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Rockefeller University

Current Projects

Sanford M. Simon, Ph.D.

Günter Blobel Professor

Many of our projects are chosen based on finding biological problems that can be studied in sufficient detail that we can do detailed biophysical modeling that can make predictions that we can test experimentally. Other projects involve developing new techniques that we believe will enable us to ask previously difficult to address questions. Some examples of each:

Fibrolamellar hepatocellular carcinoma (FLC)

We are bringing a multidisciplinary approach to the study of a specific cancer, fibrolamellar hepatocellular carcinoma (FLC), a usually lethal liver cancer that affects adolescents and young adults Why this choice? It was based on the hopes:

  • A well-defined cancer would be a homogeneous population, not a mixture of cancers.
  • A cancer in adolescents would have a relatively clean background – the patients have not had decades of life to accumulate unrelated variations in the genome.
  • A cancer in adolescents would be relatively “young” – the tumor has not had many decades to accumulate additional mutations.

If these hopes were correct, it would facilitate examining causes, progression, as well as developing diagnostics and therapeutics. This was part of the motivation behind the development of the Precision Medicine Initiative [we were honored to be invited by President Obama to introduce him, and the Precision Medicine initiative, at the launch at the White House and later to introduce Joe Biden and Pope Francis at the international launch of the Biden Cancer Initiative at The Vatican].

Validation of hopes:
We have shown that all fibrolamellar patients have a clean background with no common structural variants (inversions, amplifications, insertions/deletions) (Darcy, 2015) and only one common deletion: A loss of 400kb in one copy of chromosome 19 (Honeyman, 2014). This deletion results in the fusion gene of the first exon of DNAJB1, a heat shock protein co-factor, and the bulk of PRKACA, the catalytic subunit of protein kinase A. We have shown that the changes found in tumors, recurrences, metastases, in the coding transcriptome, the non-coding transcriptome, the proteome, the phosphoproteome, are almost indistinguishable between patients. We have shown that expression of this fusion protein, a chimera of DNAJB1-PRKACA is sufficient to produce the tumor in the livers of mice.

Working with patients:
We are actively engaged with the patient community, many of whom work with us at the bench.

  • With the patients we have established the first tissue repository for fibrolamellar with tissue, blood, from over 140 patients)
  • We work with the patients in establishing the first medical registry for fibrolamellar. This is owned and run by patients and their families. Over 200 patients have answered a collection of questions we have curated, many have uploaded their medical records, test and scans.

What have we learned?
We have shown that in all fibrolamellar patients there is a dysregulation of protein kinase A (Lalazar, 2018). In hundreds of patients, we have shown that the first exon of PRKACA, the catalytic subunit of protein kinase A, is replace with the first exon of DNAJB1, a heat shock protein co-factor (Honeyman, 2014). In one patient with fibrolamellar, we have found that they are missing a regulatory subunit of protein kinase A (Graham 2018). In three other patients the first exon of PRKACA is replaced with the first exon of ATP1B1, a sodium potassium ATPase. This dysregulation of protein kinase A produces changes in the coding and the non-coding transcriptome and the proteome that are:

  • consistent from primary to metastases
  • consistent between patients (Simon, 2015)
  • distinct between fibrolamellar and other tumors (Simon, 2015)

Expression of a functional fusion oncokinase is sufficient to recapitulate this cancer:

  • Using CRISPR in mouse liver to create the deletion, and form the fusion gene, is sufficient to produce the cancer.
  • Expressing the fusion protein on its own, without any deletions, is sufficient to produce the cancer.
  • A point mutation that eliminates the activity of the kinase, can no longer transform cells.

Where are we going?
What causes fibrolamellar?

  • What leads to the break and fusion at that particular locus?
    • We found that hundreds of patients who have a break in the first intron of DNAJB1 and the first intron of PRKACA. What is the structure of the DNA at the site of the break and fusion
    • Why does the break not seen once people age?
    • Why is it almost never seen outside the liver?
  • How does formation of the fusion protein lead to tumorigenesis? We are examining:
    • The structural dynamics of the fusion protein (Cao et al., 2019; Tomasini et al., 2018)
    • The impact of the fusion protein on the ecology of kinase signaling
    • How does expression of the fusion oncokinase affect the cell biology of the liver cells? How does it:

      •    -Alter the transcriptome (Simon et al., 2015)
         -Alter the proteome (Simon et al., 2015)
         -Alter the phosphorylation landscape

    • How do the altered liver cells interact with their environment and the immune system
    • How do the structural dynamics of the fusion protein differ from the native kinase?

Can we design ways of detecting the tumor and follow the tumor load in patients?

  • We are developing these both to help detect recurrences earlier and to follow tumor load in patients in response to therapeutics

Can we develop therapeutics for fibrolamellar. Our strategies include:

  • Targeting the fusion protein.
  • Targeting the fusion transcript.
  • Drug repurposing.
  • Harnessing the immune system.

We are doing these studies in the following model systems:

  • Patient-derived xenografts (Lalazar et al., 2021)
  • Patient-derived organoids (Narayan et al., 2020; Saltsman et al., 2020)
  • Genetically engineered mice (Kastenhuber et al., 2017)
  • Cells freshly resected from patients

Below is a selection of our papers on fibrolamellar:
Cao B, Lu TW, Martinez Fiesco JA, Tomasini M, Fan L, Simon SM, Taylor SS, Zhang P. Structures of the PKA RIα Holoenzyme with the FLHCC Driver J-PKAcα or Wild-Type PKAcα. Structure. 2019 May 7;27(5):816-828.e4. doi: 10.1016/j.str.2019.03.001. Epub 2019 Mar 21. PubMed PMID: 30905674; PubMed Central PMCID: PMC6506387.

Dao Thi VL, Wu X, Belote RL, Andreo U, Takacs CN, Fernandez JP, Vale-Silva LA, Prallet S, Decker CC, Fu RM, Qu B, Uryu K, Molina H, Saeed M, Steinmann E, Urban S, Singaraja RR, Schneider WM, Simon SM, Rice CM. Stem cell-derived polarized hepatocytes. Nat Commun. 2020 Apr 3;11(1):1677. doi: 10.1038/s41467-020-15337-2.PMID: 32245952

Darcy DG, Chiaroni-Clarke R, Murphy JM, Honeyman JN, Bhanot U, LaQuaglia MP, Simon SM. The genomic landscape of fibrolamellar hepatocellular carcinoma: whole genome sequencing of ten patients. Oncotarget. 2015 Jan 20;6(2):755-70. doi: 10.18632/oncotarget.2712. PubMed PMID: 25605237; PubMed Central PMCID: PMC4359253.

Farber BA, Lalazar G, Simon EP, Hammond WJ, Requena D, Bhanot UK, La Quaglia MP, Simon SM. Non coding RNA analysis in fibrolamellar hepatocellular carcinoma. Oncotarget. 2018 Feb 13;9(12):10211-10227. doi: 10.18632/oncotarget.23325. eCollection 2018 Feb 13. PubMed PMID: 29535801; PubMed Central PMCID: PMC5828204.

Graham RP, Lackner C, Terracciano L, González-Cantú Y, Maleszewski JJ, Greipp PT, Simon SM, Torbenson MS. Fibrolamellar carcinoma in the Carney complex: PRKAR1A loss instead of the classic DNAJB1-PRKACA fusion. Hepatology. 2018 Oct;68(4):1441-1447. doi: 10.1002/hep.29719. Epub 2018 May 11. PubMed PMID: 29222914; PubMed Central PMCID: PMC6151295.

Hammond WJ, Lalazar G, Saltsman JA, Farber BA, Danzer E, Sherpa TC, Banda CD, Andolina JR, Karimi S, Brennan CW, Torbenson MS, La Quaglia MP, Simon SM. Intracranial metastasis in fibrolamellar hepatocellular carcinoma. Pediatr Blood Cancer. 2018 Apr;65(4). doi: 10.1002/pbc.26919. Epub 2017 Dec 29. PubMed PMID: 29286561; PubMed Central PMCID: PMC6028006.

Honeyman JN, Simon EP, Robine N, Chiaroni-Clarke R, Darcy DG, Lim II, Gleason CE, Murphy JM, Rosenberg BR, Teegan L, Takacs CN, Botero S, Belote R, Germer S, Emde AK, Vacic V, Bhanot U, LaQuaglia MP, Simon SM. Detection of a recurrent DNAJB1-PRKACA chimeric transcript in fibrolamellar hepatocellular carcinoma. Science. 2014 Feb 28;343(6174):1010-4. doi: 10.1126/science.1249484. PubMed PMID: 24578576; PubMed Central PMCID: PMC4286414.

Kastenhuber ER, Lalazar G, Houlihan SL, Tschaharganeh DF, Baslan T, Chen CC, Requena D, Tian S, Bosbach B, Wilkinson JE, Simon SM, Lowe SW. DNAJB1-PRKACA fusion kinase interacts with β-catenin and the liver regenerative response to drive fibrolamellar hepatocellular carcinoma. Proc Natl Acad Sci U S A. 2017 Dec 12;114(50):13076-13084. doi: 10.1073/pnas.1716483114. Epub 2017 Nov 21. PubMed PMID: 29162699; PubMed Central PMCID: PMC5740683.

Lalazar G, Requena D, Ramos-Espiritu L, Ng D, Bhola PD, de Jong YP, Wang R, Narayan NJC, Shebl B, Levin S, Michailidis E, Kabbani M, Vercauteren KOA, Hurley AM, Farber BA, Hammond WJ, Saltsman JA, Weinberg EM, Glickman JF, Lyons BA, Ellison J, Schadde E, Hertl M, Leiting JL, Truty MJ, Smoot RL, Tierney F, Kato T, Wendel HG, LaQuaglia MP, Rice CM, Letai A, Coffino P, Torbenson MS, Ortiz MV, Simon SM. Identification of Novel Therapeutic Targets for Fibrolamellar Carcinoma Using Patient Derived Xenografts and Direct from Patient Screening. Cancer Discov. 2021 Jun 14:candisc.0872.2020. doi: 10.1158/2159-8290.CD-20-0872. Online ahead of print. PMID: 34127480

Lalazar G, Simon SM. Fibrolamellar Carcinoma: Recent Advances and Unresolved Questions on the Molecular Mechanisms. Semin Liver Dis. 2018 Feb;38(1):51-59. doi: 10.1055/s-0037-1621710. Epub 2018 Feb 22. Review. PubMed PMID: 29471565; PubMed Central PMCID: PMC6020845.

Olivieri C, Walker C, Karamafrooz A, Wang Y, Manu VS, Porcelli F, Blumenthal DK, Thomas DD, Bernlohr DA, Simon SM, Taylor SS, Veglia G. Defective internal allosteric network imparts dysfunctional ATP/substrate-binding cooperativity in oncogenic chimera of protein kinase A. Commun Biol. 2021 Mar 10;4(1):321. doi: 10.1038/s42003-021-01819-6.PMID: 33692454 Free PMC article.

Rich BS, Honeyman JN, Darcy DG, Smith PT, Williams AR, Lim II, Johnson LK, Gönen M, Simon JS, LaQuaglia MP, Simon SM. Endogenous antibodies for tumor detection. Sci Rep. 2014 May 30;4:5088. doi: 10.1038/srep05088. PubMed PMID: 24875800; PubMed Central PMCID: PMC4038850.

Saltsman JA, Hammond WJ, Narayan NJC, Requena D, Gehart H, Lalazar G, LaQuaglia MP, Clevers H, Simon S.Cancers (Basel). A Human Organoid Model of Aggressive Hepatoblastoma for Disease Modeling and Drug Testing. 2020 Sep 18;12(9):2668. doi: 10.3390/cancers12092668.PMID: 32962010

Simon EP, Freije CA, Farber BA, Lalazar G, Darcy DG, Honeyman JN, Chiaroni-Clarke R, Dill BD, Molina H, Bhanot UK, La Quaglia MP, Rosenberg BR, Simon SM. Transcriptomic characterization of fibrolamellar hepatocellular carcinoma. Proc Natl Acad Sci U S A. 2015 Nov 3;112(44):E5916-25. doi: 10.1073/pnas.1424894112. Epub 2015 Oct 21. PubMed PMID: 26489647; PubMed Central PMCID: PMC4640752.

Simon JS, Botero S, Simon SM. Sequencing the peripheral blood B and T cell repertoire – Quantifying robustness and limitations. J Immunol Methods. 2018 Dec;463:137-147. doi: 10.1016/j.jim.2018.10.003. Epub 2018 Oct 10. PubMed PMID: 30312601; PubMed Central PMCID: PMC6355145.

Tomasini MD, Wang Y, Karamafrooz A, Li G, Beuming T, Gao J, Taylor SS, Veglia G, Simon SM. Conformational Landscape of the PRKACA-DNAJB1 Chimeric Kinase, the Driver for Fibrolamellar Hepatocellular Carcinoma. Sci Rep. 2018 Jan 15;8(1):720. doi: 10.1038/s41598-017-18956-w. PubMed PMID: 29335433; PubMed Central PMCID: PMC5768683.

How does the nuclear pore function?

The nuclear pore is a massive macromolecular complex that regulates most movement in and out of the nucleus. Many groups have been generating an inventory of all of the components and the crystal structure of individual pore components. We have taken the approach of asking:

  • How do the different proteins fit together in an intact nuclear pore in a living cell?
  • Is the structure static or dynamic?

We inserted a green fluorescent protein into many different positions of one of the structural components of the nuclear pore. We excited the GFP with polarized light. By varying the angle we could find the optimal angle for exciting the dipole of the GFP. With each amino acid we added in the alpha-helix at the GFP, the angle rotated 103o. We have the crystal structure of the GFP, thus, we could determine the orientation of the protein to which it was attached. We applied similar approach to the filaments that fill the lumen of the nuclear pore, allowing us to follow their dynamics. Next we applied the approach to various core structural domains and found some domains that changed with orientation with transport through the pore. The results were quantitative enough to allow us to make computational simulations of the structural dynamics and transport of the nuclear pore that we could then experimentally test. Some examples of our work:

How do viruses assemble in the cell?

We are using assembly of a virus as a model system for how a multimolecular machine assembles. Our approach has been to study single viruses as they assemble in a living cell. We study when and how individual viral encoded molecules and molecules in the host cell interact to allow the assembly of a functional virus. Most studies examine thousands, to millions of viruses in many cells, all in different states of infection, with viruses in different states of assembly. From examining single viruses, we found that the steps of assembly are highly co-ordinated with a very prescribed order of events.  First, the genome comes to the surface, then the coat protein, Gag, assembles around it. As the Gag assembles, the membrane bends, folding around the nascent virion. Then a host protein, a member of the ESCRTIII complex, is recruited, followed within 5-10 seconds by another host protein, Vps4, an ATPase which remodels the ESCRT complex. They remain at the next of the nascent virion for under a minute, leave, and 20 seconds later the virus scissions from the host cell. The virus makes one main protein, Gag. It has a protease, which is activated upon assembly, which then cleaves Gag into separate pieces that then assemble inside the virion to mature the virus. Most recently we examined why viruses such as HIV-1 and Ebola don’t infect some species. We found that new world monkeys has a duplicated and truncate form one of the proteins of the ESCRTIII complex proteins. As result the normal ESCRT-III gets recruited, but fails to recruit the ATPase, so it falls away, but then comes back again a few times, until the ATPase is recruited and scission occurs. This produces a short few minute delay. However, it is enough for the virus to start activating its protease and cleave the Gag protein, which is then lost back into the cell, rather than being retained the virus. This few second delay does not seriously impact other ESCRT-III functions, such as cell division, but deleterious for the assembling virus. This suggests new potential therapeutic approaches to blocking the assembly of viruses such as HIV-1 and Ebola. A few examples of papers are:

How do cell-to-cell interactions in human tissue affect cellular development and activity?

We wanted a system where we can study cells in native human tissue at high resolution. Thus, we chose to study the interactions of melanocytes and keratinocytes in human skin. We study the cells, their structure and behavior, in intact human skin, as isolated individual cells, and reconstituted skin in vitro. The first example of this work is:

Check out the videos

How do molecular machines work?

Our studies with Gunter Blobel demonstrated that proteins moved across membranes through aqueous channels (see past projects). This forced the question: “What is the mechanism that moved the proteins”. This is part of a general question of how do molecular motors work. For movement of proteins across the membrane we proposed a biophysical mechanism which linked the chemical potential energy of translation to the movement of proteins, and we coined the term “Brownian Ratchet” (Simon et al. 1992) . The movement of the protein is dominated by Brownian (thermal) forces). The directionality was determined by various gradients of chemical potential in the cell. We have since worked on extrapolating this model to movement of molecules through the nuclear pore (Mincer and Simon, 2011).

How do other molecular motors function. The family of AAA+ ATPases are found in many cellular processes (we studied one member, VPS4, in our studies on viral assembly). Now we are examining ClpX, a component of a bacterial degradative machinery, akin to a proteasome. We are studying it both biochemically, but also we are pursuing single molecule studies of its activity using total internal fluorescence microscopy.

New results will be appearing here!

Developing new technologies

There have been many advances in science that have come not from a new conceptual breakthrough, but from a new technology the enables asking previously unaddressed questions. The following are a few of the new technologies we have worked on:

The immunological response in cancer:

This is a technique to facilitate our studies on cancer:

  • Dao Thi VL, Wu X, Belote RL, Andreo U, Takacs CN, Fernandez JP, Vale-Silva LA, Prallet S, Decker CC, Fu RM, Qu B, Uryu K, Molina H, Saeed M, Steinmann E, Urban S, Singaraja RR, Schneider WM, Simon SM, Rice CM. Stem cell-derived polarized hepatocytes. Nat Commun. 2020 Apr 3;11(1):1677. doi: 10.1038/s41467-020-15337-2.PMID: 32245952

Microscopy techniques:
General techniques:

Quantum dots

Some of the techniques trying to advance the use of total internal reflection:

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Laboratory of Cellular Biophysics
Kravis Research Building
The Rockefeller University
1230 York Ave.
New York, NY 10065

Sanford M. Simon
Günter Blobel Professor

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