No abstract

Endoplasmic reticulum oxidoreductin-1 alpha (ERO1α) was originally shown to be an endoplasmic reticulum (ER) resident protein undergoing oxidative cycles in concert with protein disulfide isomerase (PDI) to promote proper protein folding and to maintain homeostasis within the ER. ERO1α belongs to the flavoprotein family containing a flavin adenine dinucleotide utilized in transferring of electrons during oxidation-reduction cycles. This family is used to maintain redox potentials and protein homeostasis within the ER. ERO1α's location and function has since been shown to exist beyond the ER. Originally thought to exist solely in the ER, it has since been found to exist in the golgi apparatus, as well as in exosomes purified from patient samples. Besides aiding in protein folding of transmembrane and secretory proteins in conjunction with PDI, ERO1α is also known for formation of de novo disulfide bridges. Public databases, such as the Cancer Genome Atlas (TCGA) and The Protein Atlas, reveal ERO1α as a poor prognostic marker in multiple disease settings. Recent evidence indicates that ERO1α expression in tumor cells is a critical determinant of metastasis. However, the impact of increased ERO1α expression in tumor cells extends into the tumor microenvironment. Secretory proteins requiring ERO1α expression for proper folding have been implicated as being involved in immune escape through promotion of upregulation of programmed death ligand-1 (PD-L1) and stimulation of polymorphonuclear myeloid derived suppressor cells (PMN-MDSC's) via secretion of granulocytic colony stimulating factor (G-CSF). Hereby, ERO1α plays a pivotal role in cancer progression and potentially immune escape; making ERO1α an emerging attractive putative target for the treatment of cancer.

The flavin adenine dinucleotide containing endoplasmic reticulum oxidoreductase-1 α (ERO1α) catalyzes the de novo disulfide bond formation of secretory and transmembrane proteins and contributes toward proper protein folding. Recently, increased ERO1α expression has been shown to contribute to increased tumor growth and metastasis in multiple cancer types. In this report, we sought to define novel chemical space for targeting ERO1α function. Using the previously reported ERO1α inhibitor compound, EN-460, as a benchmark pharmacological tool, we were able to identify a sulfuretin derivative, T151742, which was approximately 2fold more potent using a recombinant enzyme assay system (IC 50 = 8.27 ± 2.33 μM) compared to EN-460 (IC 50 = 16.46 ± 3.47 μM). Additionally, T151742 (IC 50 = 16.04 μM) was slightly more sensitive than EN-460 (IC 50 = 19.35μM), using an MTT assay as an end point. Utilizing a cellular thermal shift assay (CETSA), we determined that the sulfuretin derivative T151742 demonstrated isozyme specificity for ERO1α as compared to that for ERO1β and showed no detectable binding to the FAD-containing enzyme LSD-1. T151742 retained activity in PC-9 cells in a clonogenicity assay, while EN-460 was devoid of activity. Furthermore, the activity of T151742 inhibition of clonogenicity was dependent on ERO1α expression as CRISPR-edited PC-9 cells were resistant to treatment with T151742. In summary, we identified a new scaffold that shows specificity for ERO1α compared to that for the closely related paralog ERO1β or the FAD-containing enzyme LSD-1 that can be used as a tool compound for the inhibition of ERO1α to allow for pharmacological validation of the role of ERO1α in cancer.

ID 27846 Poster Board 439Lung cancer is responsible for more deaths every year than breast, prostate, and colon cancers combined, yet patient treatment options are still limited. We aimed to characterize a role of a new potential therapeutic target, ERO1a, in non-small cell lung cancer (NSCLC). Physiologically, endoplasmic reticulum oxidoreductase 1 alpha (ERO1a) participates in formation of disulfide bonds crucial to protein folding in the endoplasmic reticulum (ER). However, high ERO1a expression was shown to be a poor prognostic indicator in multiple cancer types. While the contribution of high ERO1a levels to increased tumor burden and metastatic potential has been demonstrated, the downstream mechanisms are poorly understood. In this study, CRISPR strategies were utilized to knockout ERO1a in two lung cancer cell lines, PC-9 and HCC4006. The knockout variants demonstrated a significant reduction in colony and tumor sphere formation while no changes in cell proliferation and anoikis were observed. This finding correlated with increased survival and decreased tumor burden in SCID-Beige mice injected with ERO1a knockout cells compared to control cells (p<0.0045 Log Rank Test). As ERO1a is an ER resident protein, we hypothesized that ERO1a may alter the secretome produced by NSCLC. In support of our hypothesis, the reduction in colony-formation of ERO1a knockout clones can be rescued by media conditioned by control cells. Moreover, this effect is lost when conditioned media is subjected to heat denaturation. Together these data suggest that an ERO1a-dependent secreted protein is responsible for colony formation in lung cancer cells. Analysis of publicly available lung cancer proteomic data set (CPTAC) shows that high ERO1a expression correlates with enrichment in hallmarks of cancer, such as inflammatory response and epithelial-to-mesenchymal transition (EMT). Moreover, high ERO1a expression correlates with increased levels of multiple matrix proteins including Laminin 332, PLOD2, and LOXL2. These findings in primary patient specimens strongly agreed with the knockout cell line models showing that ERO1a expression is required for secretion of matrix associated proteins in both 2D and 3D systems. We are currently exploring whether the reduced expression of LOXL2 or LAMC2 is responsible for decreased colony formation observed in ERO1a-depleted cells. Taken together, our data indicate that ERO1a modifies the local tumor microenvironment and is an attractive target for therapeutic intervention in non-small cell lung cancer.

Endoplasmic Reticulum Oxidoreductase-1 alpha (ERO1α) is an endoplasmic reticulum (ER) resident protein responsible for oxidation of Protein Disulfide Isomerase (PDI). The molecular interactions between ERO1α and PDI allow for proper protein folding to occur. Recent reports indicate that ERO1α expression is a poor prognostic indicator of survival in multiple cancer types including multiple myeloma, lung cancer, esophageal carcinoma, hepatocellular carcinoma, and diffuse large B-cell lymphoma. In this study we utilized genetic strategies to determine the role of ERO1α in EGFR driven Non-Small Cell Lung Cancer (NSCLC). Our data indicate that CRISPR knockout of ERO1α leads to decreased clonogenicity in both PC-9 and HCC4006 cell lines. The phenotype can be partially rescued upon addition of conditioned media derived from the control cell lines. The data suggest that ERO1α expression is required for correct protein folding of a secreted factor that contributes to clonogenicity and growth. PC-9 ERO1α knockout cells injected into SCID-Beige animals led to increased overall survival (OS) compared to animals injected with PC-9 control cells. (PC-9 Control OS = 100 days, PC-9 ERO1α Knockout OS =154 days p=0.0045 **). Interestingly, ERO1α knockout cell lines still maintained sensitivity to standard of care agent Osimertinib. We identified T151742 as an ERO1α inhibitor that was approximately 2-fold more potent compared to the previously published ERO1α inhibitor EN460. A cellular thermal shift assay was performed to determine specificity of T151742 toward ERO1α compared to other FAD containing enzymes. We show that T151742 had a higher specificity towards ERO1α in comparison to other flavoenzymes such as ERO1β and LSD-1. Finally, our data indicate that PC-9 ERO1α knockout cell lines were ~2-fold resistant to T151742 by MTT but were completely resistant when using a 3D cell culture model suggesting T151742 is reliant on ERO1α in a 3D system. Finally, treatment of PC9 and HCC4006 cells with T151742 in combination with Osimertinib was synergistic in vitro. Taken together, this suggests ERO1α may be a viable target for EGFR driven NSCLC and that future studies will elucidate the use of T157142 in vivo as a single agent and in combination with EGFR inhibitors using in vivo studies. Citation Format: Brennan D. Johnson, Wei-Chih Chen, Werner J. Geldenhuys, Lori A. Hazlehurst. ERO1α as a potential target in EGFR driven non-small cell lung cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 3993.