Rockefeller University

2011

Analysis of the immunology, imaging and cell biology of fibrolamellar – continued

Goal: Support series of projects exploring the role of antibody involvement in FLC tumors

Principal Investigator: Sandy Simon, PhD

Grant length: Two years

Study overview: This effort continued the encouraging early work begun on mice to see if researchers could take advantage of the immune system to identify potential ways to detect and eventually treat fibrolamellar. In particular, the team focused on identifying whether accumulation of antibodies within malignant tissue could provide a way to identify and track tumor cells as they grow and spread.

Specific activities included:

  • Analyzing cancer tumors in mice, using slices of tissue. The goal of this effort was to characterize the accumulation of antibodies within tumors using tissue slices resected from a small animal models of different types of cancer.
  • Analyzing tumors in mice, using whole animal imaging. Since a long-term goal is to use the antibody technology to detect and treat tumors in humans, this effort was focused on developing approaches that allow antibody observations to occur in living animals.
  • Developing various nanoprobes to improve the whole animal imaging. These nanoprobes are molecules used to detect and visualize specific biological processes in living organisms. They are usually labeled with a radioactive or fluorescent tag, which allows them to be detected by imaging techniques such as positron emission tomography (PET) or fluorescence microscopy.
  • Applying the findings from the mouse tumor work to human cancers, in particular FLC.

Results: The study analyzed the accumulation of a mouse’s antibodies (specifically an antibody known as IgG, or immunoglobin G) in their tumors. The models analyzed included several mouse models for liver cancer (HCC), two models for prostate cancer, two models for breast cancer and a model of skin cancer. Both transgenic models (generated by altering the genome of the mouse) and xenograft models (using tumors transplanted from one animal to another) were used.

The study clearly demonstrated that there is a very significant concentration of a patient’s (or
animal’s) antibodies across tumor types. Similar to the results in mice tumors, the study showed increased levels of a human patient’s IgG in fibrolamellar carcinoma tumors relative to the levels in the adjacent normal tissue.

Results of the antibody accumulation analysis were published in May 2014 in Nature’s Scientific Reports. The full text of the published article – “Endogenous Antibodies for Tumor Detection” – can be accessed here.

In whole animal imaging, the team examined both PET and fluorescence imaging approaches. While each alternative had different strengths and weaknesses, much work remains to identify an imaging approach that gives the maximum sensitivity, the fewest false positives and the fewest false negatives. Additional analyses, equipment improvements and probe modification efforts were identified for future investigation.

Implications: The study demonstrated that the enrichment of antibodies within malignant tissue, including FLC tissue, provides a potential means of identifying and tracking cancer cells as they mutate and diversify. Exploiting these antibodies for diagnostic and therapeutic purposes is therefore possible by using agents that bind to those antibodies.

2010

Analysis of the immunology, imaging and cell biology of fibrolamellar

Goal: Build understanding of the immunology and cell biology of FLC using mouse models of cancer

Principal Investigator: Sandy Simon, PhD

Grant length: One year

Study background: This early study was designed to determine if researchers could take advantage of the immune system to identify potential ways to detect and eventually treat fibrolamellar.

The immune system is constantly screening bodies for transformed cells. Usually it recognizes these cells as foreign and triggers a programmed pathway of cell death called apoptosis. However, even though the immune system still can recognize tumor cells, mutations in tumor cells could disable the cell death pathway, so the immune system loses the ability to kill the tumor cell.

The study team’s hypothesis was that this mechanism could potentially be leveraged in two ways:

  • To use the immune system, and in particular antibodies, to detect micrometastases in the body
  • To use the antibodies to deliver toxins directly to the tumors, instead of using general systemic chemotherapy approaches.

Results: During the initial effort, the team completed testing on:

  • A genetically induced mouse model for hepatocellular carcinoma
  • A chemically induced mouse model for hepatocellular carcinoma
  • A genetically induced mouse model for prostate cancer.
  • Four human patients samples of fibrolamellar heptocellular carcinoma.

The work was continued with a follow-on grant.

2013

High priority FLC investigations

Goal: Support series of high priority FLC investigations

Principal Investigator: Sandy Simon, PhD

Grant length: One year

Study background: In previous work (including the initiatives funded in 2010 and 2011), the study team identified an increase of a patient’s antibodies, in particular the antibody known as IgG (immunoglobin G), in fibrolamellar, sarcomas, and a number of other tumor types.

This 2013 funding agreement was originally meant to expand the previous work in three directions:

  • Further develop and refine of the analysis of the accumulation of antibodies in human tumors.
  • Develop nanoprobes that improve whole animal imaging while reducing background non-specific signals in the liver, including synthesizing new constructs, and testing them in liver slices and mice.
  • Determine if FLC antibodies purified from a patient’s own blood could target their own tumors.

Funding reallocation: Instead, based on the results of the genomic initiative, the funding provided from this grant was diverted to follow up and further elaborate on the chimeric fusion discovery, with the goal of supporting the eventual development of effective diagnostics and therapeutics based on that insight.

Results: The bulk of the FCF funding provided in this grant was used to characterize all other changes in the DNA, the RNA (transcriptome) and the proteins (proteome) in FLC tumors.

The whole genome work, based on the analysis of tumors from 10 FLC patients, determined that the only consistent mutation (seen in all tumors analyzed) was the DNAJB1-PRKACA fusion. There were few other recurrent mutations. Other mutated genes that were found in more than one patient included MUC4, GOLGA6L2, DSPP, FOXO6 and HLA-DRB1. MUC4 was the most common additional alteration, found. It was altered in four patients and has been implicated in several other types of epithelial cancers. The study concluded that FLC has a very low tumor mutational burden in comparison to other adult and pediatric cancers. It has a relatively stable genome, with a single recurrent deletion found in all patients studied, and few additional mutations. Since the only consistent mutation found was the DNAJB1-PRKACA fusion, it concluded that it is unlikely that a “second hit” or other mutations are required for tumorigenesis.

This genomic sequencing work was published in the journal Oncotarget in December 2014. The full text of that article can be accessed here.

The transcriptome work demonstrated that the DNAJB1-PRKACA fusion results in many changes in the expression of different genes. Changes in the expression of genes in FLC tumors (compared to adjacent normal tissue) were detected for over 3,500 genes.  Although some expression changes were similar to those in HCC, many were distinct, suggesting that FLC is a unique disease rather than a variant of HCC. It also demonstrated that the transcriptome of any FLC tumor is more like that of another patient’s FLC tumor than it is of the adjacent tissue in the same patient. This strongly demonstrates that FLC is a single disease. In addition, the FLC tissue showed increased expression of oncogenes associated with various cancers including pathways found in breast cancer: ErbB2 (EGF pathway), Aurora Kinase A (AURKA) and E2F3 (cell cycle), and CYP19A1 (estrogen synthesis pathway). These dysregulated genes could potentially serve as targets for treatment of the disease.

The transcriptome work was published in October 2015 in the Proceedings of the National Academy of Sciences (PNAS), a peer reviewed journal of the National Academy of Sciences (NAS). That article – “Transcriptomic characterization of fibrolamellar hepatocellular carcinoma” – can be read here.

Implications: The key insights from this research include:

  • FLC is not a genetically inherited disease. The chimeric alternation was found in the tumor but not in the adjacent tissue. It is the result of a one-time “glitch” in a patient’s DNA.
  • Fibrolamellar is a single disease, not a collection of different diseases. The patterns of expression of genes in FLC tumors are consistent from patient to patient.
  • FLC is distinct from HCC, a finding that should be considered in planning treatment approaches.
  • FLC tumors over express several oncogenes (genes that have the potential to cause cancer) already associated with other cancers, which could lead to the identification of new targets for treatment.

In addition, the identification of the role of the Aurora Kinase A pathway in FLC by this study provided justification for a FLC-specific clinical trial of ENMD-2076, a small-molecule inhibitor of Aurora and angiogenic kinases. That multi-center trial, sponsored by CASI Pharmaceuticals with the support of Ghassan Abou-Alfa, MD of MSKCC and other investigators, was conducted from 2016 to 2018. Unfortunately, while ENMD‐2076 showed a favorable toxicity profile in FLC patients, the clinical trial results did not support further evaluation of ENMD‐2076 as single agent to treat FLC. The full results of that study were reported in The Oncologist in 2020.

2013

Creation of Fibrolamellar Tissue Repository at The Rockefeller University

Goal: Support costs related to establishment of new FLC biospecimen storage facility

Principal Investigator: Sandy Simon, PhD

Grant length: One year

Study background: Access to tumor tissue is critical to help cancer researchers understand what drives a disease and how the cancer responds to treatments. Because fibrolamellar is so rare, there has long been a shortage of FLC tissue available for research.

This funding request was to support the establishment of a new tissue repository for fibrolamellar carcinoma at The Rockefeller University, in new lab space built specifically for this purpose. The lab needed a new secure freezer to store the FLC samples and a secure computer to house de-identified patient data, which could then be correlated with the tissue samples to benefit research on outcomes and recurrence.

Results: The equipment was purchased and the new tissue repository successfully developed. These samples and data will be made available to collaborators and labs that are researching fibrolamellar, so that progress can be made without sequestration of resources or overlapping efforts.

2013

Genomic initiative on Fibrolamellar Hepatocellular Carcinoma

Goal: Identify genetic mutations in FLC

Principal Investigator: Sandy Simon, PhD

Grant length: One year

Study background: The goal of this project was to identify genetic mutations present in FLC. Identification of mutations common in FLC tumors is critical to enhancing the understanding of the underlying biology of this tumor and allow for development of novel therapeutics.

Specifically, the study planned to:

  • Sequence the whole genome (the DNA) of ten fibrolamellar tumors and, for the same ten tumors, the DNA of
    the adjacent normal tissue.
  • For both the tumor and the adjacent normal tissue, sequence the messages (mRNA) that encode protein – in a technique referred to as RNAseq.
  • For both the tumor and the adjacent normal tissue, sequence the small RNA molecules that do not encode for proteins, but that are involved in regulation of gene expression – and which have been shown to be critical in many cancers.

Specific aims of the study included:

  • Comparing tumor and adjacent tissue to reveal alterations that are specific to the transformation of a fibrolamellar tumor cell.
  • Comparing fibrolamellar tumors with classic viral- or cirrhosis-related HCC to reveal to what extent these are completely independent diseases, or if they are somehow related.
  • Identifying any common alterations in the fibrolamellar tumors that may help to either identify markers to look for in patients to help follow or localize the disease and suggest therapeutic interventions for either targeting the tumor cells or potentially turning off the alteration driving the tumor.

The sequencing work leveraged the capabilities of the New York Genome Center.

Results: This research determined that a unique genetic mutation, a chimeric gene fusion, was common across all FLC tissue samples studied. Specifically, the study:

  • identified (from whole-genome sequencing) a 400 kb heterozygous deletion on chromosome 19 in ten out of ten FLC patients tested, and
  • detected (via whole-transcriptome analysis) a chimeric DNAJB1- PRKACA RNA transcript in 12 of 12 patients tested.

The identified fusion transcript was shown to encode a chimeric DNAJB1-PRKACA protein that couples a segment of the heat shock protein, DNAJB1, with the catalytic domain of protein kinase A (PKA) and exhibits full retention of PKA activity. Neither the genomic deletion, the chimeric transcript, nor the chimera protein were present in any matched normal liver samples tested.

This research was conducted at the Tucker Davis Research Facility at Rockefeller University, led by Dr. Sandy Simon. His daughter Elana, a fibrolamellar patient, was a lead researcher. The results were published in the preeminent medical journal Science and reported in The Wall Street Journal, US News and World Report, AP, The Today Show, NBC Nightly News, and presented to President Obama.

Click here to read or download the resulting article “Detection of a Recurrent DNAJB1-PRKACA Chimeric Transcript in Fibrolamellar Hepatocellular Carcinoma”, published in Science in February 2014.

Implications: This research ultimately resulted in the game-changing discovery that a unique genetic mutation, a chimeric gene, was common to all fibrolamellar tissues studied. While the role of the DNAJB1-PRKACA chimera in the pathogenesis of FLC was not confirmed until later studies, this effort raised the possibility that it contributes to the pathogenesis of the tumor and may represent an important therapeutic target.

Shortly after the publication of the study, Sandy Simon commented on the implications of the research:

“Thanks to the support of the Fibrolamellar Cancer Foundation, we have identified a consistent alteration in the DNA of the fibrolamellar tumor – an alteration that was found in the tumors of every patient tested. The next necessary steps are to develop both diagnostics and therapeutics. The results demonstrate that there is a single genetic alteration which is found only in the tumor. It is not genetically inherited. Thus, if it is detected early enough and fully cut out, the cancer may not recur. “

Sandy Simon, PhD

2016

Developing therapeutics for fibrolamellar hepatocellular carcinoma

Goal: Develop new therapeutics for FLC

Principal Investigator: Sandy Simon, PhD

Investigator Collaboration: Barbara Lyons, PhD (New Mexico State University)

Grant length: Two years

Study overview: The goal of this work was to identify potential therapeutic strategies to target FLC. The team identified three independent approaches to analyze based on their laboratory’s work showing that a single common DNA alteration is found in all fibrolamellar tumors leading to the DNAJB1-PRKACA fusion, and that this chimera is sufficient to cause fibrolamellar. The three sub-projects covered included:

  1. A high-throughput screen for molecules that directly block the chimera
  2. A screen to identify molecules that are directly phosphorylated by the chimera.
  3. An analysis of the structural dynamics of the chimera using a molecular dynamics simulation to identify sites on the chimera that would be appropriate for targeting therapeutics.

Key Findings: The study team purified the native and the chimera protein and observed the same level of kinase activity for both the proteins, which indicates that pan-kinase inhibitors may not work for FLC. Subsequently, they screened a library of approved bioactive compounds and identified a small molecule with some efficacy against the disease.

During the funding period, most progress was made against the third sub-project – characterizing the molecular dynamics of the chimera. Using molecular dynamics simulations and NMR, they found an ensemble of conformations of native and chimeric kinase. Because the conserved core of the wild-type enzyme and the chimeric fusion show little structural differences and the canonical function is not affected by the J-domain appendix, the presence of alternate conformations may create a way to target the chimera selectively. This opens up the possibility to develop novel small molecule inhibitors directed at the region of the fusion.

Results of the conformational analysis were published in Nature’s Scientific Reports, in January 2018. The text of that article – “Conformational Landscape of the PRKACA-DNAJB1 Chimeric Kinase, the Driver for Fibrolamellar Hepatocellular Carcinoma” – can be read here.