SUMMARY An important milestone in the treatment of non-small cell lung cancer (NSCLC) is the discovery of epidermal growth factor receptor (EGFR) activating mutations as an effective therapeutic target and the successful development of EGFR tyrosine kinase inhibitors (EGFR-TKIs). AZD9291 (TAGRISSOTM or osimertinib) represents a 3rd generation EGFR-TKI and an FDA-approved drug for patients with EGFR mutant NSCLC that has become resistant to 1st generation EGFR-TKIs through the T790M mutation and for EGFR mutation-positive advanced NSCLC as a front line treatment. Unfortunately, patients eventually relapse and become resistant to AZD9291 in the clinic, resulting in disease progression. Thus, a better understanding of the underlying mechanisms and development of effective strategies to overcome AZD9291 resistance is an urgent and critical area of unmet need in the clinic. The C-MYC gene was the first Myc family member found in the human genome and its product, c-Myc, functions as a transcription factor to regulate the expression of many genes whose products are involved in the regulation of various physiological processes, such as cell survival, proliferation, differentiation, metabolism and host immunity. MYC is genetically activated and/or overexpressed in most types of human cancer including lung cancer and thus is a central driver of malignant cellular growth and proliferation. c-Myc expression is associated with response or sensitivity of cancer cells to chemotherapy or radiotherapy. However, no study has linked c-Myc to targeted therapy by third generation EGFR-TKIs. Our strong preliminary data support the overall hypothesis that c-Myc modulation may play a critical role in mediating therapeutic efficacy of AZD9291 against EGFRm NSCLC and the development of acquired resistance to AZD9291; accordingly, targeting c-Myc will be an effective strategy to overcome acquired resistance to AZD9291 and other EGFR-TKIs. This hypothesis will be tested by accomplishing the following 3 specific aims: 1) To understand the mechanism(s) by which c-Myc is reduced in EGFR mutant NSCLC cells by AZD9291 or other EGFR-TKIs; 2) To demonstrate the biological significance of c-Myc suppression in EGFR-targeted therapy; and 3) To develop effective therapeutic regimens to overcome acquired resistance to AZD9291 by targeting c-Myc. The objectives of this study are to understand the mechanisms by which AZD9291 and other EGFR-TKIs decrease c-Myc levels, to demonstrate the biological significance of c-Myc suppression during EGFR-targeted cancer therapy; and to develop effective strategies to overcome acquired resistance to AZD9291 by targeting c-Myc. This proposal is of high scientific and translational significance. The outcomes of this study can be immediately translated to the clinical treatment of NSCLC patients with acquired resistance to AZD9291 or other EGFR-TKIs. 1