Unraveling Systemic Responses to NQO1-Activated IB-DNQand Rucaparib Single and Dual Agent Therapy in Triple-Negative Breast Cancers

Triple negative breast cancer (TNBC) is a highly aggressive breast cancer that is unresponsive to hormonal therapies. One potential TNBC-specific therapeutic target is NQO1, as it is highly expressed in many TNBC patients and lowly expressed in non-cancer tissues. Here we use a derivative of DNQ, isobutyl-deoxynyboquinone (IB-DNQ) that is more potent and specific in killing TNBC cells than NQO1-activator {beta}-lapachone while displaying strong NQO1-dependence. We evaluated the cellular signaling changes that occur following 4-hour treatment of TNBC cells with either single agent or combination IB-DNQ and / or PARP inhibitor (Rucaparib). Short treatments (4 hours) with IB-DNQ alone or combined with the PARP inhibitor Rucaparib revealed few changes in protein abundance but significant rapid alterations in protein phosphorylation and thermal stability, with clear synergy in the combination treatment. Key phosphorylated targets linked to RNA Polymerase II inhibition and DNA damage response were altered during our short time treatment. Thermal proteome profiling (TPP) identified novel, combination-specific changes in protein biophysical state suggesting new therapeutic vulnerabilities in TNBC cells. Our findings highlight how even brief treatments can uncover distinct biophysical protein changes via TPP, offering a resource for mechanistic studies of IB-DNQ mechanism of action and the development of NQO1-activated therapeutics for TNBC treatment.