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Princess Maxima Center for Pediatric Oncology

2023

CRISPR-engineering of human liver organoids to study fibrolamellar carcinoma

Goals: Model development and understanding the requirements for FLC tumor formation

Principal Investigators: Benedetta Artegiani, PhD; and Delilah Hendriks, PhD

Grant length: Two years

Study overview: In comparison with more common cancers, research models of FLC have been extremely limited. Drs. Artegiani and Hendriks have addressed this problem by creating “synthetic” models of FLC, generating cultures of normal cells as organoids (3D mini-organs). Specifically, they developed both liver cell organoids and techniques to genetically modify these organoids using CRISPR technology by which specific genetic changes can be introduced into normal cells.

In previous work, these investigators “knocked-in” the DNAJB1-PRKACA fusion gene into organoids. They found that this altered the normal hepatic cells to resemble FLC cancer cells in some respects, especially by changing patterns of gene expression in ways consistent with actual tumor cells. However, these gene-modified organoids did not fully transform into malignant cancer cells. While the DNAJB1-PRKACA fusion is clearly known to be the driver of FLC, and essential for the growth and survival of FLC tumor cells, there have been other suggestions that this fusion gene alone if not enough for the complete transformation to a robust FLC-like cancer. Similar observations were reported previously in studies of knock-in of the fusion oncogene into livers of young mice; in this case, at least one additional genetic change was required to yield aggressive tumor growth.

In this current study, the applicants propose to extend their initial findings in several ways. A major goal is to determine whether the introduction of additional mutations in cells containing the DNAJB1-PRKACA fusion can generate biological features more closely resembling fully transformed FLC cancer cells. The team will also determine whether introduction of the fusion gene and other genetic changes into normal ductal organoids gives more complete FLC-like transformation than in liver organoids. Finally, they propose to study influences of the tumor microenvironment on cells with the characteristic mutations of FLC by establishing co-cultures of the genetically modified cells with “mini-liver” cultures containing multiple cell types.

Successful accomplishment of the goals of this study should:

  • reveal drivers of FLC that act in coordination with the fusion gene, which could lead to the discovery and design of new therapeutic approaches.
  • build an understanding of the cause of observed heterogeneity among FLC patients
  • create new disease models useful for the testing and development of FLC therapeutics.

2021

CRISPR-engineering of human liver organoids to study fibrolamellar carcinoma

Goal: Model development

Principal Investigator: Benedetta Artegiani, PhD

Grant length: Two years

Study Overview: Research into FLC is complicated because of the limited experimental tools that can be used to study this rare cancer. While tests in laboratory animals such as mice are useful tools, they do not reflect the human disease due to inherent differences between mice and humans. Consequently, experimental models that can provide insights into human disease development and progression are highly sought after.

The overall goal of this research project was to build new models using human cells to study FLC. The researchers made use of lab-grown three-dimensional human mini-livers, so-called “organoids”. These organoids were grown from the two most important cells in the liver: the ductal cells and the hepatocytes. The researchers then precisely modified the DNA of the organoids using CRISPRCas9 technology, also called molecular scissors, to mimic the changes in the DNA that have been found in FLC.

By constructing and using such FLC models, the researchers hope to increase our current understanding of the origin of FLC tumors, and eventually create useful model systems for performing drug screens to identify new therapies.

Key Findings: In this study, the investigators successfully “knocked-in” FLC’s characteristic DNAJB1-PRKACA fusion gene into organoids. They found that this altered the normal hepatic cells to resemble FLC cancer cells in some respects, especially by changing patterns of gene expression in ways consistent with actual tumor cells. However, these gene-modified organoids did not fully transform into malignant cancer cells. While the DNAJB1-PRKACA fusion is clearly known to be the driver of FLC, and essential for the growth and survival of FLC tumor cells, in this study at least one additional genetic change was required to yield aggressive tumor growth.

Artegiani and Hendriks found that using CRISPR to inactivate two additional genes in liver cell organoids generated fully transformed cancer cells – the inactivation of both copies of BAP1, known as a tumor suppressor gene, and both copies of PRKAR2A. The latter codes for the regulatory subunit of protein kinase A (PKA). Its loss leads to hyperactivity of PKA, the same enzyme that is dysregulated by the DNAJB1-PRKACA mutation. PRKACA codes for the active catalytic subunit of PKA, which normally is turned off by the regulatory (R) subunit and activated when the “second messenger” cyclic-AMP (cAMP) binds to R. In the absence of R subunits, the catalytic (C) subunit is active even without cAMP. The rare genetic loss of PRKAR2A (the Carney complex) has been associated with the development of the exceptional cases of FLC (approximately 1%) that do not have the classic fusion gene. An important feature of the organoids altered by inactivating BAP1 and PRKAR2A is that they more closely resemble primary FLC tumor samples and have properties in common with primitive progenitor cells found in biliary ducts.

The researchers concluded that although mutations in the PKA genes are crucial to the formation of FLC, they may not completely explain the disease development and progression. Their findings were published In May 2023 in Nature Communications. Click here to read and download the published journal article.

After the conclusion of this study, FLC funded a follow-on grant to determine whether the introduction of additional mutations in cells containing the DNAJB1-PRKACA fusion can generate biological features more closely resembling fully transformed FLC cancer cells. 

Benedetta Artegiani’s and Delilah Hendriks’ lab activites were also described in a general press articles in The Scientist in March 2023.