Eukaryotic elongation factor 2 protein kinase (eEF2K) is a ubiquitously expressed protein that belongs to a family of alpha kinases characterized by an "atypical" kinase domain. Our recent findings demonstrate that inhibition or genetic inactivation of eEF2K protects normal tissues from cytotoxic effects of ionizing radiation and chemotherapeutic agents. We have previously generated eEF2K-deficient animals that have no adverse phenotypes but demonstrate increased resistance to both lethal doses of radiation and doxorubicin-induced cytotoxicity. This radio- and chemoresistant phenotype is accompanied by decreased levels of apoptosis in proliferating tissues. Moreover, siRNA-mediated knockdown of eEF2K sensitizes cancer cells to nutrient deprivation and to treatment with chemotherapeutic agent doxorubicin and suppresses tumor growth in the mouse model of breast cancer. Thus, we expect eEF2K inhibitors to exert dual effects in protecting normal tissues while enhancing tumor killing during chemotherapy, thereby significantly enhancing the therapeutic index of conventional chemotherapy. Several attempts have been made to develop chemical inhibitors of eEF2K, but the resulting compounds suffered from various limitations, such as low specificity for eEF2K, insufficient potency, high serum binding, or high toxicity, precluding their application in the clinic. We have developed a novel screening platform enabling rapid and accurate determination of eEF2K phosphorylation activity in vitro. Using this platform, we have generated a series of new eEF2K inhibitors that can be produced with high yields and purity and exhibit high specificity, improved potency, low serum binding, and low toxicity. Our preliminary data show that this series of eEF2K inhibitors effectively mimics the effects of siRNA-mediated knockdown of eEF2K expression, sensitizing cancer cells to nutrient deprivation and doxorubicin. Moreover, our lead inhibitor compound reduced doxorubicin toxicity in vivo, as evidenced by a reduction of a biomarker of tissue damage. In this grant we propose to further optimize our eEF2K inhibitor series and to carry out a panel of proof-of-concept experiments testing the efficacy of these inhibitors in sensitizing cancer cells to nutrient deprivation and doxorubicin treatment and suppressing tumor growth in a mouse breast cancer model. It is anticipated that upon completion of this project we will identify an inhibitor of eEF2K that will potentiate tumor killing by chemotherapy while protecting normal tissues from the associated toxicity, thereby representing an unprecedented approach to cancer therapy.
Public Health Relevance Statement: Public Health Relevance: Environment within solid tumors is characterized by low vasculature and nutrient deprivation, and ability of tumor cells to adapt to these conditions is associated with enhanced malignant potential. Our ability to fight these tumors is further limited by the toxic side effects of existing chemotherapeutics. In vivo inactivation of eukaryotic elongation factor 2 kinase (eEF2K) has a two-pronged anti-tumor effect: it sensitizes tumor cells both to nutrient deprivation and to chemotherapy. We have developed highly specific inhibitors of eEF2K and the major goal of the proposed research is to test their efficacy in sensitizing cancer cells to nutrient depletion and to chemotherapeutic agent doxorubicin in cell lines and a mouse model of breast cancer.
Project Terms: Adverse effects; Animals; Apoptosis; base; Binding (Molecular Function); Biological Assay; Biological Markers; Breast Cancer Cell; Breast Cancer Model; calmodulin-dependent protein kinase III; cancer cell; Cancer cell line; cancer therapy; Cell Line; Cells; Chemicals; chemotherapeutic agent; chemotherapy; Clinic; cross reactivity; cytotoxic; cytotoxicity; Data; deprivation; design; Dose; Doxorubicin; Drug Targeting; Effectiveness; efficacy testing; Elongation Factor; Embryo; Environment; Exhibits; experience; Family; Fibroblasts; fighting; Genetic; Goals; Grant; Growth; Imagery; improved; In Vitro; in vitro activity; in vivo; in vivo imaging; inhibitor/antagonist; Inhibitory Concentration 50; Ionizing radiation; Killings; Lead; Legal patent; Luciferases; Malignant - descriptor; malignant breast neoplasm; Malignant Neoplasms; MDA MB 231; Measures; Mediating; Modification; mouse model; Mus; Neoplasm Metastasis; neoplastic cell; Normal Cell; Normal tissue morphology; novel; Nude Mice; Nutrient; osteosarcoma; Patients; Peptide Elongation Factor 2; Pharmaceutical Preparations; Phenotype; Phosphorylation; Phosphotransferases; preclinical study; Proliferating; Protein Kinase; protein kinase inhibitor; Protein Kinase Inhibitors; Proteins; public health relevance; Radiation; Relative (related person); Reporting; Research; research study; Resistance; response; screening; Series; Serum; Small Interfering RNA; Solid Neoplasm; Specificity; Structure-Activity Relationship; System; Testing; Therapeutic Index; Tissues; Toxic effect; Translations; triple-negative invasive breast carcinoma; tumor; Tumor Burden; tumor growth; tumor xenograft
