ABSTRACT Our long-term goal is to develop novel antitumor therapies to treat cancer with elevated (> 100-fold) levels of NAD(P)H:quinone oxidoreductase 1 (NQO1). β-Lapachone (β-lap), an NQO1 bioactivatable drug, selectively targets NQO1+ tumors and is activated by NQO1 to generate reactive oxygen species (ROS), leading to extensive DNA damage and PARP1-driven tumor programmed necrosis. Our preliminary studies demonstrate that both neutrophil-mediated innate immunity and CD8-mediated adaptive immunity are required for antitumor efficacy of β-lap in vivo. Furthermore, our previous studies reveal that targeting NQO1 potently triggers innate sensing within tumor microenvironment (TME) that synergizes with immunotherapy to overcome adaptive resistance. Our objective here is to define and delineate the mechanism(s) of tumor-specific ROS and DNA damage induced by β-lap that stimulates antitumor immunity, and determine how β-lap synergizes with immune checkpoint blockade therapy. Our central hypothesis is that (i) β-lap treatment triggers immunogenic cell death (ICD) and induces damage-associated molecular patterns (DAMPs) release;; (ii) phagocytes/antigen-presenting cells (APCs) recruitment promotes cross-priming of cytotoxic T cells (CTLs) for suppression of tumor by increasing antigen/DNA uptake and type I interferons (IFNs) production;; and (iii) upregulated PD-L1 within TME contributes to tumor relapse and provides therapeutic window for combination therapy of β-lap with immune checkpoint blockade. We propose the following Specific Aims. AIM 1: Elucidate the mechanism of β-lap- triggered ICD for innate immune sensing. Our working hypothesis is that β-lap triggers ICD for innate sensing via the release of DAMPs. We will assess the ability of β-lap to stimulate tumor ICD in vivo by therapeutic vaccine assay. We will also determine which type(s) of tumor DNA (genomic or mitochondrial) is the major source(s) of IFNs production after β-lap treatment. AIM 2: Define how tumor cells and immune cells cross-talk occurs in β-lap-induced antitumor immunity. Our working hypothesis is that β-lap-induced neutrophils cross-prime T cells directly or interact with DCs/macrophages to prime T cells. We will determine the effects of β-lap treatment on cGAS/STING/IFNs involved in T cells cross-priming. AIM 3: Determine the mechanism by which β-lap synergizes with immune checkpoint blockade therapy to efficaciously kill NQO1+ tumors. Our working hypothesis is that increased PD-L1 within TME contributes to tumor relapse of large tumors after initial responses to β-lap. We will determine in which type(s) of cells PD-L1 expression is upregulated within TME. We will also determine which type(s) of PD-L1-expressing cells is essential for the synergistic e...