Targeting DNA repair in KRAS mutated lung cancer by chemical screening In the 7 x 7-inch space below, summarize concisely your proposed research, outlining background, objective/hypothesis, specific aims, study design, and relevance to the cancer problem. You will prepare the abstract as a separate file when you electronically submit your application. Refer to Application Instructions. If the application is funded, this Abstract will become public information. Therefore, do not include proprietary/confidential information. Background: Lung cancer is the leading cause of cancer death in the US. Around 30% lung adenocarcinoma carries KRAS mutation, which lacks targeted therapies. Chemotherapy remains the mainstay treatment for KRAS mutated lung cancers. Many chemotherapy kills cancer cells by causing massive DNA damage, particularly double strand breaks (DSBs). However, cells also evolved protective mechanisms (DNA damage response and repair) to evade the cell killing effect of chemotherapy. Hence, small molecules that inhibit DNA damage response and DSB repair can be repurposed into effective chemo-sensitizers for KRAS mutated lung cancers. Preliminary Data: Natural products display a wide variety of structural complexity and diversity, representing a rich resource for drug discovery. To identify chemo-sensitizers for KRAS mutated lung cancer, we screened a natural product library (~1000 compounds with various structural types) and identified cardiac glycosides as potent DNA damage response inhibitors. We demonstrate that cardiac glycosides specifically inhibit the 5' to 3' DSB end resection, a process that is required for the activation of DNA damage response and faithful DSB repair. Cardiac glycosides strongly enhanced the growth inhibition effect of DSB-inducing drugs on KRAS mutant lung cancer cells while having much less effect on normal lung fibroblasts, indicating a cancer specific effect of these compounds. This therapy sensitizing effect was confirmed in xenografted lung cancers in mice. Objective: The goal of this project is to determine the molecular targets and detailed mechanisms by which cardiac glycosides sensitize chemotherapy in KRAS mutated lung cancers. Study Design:
In Aim 1, we will determine how cardiac glycosides inhibit the 5' to 3' DSB end resection. DSB end resection is controlled by many proteins including 53BP1, BRCA1, UHRF1, etc. We hypothesize that cardiac glycosides inhibit DSB end resection by regulating expression levels of these critical DSB genes. Through whole genome sequencing and stable isotope labeling with amino acids (SILAC), we identified UHRF1 as the top candidate as UHRF1 plays a critical role in promoting the 5' to 3' end resection of DSBs and inhibition of UHRF1 suppresses DSB end resection. Here we will determine (1) how UHRF1 mediates DNA damage response and DSB repair in the presence of cardiac glycosides, and (2) the molecular details by which cardiac glycosides regulate the expression level of UHRF1, and therefore cell sensitivity to DSB-inducing anticancer drugs.
In Aim 2, we will identify cellular targets by which cardiac glycosides directly act on through chemical proteomic analysis (i.e., molecular capturing followed by mass spectrometry). We identified Cdc20, the activating factor for the mitotic E3 ubiquitin ligase anaphase promoting complex/cyclosome (APC/C), as a candidate because UHRF1 was reported to be degraded likely dependent on Cdc20. (1) We will purify various Cdc20 recombinant proteins to perform (a) surface plasmon resonance (SPR) to determine the binding kinetics, and (b) isothermal titration calorimetry to determine the binding stoichiometry, affinity and enthalpy of cardiac glycosides with Cdc20. (2) We will use cell thermal shift assay to determine the interaction of AT2 with Cdc20 in cultured cells. (3) We will further determine the impact of the interaction of cardiac glycosides with Cdc20 on UHRF1 degradation.
In Aim 3, we will perform structure-activity relationship studies to characterize and validate cardiac glycoside derivatives that have highest possible solubility and DNA damage inhibition activity while reducing the cardiac toxicity. These compounds will be the leads for further clinical applications.
In Aim 4, we will determine the therapeutic potential of cardiac glycosides in enhancing the effect of chemotherapy using identify, KRAS mutated orthotopic lung cancer patient-derived xenografts and genetically modified mouse lung cancer models. These studies will provide the foundation for testing the combination of radiotherapy or chemotherapy with cardiac glycosides in lung cancer treatment in the clinic. Cancer Relevance: Lung cancer is a devastating disease. Identification of therapy sensitizers to enhance the effect of chemotherapy in KRAS mutated lung cancer is highly desirable, which will improve the survival of lung cancer patients, especially those with KRAS mutations, in the US.
The overall goal of this proposal is to illustrate molecular mechanisms and biological actions of cardiac glycosides on inhibiting KRAS mutant lung cancers. Knowledge gained through the support of this project may lead to new discovery of unique approaches to treat KRAS mutant lung cancers, which lack effective treatment to date. These studies will improve the lifespan of patients with KRAS mutant lung cancers in the US.