RESEARCH PROJECT 1 - SUMMARY Antibody-drug conjugates (ADCs) are a rapidly growing class of chemotherapy drugs designed to deliver cancer cell specific cytotoxic payloads. Trastuzumab-deruxtecan (DS-8201a) is a pioneer next generation ADC comprised of trastuzumab attached to a topoisomerase I (TOP1) inhibitor payload via a unique cleavable linker. DS-8201a is under active investigation for the treatment of multiple cancer types, and has received FDA approval for several indications, including advanced HER2-positive (IHC 3+ or ISH+) and more recently the HER2-low (IHC 1+, 2+/ISH-) breast cancer (BC). However, a subset of patients with HER2 IHC 0 BC also responded to DS-8201a in clinical trials. The immunohistochemistry (IHC) assay routinely used in the clinic is inadequate in distinguishing the lower ranges of HER2 expression. Development of a more quantitative HER2 assay is an unmet need. Using multiple-reaction monitoring mass spectrometry (MRM-MS) proteomic approach, our collaborator Dr. Amanda Paulovich has developed a CLIA assay which is capable of detecting a large range of HER2 expression in BCs that are HER2-low or HER2 0 by conventional IHC. In this proposal, we will examine the utility of this assay in predicting DS-8201a efficacy in “HER2 low” BC, defined by HER2 IHC 0-2+/ISH-. We hypothesize that HER2 expression quantified by the MRM-MS could predict DS-8201a activity. Another area of unmet clinical need that this project addresses is to develop mechanism-based strategies to improve the efficacy of DS-8201a for HER2 low tumors since they often derive less benefit from DS-8201a monotherapy. Based on our preliminary data, we hypothesize that inhibitors against components of the DNA damage response and repair pathway (DDRi) could enhance the anti-tumor effect of DS-8201a. In Aim 1, we will examine whether the HER2 expression by MRM-MS and DDR pathway alterations predict response to single agent DS-8201a using 100 HER2 low BC PDX derived organoid models. Drug response in organoids will be validated in selected PDX models in vitro. In Aim 2, we will determine whether DDRi, including the PARPi (olaparib), ATRi (AZD6738), ATMi (AZ1930), BERi (TRC-102), and DNA-PKi (M3814), enhances the anti-tumor effect of DS-8201a in HER2 low BC in vitro and in vivo. We will investigate treatment effects of DDRi and DS-8201a, alone or in combination, on HER2 level, markers of DNA damage, apoptosis, and on the transcriptome and proteome/phosphoproteome. We will also perform spatial transcriptomics and snRNA sequencing of selected PDX models to analyze intra- tumor heterogeneity in treatment response. In Aim 3, we will examine whether DDRi enhances the anti-tumor activity of DS-8201a in HER2 low colorectal cancer, lung cancer, and pancreatic adenocarcinoma. By assessing the utility of a quantitative HER2 assay and proteogenomic analyses of DDR pathway, we aim to improve the selection of patients likely to respond to DS-8201a in future trials. Results from the ...