ABSTRACT MYC oncoproteins (including c-MYC, L-MYC and N-MYC) play critical roles in the initiation, progression and recurrence of many human malignancies. Extensive studies indicate that MYC is required to maintain tumor cell survival and proliferation. We have recently used a novel approach that combined computer-aided modeling with a rapid in vivo screen to develop a new series of direct small molecule inhibitors (MYCi’s) that show excellent selectivity, potency and tolerability in multiple MYC-driven cancer models. These compounds demonstrate a dual mechanism of action. First, direct binding of MYCi to MYC in the basic helix-loop-helix (bHLH) region disrupts complex formation with MYC which is required for MYC transcriptional activity. Secondly, binding of MYCi enhances MYC phosphorylation on threonine-58 (pT58) which promotes MYC degradation via the ubiquitin-proteasome pathway. However the key downstream effectors of these events and how they might impact cellular function are unknown. Reduction of MYC protein and enhanced pT58MYC may be expected to have profound effects on MYC family protein interactions with each other and with chromatin. In this regard, we have observed in preliminary studies that MYCi leads to selective loss of MYC at genomic loci enriched for master chromatin regulators (CTCF and FOX), suggesting disruption of the 3D architecture of the MYC-bound genome in response to MYCi. Additionally, unfolded MYC due to MYCi binding and/or enhanced MYC degradation may provoke a cellular stress response. Using unbiased ATAC-seq and RNA-seq approaches, we found that MYCi treatment activates the ATF4/CHOP stress response pathway. Importantly, activation of ATF4/CHOP by MYCi is an on-target, MYC-dependent effect. ATF4 mediates MYCi antitumor activity as ATF4 depletion partially ameliorates the antitumor effects of MYCi. Furthermore, we propose that MYCi-induced ATF4 cytokines modulate the tumor microenvironment. Activation of the ATF4 pathway by MYCi exposes potential therapeutic vulnerabilities for rational combination approaches, such as combination of MYCi with proteasome inhibitors that activates ATF4. Based on the preliminary findings, our central hypotheses is that MYCi inhibits MYC-dependent tumorigenesis by a dual-pronged mode of action. First, MYCi affects MYC family target gene expression by disrupting MYC/MAX interaction and by promoting MYC degradation. Secondly, binding of MYCi to MYC and/or MYC degradation activates an ATF4/CHOP stress response pathway that suppresses tumor cell viability. We propose the following specific aims to test these hypotheses: Aim 1). To investigate the mechanisms by which MYC inhibitor modulates MYC transcriptional activity and the epigenetic landscape. We will investigate the consequences of MYCi treatment on the recruitment of MYC, pT58MYC, and associated factors to chromatin; changes to 3D chromatin architecture; as well as the effects on MYC-driven transcriptional output in tumor cells vitro...