The c-Myc oncogene (MYC) drives malignant progression, but also induces robust anabolic and proliferative programs leading to intrinsic stress. The mechanisms enabling adaptation to MYC-induced stress are not fully understood. We have uncovered an essential role for the transcription factor ATF4 in survival following MYC activation. MYC upregulates ATF4 by activating GCN2 kinase through uncharged tRNAs. Subsequently, ATF4 co-occupies promoter regions of over 30 MYC target genes, primarily those regulating amino acid and protein synthesis, including 4E-BP1, a negative regulator of translation. 4E-BP1 is essential to balance protein synthesis, relieving MYC-induced proteotoxic stress. 4E-BP1 activity is negatively regulated by mTORC1-dependent phosphorylation and inhibition of mTORC1 signaling rescues ATF4 deficient cells from MYC-induced ER stress. Acute deletion of ATF4 significantly delays MYC-driven tumor progression and increases survival in mouse models. Our results establish ATF4 as a cellular rheostat of MYC-activity, ensuring enhanced translation rates are compatible with survival and tumor progression.

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Resistance to BRAF and MEK inhibitors (BRAFi + MEKi) in BRAF-mutant tumors occurs through heterogeneous mechanisms, including ERK reactivation and autophagy.Little is known about the mechanisms by which ERK reactivation or autophagy is induced by BRAFi + MEKi. Here, we report that in BRAF -mutant melanoma cells, BRAFi + MEKi induced SEC61-dependent endoplasmic reticulum (ER) translocation of the MAPK pathway via GRP78 and KSR2. Inhibition of ER translocation prevented ERK reactivation and autophagy. Following ER translocation, ERK exited the ER and was rephosphorylated by PERK. Reactivated ERK phosphorylated ATF4, which activated cytoprotective autophagy. Upregulation of GRP78 and phosphorylation of ATF4 were detected in tumors of patients resistant to BRAFi + MEKi. ER translocation of the MAPK pathway was demonstrated in therapy-resistant patient-derived xenografts. Expression of a dominant-negative ATF4 mutant conferred sensitivity to BRAFi + MEKi in vivo . This mechanism reconciles two major targeted therapy resistance pathways and identifi es druggable targets, whose inhibition would likely enhance the response to BRAFi + MEKi. SIGNIFICANCE: ERK reactivation and autophagy are considered distinct resistance pathways to BRAF + MEK inhibition (BRAFi + MEKi) in BRAF V600E cancers. Here, we report BRAFi + MEKi-induced ER translocation of the MAPK pathway is necessary for ERK reactivation, which drives autophagy. The ER translocation mechanism is a major druggable driver of resistance to targeted therapy.

Terminal differentiation opposes proliferation in the vast majority of tissue types. As a result, loss of lineage differentiation is a hallmark of aggressive cancers, including soft tissue sarcomas (STS). Consistent with these observations, undifferentiated pleomorphic sarcoma (UPS), an STS subtype devoid of lineage markers, is among the most lethal sarcomas in adults. Though tissue-specific features are lost in these mesenchymal tumors they are most commonly diagnosed in skeletal muscle, and are thought to develop from transformed muscle progenitor cells. We have found that a combination of HDAC (Vorinostat) and BET bromodomain (JQ1) inhibition partially restores differentiation to skeletal muscle UPS cells and tissues, enforcing a myoblast-like identity. Importantly, differentiation is partially contingent upon downregulation of the Hippo pathway transcriptional effector Yes-associated protein 1 (YAP1) and nuclear factor (NF)-κB. Previously, we observed that Vorinostat/JQ1 inactivates YAP1 and restores oscillation of NF-κB in differentiating myoblasts. These effects correlate with reduced tumorigenesis, and enhanced differentiation. However, the mechanisms by which the Hippo/NF-κB axis impact differentiation remained unknown. Here, we report that YAP1 and NF-κB activity suppress circadian clock function, inhibiting differentiation and promoting proliferation. In most tissues, clock activation is antagonized by the unfolded protein response (UPR). However, skeletal muscle differentiation requires both Clock and UPR activity, suggesting the molecular link between them is unique in muscle. In skeletal muscle-derived UPS, we observed that YAP1 suppresses PERK and ATF6-mediated UPR target expression as well as clock genes. These pathways govern metabolic processes, including autophagy, and their disruption shifts metabolism toward cancer cell-associated glycolysis and hyper-proliferation. Treatment with Vorinostat/JQ1 inhibited glycolysis/MTOR signaling, activated the clock, and upregulated the UPR and autophagy via inhibition of YAP1/NF-κB. These findings support the use of epigenetic modulators to treat human UPS. In addition, we identify specific autophagy, UPR, and muscle differentiation-associated genes as potential biomarkers of treatment efficacy and differentiation.

Bidirectional signalling between the tumour and stroma shapes tumour aggressiveness and metastasis. ATF4 is a major effector of the Integrated Stress Response, a homeostatic mechanism that couples cell growth and survival to bioenergetic demands. Using conditional knockout ATF4 mice, we show that global, or fibroblast-specific loss of host ATF4, results in deficient vascularization and a pronounced growth delay of syngeneic melanoma and pancreatic tumours. Single-cell transcriptomics of tumours grown in Atf4Δ/Δ mice uncovered a reduction in activation markers in perivascular cancer-associated fibroblasts (CAFs). Atf4Δ/Δ fibroblasts displayed significant defects in collagen biosynthesis and deposition and a reduced ability to support angiogenesis. Mechanistically, ATF4 regulates the expression of the Col1a1 gene and levels of glycine and proline, the major amino acids of collagen. Analyses of human melanoma and pancreatic tumours revealed a strong correlation between ATF4 and collagen levels. Our findings establish stromal ATF4 as a key driver of CAF functionality, malignant progression and metastasis.

Development and growth of a tumor as well as its ability to metastasize involves a complex relationship with its tissue microenvironment. A proliferating tumor encounters several microenvironmental stress conditions such as hypoxia, lack of nutrients and acidosis. To cope with these conditions, cancer cells have developed elaborate cytoprotective mechanisms which provide them with distinct advantages to thrive. Thus, deciphering the signaling pathways which get activated in the tumor microenvironment has been paramount to develop new therapeutic strategies for treatment. The Unfolded Protein Response (UPR) is an adaptive prosurvival pathway elicited by stresses in the tumor microenvironment (e.g., hypoxia, low glucose) and involves translational and transcriptional activation of effector genes which act to relieve cellular stress and block cancer cell death. We developed a strategy to comprehensively analyze critical mediators of cell fate in response to UPR activation. We have delivered a lentiviral genome-scale CRISPR Cas9 knockout (GeCKOv2) library to Sq20B cells (human squamous head and neck carcinoma) and A375 (human melanoma) cells. The library is targeting 19,050 genes with 123,411 unique guide sequences and enables both negative and positive selection screening. We used the GeCKO v2 library to identify genes essential for triggering the UPR in response to thapsigargin and tunicamycin, known specific activators of ER stress. Our highest-ranking candidates include BIRC5/Survivin, a well-studied molecule that acts as an inhibitor of apoptosis and is highly expressed in cancer cells and eukaryotic translation initiation factor eIF6, whose overexpression increases motility and invasiveness of cancer cells. Our preliminary results indicate that loss of Survivin and eIF6 dramatically enhance sensitization of cells to various ER stress conditions. Moreover, this synergistic outcome is observed when cells are treated with YM155, a small molecule that selectively suppresses Survivin and is used in phase I/II clinical trials. Lastly, morphological changes like endoreduplication are observed after long term absence of Survivin indicating its endogenous ER stress inducing role. Taken together, Survivin and eIF6 are important mediators of survival following ER stress and characterizing the pathways involved can lead to the development of novel targeted agents and therapeutic approaches. Citation Format: Nektaria Maria Leli, Souvik Dey, Lauren Brady, Constantinos Koumenis. Identifying novel regulators of the Unfolded Protein Response (UPR) by genome-scale CRISPR-Cas9 knockout screens [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 1247. doi:10.1158/1538-7445.AM2017-1247

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