Andrew A. Badachhape

Hepatoblastoma (HB) is the most common pediatric liver malignancy. However, its cellular origin and molecular drivers remain poorly defined. Using single-nuclear RNA sequencing (snRNA-seq), we identified a proliferative, hepatocyte-derived tumor cell population (cycling Hep ^T ) enriched for Enhancer of Zeste Homolog 2 ( EZH2) expression, particularly in the aggressive embryonal subtype. Integrative genomic and transcriptomic profiling confirmed EZH2 overexpression. Disruption of the PRC2 complex was evident through mislocalization and reduced expression of SUZ12, a core component. EZH2 overexpression correlated with upregulation of mitotic regulators such as AURKB and Ki67 in human HB gene expression analysis as compared to background liver. Targeted sequencing identified variants of uncertain significance in EZH2 and SUZ12 in 11 of 11 patient tumors. Pharmacologic inhibition of EZH2 with EPZ-6438 reduced proliferation and sensitized HB cells to cisplatin through gene regulation, potentially modulating platinum accumulation both in vitro and in vivo . In summary, EZH2 promotes HB progression through epigenetic silencing and noncanonical signaling pathways. These findings support EZH2’s contribution to HB pathogenesis, therefore identifying it as a novel therapeutic target. STATEMENT OF SIGNIFICANCE: EZH2 is enriched in hepatoblastoma, suggesting its role in early tumorigenesis. Its inhibition, combined with cisplatin, produces synergistic anti-tumor effects, supporting EZH2 as both an oncogenic and therapeutic target.

Background: Amyloid plaques are a pathological hallmark of Alzheimer’s disease (AD). In a recent pre‐clinical study, we showed an amyloid‐targeted liposomal macrocyclic gadolinium (Gd) contrast agent, ADx‐001, for in vivo MRI‐based detection of amyloid plaques in mouse. Although ADx‐001 showed high sensitivity at a high dose (0.2 mmol Gd/kg), the performance was sub‐optimal (<70%) at lower doses (0.1 and 0.15 mmol Gd/kg). In this work, we investigated if nano‐radiomics (radiomic analysis of nanoparticle contrast‐enhanced images) would increase sensitivity of ADx‐001 at lower dose levels.Method: In vivo studies were performed in the APP/PSEN1 mouse model of amyloid pathology. The efficacy of ADx‐001‐ enhanced MRI was studied at three dose‐levels: 0.10, 0.15, and 0.20 (mmol Gd/kg). Pre‐ and post‐contrast MRI was performed in transgenic (n = 6/dose) and wild‐type mice (n = 6/dose) using a T1‐weighted spin‐echo sequence. Semi‐automatic 3D segmentation of hippocampal and cortical regions was performed using a mouse brain MR atlas (Fig.1). Radiomic analysis was executed on the hippocampus and cortex regions of ADx‐001‐enhanced MR images 900 radiomic features (RFs). RF selection was completed using a non‐parametric neighborhood component method. 5‐fold cross‐validation was performed using a set of linear and non‐linear classifiers to confirm the accuracy of group separation.Result: Seven RFs (three RFs for cortex and four RFs for hippocampus) were identified that differentiated amyloid‐positive transgenic mice from amyloid‐negative wild‐type mice based on ADx‐001‐enhanced MRI. The best performing nearest‐neighbor classification model was trained simultaneously on all ADx‐001 dose groups. Nano‐radiomic analysis of ADx‐001‐enhanced MRI demonstrated 100% accuracy, specificity, and sensitivity for dose levels of 0.2 and 0.15 mmol Gd/kg. For low dose level (0.1 mmol Gd/kg), radiomics achieved accuracy of 91.6% and sensitivity of 83.3%, while maintaining specificity at 100%. The new results were superior when compared to previously reported results based on global signal enhancement analysis.Conclusion: Nano‐radiomic analysis of ADx‐001‐enhanced MRI improved sensitivity of ADx‐001 at lower dose levels for the detection of amyloid pathology and demonstrated excellent for intermediate and high dose. Our study demonstrates that radiomic analysis of contrast‐enhanced MR images could boost the performance of targeted molecular imaging agents for early detection of AD.

Introduction: Placenta accreta spectrum (PAS) occurs when the placenta is pathologically adherent to the myometrium. An intact retroplacental clear space (RPCS) is a marker of normal placentation, but visualization with conventional imaging techniques is a challenge. In this study, we investigate use of an FDA-approved iron oxide nanoparticle, ferumoxytol, for contrast-enhanced magnetic resonance imaging of the RPCS in mouse models of normal pregnancy and PAS. We then demonstrate the translational potential of this technique in human patients presenting with severe PAS (FIGO Grade 3C), moderate PAS (FIGO Grade 1), and no PAS. Methods: A T1-weighted gradient recalled echo (GRE) sequence was used to determine the optimal dose of ferumoxytol in pregnant mice. Pregnant Gab3 ^-/- mice, which demonstrate placental invasion, were then imaged at day 16 of gestation alongside wild-type (WT) pregnant mice which do not demonstrate invasion. Signal-to-noise ratio (SNR) was computed for placenta and RPCS for all fetoplacental units (FPUs) with ferumoxytol-enhanced magnetic resonance imaging (Fe-MRI) and used for the determination of contrast-to-noise ratio (CNR). Fe-MRI was also performed in 3 pregnant subjects using standard T1 and T2 weighted sequences and a 3D magnetic resonance angiography (MRA) sequence. RPCS volume and relative signal were calculated in all three subjects. Results: Ferumoxytol administered at 5 mg/kg produced strong T1 shortening in blood and led to strong placental enhancement in Fe-MRI images. Gab3 ^-/- mice demonstrated loss of hypointense region characteristic of the RPCS relative to WT mice in T1w Fe-MRI. CNR between RPCS and placenta was lower in FPUs of Gab3 ^-/- mice compared to WT mice, indicating higher degrees of vascularization and interruptions throughout the space. In human patients, Fe-MRI at a dose of 5 mg/kg enabled high uteroplacental vasculature signal and quantification of the volume and signal profile in severe and moderate invasion of the placenta relative to a non-PAS case. Discussion: Ferumoxytol, an FDA-approved iron oxide nanoparticle formulation, enabled visualization of abnormal vascularization and loss of uteroplacental interface in a murine model of PAS. The potential of this non-invasive visualization technique was then further demonstrated in human subjects. Diagnosis of placental invasion using Fe-MRI may provide a sensitive method for clinical detection of PAS.

Introduction: Prior preclinical studies established the utility of liposomal nanoparticle blood-pool contrast agents in visualizing the retroplacental clear space (RPCS), a marker of normal placentation, while sparing fetuses from exposure because the agent does not cross the placental barrier. In this work we characterized RPCS disruption in a mouse model of placenta accreta spectrum (PAS) using these agents. Methods: Contrast-enhanced MRI (CE-MRI) and computed tomography (CE-CT) using liposomal nanoparticles bearing gadolinium (liposomal-Gd) and iodine were performed in pregnant Gab3 ^-/- and wild type (WT) mice at day 16 of gestation. CE-MRI was performed on a 1T scanner using a 2D T1-weighted sequence (100×100×600 µm ³ voxels) and CE-CT was performed at a higher resolution (70×70×70 µm ³ voxels). Animals were euthanized post-imaging and feto-placental units (FPUs) were harvested for histological examination. RPCS conspicuity was scored through blinded assessment of images. Results: Pregnant Gab3 ^-/- mice show elevated rates of complicated pregnancy. Contrast-enhanced imaging demonstrated frank infiltration of the RPCS of Gab3 ^-/- FPUs. RPCS in Gab3 ^-/- FPUs was smaller in volume, demonstrated a heterogeneous signal profile, and received lower conspicuity scores than WT FPUs. Histology confirmed in vivo findings and demonstrated staining consistent with a thinner RPCS in Gab3 ^-/- FPUs. Discussion: Imaging of the Gab3 ^-/- mouse model at late gestation with liposomal contrast agents enabled in vivo characterization of morphological differences in the RPCS that could cause the observed pregnancy complications. An MRI-based method for visualizing the RPCS would be valuable for early detection of invasive placentation.

Introduction: Hepatoblastoma (HB) is the most frequent liver malignancy of childhood with an annual incidence of 1.5 per million and a survival rate less than 50%. Due to significant side effects and ineffectiveness of standard chemotherapy for relapse refractory disease, the treatment options are now shifting towards targeted therapies. Cyclin-dependent kinases (CDKs) are potential cancer therapeutic targets because of their critical role in promoting cell growth and regulating transcriptional processes. This study aims to assess the therapeutic efficacy of dinaciclib, cyclin-dependent kinase inhibitor, with HB in preclinical in vitroand in vivo models. Method: Dinaciclib was tested in patient derived cell lines (PDCL) from different patient derived xenograft (PDX) models using CellTiter-Glo Luminescent Viability assays. Dinaciclib was also assayed in vitro for cytotoxicity (MTT assay) and proliferation (CCK-8 assay). Differential expression of CDKs was checked by microarray analysis between normal liver and HB patients. Results were confirmed by immunoblotting assays. Immunoblotting assays were also used to see the effect of dinaciclib on CDK 1/2/5/9 and to assess the induction of apoptosis (PARP cleavage) in vitro. Dinaciclib was also tested in our orthotopic PDX models of high-risk HB. MRI and alpha-fetoprotein (AFP) ELISA were done to evaluate tumor growth. Tumor weights and volumes, as well as, immunohistochemistry were also used to assess effects of dinaciclib in vivo. Result: Dinaciclib suppressed cell proliferation and viability in a dose-dependent manner in all HB cell lines tested with IC₅₀ in the low micromolar range (HepG2- 3.75; HepT1- 0.007; Huh6- 1.42; HB17- 0.72). Microarray analysis showed significant upregulation of CDK1 (p=0.0003), CDK2 (p=0.00023) and CDK9 (p=0.015) in HB patient samples, however, immunoblotting with dinaciclib-treated cells showed decreased expression of CDKs 1/2/5/9 as well as induction of apoptosis. Dinaciclib also showed in vivo inhibition of tumor growth compared to placebo in both PDX models (HB47: relative tumor volume p = 0.03, T/C ratio= 0.36, tumor weight p = 0.02; HB52: relative tumor volume p = 0.06, T/C ratio= 0.16, tumor weight p = 0.07). Conclusion: Dinaciclib treatment showed promising effects with preclinical HB cell lines and PDX models. Citation Format: Rohit K. Srivastava, Roma Patel, Andres F. Espinoza, Yang Yu, Richard S. Whitlock, Samuel Larson, Andrew Badachhape, Jianhua Yang, Sarah E. Woodfield, Sanjeev A. Vasudevan. Efficacy of dinaciclib in hepatoblastoma through inhibition of cyclin dependent kinases [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 3891.

MYCN is a major driver for neuroblastoma (NB) and the tyrosine hydroxylase (TH)-MYCN transgenic mouse model is extensively used for preclinical NB studies. However, spatio-temporal NB progression in the TH-MYCN model has not been studied, and questions remain about the value of implanted models as a surrogate for transgenic mice. In this work, we used magnetic resonance imaging (MRI) to study tumor progression and nanoparticle contrast-enhanced computed tomography (n-CECT) to assess tumor vascular architecture in TH-MYCN transgenic mice (2–7 weeks of age) and TH-MYCN+/+-derived orthotopic allograft and syngeneic mice (2–5 weeks post-tumor implantation). Tumors in TH-MYCN transgenic mice became evident in the abdominal paraspinal region at week 5. A delayed thoracic paraspinal mass became evident at week 6 and most mice succumbed by week 7. In allograft and syngeneic mice, single mass tumor growth was restricted to the peritoneal cavity. N-CECT revealed a predominantly microvascular network in TH-MYCN tumors while implanted tumors exhibited heterogeneous and tortuous vessels. N-CECT quantitative analysis demonstrated high vascularity (tumor fractional blood volume ~ 0.12) in all models. Multi-modal imaging of TH-MYCN transgenic and implanted models revealed differences in growth patterns and vascular architecture that should be considered in designing preclinical studies.