Understanding and targeting network-level sheddase regulation in invasive disease

Thesis (Ph. D.)--Massachusetts Institute of Technology, Dept. of Biological Engineering, 2013.Cataloged from PDF version of thesis.Includes bibliographical references (p. 197-212).Regulated cell-surface proteolysis underpins key processes of cellular growth and motility in both physiological and pathological contexts. However, comprehending how multiple proteolytic events cohesively integrate to yield context-dependent cellular behavior remains a challenge in the fields of both protease biology and systems biology in general. This work begins to address that challenge by quantitatively investigating the integrated effect of multiple diverse proteolytic events and their interaction with cell-signaling pathways from a computational network perspective, particularly focusing on A Disintegrin and Metalloproteinases (ADAMs). ADAMs have been studied for decades as the principal cell-surface "sheddases" responsible for cleaving growth factor ligands and receptor tyrosine kinase ectodomains from the cell surface. However, activity regulation, feedback, and catalytic promiscuity impede our understanding of context-dependent sheddase function, and clinical trials targeting metalloproteinases in cancer have failed in part due to a poor understanding of the complex functions they mediate. This thesis outlines a conceptual framework for studying protease network biology (Chapter 1), describes novel experimental methods designed for such a framework (Chapters 2-3), and applies both to understand protease regulation in invasive disease (Chapter 4). Using combined measurement and computational modeling, we present a paradigm for monitoring and analyzing complex networks of protease activities that interface with signaling pathways to influence cellular migration in the invasive diseases of cancer and endometriosis. We find sheddase activity integrates with signaling pathways to direct cell migration, especially through concomitant proteolysis of both ligands and receptors. We find that indirect reduction of sheddase activity through kinase inhibition can lead to an accumulation of growth-factor receptors on the cell surface, consequently producing undesired compensatory signaling feedback. Thus, here we present a novel mechanism of rapid, protease-driven resistance to kinase inhibitors, and we subsequently demonstrate strategies for overcoming resistance through drug combinations. We develop a novel microfluidic platform to study protease activities in clinical samples, and apply the technology to study the peritoneal fluid from endometriosis patients. Results indicate joint dysregulation of sheddase activity with disease. Overall, this work provides a model for measuring, understanding, and targeting networks of proteases and the kinases with which they interact.by Miles Aaron Miller.Ph.D

The Receptor AXL Diversifies EGFR Signaling and Limits the Response to EGFR-Targeted Inhibitors in Triple-Negative Breast Cancer Cells

The relationship between drug resistance, changes in signaling, and emergence of an invasive phenotype is well appreciated, but the underlying mechanisms are not well understood. Using machine learning analysis applied to the Cancer Cell Line Encyclopedia database, we identified expression of AXL, the gene that encodes the epithelial-to-mesenchymal transition (EMT)–associated receptor tyrosine kinase (RTK) AXL, as exceptionally predictive of lack of response to ErbB family receptor–targeted inhibitors. Activation of EGFR (epidermal growth factor receptor) transactivated AXL, and this ligand-independent AXL activity diversified EGFR-induced signaling into additional downstream pathways beyond those triggered by EGFR alone. AXL-mediated signaling diversification was required for EGF (epidermal growth factor)–elicited motility responses in AXL-positive TNBC (triple-negative breast cancer) cells. Using cross-linking coimmunoprecipitation assays, we determined that AXL associated with EGFR, other ErbB receptor family members, MET (hepatocyte growth factor receptor), and PDGFR (platelet-derived growth factor receptor) but not IGF1R (insulin-like growth factor 1 receptor) or INSR (insulin receptor). From these AXL interaction data, we predicted AXL-mediated signaling synergy for additional RTKs and validated these predictions in cells. This alternative mechanism of receptor activation limits the use of ligand-blocking therapies and indicates against therapy withdrawal after acquired resistance. Further, subadditive interaction between EGFR- and AXL-targeted inhibitors across all AXL-positive TNBC cell lines may indicate that increased abundance of EGFR is principally a means to transactivation-mediated signaling.United States. Dept. of Defense (Congressionally Directed Medical Research Programs, Breast Cancer Research Program (W81XWH-11-1-0088))National Science Foundation (U.S.) (Graduate Research Fellowship)Repligen Corporation (Fellowship in Cancer Research)National Cancer Institute (U.S.). Integrative Cancer Biology Program (1-U54-CA112967)David H. Koch Institute for Integrative Cancer Research at MIT (Frontier Research Program Initiator Award)National Institutes of Health (U.S.) (NIH R01-CA96504

Modular Design of Artificial Tissue Homeostasis: Robust Control through Synthetic Cellular Heterogeneity

Synthetic biology efforts have largely focused on small engineered gene networks, yet understanding how to integrate multiple synthetic modules and interface them with endogenous pathways remains a challenge. Here we present the design, system integration, and analysis of several large scale synthetic gene circuits for artificial tissue homeostasis. Diabetes therapy represents a possible application for engineered homeostasis, where genetically programmed stem cells maintain a steady population of β-cells despite continuous turnover. We develop a new iterative process that incorporates modular design principles with hierarchical performance optimization targeted for environments with uncertainty and incomplete information. We employ theoretical analysis and computational simulations of multicellular reaction/diffusion models to design and understand system behavior, and find that certain features often associated with robustness (e.g., multicellular synchronization and noise attenuation) are actually detrimental for tissue homeostasis. We overcome these problems by engineering a new class of genetic modules for ‘synthetic cellular heterogeneity’ that function to generate beneficial population diversity. We design two such modules (an asynchronous genetic oscillator and a signaling throttle mechanism), demonstrate their capacity for enhancing robust control, and provide guidance for experimental implementation with various computational techniques. We found that designing modules for synthetic heterogeneity can be complex, and in general requires a framework for non-linear and multifactorial analysis. Consequently, we adapt a ‘phenotypic sensitivity analysis’ method to determine how functional module behaviors combine to achieve optimal system performance. We ultimately combine this analysis with Bayesian network inference to extract critical, causal relationships between a module's biochemical rate-constants, its high level functional behavior in isolation, and its impact on overall system performance once integrated.National Institutes of Health (U.S.) (NIH NIGMS grant R01GM086881)National Science Foundation (U.S.) (NSF Award #1001092)National Science Foundation (U.S.) (NSF Graduate Research Fellowship Program)Swiss National Science Foundation (SystemsX.ch grant

MENA Confers Resistance to Paclitaxel in Triple-Negative Breast Cancer

Taxane therapy remains the standard of care for triple-negative breast cancer. However, high frequencies of recurrence and progression in treated patients indicate that metastatic breast cancer cells can acquire resistance to this drug. The actin regulatory protein MENA and particularly its invasive isoform, MENAINV , are established drivers of metastasis. MENAINV expression is significantly correlated with metastasis and poor outcome in human patients with breast cancer. We investigated whether MENA isoforms might play a role in driving resistance to chemotherapeutics. We find that both MENA and MENAINV confer resistance to the taxane paclitaxel, but not to the widely used DNA-damaging agents doxorubicin or cisplatin. Furthermore, paclitaxel treatment does not attenuate growth of MENAINV -driven metastatic lesions. Mechanistically, MENA isoform expression alters the ratio of dynamic and stable microtubule populations in paclitaxel-treated cells. MENA expression also increases MAPK signaling in response to paclitaxel treatment. Decreasing ERK phosphorylation by cotreatment with MEK inhibitor restored paclitaxel sensitivity by driving microtubule stabilization in MENA isoform-expressing cells. Our results reveal a novel mechanism of taxane resistance in highly metastatic breast cancer cells and identify a combination therapy to overcome such resistance

Tumour-associated macrophages act as a slow-release reservoir of nano-therapeutic Pt(IV) pro-drug

Therapeutic nanoparticles (TNPs) aim to deliver drugs more safely and effectively to cancers, yet clinical results have been unpredictable owing to limited in vivo understanding. Here we use single-cell imaging of intratumoral TNP pharmacokinetics and pharmacodynamics to better comprehend their heterogeneous behaviour. Model TNPs comprising a fluorescent platinum(IV) pro-drug and a clinically tested polymer platform (PLGA-b-PEG) promote long drug circulation and alter accumulation by directing cellular uptake toward tumour-associated macrophages (TAMs). Simultaneous imaging of TNP vehicle, its drug payload and single-cell DNA damage response reveals that TAMs serve as a local drug depot that accumulates significant vehicle from which DNA-damaging Pt payload gradually releases to neighbouring tumour cells. Correspondingly, TAM depletion reduces intratumoral TNP accumulation and efficacy. Thus, nanotherapeutics co-opt TAMs for drug delivery, which has implications for TNP design and for selecting patients into trials.National Cancer Institute (U.S.) (Grant RO1-CA034992

Phase 1 Trial of Malaria Transmission Blocking Vaccine Candidates Pfs25 and Pvs25 Formulated with Montanide ISA 51

Pfs25 and Pvs25, surface proteins of mosquito stage of the malaria parasites P. falciparum and P. vivax, respectively, are leading candidates for vaccines preventing malaria transmission by mosquitoes. This single blinded, dose escalating, controlled Phase 1 study assessed the safety and immunogenicity of recombinant Pfs25 and Pvs25 formulated with Montanide ISA 51, a water-in-oil emulsion.The trial was conducted at The Johns Hopkins Center for Immunization Research, Washington DC, USA, between May 16, 2005-April 30, 2007. The trial was designed to enroll 72 healthy male and non-pregnant female volunteers into 1 group to receive adjuvant control and 6 groups to receive escalating doses of the vaccines. Due to unexpected reactogenicity, the vaccination was halted and only 36 volunteers were enrolled into 4 groups: 3 groups of 10 volunteers each were immunized with 5 microg of Pfs25/ISA 51, 5 microg of Pvs25/ISA 51, or 20 microg of Pvs25/ISA 51, respectively. A fourth group of 6 volunteers received adjuvant control (PBS/ISA 51). Frequent local reactogenicity was observed. Systemic adverse events included two cases of erythema nodosum considered to be probably related to the combination of the antigen and the adjuvant. Significant antibody responses were detected in volunteers who completed the lowest scheduled doses of Pfs25/ISA 51. Serum anti-Pfs25 levels correlated with transmission blocking activity.It is feasible to induce transmission blocking immunity in humans using the Pfs25/ISA 51 vaccine, but these vaccines are unexpectedly reactogenic for further development. This is the first report that the formulation is associated with systemic adverse events including erythema nodosum.ClinicalTrials.gov NCT00295581

Phase 1 Study of Two Merozoite Surface Protein 1 (MSP1(42)) Vaccines for Plasmodium falciparum Malaria

OBJECTIVES: To assess the safety and immunogenicity of two vaccines, MSP1(42)-FVO/Alhydrogel and MSP1(42)-3D7/Alhydrogel, targeting blood-stage Plasmodium falciparum parasites. DESIGN: A Phase 1 open-label, dose-escalating study. SETTING: Quintiles Phase 1 Services, Lenexa, Kansas between July 2004 and November 2005. PARTICIPANTS: Sixty healthy malaria-naïve volunteers 18–48 y of age. INTERVENTIONS: The C-terminal 42-kDa region of merozoite surface protein 1 (MSP1(42)) corresponding to the two allelic forms present in FVO and 3D7 P. falciparum lines were expressed in Escherichia coli, refolded, purified, and formulated on Alhydrogel (aluminum hydroxide). For each vaccine, volunteers in each of three dose cohorts (5, 20, and 80 μg) were vaccinated at 0, 28, and 180 d. Volunteers were followed for 1 y. OUTCOME MEASURES: The safety of MSP1(42)-FVO/Alhydrogel and MSP1(42)-3D7/Alhydrogel was assessed. The antibody response to each vaccine was measured by reactivity to homologous and heterologous MSP1(42), MSP1(19), and MSP1(33) recombinant proteins and recognition of FVO and 3D7 parasites. RESULTS: Anti-MSP1(42) antibodies were detected by ELISA in 20/27 (74%) and 22/27 (81%) volunteers receiving three vaccinations of MSP1(42)-FVO/Alhydrogel or MSP1(42)-3D7/Alhydrogel, respectively. Regardless of the vaccine, the antibodies were cross-reactive to both MSP1(42)-FVO and MSP1(42)-3D7 proteins. The majority of the antibody response targeted the C-terminal 19-kDa domain of MSP1(42), although low-level antibodies to the N-terminal 33-kDa domain of MSP1(42) were also detected. Immunofluorescence microscopy of sera from the volunteers demonstrated reactivity with both FVO and 3D7 P. falciparum schizonts and free merozoites. Minimal in vitro growth inhibition of FVO or 3D7 parasites by purified IgG from the sera of the vaccinees was observed. CONCLUSIONS: The MSP1(42)/Alhydrogel vaccines were safe and well tolerated but not sufficiently immunogenic to generate a biologic effect in vitro. Addition of immunostimulants to the Alhydrogel formulation to elicit higher vaccine-induced responses in humans may be required for an effective vaccine

A communal catalogue reveals Earth's multiscale microbial diversity

Our growing awareness of the microbial world's importance and diversity contrasts starkly with our limited understanding of its fundamental structure. Despite recent advances in DNA sequencing, a lack of standardized protocols and common analytical frameworks impedes comparisons among studies, hindering the development of global inferences about microbial life on Earth. Here we present a meta-analysis of microbial community samples collected by hundreds of researchers for the Earth Microbiome Project. Coordinated protocols and new analytical methods, particularly the use of exact sequences instead of clustered operational taxonomic units, enable bacterial and archaeal ribosomal RNA gene sequences to be followed across multiple studies and allow us to explore patterns of diversity at an unprecedented scale. The result is both a reference database giving global context to DNA sequence data and a framework for incorporating data from future studies, fostering increasingly complete characterization of Earth's microbial diversity.Peer reviewe

A communal catalogue reveals Earth’s multiscale microbial diversity

Our growing awareness of the microbial world’s importance and diversity contrasts starkly with our limited understanding of its fundamental structure. Despite recent advances in DNA sequencing, a lack of standardized protocols and common analytical frameworks impedes comparisons among studies, hindering the development of global inferences about microbial life on Earth. Here we present a meta-analysis of microbial community samples collected by hundreds of researchers for the Earth Microbiome Project. Coordinated protocols and new analytical methods, particularly the use of exact sequences instead of clustered operational taxonomic units, enable bacterial and archaeal ribosomal RNA gene sequences to be followed across multiple studies and allow us to explore patterns of diversity at an unprecedented scale. The result is both a reference database giving global context to DNA sequence data and a framework for incorporating data from future studies, fostering increasingly complete characterization of Earth’s microbial diversity

Search for dark matter produced in association with bottom or top quarks in √s = 13 TeV pp collisions with the ATLAS detector

A search for weakly interacting massive particle dark matter produced in association with bottom or top quarks is presented. Final states containing third-generation quarks and miss- ing transverse momentum are considered. The analysis uses 36.1 fb−1 of proton–proton collision data recorded by the ATLAS experiment at √s = 13 TeV in 2015 and 2016. No significant excess of events above the estimated backgrounds is observed. The results are in- terpreted in the framework of simplified models of spin-0 dark-matter mediators. For colour- neutral spin-0 mediators produced in association with top quarks and decaying into a pair of dark-matter particles, mediator masses below 50 GeV are excluded assuming a dark-matter candidate mass of 1 GeV and unitary couplings. For scalar and pseudoscalar mediators produced in association with bottom quarks, the search sets limits on the production cross- section of 300 times the predicted rate for mediators with masses between 10 and 50 GeV and assuming a dark-matter mass of 1 GeV and unitary coupling. Constraints on colour- charged scalar simplified models are also presented. Assuming a dark-matter particle mass of 35 GeV, mediator particles with mass below 1.1 TeV are excluded for couplings yielding a dark-matter relic density consistent with measurements