PROJECT SUMMARY Triple negative breast cancer (TNBC) is the most aggressive breast cancer subtype. It accounts for ~15% of all breast cancer patients yet is responsible for 30% of breast cancer deaths. TNBC is treated primarily by conventional chemotherapy; however, resistance to therapy is common leading to high mortality rates. Recently, we identified hypoxia-induced ECM re-modeler, lysyl oxidase (LOX) as a key mediator of doxorubicin resistance in TNBC (Saatci et al, Nature Communications, 2020). LOX inhibition offers a unique opportunity to re-sensitize the most aggressive breast tumors to standard-of-care chemotherapeutics. The overall objectives of this project are to (i) delineate the roles of LOX in chemoresistance, (ii) determine the mechanisms through which LOX exerts these roles, (iii) and generate prototypes of potent and selective LOX inhibitors to overcome chemoresistance in TNBC. We hypothesize that (i) LOX induces resistance not only to doxorubicin but also to other chemotherapeutic drugs by its enzymatic activity; (ii) LOX exerts this effect both by increasing collagen cross-linking/fibronectin assembly (canonical LOX function) leading to reduced drug penetration and increased integrin-mediated signaling and by regulating transcription (non-canonical LOX function) via interacting and oxidizing its substrates, culminating in activation of FAK/Src signaling and cell survival; and (iii) targeting LOX activity with selective small-molecule inhibitors will overcome chemoresistance by blocking both canonical and non-canonical LOX functions in TNBC. These hypotheses will be tested by pursuing three specific aims: 1) To determine the role of canonical ECM cross-linking function of LOX in resistance to different chemotherapeutics in TNBC. We will test the general chemosensitizer role of LOX and necessity of its enzymatic activity by generating cells with CRISPR-mediated LOX knock-out and reconstitution and testing their effects on chemoresistance in vitro and in vivo. LOX-mediated ECM changes will be analyzed by advanced microscopy techniques, e.g. MP-SHG, and the resulting drug penetration will be studied by IF and MALDI-MSI. 2) To determine the role of non-canonical transcription-regulating functions of LOX in TNBC chemoresistance. We will determine if LOX controls global transcription and identify novel LOX substrates by combining transcriptomics (RNA-Seq) and proteomics (TurboID) approaches. We will generate oxidation-deficient LOX substrates and test their effects on LOX-mediated chemoresistance. 3) To characterize novel LOX enzymatic inhibitors and test their potential as chemosensitizers in TNBC. We will test the selectivity of our inhibitors in cells with LOX knock-out/reconstitution and their off-target profiles and test their chemosensitization ability in organoids. We will perform PK/PD and toxicity profiling studies and test the inhibitors for overcoming chemoresistance in TNBC PDXs. The proposed project is expected to provide...