Project Summary/Abstract The KRAS mutation (mtKRAS) occurs in approximately 25% of all human tumors and confers resistance to EGFR tyrosine kinase inhibitors (TKI). Targeting KRAS mutations with drugs has been challenging. We are developing DGD-1202 a specific degrader, not an inhibitor, of activated EGFR kinase that exhibits profound activity against mtKRAS-driven murine tumor models. Our data indicate that degradation of activated EGFR without affecting mtKRAS levels could be a novel therapy for TKI-resistant mtKRAS-driven tumors. We hypothesize that in such wtEGFR/mtKRAS tumor types, kinase-independent roles of EGFR are vital for cell survival. Therefore, degradation of activated EGFR is sufficient to kill cells, including mtKRAS expressing cells. The overall goal of our study is to confirm the efficacy and mechanism of action of DGD-1202. Accordingly, the three specific aims of this proposal are as follows. In Aim 1, we will determine the kinase-independent roles of EGFR protein in mtKRAS-driven tumors. The ablation of EGFR by siRNA or shRNA kills both EGFR-driven or mtKRAS expressing cells. This indicates that EGFR protein plays an essential role in cell survival, independent of its kinase functions. We hypothesize that kinase-dead (KD) EGFR protein expression will rescue cell death induced by EGFR ablation in these cells. In this aim, we will investigate if kinase-dead EGFR can promote cell survival and rescue DGD-1202 mediated cell death. In Aim 2, we will elucidate the mechanisms by which DGD-1202 induced EGFR degradation overcomes mtKRAS mediated TKI resistance. EGFR protein plays important scaffolding functions, and its ablation results in the loss of membrane transporters such as xCT and SGLT1. Loss of these transporters limits cellular uptake of cystine and glucose, respectively, thus reducing the synthesis of glutathione peroxidase, a key free radical scavenger. In this aim, we will investigate the effects of DGD-1202 induced EGFR degradation on xCT and SGLT1 in mtKRAS-driven cancer cells and its effect on the accumulation of reactive oxygen intermediates. Based on our preliminary data, we hypothesize that DGD-1202 will reduce xCT and SGLT1 protein levels in wtEGFR cells but have minimal effect in KD EGFR expressing cells, where DGD-1202 does not degrade EGFR. Finally, in Aim 3, we will assess the efficacy of DGD-1202 in a panel of mtKRAS-driven, EGFR TKI-resistant xenograft, and transgenic mouse models. Our preliminary data indicate the robust single-agent activity of DGD-1202 in several mtKRAS-driven cancer models. In this aim, we will determine the dose and exposure-response profiles. We will also determine the effect of DGD-1202 treatment on long-term survival. This approach will target a broad spectrum of patients and offer a therapeutic option for both treatment-naive and EGFR-TKI resistant mtKRAS-driven tumors.