Systems approaches to uncovering the contribution of environment-mediated drug resistance.
M. Creixell, H. Kim, F. Mohammadi, S. R. Peyton, & A. S. Meyer. (2022). Current Opinion in Solid State and Materials Science.
[Abstract]
Abstract: Cancer drug response is heavily influenced by the extracellular matrix (ECM) environment. Despite a clear appreciation that the ECM influences cancer drug response and progression, a unified view of how, where, and when environment-mediated drug resistance contributes to cancer progression has not coalesced. Here, we survey some specific ways in which the ECM contributes to cancer resistance with a focus on how materials development can coincide with systems biology approaches to better understand and perturb this contribution. We argue that part of the reason that environment-mediated resistance remains a perplexing problem is our lack of a wholistic view of the entire range of environments and their impacts on cell behavior. We cover a series of recent experimental and computational tools that will aid exploration of ECM reactions space, and how they might be synergistically integrated.
Dual data and motif clustering improves the modeling and interpretation of phosphoproteomic data.
M. Creixell, & A. S. Meyer. (2022). Cell Reports Methods.
[Abstract]
[Preprint]
Abstract:
Cell signaling is orchestrated in part through a network of protein kinases and phosphatases. Dysregulation of kinase signaling is widespread in diseases such as cancer and is readily targetable through inhibitors. Mass spectrometry-based analysis can provide a global view of kinase regulation but mining these data is complicated by its stochastic coverage of the proteome, measurement of substrates rather than kinases, and the scale of the data. Here, we implement a Dual Data and Motif Clustering (DDMC) strategy that simultaneously clusters peptides into similarly regulated groups based on their variation and their sequence profile. We show that this can help to identify putative upstream kinases and supply more robust clustering. We apply this clustering to clinical proteomic profiling of lung cancer and identify conserved proteomic signatures of tumorigenicity, genetic mutations, and immune infiltration. We propose that DDMC provides a general and flexible clustering strategy for the analysis of phosphoproteomic data.
Integrated proteomics-based physical and functional mapping of AXL kinase signaling pathways and
inhibitors define its role in cell migration.
A. Majumder, S. Hosseinian, M. J. Stroud, E. Adhikari, J. J. Saller, D. M. A. Smith, D. G. Zhang, S. Agarwal, M. Creixell, B. S. Meyer, M. F. Kinose, K. S. Bowers, B. Fang, P. A. Stewart, E. A. Welsh, T. A. Boyle, A. S. Meyer, J. M. Koomen, & E. B. Haura. (2022). Molecular Cancer Research.
[Abstract]
Abstract:
To better understand the signaling complexity of AXL, a member of the TAM receptor tyrosine
kinase family, we created a physical and functional map of AXL signaling interactions,
phosphorylation events, and target-engagement of three AXL tyrosine kinase inhibitors (TKI). We
assessed AXL protein-complexes using BioID, effects of AXL TKI on global phosphoproteins using
mass spectrometry, and target engagement of AXL TKI using activity-based protein profiling. BioID
identifies AXL-interacting proteins that are mostly involved in cell adhesion/migration. Global
phosphoproteomics show that AXL inhibition decreases phosphorylation of peptides involved in
phosphatidylinositol-mediated signaling and cell adhesion/migration. Comparison of three AXL
inhibitors reveals that TKI RXDX-106 inhibits pAXL, pAKT and migration/invasion of these cells
without reducing their viability, while Bemcentinib exerts AXL-independent phenotypic effects on
viability. Proteomic characterization of these TKIs demonstrates that they inhibit diverse
targets in addition to AXL, with Bemcentinib having the most off-targets. AXL and EGFR TKI
co-treatment did not reverse resistance in cell line models of Erlotinib-resistance. However, a
unique vulnerability was identified in one resistant clone, wherein combination of Bemcentinib
and Erlotinib inhibited cell viability and signaling. We also show that AXL is overexpressed in
30-40% of non-small but rarely in small-cell lung cancer. Cell lines have a wide range of AXL
expression, with basal activation detected rarely.
A quantitative view of strategies to engineer cell-selective ligand binding.
Z. C. Tan, B. T. Orcutt-Jahns, & A. S. Meyer. (2021). Integrative Biology.
[Abstract]
[Preprint]
Abstract:
A critical property of many therapies is their selective binding to target populations.
Exceptional specificity can arise from high-affinity binding to surface targets expressed
exclusively on target cell types. In many cases, however, therapeutic targets are only expressed
at subtly different levels relative to off-target cells. More complex binding strategies have
been developed to overcome this limitation, including multi-specific and multivalent molecules,
creating a combinatorial explosion of design possibilities. Guiding strategies for developing
cell-specific binding are critical to employ these tools. Here, we employ a uniquely general
multivalent binding model to dissect multi-ligand and multi-receptor interactions. This model
allows us to analyze and explore a series of mechanisms to engineer cell selectivity, including
mixtures of molecules, affinity adjustments, valency changes, multi-specific molecules, and
ligand competition. Each of these strategies can optimize selectivity in distinct cases, leading
to enhanced selectivity when employed together. The proposed model, therefore, provides a
comprehensive toolkit for the model-driven design of selectively binding therapies.
Tensor-structured decomposition improves systems serology analysis.
Z. C. Tan, M. Murphy, H. S. Alpay, S. D. Taylor, & A. S. Meyer. (2021). Molecular Systems Biology.
[Abstract]
[DOI]
[Preprint]
Abstract:
Systems serology provides a broad view of humoral immunity by profiling both the antigen-binding
and Fc properties of antibodies. These studies contain structured biophysical profiling across
disease-relevant antigen targets, alongside additional measurements made for single antigens or
in an antigen-generic manner. Identifying patterns in these measurements helps guide vaccine and
therapeutic antibody development, improve our understanding of diseases, and discover conserved
regulatory mechanisms. Here, we report that coupled matrix–tensor factorization (CMTF) can reduce
these data into consistent patterns by recognizing the intrinsic structure of these data. We use
measurements from two previous studies of HIV- and SARS-CoV-2-infected subjects as examples. CMTF
outperforms standard methods like principal components analysis in the extent of data reduction
while maintaining equivalent prediction of immune functional responses and disease status. Under
CMTF, model interpretation improves through effective data reduction, separation of the Fc and
antigen-binding effects, and recognition of consistent patterns across individual measurements.
Data reduction also helps make prediction models more replicable. Therefore, we propose that CMTF
is an effective general strategy for data exploration in systems serology.
Multivalency enhances the specificity of Fc-cytokine fusions.
B. Orcutt-Jahns, P. C. Emmel, E. M. Snyder, C. Posner, S. M. Carlson, & A. S. Meyer. (2021). Submitted.
[Abstract]
[Preprint]
Abstract:
The common γ-chain receptor cytokines are promising immune therapies due to their central role in
coordinating the proliferation and activity of various immune cell populations. One of these
cytokines, interleukin (IL)-2, has potential as a therapy in autoimmunity but is limited in
effectiveness by its modest specificity toward regulatory T cells (Tregs). Therapeutic ligands
are often made dimeric as antibody Fc fusions to confer desirable pharmacokinetic benefits, with
under-explored consequences on signaling. Here, we systematically profiled the signaling
responses to a panel of wild type and mutein IL-2 molecules in various Fc fusion configurations.
We used a tensor-structured dimensionality reduction scheme to decompose the responses of each
cell population to each ligand over a range of time points and cytokine concentrations. We found
that dimeric muteins are uniquely specific for Tregs at intermediate ligand concentrations. We
then compared signaling response across all treatments to a simple, two-step multivalent binding
model. Our model was able to predict cellular responses with high accuracy. Bivalent Fc fusions
display enhanced specificity and potency for Tregs through avidity effects toward IL-2Rα. We then
utilize our model to identify the potential benefits conferred by valency engineering as an
additional mechanism for cytokines with optimized therapeutic benefits. In total, these findings
represent a comprehensive analysis of how ligand properties, and their consequent effects on
surface receptor-ligand interactions, translate to selective activation of immune cell
populations. It also identifies a new route toward engineering even more selective therapeutic
cytokines.
A lineage tree-based hidden Markov model to quantify cellular heterogeneity and plasticity.
F. Mohammadi, S. Visagan, S. M. Gross, L. Karginov, J. C. Lagarde, L. M. Heiser, & A. S. Meyer. (2021). Submitted.
[Abstract]
[Preprint]
Abstract:
Cell plasticity, or the ability of cells within a population to reversibly alter their phenotype,
is an important feature of tissue homeostasis during processes such as wound healing and cancer.
Plasticity operates alongside other sources of cell-to-cell heterogeneity such as genetic
mutations and variation in signaling. Ultimately these processes prevent most cancer therapies
from being curative. The predominant methods of quantifying tumor-drug response operate on
snapshot population-level measurements and therefore lack evolutionary dynamics, which are
particularly critical for dynamic processes such as plasticity. Here we apply a tree-based
adaptation of a hidden Markov model (tHMM) that employs single cell lineages as input to learn
the characteristic patterns of single cell heterogeneity and state transitions in an unsupervised
fashion. This model enables single cell classification based on the phenotype of individual cells
and their relatives for improved specificity in pinpointing the structure and dynamics of
variability in drug response. Integrating this model with a modular interface for defining
observed phenotypes allows the model to easily be adapted to any phenotype measured in single
cells. To benchmark our model, we paired cell fate with either cell lifetimes or individual cell
cycle phase lengths (G1 and S/G2) as our observed phenotypes on synthetic data and demonstrated
that the model successfully classifies cells within experimentally tractable dataset sizes. As an
application, we analyzed experimental measurements of cell fate and phase duration in cancer cell
populations treated with chemotherapies to determine the number of distinct subpopulations. In
total, this tHMM framework allows for the flexible classification of single cell heterogeneity
across lineages.
Modeling Cell-Specific Dynamics and Regulation of the Common Gamma Chain Cytokines.
A. M. Farhat, A. C. Weiner, C. Posner, Z. S. Kim, S. M. Carlson, & A. S. Meyer. (2021). Cell Reports.
[Abstract]
[DOI]
[Preprint]
Abstract:
The γ-chain receptor dimerizes with complexes of the cytokines interleukin-2 (IL-2), IL-4, IL-7,
IL-9, IL-15, and IL-21 and their corresponding “private” receptors. These cytokines have existing
uses and future potential as immune therapies because of their ability to regulate the abundance
and function of specific immune cell populations. Here, we build a binding reaction model for the
ligand-receptor interactions of common γ-chain cytokines, which includes receptor trafficking
dynamics, enabling quantitative predictions of cell-type-specific response to natural and
engineered cytokines. We then show that tensor factorization is a powerful tool to visualize
changes in the input-output behavior of the family across time, cell types, ligands, and
concentrations. These results present a more accurate model of ligand response validated across a
panel of immune cell types as well as a general approach for generating interpretable guidelines
for manipulation of cell-type-specific targeting of engineered ligands.
Analysis and modeling of cancer drug responses using cell cycle phase-specific rate effects.
S. M. Gross, F. Mohammadi, C. Sanchez-Aguila, P. J. Zhan, A. S. Meyer, & L. M. Heiser. (2021). BioRxiv.
[Abstract]
[DOI]
[Preprint]
Abstract:
A major challenge to improving outcomes for patients with cancer is the identification of
effective therapeutic strategies that can prevent tumor cell proliferation. Here we sought to
gain a deeper understanding of how anti-cancer agents modulate cell cycle progression in HER2+
breast cancer, a disease subtype that accounts for 20% of all breast cancers. We treated HER2+
breast cancer cells with a panel of drugs and tracked changes in cell number and cell cycle
phase, which revealed drug-specific cell cycle effects that varied across time. This suggested
that a computational model that could account for cell cycle phase durations would provide a
framework to explore drug-induced changes in cell cycle changes. Toward that goal, we developed a
linear chain trick (LCT) computational model, in which the cell cycle is partitioned into
subphases that faithfully captured drug-induced dynamic responses. The model inferred
phase-specific drug effects and independent modulation of cell cycle phases, which we confirmed
experimentally. We then used our LCT model to predict the effect of unseen drug combinations that
target cells in different cell cycle phases. Experimental testing confirmed several model
predictions and identified combination treatment strategies that may improve therapeutic response
in patients with HER2+ breast cancer. Overall, this integrated experimental and modeling approach
opens new avenues for assessing drug responses, predicting effective drug combinations, and
identifying optimal drug sequencing strategies.Competing Interest StatementThe authors have
declared no competing interest.
A general model of multivalent binding with ligands of heterotypic subunits and multiple surface
receptors
.
Z. C. Tan, & A. S. Meyer. (2021). Mathematical Biosciences.
[Abstract]
[DOI]
[Preprint]
Abstract:
Multivalent cell surface receptor binding is a ubiquitous biological phenomenon with functional
and therapeutic significance. Predicting the amount of ligand binding for a cell remains an
important question in computational biology as it can provide great insight into cell-to-cell
communication and rational drug design toward specific targets. In this study, we extend a
mechanistic, two-step multivalent binding model. This model predicts the behavior of a mixture of
different multivalent ligand complexes binding to cells expressing various types of receptors. It
accounts for the combinatorially large number of interactions between multiple ligands and
receptors, optionally allowing a mixture of complexes with different valencies and complexes that
contain heterogeneous ligand units. We derive the macroscopic predictions and demonstrate how
this model enables large-scale predictions on mixture binding and the binding space of a ligand.
This model thus provides an elegant and computationally efficient framework for analyzing
multivalent binding.
Measurement and models accounting for cell death capture hidden variation in compound response.
S. Y. Bae, N. Guan, R. Yan, K. Warner, S. D. Taylor, & A. S. Meyer. (2020). Cell Death & Disease.
[Abstract]
[DOI]
[Preprint]
Abstract:
Cancer cell sensitivity or resistance is almost universally quantified through a direct or
surrogate measure of cell number. However, compound responses can occur through many distinct
phenotypic outcomes, including changes in cell growth, apoptosis, and non-apoptotic cell death.
These outcomes have divergent effects on the tumor microenvironment, immune response, and
resistance mechanisms. Here, we show that quantifying cell viability alone is insufficient to
distinguish between these compound responses. Using an alternative assay and drug-response
analysis amenable to high-throughput measurement, we find that compounds with identical viability
outcomes can have very different effects on cell growth and death. Moreover, additive compound
pairs with distinct growth/death effects can appear synergistic when only assessed by viability.
Overall, these results demonstrate an approach to incorporating measurements of cell death when
characterizing a pharmacologic response.
An engineered human Fc domain that behaves like a pH-toggle switch for ultra-long circulation
persistence
.
C.-H. Lee, T. H. Kang, O. Godon, M. Watanabe, G. Delidakis, C. M. Gillis, D. Sterlin, D. Hardy, M. Cogné, L. E. Macdonald, A. J. Murphy, N. Tu, J. Lee, J. R. McDaniel, E. Makowski, P. M. Tessier, A. S. Meyer, P. Bruhns, & G. Georgiou. (2019). Nature Communications.
[Abstract]
[DOI]
Abstract:
The pharmacokinetic properties of antibodies are largely dictated by the pH-dependent binding of
the IgG fragment crystallizable (Fc) domain to the human neonatal Fc receptor (hFcRn). Engineered
Fc domains that confer a longer circulation half-life by virtue of more favorable pH-dependent
binding to hFcRn are of great therapeutic interest. Here we developed a pH Toggle switch Fc
variant containing the L309D/Q311H/N434S (DHS) substitutions, which exhibits markedly improved
pharmacokinetics relative to both native IgG1 and widely used half-life extension variants, both
in conventional hFcRn transgenic mice and in new knock-in mouse strains. engineered specifically
to recapitulate all the key processes relevant to human antibody persistence in circulation,
namely: (i) physiological expression of hFcRn, (ii) the impact of hFcγRs on antibody clearance
and (iii) the role of competing endogenous IgG. DHS-IgG retains intact effector functions, which
are important for the clearance of target pathogenic cells and also has favorable developability.
Systems biology approaches to measure and model phenotypic heterogeneity in cancer.
A. S. Meyer, & L. M. Heiser. (2019). Current Opinion in Systems Biology.
[Abstract]
[DOI]
Abstract:
The recent wide-spread adoption of single cell profiling technologies has revealed that
individual cancers are not homogenous collections of deregulated cells, but instead are comprised
of multiple genetically and phenotypically distinct cell subpopulations that exhibit a wide range
of responses to extracellular signals and therapeutic insult. Such observations point to the
urgent need to understand cancer as a complex, adaptive system. Cancer systems biology studies
seek to develop the experimental and theoretical methods required to understand how biological
components work together to determine how cancer cells function. Ultimately, such approaches will
lead to improvements in how cancer is managed and treated. In this review, we discuss recent
advances in cancer systems biology approaches to quantify, model, and elucidate mechanisms of
heterogeneity.
Versatile targeting system for lentiviral vectors involving biotinylated targeting molecules.
K. Situ, B. A. Chua, S. Y. Bae, A. S. Meyer, & K. Morizono. (2018). Virology.
[Abstract]
[DOI]
Abstract:
Conjugating certain types of lentiviral vectors with targeting ligands can redirect the vectors
to specifically transduce desired cell types. However, extensive genetic and/or biochemical
manipulations are required for conjugation, which hinders applications for targeting lentiviral
vectors for broader research fields. We developed envelope proteins fused with biotin-binding
molecules to conjugate the pseudotyped vectors with biotinylated targeting molecules by simply
mixing them. The envelope proteins fused with the monomeric, but not tetrameric, biotin-binding
molecules can pseudotype lentiviral vectors and be conjugated with biotinylated targeting
ligands. The conjugation is stable enough to redirect lentiviral transduction in the presence of
serum, indicating their potential in in vivo . When a signaling molecule is conjugated with the
vector, the conjugation facilitates transduction and signaling in a receptor-specific manner.
This simple method of ligand conjugation and ease of obtaining various types of biotinylated
ligands will make targeted lentiviral transduction easily applicable to broad fields of research.
Systems Modeling Identifies Divergent Receptor Tyrosine Kinase Reprogramming to MAPK Pathway
Inhibition
.
A. M. Claas, L. Atta, S. Gordonov, A. S. Meyer, & D. A. Lauffenburger. (2018). Cellular and Molecular Bioengineering.
[Abstract]
[DOI]
Abstract:
Targeted cancer therapeutics have demonstrated more limited clinical efficacy than anticipated,
due to both intrinsic and acquired drug resistance. Underlying mechanisms have been largely
attributed to genetic changes, but a substantial proportion of resistance observations remain
unexplained by genomic properties. Emerging evidence shows that receptor tyrosine kinase (RTK)
reprogramming is a major alternative process causing targeted drug resistance, separate from
genetic alterations. Hence, the contributions of mechanisms leading to this process need to be
more rigorously assessed.
Human induced pluripotent stem cell-derived glial cells and neural progenitors display divergent
responses to Zika and dengue infections
.
J. Muffat, Y. Li, A. Omer, A. Durbin, I. Bosch, G. Bakiasi, E. Richards, A. Meyer, L. Gehrke, & R. Jaenisch. (2018). Proceedings of the National Academy of Sciences.
[Abstract]
[DOI]
Abstract:
Maternal Zika virus (ZIKV) infection during pregnancy is recognized as the cause of an epidemic
of microcephaly and other neurological anomalies in human fetuses. It remains unclear how ZIKV
accesses the highly vulnerable population of neural progenitors of the fetal central nervous
system (CNS), and which cell types of the CNS may be viral reservoirs. In contrast, the related
dengue virus (DENV) does not elicit teratogenicity. To model viral interaction with cells of the
fetal CNS in vitro, we investigated the tropism of ZIKV and DENV for different induced
pluripotent stem cell-derived human cells, with a particular focus on microglia-like cells. We
show that ZIKV infected isogenic neural progenitors, astrocytes, and microglia-like cells
(pMGLs), but was only cytotoxic to neural progenitors. Infected glial cells propagated ZIKV and
maintained ZIKV load over time, leading to viral spread to susceptible cells. DENV triggered
stronger immune responses and could be cleared by neural and glial cells more efficiently. pMGLs,
when cocultured with neural spheroids, invaded the tissue and, when infected with ZIKV, initiated
neural infection. Since microglia derive from primitive macrophages originating in proximity to
the maternal vasculature, they may act as a viral reservoir for ZIKV and establish infection of
the fetal brain. Infection of immature neural stem cells by invading microglia may occur in the
early stages of pregnancy, before angiogenesis in the brain rudiments. Our data are also
consistent with ZIKV and DENV affecting the integrity of the blood–brain barrier, thus allowing
infection of the brain later in life.
Dissecting FcγR Regulation Through a Multivalent Binding Model.
R. A. Robinett, N. Guan, A. Lux, M. Biburger, F. Nimmerjahn, & A. S. Meyer. (2018). Cell Systems.
[Abstract]
[DOI]
Abstract:
Many immune receptors transduce activation across the plasma membrane through their clustering.
With Fcγ receptors (FcγRs), this clustering is driven by binding to antibodies of differing
affinities that are in turn bound to multivalent antigen. As a consequence of this activation
mechanism, accounting for and rationally manipulating immunoglobulin (Ig)G effector function is
complicated by, among other factors, differing affinities between FcγR species and changes in the
valency of antigen binding. In this study, we show that a model of multivalent receptor-ligand
binding can effectively account for the contribution of IgG-FcγR affinity and immune complex
valency. This model in turn enables us to make specific predictions about the effect of immune
complexes of defined composition. In total, these results enable both rational immune complex
design for a desired IgG effector function and the deconvolution of effector function by immune
complexes.
Apoptotic Bodies Elicit Gas6-mediated Migration of AXL-expressing Tumor Cells.
A. Zweemer, C. B. French, J. Mesfin, S. Gordonov, A. S. Meyer, & D. A. Lauffenburger. (2017). Molecular Cancer Research.
[Abstract]
[DOI]
Abstract:
Metastases are a major cause of cancer mortality. AXL, a receptor tyrosine kinase (RTK)
aberrantly expressed in many tumors, is a potent oncogenic driver of metastatic cell motility and
has been identified as broadly relevant in cancer drug resistance. Despite its frequent
association with changes in cancer phenotypes, the precise mechanism leading to AXL activation is
incompletely understood. In addition to its ligand growth arrest specific-6 (Gas6), activation of
AXL requires the lipid moiety phosphatidylserine (PS). PS is only available to mediate AXL
activation when it is externalized on cell membranes, an event that occurs during certain
physiologic processes such as apoptosis. Here it is reported that exposure of cancer cells to
PS-containing vesicles, including synthetic liposomes and apoptotic bodies, contributes to
enhanced migration of tumor cells via a PS-Gas6-AXL signaling axis. These findings suggest that
anti-cancer treatments that induce fractional cell killing enhance the motility of surviving
cells in AXL-expressing tumors, which may explain the widespread role of AXL in limiting
therapeutic efficacy. Implications: This study demonstrates that motility behavior of
AXL-expressing tumor cells can be elicited by Gas6-bearing apoptotic bodies generated from tumor
treatment with therapeutics that produce killing of a portion of the tumor cells present but not
all, hence generating potentially problematic invasive and metastatic behavior of the surviving
tumor cells.
A Biomaterial Screening Approach to Reveal Microenvironmental Mechanisms of Drug Resistance.
A. D. Schwartz, L. E. Barney, L. E. Jansen, T. V. Nguyen, C. L. Hall, A. S. Meyer, & S. Peyton. (2017). Integrative Biology.
[Abstract]
[DOI]
Abstract:
Traditional drug screening methods lack features of the tumor microenvironment that can
contribute to resistance. There remains a gap in whether extracellular signals, such as
stiffness, dimensionality, and cell-cell contacts act independently, or are integrated within a
cell, to affect drug sensitizations or resistance. This is critically important, as adaptive
resistance is mediated, at least in part, by the extracellular matrix (ECM) of the tumor
microenvironment. We developed an approach to screen drug responses in cells cultured on 2D and
in 3D biomaterial environments to explore how key features of ECM mediate drug response. This
approach uncovered that cells on 2D hydrogels and as spheroids encapsulated in 3D hydrogels were
less responsive to receptor tyrosine kinase (RTK)-targeting drugs sorafenib and lapatinib, but
not cytotoxic drugs, compared to single cells in hydrogels and cells on plastic. Transcriptomic
differences between these in vitro models and tumor xenografts did not reveal mechanisms of
ECM-mediated resistance. However, a systems biology analysis of phospho-kinome data suggested
that MEK phosphorylation was associated with RTK-targeted drug resistance. Using sorafenib as a
model drug, we found that co-administration with a MEK inhibitor decreased ECM-mediated
resistance in vitro and reduced in vivo tumor burden compared to sorafenib alone. In sum, we
provide a novel strategy for identifying and overcoming ECM-mediated resistance mechanisms by
performing drug screening, phospho-kinome analysis, and systems biology across multiple
biomaterial environments.
Systems Approaches to Cancer Biology.
T. C. Archer, E. J. Fertig, S. J. C. Gosline, M. Hafner, S. K. Hughes, B. A. Joughin, A. S. Meyer, S. R. Piccolo, & A. N. Shajahan-Haq. (2016). Cancer Research.
[Abstract]
[DOI]
Abstract:
Cancer systems biology aims to understand cancer as an integrated system of genes, proteins,
networks, and interactions rather than an entity of isolated molecular and cellular components.
The inaugural Systems Approaches to Cancer Biology Conference, cosponsored by the Association of
Early Career Cancer Systems Biologists and the National Cancer Institute of the NIH, focused on
the interdisciplinary field of cancer systems biology and the challenging cancer questions that
are best addressed through the combination of experimental and computational analyses. Attendees
found that elucidating the many molecular features of cancer inevitably reveals new forms of
complexity and concluded that ensuring the reproducibility and impact of cancer systems biology
studies will require widespread method and data sharing and, ultimately, the translation of
important findings to the clinic.
JNK Pathway Activation Modulates Acquired Resistance to EGFR/HER2-Targeted Therapies.
S. Manole, E. J. Richards, & A. S. Meyer. (2016). Cancer Research.
[Abstract]
[DOI]
Abstract:
Resistance limits the effectiveness of receptor tyrosine kinase (RTK)-targeted therapies.
Combination therapies targeting resistance mechanisms can considerably improve response, but will
require an improved understanding of when particular combinations will be effective. One common
form of resistance is bypass signaling, wherein RTKs not targeted by an inhibitor can direct
reactivation of pathways essential for survival. Although this mechanism of resistance is well
appreciated, it is unclear which downstream signaling events are responsible. Here, we apply a
combined experimental- and statistical modeling-based approach to identify a set of pathway
reactivation essential for RTK-mediated bypass resistance. Differences in the downstream pathway
activation provided by particular RTKs lead to qualitative differences in the capacity of each
receptor to drive therapeutic resistance. We identify and validate that the JNK pathway is
activated during and strongly modulates bypass resistance. These results identify effective
therapeutic combinations that block bypass-mediated resistance and provide a basic understanding
of this network-level change in kinase dependence that will inform the design of prognostic
assays for identifying effective therapeutic combinations in individual patients.
Reduced Proteolytic Shedding of Receptor Tyrosine Kinases Is a Post-Translational Mechanism of
Kinase Inhibitor Resistance
.
M. A. Miller, M. J. Oudin, R. J. Sullivan, S. J. Wang, A. S. Meyer, H. Im, D. T. Frederick, J. Tadros, L. G. Griffith, H. Lee, R. Weissleder, K. T. Flaherty, F. B. Gertler, & D. A. Lauffenburger. (2016). Cancer Discovery.
[Abstract]
[DOI]
Abstract:
Kinase inhibitor resistance often involves upregulation of poorly understood “bypass” signaling
pathways. Here, we show that extracellular proteomic adaptation is one path to bypass signaling
and drug resistance. Proteolytic shedding of surface receptors, which can provide negative
feedback on signaling activity, is blocked by kinase inhibitor treatment and enhances bypass
signaling. In particular, MEK inhibition broadly decreases shedding of multiple receptor tyrosine
kinases (RTK), including HER4, MET, and most prominently AXL, an ADAM10 and ADAM17 substrate,
thus increasing surface RTK levels and mitogenic signaling. Progression-free survival of patients
with melanoma treated with clinical BRAF/MEK inhibitors inversely correlates with RTK shedding
reduction following treatment, as measured noninvasively in blood plasma. Disrupting protease
inhibition by neutralizing TIMP1 improves MAPK inhibitor efficacy, and combined MAPK/AXL
inhibition synergistically reduces tumor growth and metastasis in xenograft models. Altogether,
extracellular proteomic rewiring through reduced RTK shedding represents a surprising mechanism
for bypass signaling in cancer drug resistance.
A requirement for filopodia extension toward Slit during Robo-mediated axon repulsion.
R. E. McConnell, J. E. V. Veen, M. Vidaki, A. V. Kwiatkowski, A. S. Meyer, & F. B. Gertler. (2016). Journal of Cell Biology.
[Abstract]
[DOI]
Abstract:
Axons navigate long distances through complex 3D environments to interconnect the nervous system
during development. Although the precise spatiotemporal effects of most axon guidance cues remain
poorly characterized, a prevailing model posits that attractive guidance cues stimulate actin
polymerization in neuronal growth cones whereas repulsive cues induce actin disassembly. Contrary
to this model, we find that the repulsive guidance cue Slit stimulates the formation and
elongation of actin-based filopodia from mouse dorsal root ganglion growth cones. Surprisingly,
filopodia form and elongate toward sources of Slit, a response that we find is required for
subsequent axonal repulsion away from Slit. Mechanistically, Slit evokes changes in filopodium
dynamics by increasing direct binding of its receptor, Robo, to members of the actin-regulatory
Ena/VASP family. Perturbing filopodium dynamics pharmacologically or genetically disrupts
Slit-mediated repulsion and produces severe axon guidance defects in vivo. Thus, Slit locally
stimulates directional filopodial extension, a process that is required for subsequent axonal
repulsion downstream of the Robo receptor.
Selectivity in Subunit Composition of Ena/VASP Tetramers.
D. N. Riquelme, A. S. Meyer, M. Barzik, A. Keating, & F. Gertler. (2015). Bioscience Reports.
[Abstract]
[DOI]
Abstract:
The members of the actin regulatory family of Ena/VASP proteins form stable tetramers. The
vertebrate members of the Ena/VASP family, VASP, Mena, and EVL, have many overlapping properties
and expression patterns, but functional and regulatory differences between paralogs have been
observed. The formation of mixed oligomers may serve a regulatory role to refine Ena/VASP
activity. While it has been assumed that family members can form mixed oligomers, this
possibility has not been investigated systematically. Using cells expressing controlled
combinations of VASP, Mena, and EVL, we evaluated the composition of Ena/VASP oligomers and found
that VASP forms oligomers without apparent bias with itself, Mena, or EVL. However, Mena and EVL
showed only weak hetero-oligomerization, suggesting specificity in the association of Ena/VASP
family members. Co-expression of VASP increased the ability of Mena and EVL to form mixed
oligomers. Additionally, we found that the tetramerization domain at the C-termini of Ena/VASP
proteins conferred the observed selectivity. Finally, we demonstrate that replacement of the TD
with a synthetic tetramerizing coiled-coil sequence supports homo-oligomerization and normal VASP
subcellular localization.
Targeting autocrine HB-EGF signaling with specific ADAM12 inhibition using recombinant ADAM12
prodomain
.
M. A. Miller, M. L. Moss, G. Powell, R. Petrovich, L. Edwards, A. S. Meyer, L. G. Griffith, & D. A. Lauffenburger. (2015). Scientific Reports.
[Abstract]
Abstract:
Dysregulation of ErbB-family signaling underlies numerous pathologies and has been
therapeutically targeted through inhibiting ErbB-receptors themselves or their cognate ligands.
For the latter, “decoy” antibodies have been developed to sequester ligands including
heparin-binding epidermal growth factor (HB-EGF); however, demonstrating sufficient efficacy has
been difficult. Here, we hypothesized that this strategy depends on properties such as
ligand-receptor binding affinity, which varies widely across the known ErbB-family ligands.
Guided by computational modeling, we found that high-affinity ligands such as HB-EGF are more
difficult to target with decoy antibodies compared to low-affinity ligands such as amphiregulin
(AREG). To address this issue, we developed an alternative method for inhibiting HB-EGF activity
by targeting its cleavage from the cell surface. In a model of the invasive disease
endometriosis, we identified A Disintegrin and Metalloproteinase 12 (ADAM12) as a protease
implicated in HB-EGF shedding. We designed a specific inhibitor of ADAM12 based on its
recombinant prodomain (PA12), which selectively inhibits ADAM12 but not ADAM10 or ADAM17. In
endometriotic cells, PA12 significantly reduced HB-EGF shedding and resultant cellular migration.
Overall, specific inhibition of ligand shedding represents a possible alternative to decoy
antibodies, especially for ligands such as HB-EGF that exhibit high binding affinity and
localized signaling.
The AXL Receptor Is a Sensor of Ligand Spatial Heterogeneity.
A. S. Meyer, A. J. M. Zweemer, & D. A. Lauffenburger. (2015). Cell Systems. doi: 10.1016/j.cels.2015.06.002
[Abstract]
[DOI]
Abstract:
The AXL receptor is a TAM (Tyro3, AXL, MerTK) receptor tyrosine kinase (RTK) important in
physiological inflammatory processes such as blood clotting, viral infection, and innate
immune-mediated cell clearance. Overexpression of the receptor in a number of solid tumors is
increasingly appreciated as a key drug resistance and tumor dissemination mechanism. Although the
ligand-receptor (Gas6-AXL) complex structure is known, literature reports on ligand-mediated
signaling have provided conflicting conclusions regarding the influence of other factors such as
phosphatidylserine binding, and a detailed, mechanistic picture of AXL activation has not
emerged. Integrating quantitative experiments with mathematical modeling, we show here that AXL
operates to sense local spatial heterogeneity in ligand concentration, a feature consistent with
its physiological role in inflammatory cell responses. This effect arises as a result of an
intricate reaction-diffusion interaction. Our results demonstrate that AXL functions distinctly
from other RTK families, a vital insight for the envisioned design of AXL-targeted therapeutic
intervention.
The Receptor AXL Diversifies EGFR Signaling and Limits the Response to EGFR-Targeted Inhibitors
in Triple-Negative Breast Cancer Cells
.
A. S. Meyer, M. A. Miller, F. B. Gertler, & D. A. Lauffenburger. (2013). Science Signaling.
[Abstract]
[DOI]
Abstract:
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.
ADAM-10 and -17 regulate endometriotic cell migration via concerted ligand and receptor shedding
feedback on kinase signaling.
.
M. A. Miller, A. S. Meyer, M. T. Beste, Z. Lasisi, S. Reddy, K. W. Jeng, C.-H. Chen, J. Han, K. Isaacson, L. G. Griffith, & D. A. Lauffenburger. (2013). Proc Natl Acad Sci U S A.
[Abstract]
[DOI]
Abstract:
A Disintegrin and Metalloproteinases (ADAMs) are the principal enzymes for shedding receptor
tyrosine kinase (RTK) ectodomains and ligands from the cell surface. Multiple layers of activity
regulation, feedback, and catalytic promiscuity impede our understanding of context-dependent
ADAM "sheddase" function and our ability to predictably target that function in disease. This
study uses combined measurement and computational modeling to examine how various growth factor
environments influence sheddase activity and cell migration in the invasive disease of
endometriosis. We find that ADAM-10 and -17 dynamically integrate numerous signaling pathways to
direct cell motility. Data-driven modeling reveals that induced cell migration is a quantitative
function of positive feedback through EGF ligand release and negative feedback through RTK
shedding. Although sheddase inhibition prevents autocrine ligand shedding and resultant EGF
receptor transactivation, it also leads to an accumulation of phosphorylated receptors (HER2,
HER4, and MET) on the cell surface, which subsequently enhances Jnk/p38 signaling. Jnk/p38
inhibition reduces cell migration by blocking sheddase activity while additionally preventing the
compensatory signaling from accumulated RTKs. In contrast, Mek inhibition reduces ADAM-10 and -17
activities but fails to inhibit compensatory signaling from accumulated RTKs, which actually
enhances cell motility in some contexts. Thus, here we present a sheddase-based mechanism of
rapidly acquired resistance to Mek inhibition through reduced RTK shedding that can be overcome
with rationally directed combination inhibitor treatment. We investigate the clinical relevance
of these findings using targeted proteomics of peritoneal fluid from endometriosis patients and
find growth-factor-driven ADAM-10 activity and MET shedding are jointly dysregulated with
disease.
2D protrusion but not motility predicts growth factor–induced cancer cell migration in 3D
collagen
.
A. S. Meyer, S. K. Hughes-Alford, J. E. Kay, A. Castillo, A. Wells, F. B. Gertler, & D. A. Lauffenburger. (2012). The Journal of Cell Biology.
[Abstract]
[DOI]
Abstract:
Growth factor–induced migration is a critical step in the dissemination and metastasis of solid
tumors. Although differences in properties characterizing cell migration on two-dimensional (2D)
substrata versus within three-dimensional (3D) matrices have been noted for particular growth
factor stimuli, the 2D approach remains in more common use as an efficient surrogate, especially
for high-throughput experiments. We therefore were motivated to investigate which migration
properties measured in various 2D assays might be reflective of 3D migratory behavioral
responses. We used human triple-negative breast cancer lines stimulated by a panel of receptor
tyrosine kinase ligands relevant to mammary carcinoma progression. Whereas 2D migration
properties did not correlate well with 3D behavior across multiple growth factors, we found that
increased membrane protrusion elicited by growth factor stimulation did relate robustly to
enhanced 3D migration properties of the MDA-MB-231 and MDA-MB-157 lines. Interestingly, we
observed this to be a more reliable relationship than cognate receptor expression or activation
levels across these and two additional mammary tumor lines.
Fluorinert, an oxygen carrier, improves cell culture performance in deep square 96-well plates by
facilitating oxygen transfer
.
A. Meyer, R. G. G. Condon, G. Keil, N. Jhaveri, Z. Liu, & Y.-S. Tsao. (2012). Biotechnology Progress.
[Abstract]
[DOI]
Abstract:
In bioprocess development, the 96-well plate format has been widely used for high-throughput
screening of production cell line or culture conditions. However, suspension cell cultures in
conventional 96-well plates often fail to reach high cell density under normal agitation
presumably due to constraints in oxygen transfer. Although more vigorous agitation can improve
gas transfer in 96-well plate format, it often requires specialized instruments. In this report,
we employed Fluorinert, a biologically inert perfluorocarbon, to improve oxygen transfer in
96-well plate and to enable the growth of a Chinese Hamster Ovary cell line expressing a
recombinant monoclonal antibody. When different amounts of Fluorinert were added to the cell
culture medium, a dose-dependent improvement in cell growth was observed in both conventional and
deep square 96-well plates. When sufficient Fluorinert was present in the culture, the cell
growth rate, the peak cell density, and recombinant protein production levels achieved in deep
square 96-wells were comparable to cultures in ventilated shake flasks. Although Fluorinert is
known to dissolve gases such as oxygen and CO2, it does not dissolve nor extract medium
components, such as glucose, lactate, or amino acids. We conclude that mixing Fluorinert with
culture media is a suitable model for miniaturization of cell line development and process
optimization. Proper cell growth and cellular productivity can be obtained with a standard shaker
without the need for any additional aeration or vigorous agitation. \copyright 2011 American
Institute of Chemical Engineers Biotechnol. Prog., 2012
Signaling network state predicts Twist-mediated effects on breast cell migration across diverse
growth factor contexts
.
H.-D. Kim, A. S. Meyer, J. P. Wagner, S. K. Alford, A. Wells, F. B. Gertler, & D. A. Lauffenburger. (2011). Molecular & Cellular Proteomics.
[Abstract]
[DOI]
Abstract:
Epithelial-mesenchymal transition (EMT), whether in developmental morphogenesis or malignant
transformation, prominently involves modified cell motility behavior. While major advances have
transpired in understanding the molecular pathways regulating the process of EMT induction per se
by certain environmental stimuli, an important outstanding question is how the activities of
signaling pathways governing motility yield the diverse movement behaviors characteristic of
pre-induction versus post-induction states across a broad landscape of growth factor contexts.
For the particular case of EMT induction in human mammary cells by ectopic expression of the
transcription factor Twist, we found the migration responses to a panel of growth factors (EGF,
HRG, IGF, HGF) dramatically disparate between confluent pre-Twist epithelial cells and sparsely
distributed post-Twist mesenchymal cells - but that a computational model quantitatively
integrating multiple key signaling node activities could nonetheless account for this full range
of behavior. Moreover, motility in both conditions was successfully predicted a priori for an
additional growth factor treatment (PDGF). While this signaling network state model could
comprehend motility behavior globally, modulation of the network interactions underlying the
altered pathway activities was identified by ascertaining differences in quantitative topological
influences among the nodes between the two conditions.
Integrated Pathway Analysis of Genome-Wide Expression Changes Associated with Serum-Free
Suspension Adaptation of an Antibody-Producing Chinese Hamster Ovary (CHO) Cell Line
.
Y.-S. Tsao, A. A. Merchant, A. Meyer, Z. Liu, M. Smith, D. Levitan, & E. Gustafson. (2010). In Animal Cell Technology: Basic & Applied Aspects.
[DOI]
Concentration of mammalian genomic DNA using two-phase aqueous micellar systems.
F. Mashayekhi, A. S. Meyer, S. A. Shiigi, V. Nguyen, & D. T. Kamei. (2009). Biotechnology and Bioengineering.
[Abstract]
[DOI]
Abstract:
The concentration of biomarkers, such as DNA, prior to a subsequent detection step may facilitate
the early detection of cancer, which could significantly increase chances for survival. In this
study, the partitioning behavior of mammalian genomic DNA fragments in a two-phase aqueous
micellar system was investigated using both experiment and theory. The micellar system was
generated using the nonionic surfactant Triton X-114 and phosphate-buffered saline (PBS).
Partition coefficients were measured under a variety of conditions and compared with our
theoretical predictions. With this comparison, we demonstrated that the partitioning behavior of
DNA fragments in this system is primarily driven by repulsive, steric, excluded-volume
interactions that operate between the micelles and the DNA fragments, but is limited by the
entrainment of micelle-poor, DNA-rich domains in the macroscopic micelle-rich phase. Furthermore,
the volume ratio, that is, the volume of the top, micelle-poor phase divided by that of the
bottom, micelle-rich phase, was manipulated to concentrate DNA fragments in the top phase.
Specifically, by decreasing the volume ratio from 1 to 1/10, we demonstrated proof-of-principle
that the concentration of DNA fragments in the top phase could be increased two- to nine-fold in
a predictive manner.
Enhancing the Detection of Urinary Tract Infections Using Two-Phase Aqueous Micellar Systems.
S. A. Shiigi, A. S. Meyer, & D. T. Kamei. (2009). The UCLA USJ.
[Abstract]
Abstract:
Urinary tract infections (UTIs) are a leading cause of health expenditures, in part due to the
expensive and lengthy diagnostic method that involves culturing bacteria. A new technique has
been developed allowing for faster UTI diagnosis through an electrochemical chip to measure
unique ribosomal RNA (rRNA) found in bacteria associated with UTIs. Although this technique has
had success, concentrating the bacterial rRNA through the use of two-phase aqueous micellar
systems in the urine sample prior to utilizing the chip may provide increased sensitivity. This
approach is appealing because these systems are relatively inexpensive, easily scalable, and
simple to use. In this study, we examined the partitioning behavior of bacterial RNA fragments,
puri ed from Enteroccocus faecalis, in a two-phase micellar system comprised of the nonionic
surfactant Triton X-114 and phosphate-buffered saline. Experimentally measured results were
compared to theoretical values to determine the governing factors involved in RNA partitioning.
We demonstrated that RNA fragments partition primarily due to steric, excluded-volume
interactions that exist between the RNA and micelles. However, the presence of entrained
micelle-poor, RNA-rich domains in the macroscopic micelle-rich, RNA-poor phase limits the extent
of RNA partitioning in this system. Additionally, by manipulating the volume ratio, or the volume
of the top, micelle-poor phase divided by that of the bottom, micelle-rich phase, we demonstrated
that RNA fragments can be concentrated up to four-fold in a predictive manner. Concentrating the
bacterial RNA with these two-phase micellar systems prior to detection with the UTI chip may
facilitate the earlier detection of UTIs.