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Notes from Opening Plenary Session – The Genome and Beyond from the 2015 AACR Meeting in Philadelphia PA; Sunday April 19, 2015

 

Reporter: Stephen J. Williams, Ph.D.

The following contain notes from the Sunday April 19, 2015 AACR Meeting (Pennsylvania Convention Center, Philadelphia PA) 9:30 AM Opening Plenary Session

The Genome and Beyond

Session Chairperson: Lewis C. Cantley, Ph.D.

Speakers: Michael R. Stratton, Tyler Jacks, Stephen B. Baylin, Robert D. Schreiber, Williams R. Sellers

  1. A) Insights From Cancer Genomes: Michael R. Stratton, Ph.D.; Director of the Wellcome Trust Sanger Institute
  • How do we correlate mutations with causative factors of carcinogenesis and exposure?
  • Cancer was thought as a disease of somatic mutations
  • UV skin exposure – see C>T transversion in TP53 while tobacco exposure and lung cancer see more C>A transversion; Is it possible to determine EXPOSURE SIGNATURES?
  • Use a method called non negative matrix factorization (like face pattern recognition but a mutation pattern recognition)
  • Performed sequence analysis producing 12,000 mutation catalogs with 8,000 somatic mutation signatures
  • Found more mutations than expected; some mutation signatures found in all cancers, while some signatures in half of cancers, and some signatures not found in cancer
  • For example found 3 mutation signatures in ovarian cancer but 13 for breast cancers (80,000 mutations); his signatures are actually spectrums of mutations
  • kataegis: defined as localized hypermutation; an example is a signature he found related to AID/APOBEC family (involved in IgG variability); kataegis is more prone in hematologic cancers than solid cancers
  • recurrent tumors show a difference in mutation signatures than primary tumor before drug treatment

 

  1. B) Engineering Cancer Genomes: Tyler Jacks, Ph.D.; Director, Koch Institute for Integrative Cancer Research
  • Cancer GEM’s (genetically engineered mouse models of cancer) had moved from transgenics to defined oncogenes
  • Observation that p53 -/- mice develop spontaneous tumors (lymphomas)
  • then GEMs moved to Cre/Lox systems to generate mice with deletions however these tumor models require lots of animals, much time to create, expensive to keep;
  • figured can use CRSPR/Cas9 as rapid, inexpensive way to generate engineered mice and tumor models
  • he used CRSPR/Cas9 vectors targeting PTEN to introduce PTEN mutations in-vivo to hepatocytes; when they also introduced p53 mutations produced hemangiosarcomas; took ONLY THREE months to produce detectable tumors
  • also produced liver tumors by using CRSPR/Cas9 to introduce gain of function mutation in β-catenin

 

See an article describing this study by MIT News “A New Way To Model Cancer: New gene-editing technique allows scientists to more rapidly study the role of mutations in tumor development.”

The original research article can be found in the August 6, 2014 issue of Nature[1]

And see also on the Jacks Lab site under Research

  1. C) Above the Genome; The Epigenome and its Biology: Stephen B. Baylin
  • Baylin feels epigenetic therapy could be used for cancer cell reprogramming
  • Interplay between Histone (Movers) and epigenetic marks (Writers, Readers) important for developing epigenetic therapy
  • Difference between stem cells and cancer: cancer keeps multiple methylation marks whereas stem cells either keep one on or turn off marks in lineage
  • Corepressor drugs are a new exciting class in chemotherapeutic development
  • (Histone Demythylase {LSD1} inhibitors in clinical trials)
  • Bromodomain (Brd4) enhancers in clinical trials
  1. D) Using Genomes to Personalize Immunotherapy: Robert D. Schreiber, Ph.D.,
  • The three “E’s” of cancer immunoediting: Elimination, Equilibrium, and Escape
  • First evidence for immunoediting came from mice that were immunocompetent resistant to 3 methylcholanthrene (3mca)-induced tumorigenesis but RAG2 -/- form 3mca-induced tumors
  • RAG2-/- unedited (retain immunogenicity); tumors rejected by wild type mice
  • Edited tumors (aren’t immunogenic) led to tolerization of tumors
  • Can use genomic studies to identify mutant proteins which could be cancer specific immunoepitopes
  • MHC (major histocompatibility complex) tetramers: can develop vaccines against epitope and personalize therapy but only good as checkpoint block (anti-PD1 and anti CTLA4) but personalized vaccines can increase therapeutic window so don’t need to start PD1 therapy right away
  • For more details see references Schreiker 2011 Science and Shankaran 2001 in Nature
  1. E) Report on the Melanoma Keynote 006 Trial comparing pembrolizumab and ipilimumab (PD1 inhibitors)

Results of this trial were published the morning of the meeting in the New England Journal of Medicine and can be found here.

A few notes:

From the paper: The anti–PD-1 antibody pembrolizumab prolonged progression-free survival and overall survival and had less high-grade toxicity than did ipilimumab in patients with advanced melanoma. (Funded by Merck Sharp & Dohme; KEYNOTE-006 ClinicalTrials.gov number, NCT01866319.)

And from Twitter:

Robert Cade, PharmD @VTOncologyPharm

KEYNOTE-006 was presented at this week’s #AACR15 conference. Pembrolizumab blew away ipilimumab as 1st-line therapy for metastatic melanoma.

2:02 PM – 21 Apr 2015

Jeb Keiper @JebKeiper

KEYNOTE-006 at #AACR15 has pembro HR 0.63 in OS over ipi. Issue is ipi is dosed only 4 times over 2 years (per label) vs Q2W for pembro. Hmm

11:55 AM – 19 Apr 2015

OncLive.com @OncLive

Dr Antoni Ribas presenting data from KEYNOTE-006 at #AACR15 – Read more about the findings, at http://ow.ly/LMG6T 

11:25 AM – 19 Apr 2015

Joe @GantosJ

$MRK on 03/24 KEYNOTE-006 vs Yervoy Ph3 stopped early for meeting goals of PFS, OS & full data @ #AACR15 now back to weekend & family

9:05 AM – 19 Apr 2015

Kristen Slangerup @medwritekristen

Keytruda OS benefit over Yervoy in frontline #melanoma $MRK stops Ph3 early & data to come @ #AACR15 #immunotherapy http://yhoo.it/1EYwwq8 

2:40 PM – 26 Mar 2015

Yahoo Finance @YahooFinance

Merck’s Pivotal KEYNOTE-006 Study in First-Line Treatment for…

Merck , known as MSD outside the United States and Canada, today announced that the randomized, pivotal Phase 3 study investigating KEYTRUDA® compared to ipilimumab in the first-line treatment of…

View on web

 

Stephen J Williams @StephenJWillia2

Progression free survival better for pembrolzumab over ipilimumab by 2.5 months #AACR15 @Pharma_BI #Cancer #Immunotherapy

11:56 AM – 19 Apr 2015

 

Stephen J Williams @StephenJWillia2

Melanoma Keynote 006 trial PD1 inhibitor #Immunotherapy 80% responders after 1 year @Pharma_BI #AACR15

 

References

  1. Xue W, Chen S, Yin H, Tammela T, Papagiannakopoulos T, Joshi NS, Cai W, Yang G, Bronson R, Crowley DG et al: CRISPR-mediated direct mutation of cancer genes in the mouse liver. Nature 2014, 514(7522):380-384.

 

Other related articles on Cancer Genomics and Social Media Coverage were published in this Open Access Online Scientific Journal, include the following:

Cancer Biology and Genomics for Disease Diagnosis

Introduction – The Evolution of Cancer Therapy and Cancer Research: How We Got Here?

Methodology for Conference Coverage using Social Media: 2014 MassBio Annual Meeting 4/3 – 4/4 2014, Royal Sonesta Hotel, Cambridge, MA

List of Breakthroughs in Cancer Research and Oncology Drug Development by Awardees of The Israel Cancer Research Fund

2013 American Cancer Research Association Award for Outstanding Achievement in Chemistry in Cancer Research: Professor Alexander Levitzki

Genomics and Epigenetics: Genetic Errors and Methodologies – Cancer and Other Diseases

Cancer Genomics – Leading the Way by Cancer Genomics Program at UC Santa Cruz

Genomics and Metabolomics Advances in Cancer

Pancreatic Cancer: Genetics, Genomics and Immunotherapy

Multiple Lung Cancer Genomic Projects Suggest New Targets, Research Directions for Non-Small Cell Lung Cancer

 

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The SCID Pig II: Researchers Develop Another SCID Pig, And Another Great Model For Cancer Research

 

Writer. Reporter: Stephen J. Williams, Ph.D.

gottingen minipig2

 

 

The choice of suitable animal model of disease may define future success or failure for drug development, basic and translational research, or biomarker discovery projects.   Indeed, as highlighted in one of my earlier posts “Heroes in Medical Research: Developing Models for Cancer Research”, the choice of animal to model a human disease can have drastic implications in the basic researchers ability to understand metabolic and genetic factors causally associated with disease development. As described in that post the King rat model led to our understanding of the genetics of early development and sex determination while early mouse models helped us to understand the impact of microenvironment on cell fate and the discovery of stem cells. In addition, transgenic and immunodeficient mice resulted in transformational studies on our understanding of cancer. Small rodent models are ideal for following reasons:

  • Ease of genetic manipulation
  • Availability of well-defined models
  • Ease of low cost of use

Regardless of these benefits many investigators in industry and academia are looking to models of human disease in animals more closely resembling human anatomy, physiology, and genetics.

 

There is a growing need for alternative animal models in cancer research.

 

As I had discussed in another of my earlier posts “The SCID Pig: How Pigs are becoming a Great Alternate Model for Cancer Research”, the pig is gaining notoriety and acceptance as a very suitable animal to model human disease as minipigs and humans have:

  • Similar physiology
  • Similar genetics: >90% homology
  • Similar anatomic dimensions: i.e. Adult Gottingen minipigs are 70kg (adult human male weight)
  • Similar organ size and structure to humans organ size and structure
  • Pig genome sequencing project nearly complete
  • Ability to manipulate pig genetics

The post had discussed the development of a severe combined immunodeficient (SCID) pig by investigators at Iowa State and Kansas State University. This line of pigs, selected on a specific diet, could act as recipients for human cancer cell lines, a proof of their SCID phenotype.

A report featured on Fierce Biotech Research “MU Scientists Successfully Transplant, Grow Stem Cells in Pigs” discussed the development of a new genetically-modified immunodeficient porcine model by researchers at the University of Missouri, recently published in Proceedings of the National Academy of Sciences[1].

These pigs are available from the National Swine Resource and Research Center (http://nsrrc.missouri.edu).

For the report on Fierce Biotech Research please follow the link below:

http://www.fiercebiotechresearch.com/press-releases/mu-scientists-successfully-transplant-grow-stem-cells-pigs

 

The report in FierceBiotech highlights the type of studies an immunocompromised pig model would be useful for including:

  • Regenerative medicine
  • Xenotransplantation
  • Tumor growth and efficacy studies

 

Comments in the post from the investigators explained the benefits of developing such a porcine model system including:

“The rejection of transplants and grafts by host bodies is a huge hurdle for medical researchers,” said R. Michael Roberts, Curators Professor of Animal Science and Biochemistry and a researcher in the Bond Life Sciences Center. “By establishing that these pigs will support transplants without the fear of rejection, we can move stem cell therapy research forward at a quicker pace.”

The studies main investigators, Drs. Randall Prather and R. Michael Roberts, both of University of Missouri, along with first authors Kiho Lee, Deug-Nam Kwon and Toshihiko Ezashi, used biallellic mutation of the RAG2 gene in Gottingen minipig fibroblasts and then subsequent somatic cell nuclear transfer (SCNT) to produce the RAG2-/- animals. (Rag2 is a protein involved in V(D)J recombination of antibodies during early B and T cell development. See GeneCard link above)

As proof of their SCID phenotype the authors showed that

  1. these RAG2-/- animals could act as host for human induced pluripotent stem cells
  2. act as recipient for allogeneic porcine stem cells
  3. reduced levels of (CD21+) B cells and (CD3+) T cells
  4. growth retardation if housed under standard, non-sterile conditions

Details of the study are given below:

Methodology Used

For Production of Gottingen minipigs carrying the RAG2 mutation

To produce targeted mutations in RAG2:

  • TALENS () were constructed to produced mutation in exon 2 of RAG2
  • Constructed TALENS and reporter electroporated in fetal-derived pig fibroblasts
  • SCNT used to transfer RAG2 mutant nuclei to donor oocytes
  • 9 embryo transfers resulted in 22 live piglets
  • Piglets genotyped as either monoallelic or biallelic RAG2 mutant
  • RAG2wild-type and mutants housed in either pathogen-free or normal housing conditions

To verify SCID phenotype of litter by either

  1. Graft acceptance of human iPSCs and teratoma formation

–          Fibroblasts from human umbilical cord reprogrammed to pluripotency; verified by pluripotent markers POUSF1, NANOG, SSEA-3)

–          Two human and porcine iPSC lines with trophoblastic properties[2] were injected subcutaneously in ear or flank

–          Tumor formation analyzed by immunohistochemistry using markers:

CTNNBI (B-catenin)

VWF (von Willebrand

DES and ACTG2

GFAP and ENO2

Human specific MFN1 (both antibody and gene primers)

  1. Flow Cytometry

–          Analysis of piglet spleen cells for B cell population (CD21)

–          Analysis of piglet spleen cell for T cell population (CD3)

C.    Histology

– histo evaluation of thymus, spleen

– marker evaluation of spleen using anti-CD79A (B cells), CD3 (T cells),

CD335 (NK cells)

Results

TALEN produced a variety of indels (insertion/deletions) and three RAG2 mutatnt colonies (containing monoallelic, mix of mono and biallelic) used for SCNT.

Three litters produced 16 piglets (eight survived [four mono and four biallelic]

Biallelic RAG2 mutants showed slower weight gain than wild type or monoallelic mutants with signs of inflammation and apoptosis in spleen and designated “failure to thrive” in standard housing…needed a clean environment to thrive.

Biallelic mutant pigs lacked mature CD21 B cells and CD3 T cells but contained macrophages and NK cells.

Implantation of human and allogenic porcine pluripotent stem cells (trophoblastic) showed rapid development of teratomas.
References

  1. Lee K, Kwon DN, Ezashi T, Choi YJ, Park C, Ericsson AC, Brown AN, Samuel MS, Park KW, Walters EM et al: Engraftment of human iPS cells and allogeneic porcine cells into pigs with inactivated RAG2 and accompanying severe combined immunodeficiency. Proceedings of the National Academy of Sciences of the United States of America 2014, 111(20):7260-7265.
  2. Ezashi T, Matsuyama H, Telugu BP, Roberts RM: Generation of colonies of induced trophoblast cells during standard reprogramming of porcine fibroblasts to induced pluripotent stem cells. Biology of reproduction 2011, 85(4):779-787.

 

Other posts on this site related to Cancer Research Tools include

The SCID Pig: How Pigs are becoming a Great Alternate Model for Cancer Research

Heroes in Medical Research: Developing Models for Cancer Research

Reprogramming Induced Pleuripotent Stem Cells

The Cancer Research Concentration @ Leaders in Pharmaceutical Business Intelligence

A Synthesis of the Beauty and Complexity of How We View Cancer

Guidelines for the welfare and use of animals in cancer research

Gene Therapy and the Genetic Study of Disease: @Berkeley and @UCSF – New DNA-editing technology spawns bold UC initiative as Crispr Goes Global

 

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Heroes in Medical Research: Developing Models for Cancer Research

Author, Curator: Stephen J. Williams, Ph.D.

 

The current rapid progress in cancer research would have never come about if not for the dedication of past researchers who had developed many of the scientific tools we use today. In this issue of Heroes in Medical Research I would like to give tribute to the researchers who had developed the some of the in-vivo and in-vitro models which are critical for cancer research.

 

The Animal Modelers in Cancer Research

Helen Dean King, Ph.D. (1869-1955)

Helen Dean King

Helen Dean King, Ph.D. from www.ExplorePAhistory.com; photo Courtesy of the Wistar Institute Archive Collection, Philadelphia, PA

 

 

The work of Dr. Helen Dean King on rat inbreeding led to development of strains of laboratory animals. Dr. King taught at Bryn Mawr College, then worked at University of Pennsylvania and the Wistar Institute under famed geneticist Thomas Hunt Morgan, researching if inbreeding would produce harmful genetic traits.   At University of Pennsylvania she examined environmental and genetic factors on gender determination.

 

 

 

 

Important papers include [1-6]as well as the following contributions:

“Studies in Inbreeding”, “Life Processes in Gray Norway Rats During Fourteen Years in Captivity”, doctoral thesis on embryologic development in toads (1899)

 

Milestones include:

 

1909    started albino rat breeding and bred 20 female and male from same litter (King colony) to 25

successive generations (inbreeding did not cause harmful traits)

 

1919     started to domesticate the wild Norwegian rats that ran thru Philadelphia (six pairs Norway rats

thru 28 generations)

A good reference for definitions of rat inbreeding versus line generation including a history of Dr. King’s work can be found at the site: Munificent Mischief Rattery and a brief history here.[7] In addition, Dr. King had investigated using rat strains as a possible recipient for tumor cells. The work was an important advent to the use of immunodeficient models for cancer research.

 

As shown below Philadelphia became a hotbed for research into embryology, development, genetics, and animal model development.

 

Beatrice Mintz, Ph.D.

(Beatrice Minz, Ph.D.; photo credit Fox Chase Cancer Center, www.pubweb.fccc.edu) Mintz

Dr. Mintz, an embryologist and cancer researcher from Fox Chase Cancer Center in Philadelphia, PA, contributed some of the most seminal discoveries leading to our current understanding of genetics, embryo development, cellular differentiation, and oncogenesis, especially melanoma, while pioneering techniques which allowed the development of genetically modified mice.

If you get the privilege of hearing her talk, take advantage of it. Dr. Mintz is one of those brilliant scientists who have the ability to look at a clinical problem from the viewpoint of a basic biological question and, at the same time, has the ability to approach the well-thought out questions with equally well thought out experimental design. For example, Dr. Mintz asked if a cell’s developmental fate was affected by location in the embryo. This led to her work by showing teratocarcinoma tumor cells in the developing embryo could revert to a more normal phenotype, essentially proving two important concepts in development and tumor biology:

  1. The existence of pluripotent stem cells
  2. That tumor cells are affected by their environment (which led to future concepts of the importance of tumor microenvironment on tumor growth

Other seminal discoveries included:

  • Development of the first mouse chimeras using novel cell fusion techniques
  • With Rudolf Jaenisch in 1974, showed integration of viral DNA from SV40, could be integrated into the DNA of developing mice and persist into adulthood somatic cells, the first transgenesis in mice which led ultimately to:
  • Development of the first genetically modified mouse model of human melanoma in 1993

Her current work, seen on the faculty webpage here, is developing mice with predisposition to melanoma to uncover risk factors associated with the early development of melanoma.

In keeping with the Philadelphia tradition another major mouse model which became seminal to cancer drug discovery was co-developed in the same city, same institute and described in the next section.

It is interesting to note that the first cloning of an animal, a frog, had taken place at the Institute for Cancer Research, later becoming Fox Chase Cancer Center, which was performed by Drs. Robert Briggs and Thomas J. King and reported in the 152 PNAS paper Transplantation of Living Nuclei From Blastula Cells into Enucleated Frogs’ Eggs.[8]

 

 The Immunodeficient Animal as a Model System for Cancer Research – Dr. Mel Bosma, Ph.D.

 

Bosma

Melvin J. Bosma, Ph.D.; photo credit Fox Chase Cancer Center

In the summer of 1980 at Fox Chase Cancer Center, Dr. Melvin J. Bosma and his co-researcher wife Gayle discovered mice with deficiencies in common circulating antibodies and since, these mice were littermates, realized they had found a genetic defect which rendered the mice immunodeficient (upon further investigation these mice were unable to produce mature B and T cells). These mice were the first scid (severe combined immunodeficiency) colony. The scid phenotype was later found to be a result of a spontaneous mutation in the enzyme Prkdc {protein kinase, DNA activated, catalytic polypeptide} involved in DNA repair, and ultimately led to a defect in V(D)J recombination of immunoglobulins.

The emergence of this scid mouse was not only crucial for AIDS research but was another turning point in cancer research , as researchers now had a robust in-vivo recipient for human tumor cells. The orthotopic xenograft of human tumor cells now allowed for studies on genetic and microenvironmental factors affecting tumorigenicity, as well as providing a model for chemotherapeutic drug development (see Suggitt for review and references)[9]. A discussion of the pros and cons of the xenograft system for cancer drug discovery would be too voluminous for this post and would warrant a full review by itself. But before the advent of such scid mouse systems researchers relied on spontaneous and syngeneic mouse tumor models such as the B16 mouse melanoma and Lewis lung tumor model.

Other scid systems have been developed such as in the dog, horse, and pig. Please see the following post on this site The SCID Pig: How Pigs are becoming a Great Alternate Model for Cancer Research. The athymic (nude) mouse (nu/nu) also is a popular immunodeficient mouse model used for cancer research

Two other in-vivo tumor models: Patient Derived Xenografts (PDX) and Genetically Engineered Mouse models (GEM) deserve their own separate discussion however the success of these new models can be attributed to the hard work of the aforementioned investigators. Therefore I will post separately and curate PDX and GEM models of cancer and highlight some new models which are having great impact on cancer drug development.

 

References

1.         Loeb L, King HD: Transplantation and Individuality Differential in Strains of Inbred Rats. The American journal of pathology 1927, 3(2):143-167.

2.         Lewis MR, Aptekman PM, King HD: Retarding action of adrenal gland on growth of sarcoma grafts in rats. J Immunol 1949, 61(4):315-319.

3.         Aptekman PM, Lewis MR, King HD: Tumor-immunity induced in rats by subcutaneous injection of tumor extract. J Immunol 1949, 63(4):435-440.

4.         Lewis MR, Aptekman PM, King HD: Inactivation of malignant tissue in tumor-immune rats. J Immunol 1949, 61(4):321-326.

5.         Lewis MR, King HD, et al.: Further studies on oncolysis and tumor immunity in rats. J Immunol 1948, 60(4):517-528.

6.         Aptekman PM, Lewis MR, King HD: A method of producing in inbred albino rats a high percentage of immunity from tumors native in their strain. J Immunol 1946, 52:77-86.

7.         Ogilvie MB: Inbreeding, eugenics, and Helen Dean King (1869-1955). Journal of the history of biology 2007, 40(3):467-507.

8.         Briggs R, King TJ: Transplantation of Living Nuclei From Blastula Cells into Enucleated Frogs’ Eggs. Proceedings of the National Academy of Sciences of the United States of America 1952, 38(5):455-463.

9.         Suggitt M, Bibby MC: 50 years of preclinical anticancer drug screening: empirical to target-driven approaches. Clinical cancer research : an official journal of the American Association for Cancer Research 2005, 11(3):971-981.

 

Other posts on this site about Cancer, Animal Models of Disease, and other articles in this series include:

The SCID Pig: How Pigs are becoming a Great Alternate Model for Cancer Research

A Synthesis of the Beauty and Complexity of How We View Cancer

Guidelines for the welfare and use of animals in cancer research

Importance of Funding Replication Studies: NIH on Credibility of Basic Biomedical Studies

FDA Guidelines For Developmental and Reproductive Toxicology (DART) Studies for Small Molecules

Report on the Fall Mid-Atlantic Society of Toxicology Meeting “Reproductive Toxicology of Biologics: Challenges and Considerations:

What`s new in pancreatic cancer research and treatment?

Heroes in Medical Research: Dr. Carmine Paul Bianchi Pharmacologist, Leader, and Mentor

Heroes in Medical Research: Dr. Robert Ting, Ph.D. and Retrovirus in AIDS and Cancer

Heroes in Medical Research: Barnett Rosenberg and the Discovery of Cisplatin

Richard Lifton, MD, PhD of Yale University and Howard Hughes Medical Institute: Recipient of 2014 Breakthrough Prizes Awarded in Life Sciences for the Discovery of Genes and Biochemical Mechanisms that cause Hypertension

Reuben Shaw, Ph.D., a geneticist and researcher at the Salk Institute: Metabolism Influences Cancer

 

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