In Vivo Screen Identifies Liver X Receptor Alpha Agonism Potentiates Sorafenib Killing of Hepatocellular Carcinoma

Gastro Hep Advances · 2022 · 12 citations

• Political Science
• Cancer research
• Medicine

Hepatocellular carcinoma (HCC) is the most prevalent cancer of the liver and is a leading cause of cancer deaths worldwide. The multikinase inhibitor sorafenib has long been used to treat HCC because it was shown to extend survival by 3 months.1Llovet J.M. et al.N Engl J Med. 2008; 359: 378-390Crossref PubMed Scopus (9030) Google Scholar Although other drugs have recently been approved for therapy of HCC based on slightly improved outcomes,2Finn R.S. et al.N Engl J Med. 2020; 382: 1894-1905Crossref PubMed Scopus (2270) Google Scholar there is still a pressing need for new and effective treatments. Sequencing technologies have identified hundreds of prognostic genes in HCC.3Uhlen M. et al.Science. 2017; 357: eaan2507Crossref PubMed Scopus (1746) Google Scholar Nevertheless, correlation does not equate to functionality. We used an innovative approach to perform genetic screens in the native environment of the mouse liver to interrogate gene function in the presence or absence of sorafenib, designed to identify potential combination treatments for HCC. We validate a drug combination—sorafenib plus a Liver X Receptor alpha (LXR⍺) agonist—with enhanced killing of HCC in multiple cell lines. LXR⍺ agonists are currently in clinical trials and may have acceptable side effect profiles for patients with advanced HCC. We engineered a plasmid library of 43 complementary DNA (cDNAs) important in liver growth and function to conduct genetic screening in the livers of Fah−/− mice, a model of liver injury and repopulation.4Wangensteen K.J. et al.Genes Dev. 2015; 29: 904-909Crossref PubMed Scopus (23) Google Scholar Fah−/− mice hydrodynamically injected into the tail vein with the plasmid library developed visible HCCs as early as 6 weeks postinjection, which were confirmed to be HCCs linked to plasmids from the library (Figure 1A and Figure A1). We hypothesized that a screening approach could be used to discover genes that confer sensitivity or resistance to antitumor drug therapy with sorafenib. Therefore, we injected Fah−/− mice with the plasmid library and allowed the mice to form tumors for 6 weeks, then began daily treatment with either 30 mg/kg sorafenib or vehicle as a control for 2 months (Figure 1B and C). Sorafenib treatment significantly reduced the liver weight to body weight ratio and tumor burden compared with vehicle (Figure 1D and Table A1). To identify genes correlated with sorafenib sensitivity, we analyzed cDNAs present or absent in HCCs that developed in the sorafenib-treated group. Strikingly, the oncogene Myc was linked to all tumors of vehicle- or sorafenib-treated mice. In contrast, the Nr1h3 transgene, which encodes LXR⍺, was present in a number of vehicle-treated tumors as expected by chance but was completely absent from sorafenib-resistant tumors (Figure 1E and F). These results suggested that LXR⍺ activity is incompatible with tumor growth in the presence of sorafenib. Based on the result of the screen, we asked whether activating LXR⍺ can increase the response to sorafenib. We treated 3 previously established human HCC cell lines (Hep3B and Huh7), a primary human HCC cell line (PGM898), and a murine hepatoma cell line (Hepa1-6) with varying concentrations of the small-molecule LXR⍺ agonists GW3965 and T0901317 and sorafenib (Figure 2A and Figure A2). In each cell line, there was a 30%–50% reduction in cell viability with the combination treatment at a minimal concentration of 5 μM GW3965 and 2 μM sorafenib compared with sorafenib-only treatment, whereas treatment with LXR⍺ agonist alone did not affect cell viability. At these concentrations, the combination treatment led to only a 20% reduction from baseline in a murine hepatocyte-like cell line (Figure A2B), suggesting treatment preferentially affects HCC cells. Taken together, the results indicate that LXR⍺ activation improves the efficacy of sorafenib in multiple HCC cell lines. We queried the mechanism by which the combination treatment targets HCC by performing RNA sequencing (RNAseq) of Hep3B and Huh7 cells treated with drugs for 24 hours (Figure 2B and Figure A3A). Principal component analysis of the RNAseq data indicated a clear separation between the cell lines and treatment groups (Figure A3B). Differential expression analysis comparing treatment to DMSO control groups showed marked changes in expression with the combination. Among genes altered congruently in both cell lines: 19 genes were differentially expressed in response to GW3965, 190 to sorafenib, and 915 to the combination treatment, reflecting a remarkable synergistic effect on gene expression. We confirmed the specificity of sorafenib and GW3965 treatment by assessing gene expression levels of known markers of drug activity (Figure A3C). The gene expression changes with combined LXR⍺ agonist and sorafenib treatment were linked to alterations in 472 gene ontology pathways. The top 10 altered pathways, based on gene ratio, are displayed in Figure 2C. The regulation of cell death was the most altered pathway, and cell cycle pathways were also altered (red boxes). When investigating steady-state messenger RNA levels of cell cycle and apoptotic regulators, we found downregulation of many proliferation markers such as various cyclins (CCNE1 and CCND1), minichromosome maintenance complex proteins (MCM2, MCM4, MCM5, and MCM6), proliferating cell nuclear antigen, and cyclin-dependent kinase 2 (Figure 2B). Western blotting confirmed the downregulation of cell cycle regulators Cyclin D1 and proliferating cell nuclear antigen with combination therapy (Figure A3D). Differentially expressed genes associated with apoptosis included (BBC3, PMAIP1, JUN, and DDIT3; Figure 2B). Western blotting confirmed the upregulation of apoptosis regulator JUN with sorafenib and the combination treatment (Figure A3E). In short, combination treatment led to diminished expression of cell cycle genes and upregulation in apoptosis markers compared with sorafenib or LXR agonist alone, highlighting the cooperativity of these compounds. In summary, our in vivo genetic screen documents that LXRα is a key signal to determine the response to sorafenib of MYC-driven tumors in a model of liver injury and repopulation. We show that LXR⍺ agonists enhance the vulnerability of HCC cell cultures to sorafenib. The beneficial action of LXR⍺ agonism we reported here is in agreement with LXR⍺ expression correlating with better HCC prognosis5Long H. et al.Pathol Oncol Res. 2018; 24: 339-344Crossref PubMed Scopus (18) Google Scholar and with diminished LXR⍺ expression in sorafenib-resistant HCC cells.6Tovar V. et al.Gut. 2017; 66: 530-540Crossref PubMed Scopus (126) Google Scholar Recent preclinical assays in mice of LXR⍺ agonists and sorafenib support the benefit of combinatorial treatment and showed enhanced antitumor activity in both xenograft- and oncogene-driven hydrodynamic tail vein injection models.7Rudalska R. et al.Nat Cancer. 2021; 2: 201-217Crossref PubMed Scopus (12) Google Scholar,8Shao W. et al.Neoplasia. 2020; 22: 1-9Crossref PubMed Scopus (11) Google Scholar Further studies are needed to identify HCC subgroups that may benefit most from this combination. Collectively, our studies document how in vivo genetic screening in combination with drug therapies could identify cancer drug dependency and susceptibility patterns, which could be scaled to high-throughput screens.9Kieckhaefer J.E. et al.Semin Liver Dis. 2019; 39: 261-274Crossref PubMed Scopus (14) Google Scholar The authors thank Anil Rustgi, MD, and Flavio Maina, PhD, for help with editing the article. Download .xlsx (.02 MB) Help with xlsx files Table S1 Download .docx (.03 MB) Help with docx files Online Methods Download .pptx (33.08 MB) Help with pptx files Figure A1 Download .pptx (7.18 MB) Help with pptx files Figure A2 Download .pptx (14.98 MB) Help with pptx files Figure A3

Variability in biopsy quality informs translational research applications in hepatocellular carcinoma

Scientific Reports · 2021 · 11 citations

• Medicine
• Internal medicine
• Radiology

In the era of precision medicine, biopsies are playing an increasingly central role in cancer research and treatment paradigms; however, patient outcomes and analyses of biopsy quality, as well as impact on downstream clinical and research applications, remain underreported. Herein, we report biopsy safety and quality outcomes for percutaneous core biopsies of hepatocellular carcinoma (HCC) performed as part of a prospective clinical trial. Patients with a clinical diagnosis of HCC were enrolled in a prospective cohort study for the genetic, proteomic, and metabolomic profiling of HCC at two academic medical centers from April 2016 to July 2020. Under image guidance, 18G core biopsies were obtained using coaxial technique at the time of locoregional therapy. The primary outcome was biopsy quality, defined as tumor fraction in the core biopsy. 56 HCC lesions from 50 patients underwent 60 biopsy events with a median of 8 core biopsies per procedure (interquartile range, IQR, 7-10). Malignancy was identified in 45/56 (80.4%, 4 without pathology) biopsy events, including HCC (40/56, 71.4%) and cholangiocarcinoma (CCA) or combined HCC-CCA (5/56, 8.9%). Biopsy quality was highly variable with a median of 40% tumor in each biopsy core (IQR 10-75). Only 43/56 (76.8%) and 23/56 (41.1%) samples met quality thresholds for genomic or metabolomic/proteomic profiling, respectively, requiring expansion of the clinical trial. Overall and major complication rates were 5/60 (8.3%) and 3/60 (5.0%), respectively. Despite uniform biopsy protocol, biopsy quality varied widely with up to 59% of samples to be inadequate for intended purpose. This finding has important consequences for clinical trial design and highlights the need for quality control prior to applications in which the presence of benign cell types may substantially alter findings.

Functional Genetic Screening Enables Theranostic Molecular Imaging in Cancer

Clinical Cancer Research · 2020 · 8 citations

• Computational biology
• Medicine
• Biology

PURPOSE: Targeted therapies for cancer have accelerated the need for functional imaging strategies that inform therapeutic efficacy. This study assesses the potential of functional genetic screening to integrate therapeutic target identification with imaging probe selection through a proof-of-principle characterization of a therapy-probe pair using dynamic nuclear polarization (DNP)-enhanced magnetic resonance spectroscopic imaging (MRSI). EXPERIMENTAL DESIGN: -weighted segmentations quantified tumor growth. RESULTS: < 0.001). CONCLUSIONS: C]-pyruvate is a theranostic strategy for hepatocellular carcinoma, enabling quantification of intratumoral LDHi pharmacodynamics and therapeutic efficacy prediction. This work lays the foundation for a novel theranostic platform wherein functional genetic screening informs imaging probe selection to quantify therapeutic efficacy on a cancer-by-cancer basis.

Image-based marker tracking and registration for intraoperative 3D image-guided interventions using augmented reality

2020 · 14 citations

• Computer Science
• Artificial Intelligence
• Computer vision

Augmented reality (AR) can enable physicians to “see” inside of patients by projecting cross-sectional imaging directly onto the patient during procedures. In order to maintain workflow, imaging must be quickly and accurately registered to the patient. We describe a method for automatically registering a CT image set projected from an augmented reality headset to a set of points in the real world as a first step towards real-time registration of medical images to patients. Sterile, radiopaque fiducial markers with unique optical identifiers were placed on a patient prior to acquiring a CT scan of the abdomen. For testing purposes, the same fiducial markers were then placed on a tabletop as a representation of the patient. Our algorithm then automatically located the fiducial markers in the CT image set, optically identified the fiducial markers on the tabletop, registered the markers in the CT image set with the optically detected markers and finally projected the registered CT image set onto the real-world markers using the augmented reality headset.The registration time for aligning the image set using 3 markers was 0.9 &plusmn; 0.2 seconds with an accuracy of 5 &plusmn; 2 mm. These findings demonstrate the feasibility of fast and accurate registration using unique radiopaque markers for aligning patient imaging onto patients for procedural planning and guidance.

Targeting glutamine metabolism slows soft tissue sarcoma growth

Nature Communications · 2020 · 99 citations

• Cancer research
• Biology
• Pathology

Tumour cells frequently utilize glutamine to meet bioenergetic and biosynthetic demands of rapid cell growth. However, glutamine dependence can be highly variable between in vitro and in vivo settings, based on surrounding microenvironments and complex adaptive responses to glutamine deprivation. Soft tissue sarcomas (STSs) are mesenchymal tumours where cytotoxic chemotherapy remains the primary approach for metastatic or unresectable disease. Therefore, it is critical to identify alternate therapies to improve patient outcomes. Using autochthonous STS murine models and unbiased metabolomics, we demonstrate that glutamine metabolism supports sarcomagenesis. STS subtypes expressing elevated glutaminase (GLS) levels are highly sensitive to glutamine starvation. In contrast to previous studies, treatment of autochthonous tumour-bearing animals with Telaglenastat (CB-839), an orally bioavailable GLS inhibitor, successfully inhibits undifferentiated pleomorphic sarcoma (UPS) tumour growth. We reveal glutamine metabolism as critical for sarcomagenesis, with CB-839 exhibiting potent therapeutic potential.