Career Enhancement Program Abstracts

The Career Enhancement Program supports several ongoing research projects. These projects currently include:

  • Genetic and Epigenetic Drivers of HCC in the Setting of NAFLD
  • PD-L1 Positive Hepatic Macrophages in Cholangiocarcinoma
  • Gene Therapy-Induced Alloreactivity for Treatment of Hepatocellular Carcinoma

Genetic and Epigenetic Drivers of HCC in the Setting of NAFLD

The principal investigator of this project is Samuel O. Antwi, Ph.D., a researcher at Mayo Clinic in Jacksonville, Florida, who focuses on pancreas cancer and liver cancer.

The goal of this career enhancement application is to promote the career development of Dr. Antwi in translational hepatobiliary cancer research. Dr. Antwi's proposal seeks to investigate the applications of host genetic differences and epigenetic alterations for early detection of hepatocellular cancer (HCC) in the setting of nonalcoholic fatty liver disease (NAFLD). During the one-year award period, he will take formal coursework in epigenetics, participate in a scientific workshop focused on epigenetics, and conduct translational research in early detection of hepatocellular cancer.

Several biological markers have been explored for early detection of hepatocellular cancer, but there is currently no reliable early detection marker, apart from alpha-fetoprotein (AFP), which doesn't have optimal sensitivity or specificity. Emerging data suggest that host genetic differences (risk variants or mutations) and epigenetic alterations (such as DNA methylation) may be useful for early detection of hepatocellular cancer. Inherited genetic differences can be used for genetic risk stratification and targeted screening of people at high risk, while aberrant DNA methylation can be useful for early detection based on the principle that changes in circulating DNA that are caused by the presence of an asymptomatic hepatocellular cancer tumor can be used to detect the tumor early for resection.

Hepatocellular cancer is often fatal, and NAFLD-related hepatocellular cancer cases are expected to increase in the United States. About one-third of patients diagnosed in the U.S. don't have a history of viral hepatitis or alcohol abuse but often have features of nonalcoholic fatty liver disease. Because hepatitis B and C are associated with both nonalcoholic fatty liver disease and hepatocellular cancer, it's critical to assess NAFLD-related hepatocellular cancer in the absence of these viral infections (nonviral and nonalcoholic hepatocellular cancer) to optimally reduce confounding.

Genetic and epigenetic differences in the one-carbon metabolism pathway have been strongly implicated in susceptibility to hepatocellular cancer in the setting of nonalcoholic fatty liver disease. In animals, loss-of-function mutations in some one-carbon metabolism genes (BHMT) lead to NAFLD development and progression to hepatocellular adenomas or hepatocellular cancer in 64% of murine models with these mutations.

In humans, genetic variations in certain one-carbon metabolism genes (PNPLA3) have been associated with an increased risk of NAFLD and hepatocellular cancer in a few small studies of candidate genes. These initial observations underscore the need for a comprehensive approach to study the role of one-carbon genes in hepatocellular cancer development in patients with NAFLD.

Functionally, the one-carbon metabolism pathway mediates DNA methylation through the transfer of methyl groups to the fifth carbon of cytosine in DNA to form 5-methylcytosine (5mC, that is, DNA methylation). However, 5mC is not stable; it is converted to 5-hydroxymethylcytosine (5hmC) by TET enzymes. Now 5hmC is recognized as an epigenetic marker that is distinct from 5mC. Our preliminary data, along with data from other researchers, suggest that 5mC and 5hmC play plausible roles in hepatocellular cancer development and could serve as markers for use independently or integratively with other tumor markers or patient characteristics for early detection of HCC.

This career enhancement application is focused on the detection of one-carbon genetic risk variants or mutations that predispose to hepatocellular cancer and the applications of 5mC and 5hmC as early detection markers for hepatocellular cancer. Ultimately, this proposal seeks to develop an integrative model that simultaneously incorporates genetic risk variants and mutations, aberrant DNA methylation, AFP, and patient characteristics for early detection of hepatocellular cancer.

PD-L1 Positive Hepatic Macrophages in Cholangiocarcinoma

The principal investigator of this project is Sumera I. Ilyas, M.B.B.S., an assistant professor of immunology and of medicine at Mayo Clinic College of Medicine and Science in Rochester, Minnesota.

The liver has a unique immune microenvironment with abundant resident macrophages called Kupffer cells, which contribute to the liver's ability to induce immune tolerance. This tolerogenic capability of liver macrophages is especially important in tumor biology because it may promote tumor progression in the liver by mediating cytotoxic T lymphocyte (CTL) exhaustion, a state of reduced T cell effector function. CTLs express multiple inhibitory receptors, such as programmed cell death 1 (PD-1), and lose their effector functions when these receptors are stimulated by ligands, such as programmed cell death 1 ligand 1 (PD-L1).

Cholangiocarcinoma is a prototypic model to explore the liver's immunologic response to tumors given its rich multicellular, stromal interactions. Although the role of PD-L1 in tumor biology is under intense investigation given the therapeutic success of checkpoint blockade, the majority of the existing body of work has focused on PD-L1 expression on cancer cells and its role in CTL exhaustion.

However, our preliminary data unexpectedly demonstrated that PD-L1 knockout animals have a significant reduction in tumor burden despite expression of PD-L1 by implanted cancer cells. This suggests that PD-L1 expression on host cells in the tumor microenvironment (macrophages) is more potent in facilitating CTL exhaustion than PD-L1 expression on cancer cells.

To this end, we have generated considerable preliminary data. First, we established a unique syngeneic, orthotopic murine transplantation model to study cholangiocarcinoma progression to permit examination of the immune response. Second, we identified PD-L1 expression by hepatic tumor-associated macrophages (TAMs) in cholangiocarcinoma; interestingly, a subset of these macrophages also expresses Clec4f, a marker specific for murine Kupffer cells. Moreover, we demonstrated increased expression of PD-L1 on TAMs compared with tumor cells in resected human cholangiocarcinoma specimens.

Finally, our data indicate that macrophage-targeted therapy with colony-stimulating factor 1 receptor (CSF1R) inhibition or checkpoint inhibition with PD-L1 blockade is tumor suppressive in our model of tumor progression.

Gene Therapy-Induced Alloreactivity for Treatment of Hepatocellular Carcinoma

The principal investigator of this project is Bolni (Marius) M. Nagalo, Ph.D., a cancer researcher at Mayo Clinic in Phoenix, Arizona.

Targeted immunotherapies have shown great promise across a wide spectrum of cancers, including hepatocellular carcinoma (HCC). However, many tumors lack expression of neoantigens able to elicit tumor-specific-cytotoxic T lymphocyte responses. In addition, the presence of immunosuppressive and anergic mechanisms in cancer represent a limiting factor to the success of immunotherapy. As the mechanisms of cancer immunotherapy resistance are being elucidated, strategies that aim to increase tumor immunogenicity and overcome immune tolerance represent high translational initiatives and could improve clinical outcomes for patients.

In ongoing experiments, we have found that using gene therapy viral vector nonself antigens can be exposed on the surface of tumor cells to induce a specific immune response capable of destroying cancer cells. These data suggest that the immune system's inherent ability to identify and reject transplanted organ or tissue based on host-donor alloantigen incompatibility can be redirected into a treatment for advanced hepatocellular carcinoma.

We therefore hypothesize that tumor-specific viral-mediated delivery of human truncated, nonsignaling alloantigens such as human leukocyte antigens (HLAs) in HCC cells using transcriptional regulation of an ultraspecific, high-efficiency tumor promoter will not only elicit strong immune-mediated cancer cell destruction, but will also increase the therapeutic index of treatment by significantly reducing viral titers required to achieve transgene expression at therapeutic levels.

To achieve this goal, we propose to use a set of adeno-associated virus (AAV) and single-cycle adenovirus (SC-Ad) to deliver an HLA class I alloantigen to tumor cells using mouse telomerase reverse transcriptase (mTERT) promoters engineered to harbor single nucleotide polymorphism (SNP) mutations that significantly enhance transcriptional activity by greater than 30fold in hepatocellular carcinoma cells compared with normal hepatocytes.