNA hybrids that increase in abundance upon CPT treatment and contribute to genome instability14–16. Faced with these data, I hypothesize that ATR signaling varies between drug treatments and cancer cell types, and that non-canonical ATR signaling contributes to CPT resistance via regulation of DNA:RNA hybrid processing. I aim to expand signaling barcodes for CPT and Olaparib-induced ATR responses several fold via scaled up phosphoproteomic screens, curating a synthetic peptide library from events uncovered. This library will enable high-throughput targeted barcoding of ATR signaling responses across cancers differentially resistant to CPT, Olaparib, and ATR inhibitor combination therapy, allowing for correlation of chemotherapy resistance with cancer-specific ATR signaling. I also aim to investigate the role of novel resection-independent ATR signaling events in cancer resistance to CPT by using genetic, biochemical, and cell biological techniques. Overall, results will delineate drug- and cancer-specific ATR responses and establish the contribution of a new mode of ATR signaling to chemotherapy outcomes. Generated knowledge will serve as a framework for systems-wide dissection of the mechanisms underlying ATR inhibitor chemotherapeutic synergies and resistances, potentially revealing drug targets and opportunities for more selective combination therapies.