Amy Jackson-Fisher

Neural and mammary gland defects in ErbB4 knockout mice genetically rescued from embryonic lethality

Mice lacking the epidermal growth factor receptor family member ErbB4 exhibit defects in cranial neural crest cell migration but die by embryonic day 11 because of defective heart development. To examine later phenotypes, we rescued the heart defects in ErbB4 mutant mice by expressing ErbB4 under a cardiac-specific myosin promoter. Rescued ErbB4 mutant mice reach adulthood and are fertile. However, during pregnancy, mammary lobuloalveoli fail to differentiate correctly and lactation is defective. Rescued mice also display aberrant cranial nerve architecture and increased numbers of large interneurons within the cerebellum.

ErbB3 is required for ductal morphogenesis in the mouse mammary gland.

IntroductionThe receptor ErbB3/HER3 is often over-expressed in human breast cancers, frequently in conjunction with over-expression of the proto-oncogene ERBB2/HER2/NEU. Although the prognostic/predictive value of ErbB3 expression in breast cancer is unclear, ErbB3 is known to contribute to therapeutic resistance. Understanding ErbB3 functions in the normal mammary gland will help to explain its role in cancer etiology and as a modulator of signaling responses to the mammary oncogene ERBB2.MethodsTo investigate the roles of ErbB3 in mouse mammary gland development, we transplanted mammary buds from ErbB3-/- embryos into the cleared mammary fat pads of wild-type immunocompromised mice. Effects on ductal outgrowth were analyzed at 4 weeks, 7 weeks and 20 weeks after transplantation for total ductal outgrowth, branch density, and number and area of terminal end buds. Sections of glands containing terminal end buds were analyzed for number and epithelial area of terminal end buds. Terminal end buds were also analyzed for presence of mitotic figures, apoptotic figures, BrdU incorporation, and expression of E-cadherin, P-cadherin, α-smooth muscle actin, and cleaved caspase-3.ResultsThe mammary ductal trees developed from ErbB3-/- buds only partly filled the mammary fat pad. In contrast to similar experiments with ErbB2-/- mammary buds, this phenotype was maintained through adulthood, pregnancy, and parturition. In addition, and in contrast to similar work with ErbB4-/- mammary buds, lobuloalveolar development of ErbB3-/- transplanted glands was normal. The ErbB3-/- mammary outgrowth defect was associated with a decrease in the size of the terminal end buds, and with increases in branch density, in the number of terminal end buds, and in the number of luminal spaces. Proliferation rates were not affected by the lack of ErbB3, but there was an increase in apoptosis in ErbB3-/- terminal end buds.ConclusionsEndogenous ErbB3 regulates morphogenesis of mammary epithelium.

GLI2 inhibition abrogates human leukemia stem cell dormancy.

BackgroundDormant leukemia stem cells (LSC) promote therapeutic resistance and leukemic progression as a result of unbridled activation of stem cell gene expression programs. Thus, we hypothesized that 1) deregulation of the hedgehog (Hh) stem cell self-renewal and cell cycle regulatory pathway would promote dormant human LSC generation and 2) that PF-04449913, a clinical antagonist of the GLI2 transcriptional activator, smoothened (SMO), would enhance dormant human LSC eradication.MethodsTo test these postulates, whole transcriptome RNA sequencing (RNA-seq), microarray, qRT-PCR, stromal co-culture, confocal fluorescence microscopic, nanoproteomic, serial transplantation and cell cycle analyses were performed on FACS purified normal, chronic phase (CP) chronic myeloid leukemia (CML), blast crisis (BC) phase CML progenitors with or without PF-04449913 treatment.ResultsNotably, RNA-seq analyses revealed that Hh pathway and cell cycle regulatory gene overexpression correlated with leukemic progression. While lentivirally enforced GLI2 expression enhanced leukemic progenitor dormancy in stromal co-cultures, this was not observed with a mutant GLI2 lacking a transactivation domain, suggesting that GLI2 expression prevented cell cycle transit. Selective SMO inhibition with PF-04449913 in humanized stromal co-cultures and LSC xenografts reduced downstream GLI2 protein and cell cycle regulatory gene expression. Moreover, SMO inhibition enhanced cell cycle transit and sensitized BC LSC to tyrosine kinase inhibition in vivo at doses that spare normal HSC.ConclusionIn summary, while GLI2, forms part of a core HH pathway transcriptional regulatory network that promotes human myeloid leukemic progression and dormant LSC generation, selective inhibition with PF-04449913 reduces the dormant LSC burden thereby providing a strong rationale for clinical trials predicated on SMO inhibition in combination with TKIs or chemotherapeutic agents with the ultimate aim of obviating leukemic therapeutic resistance, persistence and progression.

Antitumor Efficacy of the Dual PI3K/mTOR Inhibitor PF-04691502 in a Human Xenograft Tumor Model Derived from Colorectal Cancer Stem Cells Harboring a PIK3CA Mutation

PIK3CA (phosphoinositide-3-kinase, catalytic, alpha polypeptide) mutations can help predict the antitumor activity of phosphatidylinositol-3-kinase (PI3K)/mammalian target of rapamycin (mTOR) pathway inhibitors in both preclinical and clinical settings. In light of the recent discovery of tumor-initiating cancer stem cells (CSCs) in various tumor types, we developed an in vitro CSC model from xenograft tumors established in mice from a colorectal cancer patient tumor in which the CD133+/EpCAM+ population represented tumor-initiating cells. CD133+/EpCAM+ CSCs were enriched under stem cell culture conditions and formed 3-dimensional tumor spheroids. Tumor spheroid cells exhibited CSC properties, including the capability for differentiation and self-renewal, higher tumorigenic potential and chemo-resistance. Genetic analysis using an OncoCarta™ panel revealed a PIK3CA (H1047R) mutation in these cells. Using a dual PI3K/mTOR inhibitor, PF-04691502, we then showed that blockage of the PI3K/mTOR pathway inhibited the in vitro proliferation of CSCs and in vivo xenograft tumor growth with manageable toxicity. Tumor growth inhibition in mice was accompanied by a significant reduction of phosphorylated Akt (pAKT) (S473), a well-established surrogate biomarker of PI3K/mTOR signaling pathway inhibition. Collectively, our data suggest that PF-04691502 exhibits potent anticancer activity in colorectal cancer by targeting both PIK3CA (H1047R) mutant CSCs and their derivatives. These results may assist in the clinical development of PF-04691502 for the treatment of a subpopulation of colorectal cancer patients with poor outcomes.

Abstract 4504: PF-04449913, a small molecule inhibitor of Hedgehog signaling, is effective in inhibiting tumor growth in preclinical models

Aberrant activation of the Hedgehog (Hh) signaling pathway has been implicated in several human cancers. Mutations in the Patched (PTCH1) gene are responsible for basal cell nevus syndrome, and are commonly found in sporadic basal cell carcinoma and in medulloblastoma. In this study we evaluated PF-04449913, an inhibitor of the Hh signaling pathway, in a Ptch1+/-p53 mouse model of medulloblastoma and in human patient derived xenograft models. Treatment of Ptch1+/-p53+/- or Ptch1+/-p53-/- medulloblastoma allografts with PF-04449913 produced potent dose-dependent inhibition of Hh pathway activity resulting in stable tumor regression. Using Gli1 transcript levels as a surrogate for Hh pathway activity, the pharmacodynamic effects of PF-04449913 were evaluated in medulloblastoma allografts following single dose and multi dose administrations of compound. PF-04449913 treated medulloblastoma allografts had reduced levels of Gli1 gene expression and down regulation of genes linked to the Hh signaling pathway. PF-04449913 was also effective when combined with a chemotherapeutic agent in a colon patient derived xenograft model and a pancreatic patient derived xenograft model, resulting in 63% and 73% tumor growth inhibition respectively. Collectively, our study demonstrates the therapeutic efficacy of a small molecule inhibitor of Hh pathway in preclinical models of multiple cancer types in either single or combination treatments. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 4504. doi:10.1158/1538-7445.AM2011-4504

Abstract 1958: Interrogating hedgehog pathway and smoothened inhibition by PF-04449913 in patient-derived acute myeloid leukemia models

Aberrant activation of the Hedgehog (Hh) signaling pathway has been implicated in a variety of cancers and a small molecule inhibitor of Smoothened (SMO), Vismodegib, has been approved for the treatment of basal cell carcinoma, a disease frequently driven by Hh pathway signaling due to pathway mutations. SMO dependent Hh signaling has also been implicated in models of myeloid leukemia, primarily CML, where genetic loss of SMO or pharmacological inhibition limits disease progression in part through targeting the leukemic stem cell (LSC). Outside of BCR-ABL driven leukemia the role of Hh signaling and impact of SMO inhibition on disease progression and the LSC remains unclear. To explore the role of Hedgehog pathway signaling and interrogate responses to SMO inhibition in AML, a panel of primary AML patient-derived models was utilized to examine responses to PF-04449913, an oral small molecule SMO inhibitor currently in early phase clinical trials targeting myeloid malignancies. AML patient samples were characterized for Hh pathway expression levels and screened for responses to PF-04449913. Ex vivo treatment of AML bone marrow cells showed PF-04449913 was capable of inhibiting Hh pathway activity, reducing expression of key LSC regulators and decreasing populations of cells expressing LSC markers. Use of AML xenotransplant models to assess in vivo responses to PF-04449913 as single agent and in combination with Cytarabine have shown potential for combination efficacy of the two agents in select models suggesting patient selection strategies may be critical for SMO inhibitor-based therapies in AML. Citation Format: Amy Jackson-Fisher, Pamela Whalen, Mark Elliott, Melissa McMahon, Enhong Chen, Xianxian Zheng, Mark Ozeck, Donghui Huang, Paul Lira, Joseph Lee, Cathy Zhang, Justine Lam, Mary Spilker, Shibing Deng, Patrick Lappin, Penny Venne, Cynthia Heinlein, Annelie Schairer, Karen McLachlan, Todd VanArsdale. Interrogating hedgehog pathway and smoothened inhibition by PF-04449913 in patient-derived acute myeloid leukemia models. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 1958. doi:10.1158/1538-7445.AM2014-1958

Gemtuzumab Ozogamicin (GO) Inclusion to Induction Chemotherapy Eliminates Leukemic Initiating Cells and Significantly Improves Survival in Mouse Models of Acute Myeloid Leukemia

Gemtuzumab ozogamicin (GO) is an anti-CD33 antibody-drug conjugate for the treatment of acute myeloid leukemia (AML). Although GO shows a narrow therapeutic window in early clinical studies, recent reports detailing a modified dosing regimen of GO can be safely combined with induction chemotherapy, and the combination provides significant survival benefits in AML patients. Here we tested whether the survival benefits seen with the combination arise from the enhanced reduction of chemoresidual disease and leukemic initiating cells (LICs). Herein, we use cell line and patient-derived xenograft (PDX) AML models to evaluate the combination of GO with daunorubicin and cytarabine (DA) induction chemotherapy on AML blast growth and animal survival. DA chemotherapy and GO as separate treatments reduced AML burden but left significant chemoresidual disease in multiple AML models. The combination of GO and DA chemotherapy eliminated nearly all AML burden and extended overall survival. In two small subsets of AML models, chemoresidual disease following DA chemotherapy displayed hallmark markers of leukemic LICs (CLL1 and CD34). In vivo, the two chemoresistant subpopulations (CLL1+/CD117− and CD34+/CD38+) showed higher ability to self-renewal than their counterpart subpopulations, respectively. CD33 was coexpressed in these functional LIC subpopulations. We demonstrate that the GO and DA induction chemotherapy combination more effectively eliminates LICs in AML PDX models than either single agent alone. These data suggest that the survival benefit seen by the combination of GO and induction chemotherapy, nonclinically and clinically, may be attributed to the enhanced reduction of LICs.

Abstract 1220: A novel PTK7-targeted antibody-drug conjugate eliminates tumor-initiating cells and induces sustained tumor regressions

Disease relapse after treatment is common in triple-negative breast cancer (TNBC), ovarian cancer and non-small cell lung cancer (NSCLC). Therapies that target tumor-initiating cells (TICs) should improve patient survival by eliminating the cells that can drive tumor regrowth and metastasis. Here we identify Protein Tyrosine Kinase 7 (PTK7), a highly conserved but catalytically inactive receptor tyrosine kinase, as an antigen that is enriched on TICs in low-passage patient-derived xenografts (PDX) of TNBC, NSCLC and other tumor types. An anti-PTK7 antibody-drug conjugate (ADC) was generated from a humanized anti-PTK7 monoclonal antibody, a cleavable valine-citrulline-based linker and the Aur0101 auristatin microtubule inhibitor. The anti-PTK7 ADC induced sustained regressions of TNBC, NSCLC and ovarian cancer PDX, with improved activity over standard-of-care chemotherapy, and reduced the frequency of TICs as determined by serial transplantation experiments. Moreover, the ADC may have additional mechanisms of action, including an anti-angiogenic effect, that promote anti-tumor immune responses. Together these preclinical results indicate the potential of the anti-PTK7 ADC to improve the long-term survival of cancer patients. The ADC is currently being tested in a Phase 1 clinical trial, from which interim results will be presented. Citation Format: Marc Isaac Damelin, Alex Bankovich, Jeff Bernstein, Justin Lucas, Liang Chen, Sam Williams, Albert Park, Jorge Aguilar, Elana Ernstoff, Manoj Charati, Russell Dushin, Amy Jackson-Fisher, Monette Aujay, Christina Lee, Hanna Ramoth, Milly Milton, Johannes Hampl, Sasha Lazetic, Virginia Pulito, Douglas Armellino, Edward Rosfjord, Magali Guffroy, Hadi Falahatpisheh, Lindsay King, Frank Barletta, Robert Stull, Marybeth Pysz, Paul Escarpe, David Liu, Orit Foord, Brenda Gibson, Eric Powell, Christopher O’Donnell, Xiaohua Xin, Hans Peter Gerber, Puja Sapra, Scott Dylla. A novel PTK7-targeted antibody-drug conjugate eliminates tumor-initiating cells and induces sustained tumor regressions. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 1220.

Abstract 76: Molecular profiling of AML patient derived xenograft models with deep sequencing using a 109 AML associated gene panel and a 409 gene comprehensive cancer panel

Proceedings: AACR 106th Annual Meeting 2015; April 18-22, 2015; Philadelphia, PA We have developed and validated a 109 AML associated gene panel NGS assay based on sequence capture technologies. The overall assay sensitivity was 99.%, and overall assay specificity was 100% when sequenced with reference “gold” standard NA12878. Assay analytical accuracy was evaluated with diluted cell line samples harboring mutations in this gene panel, all diluted mutations with minor allele frequencies were detected, analytical limit of detection (LOD) is < 5%, and assay reproducibility is 96.8%. The assay also demonstrated robustness in sequencing 10 commercial AML patient samples (whole blood and bone marrow) and detecting mutations. Therefore, the AML gene panel deep sequencing assay is sensitive and accurate and is ready to be used for preclinical and clinical studies. We subsequently sequenced 15 AML patient derived xenograft (PDX) models with this assay. These models were also deep sequenced with a 409 comprehensive cancer gene panel based on multiplex amplicon technologies. The mutation profiles of each model will be summarized in this presentation using two sequencing platforms. By analysis of the mutation profile of each model, sub-clones of mutations were revealed in the early passages of these models. Two of these models with FLT-3 mutations were later treated with Quizartinib and Crenolanib separately, the treated mouse models were profiled with these two targeted gene panels; clonal evolution of mutations in these models treated with FLT-3 inhibitors was documented. Deep sequencing with target gene panel is a powerful tool to decipher the mutation sub-clones in the early passages of PDX models, and understand the acquired resistance of drug treatment. Citation Format: Stephen Huang, Paul Lira, Kai Wang, Cathy Zhang, Amy Jackson-Fisher, Keith Ching, Paul Rejto. Molecular profiling of AML patient derived xenograft models with deep sequencing using a 109 AML associated gene panel and a 409 gene comprehensive cancer panel. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 76. doi:10.1158/1538-7445.AM2015-76

Abstract A2-33: Molecular profiling of patient-derived xenograft models across cancers

Patient-Derived Xenograft (PDX) provides important preclinical model for pharmacological testing of oncology drug candidates. Molecular profiling of PDX tumors contributes to many areas of drug discovery from target discovery to development of clinical biomarker hypotheses and clinical trial design. We established a work flow to perform genomic and histopathology analyses of large numbers of PDX tumor models being made available for Pfizer internal research. To date we have generated whole-genome sequencing (WGS), whole-exome sequencing (WES) and whole transcriptome sequencing (RNA-Seq) data on PDX models spanning six cancer types including colon, pancreatic and breast cancers. Bioinformatics pipelines were developed to quantify gene expression and detect genetic alterations including mutation, copy number variations and gene fusions. A controlled evaluation study demonstrated that in silico classification of NGS reads into human/mouse origins is more effective than laboratory-based methods for removing mouse tissue contamination. Comparative analyses of molecular profiles from PDX and primary tumors of the same cancer origins suggest that important patterns of gene expression are retained by PDX models. An integrative genomic classifier was developed using the random forest algorithm, trained on primary tumor data, and shown to identify PDX cancer subtypes with high accuracy. Citation Format: Zhengyan Kan, Edward Rosfjord, James Hardwick, Ying Ding, Xianxian Zheng, Julio Fernandez, Stephanie Shi, Mark Ozeck, Hui Wang, Gabriel Troche, Eric Upeslacis, Amy Jackson-Fisher, Keith Ching, Shibing Deng, Xie Tao, John Chionis, Maruja Lira, Xiaorong Li, Konstantinos Tsaparikos, Patrick Lappin, Pamela Vizcarra, David Shields, Judy Lucas, Paul Rejto. Molecular profiling of patient-derived xenograft models across cancers. [abstract]. In: Proceedings of the AACR Special Conference on Translation of the Cancer Genome; Feb 7-9, 2015; San Francisco, CA. Philadelphia (PA): AACR; Cancer Res 2015;75(22 Suppl 1):Abstract nr A2-33.