Ashley Smith

The Bruton tyrosine kinase inhibitor PCI-32765 blocks B-cell activation and is efficacious in models of autoimmune disease and B-cell malignancy

Activation of the B-cell antigen receptor (BCR) signaling pathway contributes to the initiation and maintenance of B-cell malignancies and autoimmune diseases. The Bruton tyrosine kinase (Btk) is specifically required for BCR signaling as demonstrated by human and mouse mutations that disrupt Btk function and prevent B-cell maturation at steps that require a functional BCR pathway. Herein we describe a selective and irreversible Btk inhibitor, PCI-32765, that is currently under clinical development in patients with B-cell non-Hodgkin lymphoma. We have used this inhibitor to investigate the biologic effects of Btk inhibition on mature B-cell function and the progression of B cell-associated diseases in vivo. PCI-32765 blocked BCR signaling in human peripheral B cells at concentrations that did not affect T cell receptor signaling. In mice with collagen-induced arthritis, orally administered PCI-32765 reduced the level of circulating autoantibodies and completely suppressed disease. PCI-32765 also inhibited autoantibody production and the development of kidney disease in the MRL-Fas(lpr) lupus model. Occupancy of the Btk active site by PCI-32765 was monitored in vitro and in vivo using a fluorescent affinity probe for Btk. Active site occupancy of Btk was tightly correlated with the blockade of BCR signaling and in vivo efficacy. Finally, PCI-32765 induced objective clinical responses in dogs with spontaneous B-cell non-Hodgkin lymphoma. These findings support Btk inhibition as a therapeutic approach for the treatment of human diseases associated with activation of the BCR pathway.

Identification of Substrates of Human Protein-tyrosine Phosphatase PTPN22

Stimulation of mature T cells activates a downstream signaling cascade involving temporally and spatially regulated phosphorylation and dephosphorylation events mediated by protein-tyrosine kinases and phosphatases, respectively. PTPN22 (Lyp), a non-receptor protein-tyrosine phosphatase, is expressed exclusively in cells of hematopoietic origin, notably in T cells where it represses signaling through the T cell receptor. We used substrate trapping coupled with mass spectrometry-based peptide identification in an unbiased approach to identify physiological substrates of PTPN22. Several potential substrates were identified in lysates from pervanadate-stimulated Jurkat cells using PTPN22-D195A/C227S, an optimized substrate trap mutant of PTPN22. These included three novel PTPN22 substrates (Vav, CD3ϵ, and valosin containing protein) and two known substrates of PEP, the mouse homolog of PTPN22 (Lck and Zap70). T cell antigen receptor (TCR) ζ was also identified as a potential substrate in Jurkat lysates by direct immunoblotting. In vitro experiments with purified recombinant proteins demonstrated that PTPN22-D195A/C227S interacted directly with activated Lck, Zap70, and TCRζ, confirming the initial substrate trap results. Native PTPN22 dephosphorylated Lck and Zap70 at their activating tyrosine residues Tyr-394 and Tyr-493, respectively, but not at the regulatory tyrosines Tyr-505 (Lck) or Tyr-319 (Zap70). Native PTPN22 also dephosphorylated TCRζ in vitro and in cells, and its substrate trap variant co-immunoprecipitated with TCRζ when both were coexpressed in 293T cells, establishing TCRζ as a direct substrate of PTPN22.

Inhibition of IgE-mediated secretion from human basophils with a highly selective Bruton's tyrosine kinase, Btk, inhibitor.

The study of receptor-mediated signaling in human basophils is often limited by the availability of selective pharmacological agents. The early signaling reaction mediated by FcεRI aggregation is thought to require the activity of Brutons tyrosine kinase (btk), an enzyme that has been identified as important in B cells signaling because mutations lead to X-linked agammaglobulinemia. This study uses the btk selective irreversible inhibitor, PCI-32765, to explore the role of btk in a variety of functions associated with the activation of human basophils. Nine endpoints of basophil activation were examined: induced cell surface expression of CD63, CD203c, CD11b; induced secretion of histamine, LTC4, IL-4 and IL-13; the cytosolic calcium response; and the induced loss of syk kinase. Four stimuli were examined; anti-IgE antibody, formyl-met-leu-phe (FMLP), C5a and IL-3. For stimulation with anti-IgE, PCI-32765 inhibited CD63, histamine, LTC4 and IL-4 secretion with an IC50 of 3-6 nM (with 100% inhibition at 50 nM) and it inhibited CD203c and CD11b and the cytosolic calcium response with and IC50 of 30-40 nM. Fifty percent occupancy of btk with PCI-32765 occurred at ~10nM. Consistent with btk functioning downstream or in parallel to syk activation, PCI-32765 did not inhibit the loss of syk induced by anti-IgE in overnight cultures. Finally, PCI-32765 did not significantly inhibit basophil activation by FMLP or C5a and did not inhibit IL-13 release induced by IL-3. These results suggest that btk is specifically required for IgE-mediated activation of human basophils.

Abstract 4108: In vitro vascularized model for tumor growth and progression

Introduction: Tumor metastasis is the primary reason for mortality of cancer patients. The ability to model the microenvironment of the primary tumor and the secondary and tertiary sites is critical for advancing the treatment options for the invasive cancers. There is currently no 3D tumor model that mimics the in vivo vascular geometry and the microenvironment for monitoring progression of tumors. In this study, we report on the development of a 3D tumor model comprising of vascular cells in communication with tumor cells leading to invasion and metastasis at secondary and tertiary sites. Materials and Methods: Vascularized tumor networks comprising primary, secondary and tertiary tumor sites were developed using in vivo images and fabricated using soft lithography. Human mammary microvascular endothelial cells (hMMEC) were cultured in the vascular channels while a GFP-labeled metastatic breast cancer cell (MDA-MB-231) and a GFP-labeled non-metastatic cell (MCF-7) mixed with and without human fibroblast cell line BJ-5ta was cultured in the primary tumor site in a 3D environment using Matrigel™. The tumor networks were perfused with endothelial cell media and the growth, migration, invasion and metastasis of the tumor cells from the primary site to secondary and tertiary site was monitored for 28 days using time lapse microscopy. Results and Discussion: The aggressively metastatic MDA-MB-231/BJ-5ta tumor at the primary site was found to proliferate rapidly resulting in breakdown of the Matrigel and invasion across the endothelial cells. Secondary sites were localized with pockets of tumor colonies within 48 hours and by 120 hours had metastasized to the tertiary site. By 14 days, the primary site tumor formed a necrotic core while the tumor cells at secondary and tertiary were viable. In contrast, the non-aggressive MCF-7/BJ-5ta tumors were able to proliferate within the Matrigel scaffolding but did not break down beyond the primary site until 14 days. Culture of tumor cells without the presence of fibroblast or endothelial cells resulted in a significant difference in the invasion and metastasis pattern highlighting the importance of the native tumor microenvironment. Conclusions: We have developed a 3D, heterogenic model of invasive tumor growth and metastasis which closely mimics the in vivo microenvironment of solid tumors. This model can be used to investigate tumor progression and their underlying mechanisms using a combination of real-time techniques as well as ‘omic’ methodologies for screening and evaluation of the therapeutics. Acknowledgements: We gratefully acknowledge financial support from NIH (#HHSN261201400037C). References: B. Prabhakarpandian et al., J Control Release 2015; 201:49-55 Citation Format: Ashley Smith, Charles Garson, Shantanu Pradhan, Elizabeth Lipke, Robert Arnold, Balabhaskar Prabhakarpandian, Kapil Pant. In vitro vascularized model for tumor growth and progression. [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 4108.

Abstract 620: Microfluidic cancer-on-a-chip platform for assessing anti-cancer drug efficacies

Introduction: The tumor microenvironment is known to play an influential role in the angiogenic and metastatic progression of cancer and is regulated by different factors (stromal fibroblasts, extracellular matrix (ECM) proteins and endothelial cells) present in the complex milieu. Recapitulation of this complexity in three-dimensional (3D) tumor models is critical in understanding the processes involved in cancer progression and to provide clinically relevant efficacy data for cancer drugs. To address this challenge, we developed a microfluidic oncomimetic platform where breast cancer cells are co-cultured with fibroblasts along with a complex, intricate vascular network. We further investigated the effect of mechanical stiffness of the tumor stroma on the growth and morphology of cancer cells, migration of cancer cells into surrounding vasculature and the ability of standard cancer drugs to perfuse through the vasculature and target cancer cells. Materials and Methods: Poly(dimethyl siloxane) (PDMS)-based microfluidic devices, containing vascular networks in communication with tumor chamber, were fabricated using photolithography as described earlier 1 . Poly(ethylene glycol)-fibrinogen (PEG-Fb), used for 3D co-culture of cancer cells and fibroblasts, was prepared using established protocols 2 . Human breast tumor associated endothelial cells (hBTECs) were seeded within fibronectin-coated vascular channels and maintained under flow to develop endothelial networks. To obtain 3D cancer-fibroblast co-culture system, MCF7 or MDA-MB-231 breast cancer cells were co-encapsulated with BJ-5ta human fibroblasts in PEG-Fb hydrogel in the tumor chamber. Immunostaining with standard endothelial and cancer markers was conducted to confirm maturity and functionality of seeded cells. GFP-labelled cancer cells were co-cultured with hBTECs to visualize tumor cell migration. Cancer cells were also maintained in culture over several weeks within the devices to demonstrate applicability of this system to perform long-term drug dosing experiments. Finally, the cytotoxic effects of doxorubicin and paclitaxel at two different concentrations on cancer cells were evaluated by perfusing the drugs through the endothelial channels and cell viability quantified via Live/Dead staining. Conclusions: We have developed a novel 3D microfluidic, vascularized cancer-fibroblast co-culture platform, with the ability to predict drug efficacy for breast cancer. This platform can also be extended in future for cancer-immunotherapy based investigations and screening of novel nano-carrier based anti-cancer drugs. Acknowledgements: We gratefully acknowledge financial support from NIH (#HHSN261201400037C) and AURIC. References: 1. B. Prabhakarpandian et al., J Control Release 2015; 201:49-55 2. L. Almany et al., Biomaterials 2005; 26(15):2467-77 Citation Format: Shantanu Pradhan, Ashley M. Smith, Charles J. Garson, Iman Hassani, Kapil Pant, Robert D. Arnold, Balabhaskar Prabhakarpandian, Elizabeth A. Lipke. Microfluidic cancer-on-a-chip platform for assessing anti-cancer drug efficacies. [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 620.

Abstract 1352: High-throughput matrix screening reveals synergistic chemotherapeutic combinations with blockade of CD47 to enhance cytotoxicity in breast cancer

CD47 is a widely expressed cell surface receptor that serves as a counter-receptor for SIRPα in recognition of self by the innate immune system. On the other hand CD47 also serves as a signaling receptor for the matricellular protein thrombospondin-1 (TSP-1), regulating cell growth and survival. Clinical studies consistently show that increased expression of CD47 is an independent poor prognosis factor in several types of cancer, including breast cancer. Moreover elevated expression of CD47 is associated with development of resistance to chemotherapy by inhibiting cell death pathways. Therefore, agents targeting CD47 are attractive to overcome therapeutic resistance in breast cancer. Using a high-throughput assay in a 1536-well microplate format we screened a comprehensive set of approved and late stage development oncology drugs in combination with an anti-CD47 morpholino to find combinatorial strategies that are more cytotoxic against breast cancer cells. Potency and efficacy measurements during the primary screen identified compounds that synergized with anti-CD47 to augment their cytotoxicity effect. One of the compound groups that demonstrated increased synergism with CD47 were the anthracycline family of drugs. We further validated these results in a syngeneic model of breast cancer and demonstrated that blockade of CD47 in combination with doxorubicin improves chemotherapeutic response when compared to mice administered doxorubicin alone. Furthermore the anti-tumor response with anti-CD47 combination is associated with a reduction in glycolytic flux and a selective up regulation of mitophagy. Overall this indicates that blockade of CD47 in the clinic may synergize with chemotherapeutic strategies to improve patient curative responses. Citation Format: David D. Roberts, Ashley Smith, John M. Sipes, Adam Wilson, Lesley Mathews-Griner, Rajarshi Guha, Craig J. Thomas, Marc Ferrer, David R. Soto-Pantoja. High-throughput matrix screening reveals synergistic chemotherapeutic combinations with blockade of CD47 to enhance cytotoxicity in breast cancer. [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 1352.