Generating Mouse Models of Pancreatic Cancer

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1 Generating Mouse Models of Pancreatic Cancer Aom Isbell Spring/Summer 1, 2012 Alexandros Tzatsos, MD PhD

Bardeesy Lab: Goals and Objectives Objective: Understanding the genetic program for Pancreatic Ductal Adenocarcinoma (PDAC) initiation and progress -*Approx. 44K new cases in US in 2012 -*4.

2 Bardeesy Lab: Goals and Objectives Objective: Understanding the genetic program for Pancreatic Ductal Adenocarcinoma (PDAC) initiation and progress -*Approx. 44K new cases in US in *4.6% survival rate average (beyond 5 years) Goal: Engineering conditional knockout and overexpression mouse models to study initiation and maintenance of PDAC Aim: To generate mouse models that mimic the human disease in order to be used in therapeutic drug screening *National Cancer Institute website statistics

produced by epitheleal duct cells secreted through ducts into digestive tract, accounts for 90% of

3 The Pancreas has an exocrine and endocrine function Endocrine- hormones (insulin, glucagon) secreted directly into the bloodstream, accounts for about 10% pancreatic cancers Exocrine- digestive enzymes produced by epitheleal duct cells secreted through ducts into digestive tract, accounts for 90% of pancreatic cancers and, unlike endcorine, is extremely aggressive

4 Pancreatic Cancers Endocrine 10% Exocrine 90% PanINs, pancreatic interepithelial neoplastic lesions PDAC, pancreatic ductal adenocarcinoma

remains constitutively active which leads to cell proliferation and

6 In 90% of PDACs mutated KRAS oncogenes are present KRAS is normally inactive if bound to GDP (guanosine diphosphate) but mutated KRAS remains constitutively active which leads to cell proliferation and migration and DNA synthesis Constitutively activated KRAS drives the progression of PDAC

Lysine Specific Demethylase 2B: enzyme encoded by the KDM2B Gene Regulates epithelial cell differentiation during development Overexpression promotes bypass of senescence and immortalizes cells

7 Lysine Specific Demethylase 2B: enzyme encoded by the KDM2B Gene Regulates epithelial cell differentiation during development Overexpression promotes bypass of senescence and immortalizes cells Upregulation is associated with advanced disease in PDAC * KDM2B cooperates with KRAS to drive PDAC Deregulates cell proliferation downstream of KRAS Blocks tumor suppressors (such as p16) allowing KRAS to cause proliferated cell mutation *A. Tzatsos, P. Paskaleva et al.

8 Project Title Genotyping of GEMM (genetically engineered mouse models) Using Polymerase Chain Reaction to test for the presence of Cre, LoxP, and KRAS in selected mouse models -Cre recombinase: enzyme that carries out site specific DNA recombination (recognizes LoxP sequence) -LoxP: target sequence of DNA (locus) Using Gel Electrophoresis to visualize results Mice are being engineered to conditionally overexpress or ablate KDM2B (Dox inducibility) Mice that possess these genes can be bred to create Homozygous (knockout or overexpression) models and examined through histological methods to further determine the roles these genes have in disease initiation and progression

9 Ablation

10 Proof of Principle X KRAS X KDM2B (Deletion) Al1 + Al Cre PDX1 Or GATA1 Cre LoxP KDM2B LoxP X PDX1 is a tissue specific promoter (Pancreas) provides Spatial Control over transgene expression. -Cre will only be active in pancreas cells GATA1 is whole body promoter, will promote expression in all cells types. Successful cross breeding will yield a mouse with both alleles of the KDM2B gene deleted Homozygous Knockout Homozygous Knockout (-/-) yields little PDAC development Heterozygous Wild Type/Knockout (+/-) yields Intermediate development of PDAC Homozygous Wild Type (+/+) yields extensive PDAC development

11 The Targeting Vector (DNA Construct) 1. Homologous DNA that matches the gene (orange) orange 2. Positive selection marker-neor (neomycin resistance) ensures the vector is inserted (red) red Techniques in Molecular Biology, Spring '11 Lecture Slide

12 2. Techniques in Molecular Biology, Spring '11 Lecture slides

13 Techniques in Molecular Biology, spring '11 lecture slides

14 Hypothesis: Overexpression of KDM2B will enhance KRAS' ability to form PDAC, whereas KDM2B genetic deletion will inhibit PDAC formation in the context of KRAS Materials and Methods Extract genomic DNA -Alkaline Lysis Buffer (NaOH, dh2o,0.5m EDTA ph 8.0,-ethylenediaminetetraacetic acid) -Vortex -Neutralization Buffer (40mM Tris-HCl) -PCR program PCR Master Mixes -10x Buffer -dh20 -MgCl2+ -Genomic DNA samples -dntps -Taq Polymerase -Specific Primers (Gabra/Kozak etc.)

Materials and Methods (continued) Run PCR Program -Denaturing -Annealing -Extension -Exponential replication -Add dye and centrifuge Gel Preparation -Agarose powder -TBE (Tris base, boric acid, EDTA)

15 Materials and Methods (continued) Run PCR Program -Denaturing -Annealing -Extension -Exponential replication -Add dye and centrifuge Gel Preparation -Agarose powder -TBE (Tris base, boric acid, EDTA) -EtBr (ethidium bromide) -Gel castor and well comb Run Electrophoresis -Molecular ladder -Electrophoresis tub -TBE -Power Supply and cables Gel Imaging Interpret results oceanexplorer.noaa.gov/explorations/04e

Gel Image 500 bp Altered gene at 300 BP 100 bp Wild type at 200 BP Control Lane (far right) Molecular Ladder The altered gene is bigger than(more base pairs) the wild type due to the addition of the

16 Gel Image 500 bp Altered gene at 300 BP 100 bp Wild type at 200 BP Control Lane (far right) Molecular Ladder The altered gene is bigger than(more base pairs) the wild type due to the addition of the LoxP and thus won't travel as far through the agarose gel matrix LoxP=KDM2B=LoxP Wild type +/+ Heterozygous +/- Homozygous deletion -/- 259: (+/+) 260: (+/-) 261: (-/-) 262: (-/-) 263: (+/-) 264: (-/-) 265: (+/-) 266: (+/+) 267: (+/-) 268: (+/+) 269: (-/-) 270: (+/-) 271: (+/-) 272: (-/-) 273: (-/-) 274: (+/-) PCR gel image from 3/20/12, testing for presence of LoxP in mice 259 to 274

17 Results and Conclusions Gel image showed many of the mice tested carry the transgene, thus can be used for further analysis One step in a larger procedure: further genotyping, breeding and histological examination of mouse models needed to confirm hypothesis Data shows mice # 261, 262, 264, 269, 272, and 273 all have the LoxP on both alleles of the target gene (homozygous deletion) deletion making them suitable candidates for further cross-breeding and histological examination As MGH Cancer Center continues to examine and define the biology of PDAC through these methods, steps toward improved drug screening techniques and patient prognosis will be made Personal data helped identify suitable candidates for further transgene breeding and histological study

18 Acknowledgements Alexandros Tzatsos, MD, PhD- team leader, mentor Polina Paskaleva- mentor Nabeel Bardeesy, PhD- principal investigator, Bardeeesy Lab Special Thanks to the Lab Personnel at Bardeesy Lab, MGH Cancer Center

19 References - KDM2B controls tumorigenesis in pancreatic cancer via Polycomb-dependent and interdependent transcriptional programs ; A. Tzatsos, P. Paskaleva, F. Ferrari et al. - Tetracycline-controlled transcriptional regulation systems: advances and application in transgenic animal modeling ; Z.Zhu, T. Zheng, C. Lee et al. ; Cell and Developmental Biology, vol 13, Techniques in Molecular Biology spring 2011, (course) Transfection, Transgenic Knockout, lecture slides Arthur Lambert -Biology of Cancer, fall 2011, (course), RAS cell transduction pathways, lecture slides, Arthur Lambert -Nature.com, Natural Protocols, v6n National Cancer Institute.com, Pancreatic Cancer Big Picture -WHO.int, world health organization Pancreatic Cancer global statistics, pancreatic duct adenocarcinoma resources -Biology of Cancer, (textbook), Robert A. Weinberg, -Cancer.com, American Cancer Society, 'research and explore' resources Image Sources J Gastroentero Hepatol 2008 Buckwell Publishing - -Essential Cell Biology 3/e( Garland Science 2010) - recombination Techniques In Molecular Biology, Spring 2011: Transfection and Transgenic Knockdown Animals Lecture, Arthur Lambert -oceanexplorer.noaa.gov/explorations/04e

Doxycycline-inducible Models Doxycycline: a derivative of Tetracycline, induces a conformational change Tetracycline: an antibiotic produced by bacteria Tet O: tetracycline specific protein

21 Doxycycline-inducible Models Doxycycline: a derivative of Tetracycline, induces a conformational change Tetracycline: an antibiotic produced by bacteria Tet O: tetracycline specific protein (operator), is a synthesis inhibitor when not bound by TA TA: (transcriptional activator) binding protein that controls transcriptional flow Mini CMV: (promoter construct) derived from the human cytomegalovirus Inducible models provide temporal control over transgene expression; some transgenes can be toxic to host organisms during gestation/embryogenesis Tet O Mini CMV Cre Tet O Mini CMV Cre

22 Gene Activation Using Cre-LoxP LoxP Cre recombinase

Quantification of early stage lesions for loss of p53 should be shown in the main figures.

Quantification of early stage lesions for loss of p53 should be shown in the main figures. Reviewer #1 (Remarks to the Author): Expert in prostate cancer The manuscript "Clonal dynamics following p53 loss of heterozygosity in Kras-driven cancers" uses a number of novel genetically engineered