Using High-Throughput Screening to Identify DNA Response Elements That Sense Cancer Dysregulated Pathways

Abstract Earli is developing a highly sensitive, orthogonal approach that is designed to be delivered via IV injection and uses a genetic construct to usurp dysregulated pathways, and actively forces cancer cells to drive the expression of a detectable ‘synthetic’ biomarker. The key element of our cancer-activated construct that drives specificity and sensitivity of expression is a promoter sequence, synthetically engineered to leverage transcription factors (TFs) activated in cancer pathways. Acting as a molecular sensor for dysregulated TF levels, the DNA construct expresses a measurable biomarker in cancer cells, while remaining transcriptionally silent in normal adjacent tissues and benign lesions. Previously, we described a multiomics factor analysis (MOFA) to interrogate 200 non-small cell lung cancer (NSCLC) and matched normal adjacent tissue samples comprised of RNA-seq based transcriptional profiling, and MS-based proteomics and phospho-proteomics from the Clinical Proteomic Tumor Analysis Consortium (CPTAC) dataset, and generated a list of top TFs dysregulated in NSCLC. In this study, we show the capability to design, engineer, and test cancer specific activity of combinations of response elements using a Massively Parallel Reporter Assay (MPRA) high-throughput pooled screening (HTS) method. More than 2,000 unique sequences of homotypic or heterotypic TF binding sites corresponding to the top 100 TFs were arrayed with diverse spacing and individually barcoded for the high-throughput screens in NSCLC cell lines. To validate our top hits, we individually tested 10 sequences from the top 25% and 8 sequences from the bottom 25%. Nine of the top sequences produced signals 5-10X higher than the background, whereas only one of the eight bottom sequences produced signal higher than the background. We have individually validated 30 different response elements and found that 28 of 30 drove the expected expression, with the majority of response elements (n=20) showing expression 5-10X higher than background. Remarkably, two of the engineered response elements were able to drive expression 20-30X higher in a specific cell line activated by WNT-signaling, suggesting that these response elements can sense specific cancer dysregulated pathways. Our lead response elements are being tested across a broad range of in vitro primary tumor lines with diverse genetic backgrounds and transcriptional profiles prior to in vivo testing using our imaging platform to distinguish malignant lung nodules. In summary, we have established a robust screening platform to identify response elements that can be activated by cancer dysregulated pathways. We are currently expanding this platform to other cancer models and indications, and leveraging this to explore more complex response elements that use combinations of TFs and natural spacing. Citation Format: Elizabeth Stroebele, Yue (Wendy) Zhang, Ishan Podar, Chloe Xia, Ajda Rojc, Dariusz Wodziak, Maggie Louie, David Suhy. Using high-throughput screening to identify DNA response elements that sense cancer dysregulated pathways [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 1694.