Abstract Text Many antitubulin agents, such as paclitaxel (Taxol), have been used extensively for treatment of several types of cancer, including ovarian, lung, pancreatic, and breast cancers. Despite their wide use in cancer treatment, however, patient response is highly variable and drug resistance remains a major clinical issue. It is therefore essential to identify prognostic markers to predict the patient response and to enhance drug sensitivity. Protein kinase R (PKR) plays significant roles in innate immune response to viral infection and tumorigenesis. The biological significance of PKR in antitubulin chemotherapeutics and underlying mechanisms have yet to be defined. Through Phos-tag-based kinome-wide biochemical screens, we identified PKR as a critical regulator in antitubulin cytotoxicity. Our preliminary data suggest that inactivation of PKR confers resistance to Taxol in ovarian and breast cancer cells. Enhanced expression of PKR potentiates taxol cytotoxicity in vitro and in vivo. We further identified novel phosphorylation sites on PKR during antitubulin-mitotic arrest and in normal mitosis. Mechanistically, our findings also suggest that PKR controls Taxol chemosensitivity through modulating Bcl2 expression. Our hypothesis is that the PKR-Bcl2 axis functions as a therapeutic target for antitubulin agent-based chemotherapeutics in treatment of drug-resistant and/or recurrent patients. We will test our central hypothesis by three specific aims. Aim 1: Examine the functional significance of PKR in antitubulin chemotherapeutics in vivo; Aim 2: Determine how antitubulin drugs regulate PKR; Aim 3: Elucidate the mechanisms and downstream signaling of PKR in antitubulin chemosensitivity. The identification of new regulators and/or signaling pathways triggered by antitubulin drugs will shed light on the mechanisms underlying chemoresistance. Our study suggests that combining kinase activators (e.g., being characterized in this application) for PKR or Bcl2 inhibitors (FDA-approved) with antitubulin agents will have enhanced efficacy in treatment of drug-resistant and/or recurrent patients. Our findings also suggest that profiling PKR-Bcl2 signaling status of tumors (mRNA/protein levels and activity) can be useful to predict the patient response to antitubulin chemotherapeutics.