The efficacy of ferroptosis-inducing compounds IKE and RSL3 correlates with the expression of ferroptotic pathway regulators CD71 and SLC7A11 in biliary tract cancer cells.

INTRODUCTION: Biliary tract cancer (BTC) is a lethal disease with a bad overall survivability, partly arising from inadequate therapeutic alternatives, detection at a belated stage, and a resistance to common therapeutic approaches. Ferroptosis is a form of programmed cell death that depends on reactive oxygen species (ROS) and iron, causing excessive peroxidation of polyunsaturated fatty acids (PUFAs). Therefore, the objective of this investigation is, whether ferroptosis can be induced in BTC in vitro and whether this induction is dependent on specific molecular markers. METHODS: The study conducted resazurin assay and IC25/50 calculation to explore the possible cytotoxic outcomes of different classes of ferroptosis-inducing substances (FINs) on a comprehensive in vitro model of 11 BTC cell lines. Combinatory treatments with different cell death inhibitors were performed to evaluate the magnitude of ferroptosis induction. To ascertain whether ferroptotic cell death occurred, liperfluo and iron assay kits were employed to evaluate lipid ROS and intracellular iron abundance. Potential biomarkers of ferroptosis sensitivity were then assessed via western blot analysis, a rtPCR panel and functional assay kits. RESULTS: The study found that different FINs reduced cell viability in a cell line-dependent manner. In addition, we measured increased lipid ROS and intracellular Fe2+ levels upon exposure to FINs in BTC cells. Combining FINs with inhibitors of ferroptosis, necroptosis or apoptosis suggests the occurrence of ferroptotic events in BTC cell lines CCC-5, HuH-28 and KKU-055. Furthermore, we found that BTC cells display a heterogeneous profile regarding different molecular genes/markers of ferroptosis. Subsequent analysis revealed that sensitivity of BTC cells towards IKE and RSL3 positively correlated with CD71 and SLC7A11 protein expression. CONCLUSION: Our results demonstrate that induction of ferroptosis is a promising approach to inhibit BTC cell growth and that the sensitivity of BTC cells towards ferroptosis induction might be dependent on molecular markers such as CD71 and SLC7A11.

Discovery of YAP1/TAZ pathway inhibitors through phenotypic screening with potent anti-tumor activity via blockade of Rho-GTPase signaling.

This study describes the identification and target deconvolution of small molecule inhibitors of oncogenic Yes-associated protein (YAP1)/TAZ activity with potent anti-tumor activity in vivo. A high-throughput screen (HTS) of 3.8 million compounds was conducted using a cellular YAP1/TAZ reporter assay. Target deconvolution studies identified the geranylgeranyltransferase-I (GGTase-I) complex as the direct target of YAP1/TAZ pathway inhibitors. The small molecule inhibitors block the activation of Rho-GTPases, leading to subsequent inactivation of YAP1/TAZ and inhibition of cancer cell proliferation in vitro. Multi-parameter optimization resulted in BAY-593, an in vivo probe with favorable PK properties, which demonstrated anti-tumor activity and blockade of YAP1/TAZ signaling in vivo.

Enhancing anti-CD274 (PD-L1) targeting through combinatorial immunotherapy with bispecific antibodies and fusion proteins: from preclinical to phase II clinical trials.

INTRODUCTION: Immune checkpoint inhibitors have achieved great success in the treatment of many different types of cancer. Programmed cell death protein ligand 1 (PD-L1, CD274) is a major immunosuppressive immune checkpoint and a target for several already approved monoclonal antibodies. Despite this, novel strategies are under development, as the overall response remains low. AREAS COVERED: In this review, an overview of the current biomarkers for response to PD-L1 inhibitor treatment is given, followed by a discussion of potential novel biomarkers, including tumor mutational burden and circulating tumor DNA. Combinatorial immunotherapy is a potential novel strategy to increase the response to PD-L1 inhibitor treatment and currently, several interesting bispecific antibodies as well as bispecific fusion proteins are undergoing early clinical investigation. We focus on substances targeting PD-L1 and a secondary target, and a secondary immunomodulatory target like CTLA-4, TIGIT, or CD47. EXPERT OPINION: Overall, the presented studies show anti-tumor activity of these combinatorial immunotherapeutic approaches. However, still relatively low response rates suggest a need for better biomarkers.

Gene expression inhibitors for the treatment of liver fibrosis: drugs under preclinical and early clinical investigation.

INTRODUCTION: Liver fibrosis represents an unmet medical condition with growing incidence and only limited therapeutic options. Interfering with dysregulated gene expression was considered a specific treatment approach, and we are here reviewing the current options to modulate transcription and translation with small molecule inhibitors of involved enzymes, transcription factors or by using non-coding RNA molecules (RNA interference) or DNA antisense oligonucleotides. Despite promising results in preclinical models, only limited data are available from studies in humans. AREAS COVERED: This expert opinion provides a general overview of how to interfere with gene expression (transcription and translation) and highlighting recent achievements in liver fibrosis. EXPERT OPINION: Many compounds that were explored to modulate gene expression in liver fibrosis (models) were developed as anti-cancer agents. Their use in humans with impaired liver function is often impaired by the lack of specificity to inhibit only fibrosis-related genes in the liver and by associated general toxicity and narrow therapeutic windows. RNAi approaches show a higher degree of specificity and potentially less systemic toxicity. Clinical development in liver fibrosis requires close interaction between pharmaceutical companies and regulatory authorities to address topics like relevant (surrogate) endpoints to achieve meaningful readouts faster.

Identification of acetylshikonin as a novel tubulin polymerization inhibitor with antitumor activity in human hepatocellular carcinoma cells.

Background: Microtubules are attractive targets for anticancer drugs. However, the microtubule-targeting agents (MTAs) currently in clinical use exhibit inevitable drug resistance. Therefore, there is an urgent need to discover novel MTAs for the clinical treatment of cancer. Methods: Bioactive compounds extracted from Lithospermum erythrorhizon were assessed for in vitro anti-proliferative activities against a panel of human cancer cell lines using cell counting kit-8 (CCK-8) assay. Tubulin polymerization inhibition assay, colchicine competitive binding site assay, and immunofluorescence were used to validate the tubulin inhibition effect of acetylshikonin. Flow cytometry, Hoechst staining, and caspase-3 activity evaluation were performed to assess cell cycle arrest and cell apoptosis. 5,5',6,6'-tetrachloro-1,1',3,3'-tetramethylbenzimidazolylcarbocyanine iodide (JC-1) staining and dichloro-dihydro-fluorescein diacetate (DCFH-DA) staining were used to evaluate mitochondrial membrane potential (MMP) and reactive oxygen species (ROS), respectively. Results: Acetylshikonin exhibited potent anti-proliferative activities against a panel of human cancer cell lines (IC50 values: 1.09-7.26 µM) and displayed comparable cytotoxicity against several drug-resistant cell lines. Further mechanism studies revealed that acetylshikonin induced cell cycle arrest of MHCC-97H cells at G2/M phase, and significantly promoted apoptosis marked by a collapse of MMP and abnormal ROS accumulation. Conclusions: In this study, acetylshikonin was identified as MTA against hepatocellular carcinoma and can serve as a promising lead compound for further development of anti-cancer drug, underscoring its potential clinical significance.

Patient Selection Approaches in FGFR Inhibitor Trials-Many Paths to the Same End?

Inhibitors of fibroblast growth factor receptor (FGFR) signaling have been investigated in various human cancer diseases. Recently, the first compounds received FDA approval in biomarker-selected patient populations. Different approaches and technologies have been applied in clinical trials, ranging from protein (immunohistochemistry) to mRNA expression (e.g., RNA in situ hybridization) and to detection of various DNA alterations (e.g., copy number variations, mutations, gene fusions). We review, here, the advantages and limitations of the different technologies and discuss the importance of tissue and disease context in identifying the best predictive biomarker for FGFR targeting therapies.

Ferroptosis in Hepatocellular Carcinoma: Mechanisms, Drug Targets and Approaches to Clinical Translation.

Ferroptosis, an iron and reactive oxygen species (ROS)-dependent non-apoptotic type of regulated cell death, is characterized by a massive iron overload and peroxidation of polyunsaturated fatty acids (PUFAs), which finally results in cell death. Recent studies suggest that ferroptosis can influence carcinogenesis negatively and therefore may be used as a novel anti-cancer strategy. Hepatocellular carcinoma (HCC) is a deadly malignancy with poor chances of survival and is the second leading cause of cancer deaths worldwide. Diagnosis at an already late stage and general resistance to current therapies may be responsible for the dismal outcome. As the liver acts as a key factor in iron metabolism, ferroptosis is shown to play an important role in HCC carcinogenesis and, more importantly, may hold the potential to eradicate HCC. In this review, we summarize the current knowledge we have of the role of ferroptosis in HCC and the application of ferroptosis as a therapy option and provide an overview of the potential translation of ferroptosis in the clinical practice of HCC.

Chemoresistance and resistance to targeted therapies in biliary tract cancer: what have we learned?

INTRODUCTION: Biliary tract cancer (BTC), including intra- and extrahepatic cholangiocarcinoma and gallbladder cancer, is a rare and highly difficult to manage human malignancy. Besides late diagnosis and associated unresectability, frequently observed unresponsiveness toward and recurrence following chemotherapy or targeted therapy essentially contribute to the dismal prognosis of BTC patients. AREAS COVERED: The review provides an update on individual mechanisms involved resistance of BTC toward conventional chemotherapy as well as targeted therapies. We review the distinct mechanisms of pharmacoresistance (MPRs) which have been defined in BTC cells on a molecular basis and examine the specific consequences for the various approaches of chemo-, targeted or immunomodulatory therapies. EXPERT OPINION: Based on currently available experimental and clinical data, the present knowledge about these MPRs in BTCs are summarized. While some possible tactics for overcoming these mechanisms of resistance have been investigated, a BTC-specific and efficient approach based on comprehensive in vitro and in vivo experimental systems is not yet available. Additionally, a reliable monitoring of therapy-relevant cellular changes needs to be established which allows for choosing the optimal drug (combination) before and/or during pharmacological therapy.

Pharmacologic Antagonization of Cannabinoid Receptor 1 Improves Cholestasis in Abcb4-/- Mice.

BACKGROUND & AIMS: The endocannabinoid system is involved in the modulation of inflammatory, fibrotic, metabolic, and carcinogenesis-associated signaling pathways via cannabinoid receptor (CB)1 and CB2. We hypothesized that the pharmacologic antagonization of CB1 receptor improves cholestasis in Abcb4-/- mice. METHODS: After weaning, male Abcb4-/- mice were treated orally with rimonabant (a specific antagonist of CB1) or ACEA (an agonist of CB1) until up to 16 weeks of age. Liver tissue and serum were isolated and examined by means of serum analysis, quantitative real time polymerase chain reaction, Western blot, immunohistochemistry, and enzyme function. Untreated Abcb4-/- and Bagg Albino Mouse/c wild-type mice served as controls. RESULTS: Cholestasis-induced symptoms such as liver damage, bile duct proliferation, and enhanced circulating bile acids were improved by CB1 antagonization. Rimonabant treatment also improved Phosphoenolpyruvat-Carboxykinase expression and reduced inflammation and the acute-phase response. The carcinogenesis-associated cellular-Jun N-terminal kinase/cellular-JUN and signal transducer and activator of transcription 3 signaling pathways activated in Abcb4-/- mice were reduced to wild-type level by CB1 antagonization. CONCLUSIONS: We showed a protective effect of oral CB1 antagonization in chronic cholestasis using the established Abcb4-/- model. Our results suggest that pharmacologic antagonization of the CB1 receptor could have a therapeutic benefit in cholestasis-associated metabolic changes, liver damage, inflammation, and carcinogenesis.

Immunmodulatory Treatment Strategies of Hepatocellular Carcinoma: From Checkpoint Inhibitors Now to an Integrated Approach in the Future.

BACKGROUND: Hepatocellular carcinoma (HCC) still represents a human tumor entity with very limited therapeutic options, especially for advanced stages. Here, immune checkpoint modulating drugs alone or in combination with local ablative techniques could open a new and attractive therapeutic "door" to improve outcome and response rate for patients with HCC. METHODS: Published data on HCC experimental to pre-(clinical) treatment strategies from standard of care to novel immunomodulatory concepts were summarized and discussed in detail. RESULTS: Overall, our knowledge of the role of immune checkpoints in HCC is dramatically increased in the last years. Experimental and pre-clinical findings could be translated to phase 1 and 2 clinical trials and became standard of care. Local ablative techniques of HCC could improve the effectivity of immune checkpoint inhibitors in situ. CONCLUSIONS: This review demonstrates the importance of immunomodulatory treatment strategies of HCC, whereby the "best treatment code" of immune checkpoint drugs, combination with ablative techniques and of timing must be evaluated in coming clinical trials.

NF-κB Pathway as a Potential Target for Treatment of Critical Stage COVID-19 Patients.

Patients infected with SARS-CoV-2 show a wide spectrum of clinical manifestations ranging from mild febrile illness and cough up to acute respiratory distress syndrome, multiple organ failure, and death. Data from patients with severe clinical manifestations compared to patients with mild symptoms indicate that highly dysregulated exuberant inflammatory responses correlate with severity of disease and lethality. Epithelial-immune cell interactions and elevated cytokine and chemokine levels, i.e. cytokine storm, seem to play a central role in severity and lethality in COVID-19. The present perspective places a central cellular pro-inflammatory signal pathway, NF-κB, in the context of recently published data for COVID-19 and provides a hypothesis for a therapeutic approach aiming at the simultaneous inhibition of whole cascades of pro-inflammatory cytokines and chemokines. The simultaneous inhibition of multiple cytokines/chemokines is expected to have much higher therapeutic potential as compared to single target approaches to prevent cascade (i.e. redundant, triggering, amplifying, and synergistic) effects of multiple induced cytokines and chemokines in critical stage COVID-19 patients.

Fibroblast growth factor signaling in non-alcoholic fatty liver disease and non-alcoholic steatohepatitis: Paving the way to hepatocellular carcinoma.

Metabolic disorders are increasingly leading to non-alcoholic fatty liver disease, subsequent steatohepatitis, cirrhosis and hepatocellular carcinoma. Fibroblast growth factors and their receptors play an important role in maintaining metabolic homeostasis also in the liver and disorders in signaling have been identified to contribute to those pathophysiologic conditions leading to hepatic lipid accumulation and chronic inflammation. While specific and well tolerated inhibitors of fibroblast growth factor receptor activity are currently developed for (non-liver) cancer therapy, treatment of non-alcoholic fatty liver disease and non-alcoholic steatohepatitis is still limited. Fibroblast growth factor-mimicking or restoring approaches have recently evolved as a novel therapeutic option and the impact of such interactions with the fibroblast growth factor receptor signaling network during non-alcoholic fatty liver disease/non-alcoholic steatohepatitis development is reviewed here.

Challenges and opportunities in drug development for nonalcoholic steatohepatitis.

Nonalcoholic fatty liver disease (NAFLD) and nonalcoholic steatohepatitis (NASH) are considered major global medical burdens with high prevalence and steeply rising incidence. Despite the characterization of numerous pathophysiologic pathways leading to metabolic disorder, lipid accumulation, inflammation, fibrosis, and ultimately end-stage liver disease or liver cancer formation, so far no causal pharmacological therapy is available. Drug development for NAFLD and NASH is limited by long disease duration and slow progression and the need for sequential biopsies to monitor the disease stage. Additional non-invasive biomarkers could therefore improve design and feasibility of such. Here, the current concepts on preclinical models, biomarkers and clinical endpoints and trial designs are briefly reviewed.

Cannabinoid receptor 1 knockout alleviates hepatic steatosis by downregulating perilipin 2.

The endocannabinoid (EC) system has been implicated in the pathogenesis of several metabolic diseases, including nonalcoholic fatty liver disease (NAFLD). With the current study we aimed to verify the modulatory effect of endocannabinoid receptor 1 (CB1)-signaling on perilipin 2 (PLIN2)-mediated lipophagy. Here, we demonstrate that a global knockout of the cannabinoid receptor 1 gene (CB1-/-) reduced the expression of the lipid droplet binding protein PLIN2 in the livers of CB1-/- and hepatitis B surface protein (HBs)-transgenic mice, which spontaneously develop hepatic steatosis. In addition, the pharmacologic activation and antagonization of CB1 in cell culture also caused an induction or reduction of PLIN2, respectively. The decreased PLIN2 expression was associated with suppressed lipogenesis and triglyceride (TG) synthesis and enhanced autophagy as shown by increased colocalization of LC3B with lysosomal-associated membrane protein 1 (LAMP1) in HBs/CB1-/- mice. The induction of autophagy was further supported by the increased expression of LAMP1 in CB1-/- and HBs/CB1-/- mice. LAMP1 and PLIN2 were co-localized in HBs/CB1-/- indicating autophagy of cytoplasmic lipid droplets (LDs) i.e., lipophagy. Lipolysis of lipid droplets was additionally indicated by elevated expression of lysosomal acid lipase. In conclusion, these results suggest that loss of CB1 signaling leads to reduced PLIN2 abundance, which triggers lipophagy. Our new findings about the association between CB1 signaling and PLIN2 may stimulate translational studies analyzing new diagnostic and therapeutic options for NAFLD.

Phase 1 Dose Escalation Study of the Allosteric AKT Inhibitor BAY 1125976 in Advanced Solid Cancer-Lack of Association between Activating AKT Mutation and AKT Inhibition-Derived Efficacy.

This open-label, phase I first-in-human study (NCT01915576) of BAY 1125976, a highly specific and potent allosteric inhibitor of AKT1/2, aimed to evaluate the safety, pharmacokinetics, and maximum tolerated dose of BAY 1125976 in patients with advanced solid tumors. Oral dose escalation was investigated with a continuous once daily (QD) treatment (21 days/cycle) and a twice daily (BID) schedule. A dose expansion in 28 patients with hormone receptor-positive metastatic breast cancer, including nine patients harboring the AKT1E17K mutation, was performed at the recommended phase 2 dose (R2D) of 60 mg BID. Dose-limiting toxicities (Grades 3-4) were increased in transaminases, γ-glutamyltransferase (γ-GT), and alkaline phosphatase in four patients in both schedules and stomach pain in one patient. Of the 78 patients enrolled, one patient had a partial response, 30 had stable disease, and 38 had progressive disease. The clinical benefit rate was 27.9% among 43 patients treated at the R2D. AKT1E17K mutation status was not associated with tumor response. Genetic analyses revealed additional mutations that could promote tumor cell growth despite the inhibition of AKT1/2. BAY 1125976 was well tolerated and inhibited AKT1/2 signaling but did not lead to radiologic or clinical tumor responses. Thus, the refinement of a selection of biomarkers for AKT inhibitors is needed to improve their monotherapy activity.