Effect of Spatial Inhomogeneities on the Membrane Surface on Receptor Dimerization and Signal Initiation

Important signal transduction pathways originate on the plasma membrane, where microdomains may transiently entrap diffusing receptors. This results in a non-random distribution of receptors even in the resting state, which can be visualized as “clusters” by high resolution imaging methods. Here, we explore how spatial in-homogeneities in the plasma membrane might influence the dimerization and phosphorylation status of ErbB2 and ErbB3, two receptor tyrosine kinases that preferentially heterodimerize and are often co-expressed in cancer. This theoretical study is based upon spatial stochastic simulations of the two-dimensional membrane landscape, where variables include differential distributions and overlap of transient confinement zones (“domains”) for the two receptor species. The in silico model is parameterized and validated using data from single particle tracking experiments. We report key differences in signaling output based on the degree of overlap between domains and the relative retention of receptors in such domains, expressed as escape probability. Results predict that a high overlap of domains, which favors transient co-confinement of both receptor species, will enhance the rate of hetero-interactions. Where domains do not overlap, simulations confirm expectations that homo-interactions are favored. Since ErbB3 is uniquely dependent on ErbB2 interactions for activation of its catalytic activity, variations in domain overlap or escape probability markedly alter the predicted patterns and time course of ErbB3 and ErbB2 phosphorylation. Taken together, these results implicate membrane domain organization as an important modulator of signal initiation, motivating the design of novel experimental approaches to measure these important parameters across a wider range of receptor systems

Inactivation of the Euchromatic Histone-Lysine N-Methyltransferase 2 Pathway in Pancreatic Epithelial Cells Antagonizes Cancer Initiation and Pancreatitis-Associated Promotion by Altering Growth and Immune Gene Expression Networks

Pancreatic ductal adenocarcinoma (PDAC) is an aggressive, painful disease with a 5-year survival rate of only 9%. Recent evidence indicates that distinct epigenomic landscapes underlie PDAC progression, identifying the H3K9me pathway as important to its pathobiology. Here, we delineate the role of Euchromatic Histone-lysine N-Methyltransferase 2 (EHMT2), the enzyme that generates H3K9me, as a downstream effector of oncogenic KRAS during PDAC initiation and pancreatitis-associated promotion. EHMT2 inactivation in pancreatic cells reduces H3K9me2 and antagonizes KrasG12D-mediated acinar-to-ductal metaplasia (ADM) and Pancreatic Intraepithelial Neoplasia (PanIN) formation in both the Pdx1-Cre and P48Cre/+KrasG12D mouse models. Ex vivo acinar explants also show impaired EGFR-KRAS-MAPK pathway-mediated ADM upon EHMT2 deletion. Notably, KrasG12D increases EHMT2 protein levels and EHMT2-EHMT1-WIZ complex formation. Transcriptome analysis reveals that EHMT2 inactivation upregulates a cell cycle inhibitory gene expression network that converges on the Cdkn1a/p21-Chek2 pathway. Congruently, pancreas tissue from KrasG12D animals with EHMT2 inactivation have increased P21 protein levels and enhanced senescence. Furthermore, loss of EHMT2 reduces inflammatory cell infiltration typically induced during KrasG12D-mediated initiation. The inhibitory effect on KrasG12D-induced growth is maintained in the pancreatitis-accelerated model, while simultaneously modifying immunoregulatory gene networks that also contribute to carcinogenesis. This study outlines the existence of a novel KRAS-EHMT2 pathway that is critical for mediating the growth-promoting and immunoregulatory effects of this oncogene in vivo, extending human observations to support a pathophysiological role for the H3K9me pathway in PDAC

Exploring biological heterogeneity and its consequences at tissue and cellular scales through mathematical and computational modeling

This dissertation explores the effects of heterogeneity across different biological scales in cancer as well as normal cells. At the tissue scale, we investigated the variability present in the tumor microenvironment and its effect on patient chemotherapeutic outcomes using a mathematical model of drug transport. We found that parameters such as tumor blood perfusion and radius of blood vessel had an impact on the tumor cytotoxicity. This indicated that the physical microenvironment of the tumor is an important regulator of the tumor response to chemotherapy. At the cellular scale, we investigated the heterogeneity present on the membrane landscape of ErbB2 and ErbB3, two receptors that are upregulated in cancer, using a spatial stochastic model of receptor dimerization and phosphorylation. We found that membrane domains played an important role in regulating signaling emanating from this receptor dimer. In our next study, we developed a 3-D spatial stochastic model of pre-BCR, a receptor which is crucial in the development of B lymphocytes and also upregulated in a subset of patients with B-Cell Precursor Acute Lymphoblastic Leukemia, to investigate the effects of ligand independent (tonic signaling) originating from this receptor. We populated our model with single particle tracking data from two different leukemic cell lines which had different dimer off rates and diffusion coefficients, along with experimental measurements. Other important signaling molecules such as Lyn and Syk, which are active in this pathway, were also included in the model. We found that the variability in characteristics between the two cell lines led to differences in downstream signaling events from the receptor. The cell line with the lower dimer off rate formed higher order oligomers and had more overall molecule phosphorylation compared to the other. Thus, this spatial stochastic model was able to shed light on threshold signaling events which take place during tonic signaling

Effect of spatial inhomogeneities on the membrane surface on receptor dimerization and signal initiation

Important signal transduction pathways originate on the plasma membrane, where microdomains may transiently entrap diffusing receptors. This results in a non-random distribution of receptors even in the resting state, which can be visualized as clusters by high resolution imaging methods. Here, we explore how spatial in-homogeneities in the plasma membrane might influence the dimerization and phosphorylation status of ErbB2 and ErbB3, two receptor tyrosine kinases that preferentially heterodimerize and are often co-expressed in cancer. This theoretical study is based upon spatial stochastic simulations of the two-dimensional membrane landscape, where variables include differential distributions and overlap of transient confinement zones (domains) for the two receptor species. The in silico model is parameterized and validated using data from single particle tracking experiments. We report key differences in signaling output based on the degree of overlap between domains and the relative retention of receptors in such domains, expressed as escape probability. Results predict that a high overlap of domains, which favors transient co-confinement of both receptor species, will enhance the rate of hetero-interactions. Where domains do not overlap, simulations confirm expectations that homo-interactions are favored. Since ErbB3 is uniquely dependent on ErbB2 interactions for activation of its catalytic activity, variations in domain overlap or escape probability markedly alter the predicted patterns and time course of ErbB3 and ErbB2 phosphorylation. Taken together, these results implicate membrane domain organization as an important modulator of signal initiation, motivating the design of novel experimental approaches to measure these important parameters across a wider range of receptor systems

KrasG12D induces changes in chromatin territories that differentially impact early nuclear reprogramming in pancreatic cells

Abstract Background Pancreatic ductal adenocarcinoma initiation is most frequently caused by Kras mutations. Results Here, we apply biological, biochemical, and network biology methods to validate GEMM-derived cell models using inducible KrasG12D expression. We describe the time-dependent, chromatin remodeling program that impacts function during early oncogenic signaling. We find that the KrasG12D-induced transcriptional response is dominated by downregulated expression concordant with layers of epigenetic events. More open chromatin characterizes the ATAC-seq profile associated with a smaller group of upregulated genes and epigenetic marks. RRBS demonstrates that promoter hypermethylation does not account for the silencing of the extensive gene promoter network. Moreover, ChIP-Seq reveals that heterochromatin reorganization plays little role in this early transcriptional program. Notably, both gene activation and silencing primarily depend on the marking of genes with a combination of H3K27ac, H3K4me3, and H3K36me3. Indeed, integrated modeling of all these datasets shows that KrasG12D regulates its transcriptional program primarily through unique super-enhancers and enhancers, and marking specific gene promoters and bodies. We also report chromatin remodeling across genomic areas that, although not contributing directly to cis-gene transcription, are likely important for KrasG12D functions. Conclusions In summary, we report a comprehensive, time-dependent, and coordinated early epigenomic program for KrasG12D in pancreatic cells, which is mechanistically relevant to understanding chromatin remodeling events underlying transcriptional outcomes needed for the function of this oncogene.http://deepblue.lib.umich.edu/bitstream/2027.42/173870/1/13059_2021_Article_2498.pd

Inactivation of the Euchromatic Histone-Lysine N-Methyltransferase 2 Pathway in Pancreatic Epithelial Cells Antagonizes Cancer Initiation and Pancreatitis-Associated Promotion by Altering Growth and Immune Gene Expression Networks

Pancreatic ductal adenocarcinoma (PDAC) is an aggressive, painful disease with a 5-year survival rate of only 9%. Recent evidence indicates that distinct epigenomic landscapes underlie PDAC progression, identifying the H3K9me pathway as important to its pathobiology. Here, we delineate the role of Euchromatic Histone-lysine N-Methyltransferase 2 (EHMT2), the enzyme that generates H3K9me, as a downstream effector of oncogenic KRAS during PDAC initiation and pancreatitis-associated promotion. EHMT2 inactivation in pancreatic cells reduces H3K9me2 and antagonizes Kras(G12D)-mediated acinar-to-ductal metaplasia (ADM) and Pancreatic Intraepithelial Neoplasia (PanIN) formation in both the Pdx1-Cre and P48(Cre/+) Kras(G12D) mouse models. Ex vivo acinar explants also show impaired EGFR-KRAS-MAPK pathway-mediated ADM upon EHMT2 deletion. Notably, Kras(G12D) increases EHMT2 protein levels and EHMT2-EHMT1-WIZ complex formation. Transcriptome analysis reveals that EHMT2 inactivation upregulates a cell cycle inhibitory gene expression network that converges on the Cdkn1a/p21-Chek2 pathway. Congruently, pancreas tissue from Kras(G12D) animals with EHMT2 inactivation have increased P21 protein levels and enhanced senescence. Furthermore, loss of EHMT2 reduces inflammatory cell infiltration typically induced during Kras(G12D)-mediated initiation. The inhibitory effect on Kras(G12D)-induced growth is maintained in the pancreatitis-accelerated model, while simultaneously modifying immunoregulatory gene networks that also contribute to carcinogenesis. This study outlines the existence of a novel KRAS-EHMT2 pathway that is critical for mediating the growth-promoting and immunoregulatory effects of this oncogene in vivo, extending human observations to support a pathophysiological role for the H3K9me pathway in PDAC

Theory and Experimental Validation of a Spatio-temporal Model of Chemotherapy Transport to Enhance Tumor Cell Kill

<div><p>It has been hypothesized that continuously releasing drug molecules into the tumor over an extended period of time may significantly improve the chemotherapeutic efficacy by overcoming physical transport limitations of conventional bolus drug treatment. In this paper, we present a generalized space- and time-dependent mathematical model of drug transport and drug-cell interactions to quantitatively formulate this hypothesis. Model parameters describe: perfusion and tissue architecture (blood volume fraction and blood vessel radius); diffusion penetration distance of drug (i.e., a function of tissue compactness and drug uptake rates by tumor cells); and cell death rates (as function of history of drug uptake). We performed preliminary testing and validation of the mathematical model using <i>in vivo</i> experiments with different drug delivery methods on a breast cancer mouse model. Experimental data demonstrated a 3-fold increase in response using nano-vectored drug <i>vs</i>. free drug delivery, in excellent quantitative agreement with the model predictions. Our model results implicate that therapeutically targeting blood volume fraction, e.g., through vascular normalization, would achieve a better outcome due to enhanced drug delivery.</p><p>Author Summary</p><p>Cancer treatment efficacy can be significantly enhanced through the elution of drug from nano-carriers that can temporarily stay in the tumor vasculature. Here we present a relatively simple yet powerful mathematical model that accounts for both spatial and temporal heterogeneities of drug dosing to help explain, examine, and prove this concept. We find that the delivery of systemic chemotherapy through a certain form of nano-carriers would have enhanced tumor kill by a factor of 2 to 4 over the standard therapy that the patients actually received. We also find that targeting blood volume fraction (a parameter of the model) through vascular normalization can achieve more effective drug delivery and tumor kill. More importantly, this model only requires a limited number of parameters which can all be readily assessed from standard clinical diagnostic measurements (e.g., histopathology and CT). This addresses an important challenge in current translational research and justifies further development of the model towards clinical translation.</p></div

Illustration of transport-based hypothesis.

<p>By diffusion, a blood vessel supplies substrates to the cylindrical tissue volume surrounding the vessel. We hypothesize that at each position inside the tissue, the substrate supply is supported by the closest blood vessel. Thus, the influenced tissue surrounding a vessel can be estimated to be between a cylinder of radius <i>r</i>_b / (<i>L</i> BVF^1/2) in dimensionless form, and the vessel itself with dimensionless radius <i>r</i>_b / <i>L</i>. Theoretically, chemotherapeutic drugs delivered by a blood vessel kill the tissues immediately adjacent to the vessel, leaving some viable tissues on the far end. Here, we propose that through drug-loaded nano-carriers that can accumulate within tumors and continuously release drugs for a longer time (e.g., lasting several cell cycles), the drugs can penetrate further into the surrounding tissue volume and thus achieve a higher tumor killing ratio.</p

Testing the efficacy of drug-loaded nano-carriers in mice.

<p>Comparison of fraction of tumor killed measured across three different treatment BALB/c mice groups (n = 10 per group) over a period of 17 days (from day 14 to day 31 after 4T1 tumor cell inoculation, see <b><a href="http://www.ploscompbiol.org/article/info:doi/10.1371/journal.pcbi.1004969#sec003" target="_blank">Methods</a></b>) showing a roughly 3-fold increase in kill from nano-vectored drug vs. free drug. At each time point, tumor volume measurements from the three drug treatment groups were first normalized to the measurement from the control (PBS) group (no drug treatment), and then to the initial tumor volume for each group; <i>f</i>_kill was then calculated as (1 –normalized tumor volume).</p