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Cornell University

2025

Spatially resolving the tumor microenvironment of fibrolamellar carcinoma

Goal: Advance understanding of FLC’s unique tumor microenvironment

Principal Investigator: Praveen Sethupathy, PhD

Grant length: Two years

Study overview: Through this grant, Praveen Sethupathy, PhD, of Cornell University plans to outline the “spatial transcriptomics” of FLC. Dr. Sethupathy’s lab has been a leader in functional genomic studies, especially for liver and gastro-intestinal cancers. His group has focused on understanding the drivers of FLC to identify new potential therapies, and has conducted several studies of genome organization, gene and protein expression and tumor metabolism in FLC cells.

This new study builds upon years of work the Sethupathy lab has already conducted in FLC transcriptomics – the analysis of all the RNA molecules produced in FLC tissue at a given time. In simple terms, transcriptomics identifies which genes are expressed in a cell and are being transformed into functional products like proteins. Transcriptomics provides a snapshot of gene activity and therefore bridges the gap between the genome (what can happen in a cell) and the proteome (what does happen – what proteins are produced). Studying transcriptomics in FLC can therefore provide valuable insights into the molecular mechanisms underlying tumor development, progression, and treatment resistance.

Most recently, the team completed an effort leveraging state-of-the-art genomic technology to understand the gene activity of FLC at a “single-cell” level. While the “single-cell” effort provided the highest-resolution molecular profile of FLC completed to date, its limitation was that the spatial location of different cell types was not preserved.  

This “spatial transcriptomics” effort represents the next step in the continuum of work from the Sethupathy group – mapping RNA expression to specific locations of cells within tissues. Since biological functions depend on cell organization, understanding that spatial context is critical. This analysis should be especially useful to improving our understanding of FLC’s tumor microenvironment (TME), which is critically important to many ongoing research efforts. It could also help identify cell-to-cell communication pathways in FLC that could be exploited as drug targets.

2019

Retinoic acid-induced loss of DNAJB1-PRKACA fusion protein expression

Goal: Investigate the potential of retinoic acid therapy

Principal Investigators: Andrew Yen, PhD and Praveen Sethupathy, PhD

Grant length: One year

Study overview: FLC is driven by the DNAJ-PKAc fusion protein. A potential therapeutic strategy would be to induce loss of this key driver protein. One approach to substantially alter gene expression in cancer cells is differentiation induction therapy, which causes malignant cells to acquire more mature, specialized characteristics and to stop proliferating. The most successful differentiation therapy agent in current use is retinoic acid (RA), which has been the standard of care for acute promyelocytic leukemia (APL). RA, a metabolite of Vitamin A, induces APL cells to convert from a proliferating malignant state resembling immature white blood cells to a non-transformed, arrested state resembling the corresponding normal, mature white blood cells. Preliminary observations in a model cell line engineered to stably express DNAJ-PKAc showed that RA causes loss of the fusion protein.

This suggests the possibility that retinoic acid could have therapeutic activity against FLC by causing loss of the transforming protein for this tumor, thereby relieving the hepatic cells of the tumor phenotype. The study exploited the observation in this experimental model and extended it to primary cultured FLC cells. The project goals were to:

  1. Determine if retinoic acid causes loss of the fusion protein in FLC cells, and
  2. Characterize the molecular signature and cellular attributes of the retinoic acid-induced FLC cell response.

If successful this effort will:

  1. Demonstrate that retinoic acid, a drug already approved and used in leukemia therapy, has an off-label application for FLC, and
  2. Identify candidates to target for more sophisticated combination therapy, an emerging therapeutic modality that is proving effective in retinoic acid based therapy against other tumors.

The basic rationale is that if a drug relieves the FLC cells of the tumor causing protein, then the tumor phenotype would be relieved.

2022

Multicenter consortium to define the single-cell activity landscape of fibrolamellar carcinoma

Goals: Advance immunotherapy for FLC by defining promising immunological targets that can be translated into effective cell-based immunotherapies

Principal Investigators: Praveen Sethupathy, PhD (Cornell University); Mark Yarchoan, MD (Johns Hopkins University); Paul G. Thomas, PhD (St. Jude Children’s Research Hospital)

Grant length: Two years

Study overview: Like tumors of other cancer types, the microenvironment of FLC tumors is highly complex, comprising many different cell types. It is now well-established from investigation of other cancer types (such as lung, breast, and pancreatic cancer) that cross-talk among these different cell types can promote tumor development, growth, and spread. A recent study in the Sethupathy lab identified critical regions of the genome that are uniquely activated in FLC. These regions offer clues about the genes that might be most critical for the development of FLC. However, an important limitation of this work is that it was performed on bulk FLC tissue, which does not resolve different cell types, and instead treats tumor tissue as one whole unit. This means that the specific cell types in which these FLC genes are active is not yet known. This represents a major knowledge gap. Identification of the specific cell types in which FLC genes are active would then allow more precise study of the functions of these genes in FLC, and facilitate the development of more effective targeted therapeutics.

To help bridge this knowledge gap, the Sethupathy (Cornell), Yarchoan (Johns Hopkins), and Thomas (St. Jude Children’s) labs will participate in a collaborative research consortium to develop an FLC tumor “atlas”. They will leverage state-of-the-art genome-scale technologies to provide unprecedented resolution of the cellular and molecular landscape of FLC. This consortium brings together three groups with longstanding interests and experience in FLC research, as well as specific expertise in genomics and gene regulation (Sethupathy), clinical oncology (Yarchoan), and immunology (Thomas and Yarchoan).

2020

Identifying therapeutic vulnerabilities in fibrolamellar carcinoma

Goal: Investigate the impact on FLC growth and survival of inhibiting two specific oncogenes identified in previous works

Principal Investigator: Praveen Sethupathy, PhD

Grant length: Three years

Study overview: Based on previous epigenomic, metabolomic, and microRNA profiling, as well as initial drug studies, the study team has developed two new exciting hypotheses about therapeutic vulnerabilities in FLC. First, they hypothesize that inhibition of two candidate FLC oncogenes, CA12 or SLC16A14, independently and/or in conjunction with FDA-approved drugs, will dramatically reduce FLC cell viability, proliferation, and invasive capacity. Second, they hypothesize that the increase in LDHB promotes glycolysis and FLC tumor cell survival, which can be reversed by miR-375 mimics. In this project, the team proposes to test these hypotheses in several different disease models of FLC. The findings from the proposed studies could potentially lay the foundation for completely novel, effective strategies for molecular therapy.

2017

Micro RNAs and long non-coding RNAs role in fibrolamellar and evaluation of RNA-based therapeutics

Goal: Investigate the role of microRNAs and long non-coding RNAs in fibrolamellar carcinoma and evaluate RNA-based therapeutics

Principal Investigator: Praveen Sethupathy, PhD

Grant length: Two years

Study overview: This study aimed to leverage genome-scale approaches to discover the molecular factors most critical to FLC tumor formation, metastasis, and drug resistance. MicroRNAs and long non-coding RNAs are RNA species that do not contain instructions for protein formation yet are vital to numerous biological processes including tumor formation.

Specific study efforts included:

  • Identifying microRNAs and long non-coding RNAs that facilitate FLC tumor formation/invasion
  • Evaluating the potential of RNA-based therapeutics to inhibit their activity
  • Integration of these results with multiple large-scale genomic and metabolomic datasets to identify critical druggable pathways.

Key Findings: This investigation identified miR-375 as the most dysregulated miRNA in primary FLC tumors based on an analysis of the small RNA sequencing data from The Cancer Genome Atlas. It also demonstrated that miR-375 expression was decreased significantly in a FLC patient-derived xenograft model compared to 4 different cell populations of the liver. In addition, it showed that the introduction of DNAJB1-PRKACA in mice (by clustered regularly interspaced short palindromic repeats/CRISPR-associated protein 9 engineering and transposon-mediated somatic gene transfer) was sufficient to induce significant loss of miR-375 expression. Furthermore, the study found that overexpression of miR-375 in FLC cells inhibited several well characterized growth regulatory pathways and suppressed cell proliferation and migration.

These results suggest that miR-375 could be a good candidate for therapeutic investigations. The study team also identified miR-10b, another aberrantly elevated pro-proliferation miRNA in FLC, which is responsive to DNAJB1-PRKACA over-expression, but only in human cell models (HepG2, HEK293 cell lines) and not in mouse models (AML-12 cell line, TIB-75 cell lines), thus highlighting the importance of using human cell lines for FLC studies. Inhibition of miR-10b led to reduced proliferation in human cell lines, however the effect was modest. The team concluded that miR10-b may be a good candidate for some type of combination therapy.

Results of this study were published by Cellular and Molecular Gastroenterology and Hepatology in February 2019 and the Journal of Clinical Investigation in April 2022. The full texts of each of those articles can be accessed via the following links:

An article in the Cornell Chronicle available here further describes the February 2019 publication.

Implications: The study’s findings open a potentially new molecular therapeutic approach to FLC. Further studies are necessary to determine whether miR-375 has additional important targets in FLC and exactly how DNAJB1-PRKACA suppresses miR-375 expression.