High-risk hepatoblastoma dissemination control by oncogenic NRF2 - Hepatoblastoma (HB) is the most common liver tumor in children, believed to have the fastest rising incidence of all pediatric solid tumors. Unfortunately, HB has a poor overall survival rate in patients that present with high- risk disease, including metastatic, relapse, or treatment refractory tumors. These high-risk patients can expect a dismal survival rate of less than 50% due to the limited success of current therapies, including aggressive chemotherapy and surgery. Treatment failure in these patients is attributable to the spread of refractory circulating tumor cells (CTCs) that intravasate into blood vessels in the primary tumor, survive in systemic circulation, seed distant sites or other areas of the liver, lay dormant as minimal residual disease (MRD), and multiply after therapy is stopped. Therefore, a key unmet clinical need is to elucidate how CTCs drive this process of therapy resistance and metastasis, which critically hinders therapeutic success. Our overarching goal is to thoroughly characterize the molecular underpinnings of the metastatic process to inform the design of target- guided precision therapies. Our rigorous preliminary data demonstrates our innovative pipeline for identifying and sequencing CTCs from primary patient liquid biopsies and supports a role for NRF2 in HB vascular invasion and metastasis. We hypothesize that HB dissemination is driven by upregulation of NRF2 activity initially caused by chemotherapy exposure, which can be targeted to minimize or eliminate metastasis, MRD, and recurrence. In Aim 1, we will assess NRF2 activity differences among primary liver tumor, CTC, and lung metastasis samples and between chemotherapy naïve and treated samples with (a) co-IP, IF, proximity ligation, reporter, and mass spectrometry imaging (MSI) assays with samples collected from our novel, clinically relevant orthotopic cell line and patient-derived xenograft (PDX) murine models of HB and (b) IF, single cell RNA-seq, and MSI with primary patient samples. In Aim 2, we will manipulate NRF2 activity in vitro and in vivo with shRNA, overexpression, CRISPR, and small molecule inhibitor strategies and evaluate specific effects on phenotypes related to dissemination with assays for metabolism (MSI), migration (Incucyte) and invasion (transwells), and CTC and metastatic burden in our animal models. In Aim 3, we will test the efficacy of clinically relevant small molecule inhibitors that target oncogenic pathways activated by NRF2 (a) in vitro with assays for viability (MTT), migration (Incucyte), and invasion (transwells) and (b) in vivo with assays for tumor weight and volume (MRI), CTC burden, and incidence of metastasis. Our interdisciplinary team is uniquely positioned to perform these studies with our innovative, validated murine models and our unparalleled access to primary patient tissue and whole blood samples. Overall, these rigorous studies will lead to a thorough understanding of the role of NRF2 in HB tumor cell dissemination and inform the use of multiple targeted therapies for high-risk HB patients.
