Kym N. Lowes

Oval Cell Numbers in Human Chronic Liver Diseases Are Directly Related to Disease Severity

The risk of developing hepatocellular carcinoma is significantly increased in patients with genetic hemochromatosis, alcoholic liver disease, or chronic hepatitis C infection. The precise mechanisms underlying the development of hepatocellular carcinoma in these conditions are not well understood. Stem cells within the liver, termed oval cells, are involved in the pathogenesis of hepatocellular carcinoma in animal models and may be important in the development of hepatocellular carcinoma in human chronic liver diseases. The aims of this study were to determine whether oval cells could be detected in the liver of patients with genetic hemochromatosis, alcoholic liver disease, or chronic hepatitis C, and whether there is a relationship between the severity of the liver disease and the number of oval cells. Oval cells were detected using histology and immunohistochemistry in liver biopsies from patients with genetic hemochromatosis, alcoholic liver disease, or chronic hepatitis C. Oval cells were not observed in normal liver controls. Oval cell numbers increased significantly with the progression of disease severity from mild to severe in each of the diseases studied. We conclude that oval cells are frequently found in subjects with genetic hemochromatosis, alcoholic liver disease, or chronic hepatitis C. There is an association between severity of liver disease and increase in the number of oval cells consistent with the hypothesis that oval cell proliferation is associated with increased risk for development of hepatocellular carcinoma in chronic liver disease.

Structure-guided design of a selective BCL-XL inhibitor

The prosurvival BCL-2 family protein BCL-X(L) is often overexpressed in solid tumors and renders malignant tumor cells resistant to anticancer therapeutics. Enhancing apoptotic responses by inhibiting BCL-X(L) will most likely have widespread utility in cancer treatment and, instead of inhibiting multiple prosurvival BCL-2 family members, a BCL-X(L)-selective inhibitor would be expected to minimize the toxicity to normal tissues. We describe the use of a high-throughput screen to discover a new series of small molecules targeting BCL-X(L) and their structure-guided development by medicinal chemistry. The optimized compound, WEHI-539 (7), has high affinity (subnanomolar) and selectivity for BCL-X(L) and potently kills cells by selectively antagonizing its prosurvival activity. WEHI-539 will be an invaluable tool for distinguishing the roles of BCL-X(L) from those of its prosurvival relatives, both in normal cells and notably in malignant tumor cells, many of which may prove to rely upon BCL-X(L) for their sustained growth.

Exploiting selective BCL-2 family inhibitors to dissect cell survival dependencies and define improved strategies for cancer therapy

Selective inhibition of BCL-XL synergizes with docetaxel to inhibit the growth of solid tumors but does not inhibit granulopoiesis. A more refined antitumor strategy The BCL-2 family is a group of related proteins that regulate apoptosis in a variety of ways. The success of anticancer treatments often hinges on the ability to induce cancer cell death by apoptosis. As a result, there has been a great deal of interest in developing drugs that can inhibit the antiapoptotic members of the BCL-2 pathway. Unfortunately, some of these drugs are also associated with dose-limiting hematologic toxicities, such as neutropenia. Now, Leverson et al. have used a toolkit of BCL-2 family inhibitors with different specificities to show that specifically inhibiting BCL-XL (one member of this protein family) is effective for killing tumors, but without the common side effects seen with less selective drugs. The BCL-2/BCL-XL/BCL-W inhibitor ABT-263 (navitoclax) has shown promising clinical activity in lymphoid malignancies such as chronic lymphocytic leukemia. However, its efficacy in these settings is limited by thrombocytopenia caused by BCL-XL inhibition. This prompted the generation of the BCL-2–selective inhibitor venetoclax (ABT-199/GDC-0199), which demonstrates robust activity in these cancers but spares platelets. Navitoclax has also been shown to enhance the efficacy of docetaxel in preclinical models of solid tumors, but clinical use of this combination has been limited by neutropenia. We used venetoclax and the BCL-XL–selective inhibitors A-1155463 and A-1331852 to assess the relative contributions of inhibiting BCL-2 or BCL-XL to the efficacy and toxicity of the navitoclax-docetaxel combination. Selective BCL-2 inhibition suppressed granulopoiesis in vitro and in vivo, potentially accounting for the exacerbated neutropenia observed when navitoclax was combined with docetaxel clinically. By contrast, selectively inhibiting BCL-XL did not suppress granulopoiesis but was highly efficacious in combination with docetaxel when tested against a range of solid tumors. Therefore, BCL-XL–selective inhibitors have the potential to enhance the efficacy of docetaxel in solid tumors and avoid the exacerbation of neutropenia observed with navitoclax. These studies demonstrate the translational utility of this toolkit of selective BCL-2 family inhibitors and highlight their potential as improved cancer therapeutics.

Oval cell‐mediated liver regeneration: Role of cytokines and growth factors

Abstract   In experimental models, which induce liver damage and simultaneously block hepatocyte proliferation, the recruitment of a hepatic progenitor cell population comprised of oval cells is invariably observed. There is a substantial body of evidence to suggest that oval cells are involved in liver regeneration, as they differentiate into hepatocytes and biliary cells. Recently, bone marrow cells were shown to be a source of a stem cells with the capacity to repopulate the liver. Presently, the relationship between bone marrow cells and oval cells is unclear. Investigations will be greatly assisted by the availability of in vitro models based on a knowledge of cytokines that affect oval cells. While the cytokines, which regulate the different hematopoietic lineages, are well characterized, there is relatively little information regarding those that influence oval cells. This review outlines recent developments in the field of oval cell research and focuses on cytokines and growth factors that have been implicated in regulating oval cell proliferation and differentiation.

betaTrCP- and Rsk1/2-mediated degradation of BimEL inhibits apoptosis.

The BimEL tumor suppressor is a potent proapoptotic BH3-only protein. We found that, in response to survival signals, BimEL was rapidly phosphorylated on three serine residues in a conserved degron, facilitating binding and degradation via the F box protein betaTrCP. Phosphorylation of the BimEL degron was executed by Rsk1/2 and promoted by the Erk1/2-mediated phosphorylation of BimEL on Ser69. Compared to wild-type BimEL, a BimEL phosphorylation mutant unable to bind betaTrCP was stabilized and consequently potent at inducing apoptosis by the intrinsic mitochondrial pathway. Moreover, although non-small cell lung cancer (NSCLC) cells often become resistant to gefitinib (a clinically relevant tyrosine kinase inhibitor that induces apoptosis through BimEL), silencing of either betaTrCP or Rsk1/2 resulted in BimEL-mediated apoptosis of both gefitinib-sensitive and gefitinib-insensitive NSCLC cells. Our findings reveal that betaTrCP promotes cell survival in cooperation with the ERK-RSK pathway by targeting BimEL for degradation.

Inhibition of Plasmepsin V Activity Demonstrates Its Essential Role in Protein Export, PfEMP1 Display, and Survival of Malaria Parasites

A small molecule inhibitor of the malarial protease Plasmepsin V impairs protein export and cellular remodeling, reducing parasite survival in human erythrocytes.

Quinazoline Sulfonamides as Dual Binders of the Proteins B-Cell Lymphoma 2 and B-Cell Lymphoma Extra Long with Potent Proapoptotic Cell-Based Activity

ABT-737 and ABT-263 are potent inhibitors of the BH3 antiapoptotic proteins, Bcl-x(L) and Bcl-2. This class of putative anticancer agents invariantly contains an acylsulfonamide core. We have designed and synthesized a series of novel quinazoline-based inhibitors of Bcl-2 and Bcl-x(L) that contain a heterocyclic alternative to the acylsulfonamide. These compounds exhibit submicromolar, mechanism-based activity in human small-cell lung carcinoma cell lines in the presence of 10% human serum. This comprises the first successful demonstration of a quinazoline sulfonamide core serving as an effective benzoylsulfonamide bioisostere. Additionally, these novel quinazolines comprise only the second known class of Bcl-2 family protein inhibitors to induce mechanism-based cell death.

Upregulation of lymphotoxin β expression in liver progenitor (oval) cells in chronic hepatitis C

Background: Bipotent liver progenitor (oval) cells with the ability to differentiate into hepatocytes and biliary epithelium have recently been identified in human subjects with hepatitis C. Animal studies suggest that members of the tumour necrosis factor family, including lymphotoxin β (LT-β), regulate oval cell proliferation in liver disease, but its role in human liver disease is unclear. Aims: This study seeks to establish a role for LT-β in hepatitis C related liver injury and to provide evidence that its increased expression is related to the presence of oval cells. Methods: Liver biopsy specimens were obtained from patients with chronic hepatitis C virus (HCV) infection (n=20). Control liver samples (n=5) were obtained from liver resection or transplant surgery. LT-β expression in liver biopsy specimens was studied using quantitative real time polymerase chain reaction and immunohistochemistry. Results: LT-β mRNA levels were similar in control and HCV liver in the absence of fibrosis. In subjects with portal fibrosis, LT-β mRNA levels were elevated 2.2-fold over control liver levels (p=0.04). In subjects with bridging fibrosis, LT-β mRNA levels increased 4.4-fold over control liver levels (p=0.02). LT-β mRNA levels in subjects with established cirrhosis were increased 3.3-fold compared with controls and 2.6-fold compared with mild liver damage (p=0.02). Immunohistochemical analysis established that LT-β was expressed by oval cells, inflammatory cells, and small portal hepatocytes. Conclusions: In chronic HCV infection, LT-β expression is observed in multiple hepatic cell types, including oval cells. LT-β expression is significantly increased when fibrosis or cirrhosis is present, suggesting a role for LT-β in the pathogenesis of chronic hepatitis C and a possible role in oval cell mediated liver regeneration.

Discovery of potent and selective benzothiazole hydrazone inhibitors of Bcl-XL.

Developing potent molecules that inhibit Bcl-2 family mediated apoptosis affords opportunities to treat cancers via reactivation of the cell death machinery. We describe the hit-to-lead development of selective Bcl-XL inhibitors originating from a high-throughput screening campaign. Small structural changes to the hit compound increased binding affinity more than 300-fold (to IC50 < 20 nM). This molecular series exhibits drug-like characteristics, low molecular weights (Mw < 450), and unprecedented selectivity for Bcl-XL. Surface plasmon resonance experiments afford strong evidence of binding affinity within the hydrophobic groove of Bcl-XL. Biological experiments using engineered Mcl-1 deficient mouse embryonic fibroblasts (MEFs, reliant only on Bcl-XL for survival) and Bax/Bak deficient MEFs (insensitive to selective activation of Bcl-2-driven apoptosis) support a mechanism-based induction of apoptosis. This manuscript describes the first series of selective small-molecule inhibitors of Bcl-XL and provides promising leads for the development of efficacious therapeutics against solid tumors and chemoresistant cancer cell lines.

Structural basis for plasmepsin V inhibition that blocks export of malaria proteins to human erythrocytes.

Plasmepsin V, an essential aspartyl protease of malaria parasites, has a key role in the export of effector proteins to parasite-infected erythrocytes. Consequently, it is an important drug target for the two most virulent malaria parasites of humans, Plasmodium falciparum and Plasmodium vivax. We developed a potent inhibitor of plasmepsin V, called WEHI-842, which directly mimics the Plasmodium export element (PEXEL). WEHI-842 inhibits recombinant plasmepsin V with a half-maximal inhibitory concentration of 0.2 nM, efficiently blocks protein export and inhibits parasite growth. We obtained the structure of P. vivax plasmepsin V in complex with WEHI-842 to 2.4-Å resolution, which provides an explanation for the strict requirements for substrate and inhibitor binding. The structure characterizes both a plant-like fold and a malaria-specific helix-turn-helix motif that are likely to be important in cleavage of effector substrates for export.