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Lessons Learned in T1 Research: mouse to human PowerPoint Presentation
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Lessons Learned in T1 Research: mouse to human

Lessons Learned in T1 Research: mouse to human

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Lessons Learned in T1 Research: mouse to human

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    Slide 1:Lessons Learned in T1 Research: mouse to human

    Jean Y. Tang MD PhD Assistant Professor Department of Dermatology

    Slide 2:Mechanism of disease

    In vitro experiments Animal studies Human clinical trials Epidemiological studies New drugs

    Slide 3:Lessons learned: challenges

    Jack of all trades, Master of none Journals Conferences Students and trainees Not that many role models find a true believer and the experts Slower time to publication Grants: enthusiasm from NIH

    Slide 4:KL2/K23 Mentors: Ervin Epstein, Childrens Hospital Oakland Research Institute Mary-Margaret Chren, Dept of Dermatology, UCSF Charles McCulloch, Steve Cummings, Dept of Epidemiology and Biostatistics, UCSF

    Clinical trials in PTCH1 +/- BCNS patients Observational studies at Kaiser/UCSF Screen for drugs in cell lines and Ptch1+/- mice Translational Research in BCC

    Slide 6:Summary of chemopreventive agents against BCCs

    Needs to be repeated: vit D ; biomarker for K23 Needs to be repeated: vit D ; biomarker for K23

    Slide 7:Needs to be repeated: vit D ; biomarker for K23 Needs to be repeated: vit D ; biomarker for K23

    Slide 8:Epidemiology of BCC

    1 million BCC cases per yr in US Estimated annual incidence of 0.1% to 0.5% Rare risk of metastasis: < 0.5% 5th most costly cancer for Medicare The age-adjusted incidence per 100,000 white individuals: 475 cases in men, 250 cases in women The estimated lifetime risk of BCC in the white population is 33-39% in men and 23-28% in women. Risk of second BCC: 44% in 3 yr

    Slide 9:BCC basic science: Almost all BCCs have mutations in PTCH1 tumor suppressor gene All BCCs have increased Hedgehog signaling

    The pivotal molecular abnormality in BCC carcinogenesis is inappropriate activation of the hedgehog (HH) signaling pathway [17]. Essentially all BCC tumors have an overactive HH signaling pathway, commonly occurring by mutational inactivation of the tumor suppressor Patched1 (PTCH1) geneThe pivotal molecular abnormality in BCC carcinogenesis is inappropriate activation of the hedgehog (HH) signaling pathway [17]. Essentially all BCC tumors have an overactive HH signaling pathway, commonly occurring by mutational inactivation of the tumor suppressor Patched1 (PTCH1) gene

    Slide 10:Hedgehog signaling pathway regulates cell proliferation and growth

    This developmental pathway is best known for its role in embryonic patterning and hair follicle development [18]. In adult cells, genes responsible for cell proliferation, growth, and invasion are activated when the HH pathway is on. Ptch1 is a transmembrane receptor for the HH ligand, and is the key inhibitor of HH signaling. Ptch1 inhibits HH target genes by repressing Smoothened (Smo) rendering downstream Gli transcription factors inactive. The HH ligand binds to the Ptch receptor, thus relieving inhibition of Smo and activating Gli transcription factors. Most BCC tumors have inactivating mutations in the PTCH1 gene that cause Smo signaling to be constitutively activated [21]. Gli transcription factors are then continuously active and increase the expression of HH target genes. The key biomarker for the HH pathway is Gli mRNA levels. All BCC tumors have increased Gli levels, and molecularly targeted drugs against BCC have focused on decreasing the HH pathway and decreasing Gli mRNA [22]. One such example is cyclopamine, a plant alkaloid that inhibits Smo [23]. Model systems (in vitro and in vivo) showed that cyclopamine effectively inhibited BCCs [24], but clinical applications of cyclopamine showed severe side effects that would preclude its use as a chemopreventive agent for BCC This developmental pathway is best known for its role in embryonic patterning and hair follicle development [18]. In adult cells, genes responsible for cell proliferation, growth, and invasion are activated when the HH pathway is on. Ptch1 is a transmembrane receptor for the HH ligand, and is the key inhibitor of HH signaling. Ptch1 inhibits HH target genes by repressing Smoothened (Smo) rendering downstream Gli transcription factors inactive. The HH ligand binds to the Ptch receptor, thus relieving inhibition of Smo and activating Gli transcription factors. Most BCC tumors have inactivating mutations in the PTCH1 gene that cause Smo signaling to be constitutively activated [21]. Gli transcription factors are then continuously active and increase the expression of HH target genes. The key biomarker for the HH pathway is Gli mRNA levels. All BCC tumors have increased Gli levels, and molecularly targeted drugs against BCC have focused on decreasing the HH pathway and decreasing Gli mRNA [22]. One such example is cyclopamine, a plant alkaloid that inhibits Smo [23]. Model systems (in vitro and in vivo) showed that cyclopamine effectively inhibited BCCs [24], but clinical applications of cyclopamine showed severe side effects that would preclude its use as a chemopreventive agent for BCC

    Slide 11:Basal cell nevus syndrome

    Basal cell nevus syndrome are PTCH1+/- Humans with the rare autosomal domin dis BCNS inherit a defective copy of PTCH1. these pts have multiple tens to hundreds of BCCs. And characteristic develop abln include palmar pits, frontal bossing, jaw cysts, abn calcification. Inc risk for extracutan tumors medullobl and rhab. See CPC by Erv 2008 NEJM. 120 mutations identified, majority lead to premature stop codon. BCCs in BCNS pts 60% on sun exposued sites vs 85% of BCCs in nonsyndromic pts. Number of BCCs not related to lifetime sun exposure (Giovanna 1993). Humans with the rare autosomal domin dis BCNS inherit a defective copy of PTCH1. these pts have multiple tens to hundreds of BCCs. And characteristic develop abln include palmar pits, frontal bossing, jaw cysts, abn calcification. Inc risk for extracutan tumors medullobl and rhab. See CPC by Erv 2008 NEJM. 120 mutations identified, majority lead to premature stop codon. BCCs in BCNS pts 60% on sun exposued sites vs 85% of BCCs in nonsyndromic pts. Number of BCCs not related to lifetime sun exposure (Giovanna 1993).

    Slide 12:Ptch1+/- mice mimic BCNS phenotype: develop BCC tumors after IR or UV treatment Goodrich and Scott, Science 1997 Aszterbaum, Oro, Scott, Epstein Nature Medicine 1999

    Slide 13:Mechanism of disease

    In vitro experiments Animal models Human clinical trials Hedgehog pathway BCC cell lines Ptch1 +/- mice PTCH1+/- Basal cell nevus syndrome patients Roadmap for finding new therapeutics Epidemiological studies Patients with sporadic BCCs Elements of translational research. Necessary steps to developing new drugs/therapeutics. Why BCCs are a great model to study molecularly targeted therapeutics. All you had to do was shut down the HH pathway. Why I was drawn to studying BCCs and the Epstein lab. Elements of translational research. Necessary steps to developing new drugs/therapeutics. Why BCCs are a great model to study molecularly targeted therapeutics. All you had to do was shut down the HH pathway. Why I was drawn to studying BCCs and the Epstein lab.

    Slide 14:Summary of chemopreventive agents against BCCs

    Needs to be repeated: vit D ; biomarker for K23 Needs to be repeated: vit D ; biomarker for K23

    Genetic deletion of Cox1 or Cox2 decreases microscopic BCCs in Ptch1+/- mice IR-treated Ptch1+/- mice wild type (n=24), deleted for Cox1 (n=12) or for Cox2 (n=6). Mean and SEM. p<0.05 Cox1 and Cox 2 KO: Smithies Cell 1995 * *

    Slide 15:Ptch1 mice were bred with Cox1 and Cox2 ko mice (developed Smithies lab/Taconic). We compared microscopic BCCs in three groups: Ptch1 mice wt cox 1/cox2, Ptch1 mice deleted for Cox1, and Ptch1 mice deleted for COx2. deletion of cox1 or cox2 decreases number of bccs, signif dec BCC burden. Deletion of Cox2 seemed to sign reduce BCCx compare to cox 1. Ptch1 mice were bred with Cox1 and Cox2 ko mice (developed Smithies lab/Taconic). We compared microscopic BCCs in three groups: Ptch1 mice wt cox 1/cox2, Ptch1 mice deleted for Cox1, and Ptch1 mice deleted for COx2. deletion of cox1 or cox2 decreases number of bccs, signif dec BCC burden. Deletion of Cox2 seemed to sign reduce BCCx compare to cox 1.

    Celecoxib decreases microscropic BCC burden in Ptch1+/- mice (p<0.05)

    Slide 16:no dose response; not as dramatic at genetic deletion of cox2 enzyno dose response; not as dramatic at genetic deletion of cox2 enzy

    Slide 17:Study design: Phase II randomized, double-blinded, placebo controlled trial Subjects: 60 patients with Basal Cell Nevus Syndrome (BCNS) Treatment: oral Celecoxib at 200mg BID versus placebo for 24 months followed by 12 months of observation Primary endpoint: change in BCC numbers during study periods

    Baseline characteristics of study participants are similar in two groups (mean and SD)

    Slide 19:BCNS subject with low number of BCCs at baseline (<15 tumors)

    BCNS subject with high number of BCCs at baseline (>15 tumors)

    Slide 21:How to analyze BCC development

    Regession technique: Linear mixed models Calcuates a slope or rate (number of BCCs/yr) for each patient Compare percent change in rate of BCCs in placebo and celecoxib groups Accounts for drop-outs Adjust for age, gender, BCC at baseline

    Slide 23:Celecoxib reduces BCC development in subjects with less severe disease (< 15 BCCs)

    Slide 24:Lessons learned: importance of finding a reliable mouse model

    Ptch1+/- mice are a reliable model for testing new anti-BCC agents in humans Moderate effect of celecoxib on BCCs in mice and in BCNS patients Greater effect on tumor size rather than number in both mice and BCNS patients

    Slide 25:Lessons learned: statistics

    Statistics linear mixed models for determining slope of BCCs in RCT Regression models for tumors in mice Go to class Building a database (and managing) Go to class

    Slide 26:Lessons learned:

    Long time to publication Journals and co-authors disagree on whether to present data from mice and clinical trial study together Cancer Prevention Research

    Slide 27:Summary of chemopreventive agents against BCCs

    Needs to be repeated: vit D ; biomarker for K23 Needs to be repeated: vit D ; biomarker for K23

    Cyclopamine Smo Gli

    Slide 28:New and relatively safe agents that decrease Hedgehog signaling

    Corcoran and Scott, Proceedings of the Natl Acad Sci 2006 Bijlsma and Peppelenbosch, PLOS Biology 2007 Statins and vit D are not HH pathway specific, act on other pathways. But vit D and statins are relatively safe to take and tolerable used for many decades. Min tox profile, good as chemopreventive agentsStatins and vit D are not HH pathway specific, act on other pathways. But vit D and statins are relatively safe to take and tolerable used for many decades. Min tox profile, good as chemopreventive agents

    Slide 29:Mechanism of disease

    In vitro experiments Animal models Human clinical trials Statins blocks Hedgehog pathway BCC cell lines Ptch1 +/- mice Epidemiological studies Statin therapy and risk of subsequent BCCs in Kaiser cohort Elements of translational research. Necessary steps to developing new drugs/therapeutics. Why BCCs are a great model to study molecularly targeted therapeutics. All you had to do was shut down the HH pathway. Why I was drawn to studying BCCs and the Epstein lab. Elements of translational research. Necessary steps to developing new drugs/therapeutics. Why BCCs are a great model to study molecularly targeted therapeutics. All you had to do was shut down the HH pathway. Why I was drawn to studying BCCs and the Epstein lab.

    Slide 31:Lessons learned

    Have a good biomarker or target gene (Gli1 mRNA) Have a good way to measure bioavailability (24OHase) Have a reliable cell line

    Vitamin D3 decreases Gli mRNA in BCC cells Topical Vitamin D3 decrease BCC development by 50% (p<0.05)

    Slide 34:BCC tumors from Ptch1+/- K14-Cre-ER2 p53 fl/fl mice stain blue due to -galactosidase activity which is encoded by the lacZ gene that was inserted to replace the wildtype Ptch1 gene in Ptch1+/- mice. BCC tumors from Ptch1+/- K14-Cre-ER2 p53 fl/fl mice stain blue due to -galactosidase activity which is encoded by the lacZ gene that was inserted to replace the wildtype Ptch1 gene in Ptch1+/- mice.

    Topical vitamin D3 decreases BCC development in mice *Adjusted for gender and coat color of mouse

    Slide 35:Cedric: measure oxysterols, vitamin D3 or 25OH?, total cholesterol in skin and tumor treated samples.Cedric: measure oxysterols, vitamin D3 or 25OH?, total cholesterol in skin and tumor treated samples.

    Slide 36:Lessons learned

    Have a good biomarker or target gene (Gli1 mRNA) Have a good way to measure bioavailability (24OHase) Pilot trial of topical and oral vitamin D on human BCC

    Slide 37:Mechanism of disease

    In vitro experiments Animal models Human clinical trials Vit D3 blocks Hedgehog pathway BCC cell lines Ptch1 +/- mice PTCH1+/- Basal cell nevus syndrome patients Epidemiological studies Vit D3 levels in BCC pts Elements of translational research. Necessary steps to developing new drugs/therapeutics. Why BCCs are a great model to study molecularly targeted therapeutics. All you had to do was shut down the HH pathway. Why I was drawn to studying BCCs and the Epstein lab. Elements of translational research. Necessary steps to developing new drugs/therapeutics. Why BCCs are a great model to study molecularly targeted therapeutics. All you had to do was shut down the HH pathway. Why I was drawn to studying BCCs and the Epstein lab.

    Slide 38:Lesson Learned

    Translating from mouse studies to epidemiologic studies (skip the human clinical trial/pilot) Mouse to Epi (Ralph Gonzalez)

    Slide 39: The association of serum vitamin D with skin cancer risk in elderly men Jean Y. Tang1,3, Neeta Parimi2, Angela Wu1,3, John Boscardin1, Meg Chren1,Steven R. Cummings1,2, Ervin Epstein3, and Douglas C. Bauer1,2 1 University of California San Francisco, 2 San Francisco Coordinating Center, California Pacific Medical Center Research Institute, 3 Childrens Hospital Oakland Research Institute

    Slide 41:Table 3 Association of increasing serum 25(OH)D levels with non- melanoma skin cancer * Adjusted for age (continuous variable), BMI (continuous variable), season of blood draw, and clinic site Adjusted for age, BMI, season of blood draw, clinic site, outdoor walking activity (continuous variable), and cigarette smoking (yes/no)

    Slide 43:Summary of chemopreventive agents against BCCs

    Needs to be repeated: vit D ; biomarker for K23 Needs to be repeated: vit D ; biomarker for K23

    Cyclopamine Smo Gli Williams and Wang , Proceedings of the Natl Acad Sci 2003

    Slide 44:By screening a small molec library, Curis identified compounds inhibited Smo and decreased Gli levels in reporter assays. One compounds was formulated into a cream and a second compound was form for oral delivery Shown to inhibit microscopic BCCs in an ex vivo assay Phase II trial failed of topical formulation Minimal effect on BCCs in wildtype humans Did not decrease Gli1 mRNA in BCCs Hh Antagonist cream abandoned By screening a small molec library, Curis identified compounds inhibited Smo and decreased Gli levels in reporter assays. One compounds was formulated into a cream and a second compound was form for oral delivery Shown to inhibit microscopic BCCs in an ex vivo assay Phase II trial failed of topical formulation Minimal effect on BCCs in wildtype humans Did not decrease Gli1 mRNA in BCCs Hh Antagonist cream abandoned

    Slide 45:Placebo cream does not reduce BCC tumors

    Day 0 Day 20 Day 35

    Slide 46:Untreated BCC

    Slide 47:Topical Hh Antagonist BCCs decreases BCC tumors

    Day 0 Day 35 Day 20 No irritation on mice with w/0 BCCsNo irritation on mice with w/0 BCCs

    Slide 48:Placebo treated BCCs increased by 40% in tumor area while BCCs treated with Cur-61414 decreased in size by 40% (p<0.0001) and this correlated with a significant reduction of Gli1 expression in Cur-61414-treated BCCs. We asked whether this dec in BCC size was due to an inhibition of prolifer or induction of apoptosis in BCCs or something else Placebo treated BCCs increased by 40% in tumor area while BCCs treated with Cur-61414 decreased in size by 40% (p<0.0001) and this correlated with a significant reduction of Gli1 expression in Cur-61414-treated BCCs. We asked whether this dec in BCC size was due to an inhibition of prolifer or induction of apoptosis in BCCs or something else

    S Tu Re Re Re Tu Tu S S Tu S Tu S Tu Tu Hh Antagonist cream Placebo Tu Hh Antagonist cream decreases Gli1 mRNA in BCC tumors Lesson: benefits of collaborator

    Slide 50:Lesson: Benefits of UCSF Cancer Center core labs

    Treated BCCs with topical Cur 414 for 4 days and looked for markers of prolifer (ki67 staining) and apoposis (CC3) . We saw intense Ki67 staining, showing highly prolifer cells. Cur 414 treated tumors and marked dec in Did not activate apoptosis howev the caveat is that thse mice were deleted for p53 .Treated BCCs with topical Cur 414 for 4 days and looked for markers of prolifer (ki67 staining) and apoposis (CC3) . We saw intense Ki67 staining, showing highly prolifer cells. Cur 414 treated tumors and marked dec in Did not activate apoptosis howev the caveat is that thse mice were deleted for p53 .

    Slide 51:Topical Cur-414 treated BCC

    Slide 53:Lessons learned: mechanism of disease

    Topical and oral HH antagonists significantly reduce murine BCCs by decreasing tumor proliferation and/or inducing follicular differentiation. We know how to collect tumors, do these assays, get to mechanism of disease in future trials

    Slide 55:Investigator sponsored trial in BCC prevention

    Genentech Hh antagonist GDC 0449: 150mg daily Phase II, placebo controlled RCT in 41 Basal Cell Nevus Syndrome subjects Primary endpoint: change in BCCs at 12 mo and 18 mo

    Cyclopamine Smo Gli

    Slide 56:New and relatively safe agents that decrease Hedgehog signaling

    J Kim and P Beachy, Stanford Statins and vit D are not HH pathway specific, act on other pathways. But vit D and statins are relatively safe to take and tolerable used for many decades. Min tox profile, good as chemopreventive agentsStatins and vit D are not HH pathway specific, act on other pathways. But vit D and statins are relatively safe to take and tolerable used for many decades. Min tox profile, good as chemopreventive agents

    Slide 57:Mechanism of disease

    In vitro experiments Animal models Human clinical trials Small molecule library screen for inhibitors of Hedgehog pathway Cell based assays Ptch1 +/- mice Patients with sporadic BCCs Elements of translational research. Necessary steps to developing new drugs/therapeutics. Why BCCs are a great model to study molecularly targeted therapeutics. All you had to do was shut down the HH pathway. Why I was drawn to studying BCCs and the Epstein lab. Elements of translational research. Necessary steps to developing new drugs/therapeutics. Why BCCs are a great model to study molecularly targeted therapeutics. All you had to do was shut down the HH pathway. Why I was drawn to studying BCCs and the Epstein lab.

    Day 0, BCC A: 10mm Day 4 irritation and necrosis, 11 mm Day 11 residual BCC, 3mm Itraconazole #178

    Slide 59:Cyclodextrin #5460

    Day 0 BCC B (11 mm), BCC D (10 mm), BCC G (9mm) Day 7 BCC B (11 mm), BCC D (10 mm), BCC G (9mm)

    Slide 60:Lessons learned:

    Collaboration with another basic lab Post-doc fellows who are experts at specific assays (5 years) vs training someone new Focus on getting the first pilot clinical trial of itraconazole on human BCCs Measure Gli1mRNA in BCCs Measure Ki67 Paired t-test (at biopsy and at excision) New opportunities/markets

    Slide 61:Lessons learned:

    Easier to translate FDA approved drug or a drug manufactured and tested by Pharma - already have paid the $$$ New agent (vitamin D) investigator pays Efficacy Stability GMP grade for human IND

    Slide 62:Needs to be repeated: vit D ; biomarker for K23 Needs to be repeated: vit D ; biomarker for K23

    Slide 63:Childrens Hospital Oakland Ervin Epstein Po Lin So Tony Zheng Xiao Elana Shpall Angela Wu Kris Chang Yefim Khaimsky UCSF EpiBiostat Charles McCulloch Ralph Gonzalez Steve Hulley Steve Cummings Doug Bauer Neeta Parimi John Boscardin Michael Kohn

    Kaiser Division of Research Maryam Asgari Genentech Fred de Sauvage Tracy Tang Chris Callahan UCSF Derm Meg Chren Dan Bikle Loretta Chan Funding Sources NRCC CTSA KL2 NIAMS K23 Prevent Cancer Foundation American Skin Association