Matthew Lazzara

The present invention provides combination therapies useful for treating cancer, particularly breast cancer. The invention provides, in various embodiments, methods of treating a can cer, comprising administering to a patient afflicted therewith of an effective amount an immunoconjugate comprising a monoclonal antibody moiety and a first pro-apoptotic drug moiety linked thereto; and administering to the patient an effective amount of a second pro-apoptotic drug. The mono clonal antibody moiety of the immunoconjugate can act to target receptors of hormone-resistant breast cancer cells, such as HER2. Synergistic effects can be seen when the two pro apoptotic drugs, acting by a common molecular mechanism (vertical modulation) or different molecular mechanisms (horizontal modulation) are administered to patients afflicted by breast cancer, such as hormone-resistant breast cancer.

The tumor microenvironment in pancreatic ductal adenocarcinoma (PDAC) plays a key role in tumor progression and response to therapy. The dense PDAC stroma causes hypovascularity, which leads to hypoxia. Here, we showed that hypoxia drives long-lasting epithelial-mesenchymal transition (EMT) in PDAC primarily through a positive-feedback histone methylation-MAPK signaling axis. Transformed cells preferentially underwent EMT in hypoxic tumor regions in multiple model systems. Hypoxia drove a cell-autonomous EMT in PDAC cells which, unlike EMT in response to growth factors, could last for weeks. Furthermore, hypoxia reduced histone demethylase KDM2A activity, suppressed PP2 family phosphatase expression, and activated MAPKs to post-translationally stabilize histone methyltransferase NSD2, leading to an H3K36me2-dependent EMT in which hypoxia-inducible factors played only a …

In glioblastoma, a mesenchymal phenotype is associated with especially poor patient outcomes. Various glioblastoma microenvironmental factors and therapeutic interventions are purported drivers of the mesenchymal transition, but the degree to which these cues promote the same mesenchymal transitions and the uniformity of those transitions, as defined by molecular subtyping systems, is unknown. Here, we investigate this question by analyzing publicly available patient data, surveying commonly measured transcripts for mesenchymal transitions in glioma-initiating cells (GIC), and performing next-generation RNA sequencing of GICs. Analysis of patient tumor data reveals that TGFβ, TNFα, and hypoxia signaling correlate with the mesenchymal subtype more than the proneural subtype. In cultured GICs, the microenvironment-relevant growth factors TGFβ and TNFα and the chemotherapeutic temozolomide …

Epithelial-mesenchymal transition (EMT) is a developmental process that is aberrantly activated in cancers such as pancreatic ductal adenocarcinoma (PDAC) to promote disease progression and chemoresistance. EMT occurs heterogeneously among PDAC ductal cells, which frustrates efforts to identify the underlying signaling regulatory mechanisms. We hypothesize that cell-to-cell variation in the activities of specific signaling pathways explains EMT heterogeneity. To test this, we developed a workflow integrating: 1) iterative indirect immunofluorescence imaging (4i) to quantify the activities of up to seven purported EMT-regulating pathways with single-cell resolution and 2) a multivariate mutual information computational model to predict the pathways that are most informative of the EMT phenotype. In cultured PDAC cells, EMT was induced by treating cells with growth factors present in the tumor …

The pancreatic ductal adenocarcinoma (PDAC) microenvironment is populated by distinct subtypes of cancer-associated fibroblasts (CAFs), including myofibroblastic CAFs (myCAFs) that synthesize and remodel the stromal matrix and inflammatory CAFs (iCAFs) that secrete cytokines and growth factors. Subpopulations of CAFs reside in regions of tumor hypoxia, which may play a role in determining CAF behaviors. We tested the hypothesis that iCAFs preferentially drive ductal cell epithelial-mesenchymal transition (EMT), which promotes PDAC chemoresistance, and that hypoxia promotes the iCAF phenotype. Cell-cell communication analysis using publicly available single-cell RNA sequencing (scRNA-seq) data of human tumors indicated that iCAFs secrete growth factors predicted to promote ductal cell EMT. This inference was confirmed in experiments showing that PDAC cells exhibit stronger EMT in …

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the causative agent of COVID, replicates at intracellular membranes. Bone marrow stromal antigen 2 (BST-2; tetherin) is an antiviral response protein that inhibits transport of viral particles after budding within infected cells. RNA viruses such as SARS-CoV-2 use various strategies to disable BST-2, including use of transmembrane ‘accessory’ proteins that interfere with BST-2 oligomerization. ORF7a is a small, transmembrane protein present in SARS-CoV-2 shown previously to alter BST-2 glycosylation and function. In this study, we investigated the structural basis for BST-2 ORF7a interactions, with a particular focus on transmembrane and juxtamembrane interactions. Our results indicate that transmembrane domains play an important role in BST-2 ORF7a interactions and mutations to the transmembrane domain of BST-2 can alter these …

In the “Cell lines and propagation” subsection of “Materials and methods”, there is an error where fibroblast cells were incorrectly referred to as adipocytes. The correct sentence is: L929 murine fibroblasts engineered with a bidirectional cell-cell signaling circuit based on the synthetic notch (synNotch) receptor system [2] were generously provided by Dr. Wendell Lim (University of California, San Francisco).

The biophysical properties of ligand binding heavily influence the ability of receptors to specify cell fates. Understanding the rules by which ligand binding kinetics impact cell phenotype is challenging, however, because of the coupled information transfers that occur from receptors to downstream signaling effectors and from effectors to phenotypes. Here, we address that issue by developing an integrated mechanistic and data-driven computational modeling platform to predict cell responses to different ligands for the epidermal growth factor receptor (EGFR). Experimental data for model training and validation were generated using MCF7 human breast cancer cells treated with the high- and low-affinity ligands epidermal growth factor (EGF) and epiregulin (EREG), respectively. The integrated model captures the unintuitive, concentration-dependent abilities of EGF and EREG to drive signals and phenotypes differently, even at similar levels of receptor occupancy. For example, the model correctly predicts the dominance of EREG over EGF in driving a cell differentiation phenotype through AKT signaling at intermediate and saturating ligand concentrations and the ability of EGF and EREG to drive a broadly concentration-sensitive migration phenotype through cooperative ERK and AKT signaling. Parameter sensitivity analysis identifies EGFR endocytosis, which is differentially regulated by EGF and EREG, as one of the most important determinants of the alternative phenotypes driven by different ligands. The integrated model provides a new platform to predict how phenotypes are controlled by the earliest biophysical rate processes in signal …

In the PDAC tumor microenvironment, multiple factors initiate the epithelial-mesenchymal transition (EMT) that occurs heterogeneously among transformed ductal cells, but it is unclear if different drivers promote EMT through common or distinct signaling pathways. Here, we use single-cell RNA sequencing (scRNA-seq) to identify the transcriptional basis for EMT in pancreas cancer cells in response to hypoxia or EMT-inducing growth factors. Using clustering and gene set enrichment analysis, we find EMT gene expression patterns that are unique to the hypoxia or growth factor conditions or that are common between them. Among the inferences from the analysis, we find that the FAT1 cell adhesion protein is enriched in epithelial cells and suppresses EMT. Further, the receptor tyrosine kinase AXL is preferentially expressed in hypoxic mesenchymal cells in a manner correlating with YAP nuclear localization, which is suppressed by FAT1 expression. AXL inhibition prevents EMT in response to hypoxia but not growth factors. Relationships between FAT1 or AXL expression with EMT were confirmed through analysis of patient tumor scRNA-seq data. Further exploration of inferences from this unique dataset will reveal additional microenvironment context-specific signaling pathways for EMT that may represent novel drug targets for PDAC combination therapies.

DiscoverBMB Abstracts Abstract 1249 14-3-3 binding stabilizes and sequesters PTOV1 to the cytoplasm during G1 Katie Pennington, Longwood University Colten McEwan, James Woods, Colin Muir, AG Pramoda Sahankumari, Riley Eastmond, Eranga Balasooriya, Christina Egbert, Joshua Andersen Cells must respond to changing environments by modulating signaling and gene expression patterns to maintain homeostasis, health, and viability. Inappropriate responses lead to disease processes, such as cancer. Despite improvements in targeted treatment options, cancer remains the second leading cause of death in the United States, illustrating the need to develop new highly effective targeted therapies. 14-3-3 interacts with a network of phosphorylated proteins to support signaling pathways promoting oncogenesis, metastasis, growth, cellular survival, and chemoresistance in a variety of cancers, including …

Pancreatic ductal adenocarcinoma (PDAC) tumors are poorly vascularized and exhibit regions of hypoxia. Here, we demonstrate that this feature of the tumor microenvironment promotes epithelial-mesenchymal transition (EMT), which occurs early in PDAC and drives chemoresistance, and we identify the underlying signaling mechanism. Analysis of publicly-available human transcriptomics and proteomics demonstrated that PDAC cells or tumors enriched in mesenchymal markers were also enriched in markers of hypoxia or HIF activity. Furthermore, in lineage-traced autochthonous, orthotopic patient-derived xenograft, and orthotopic or subcutaneous implanted cell line models of PDAC, hypoxic tumor tissue regions were enriched for neoplastic cells that had undergone EMT. In cell culture experiments, PDAC cells from human and mouse tumors exhibited an ability to undergo EMT in response to 1% O2, with …

Elizabeth Bloch, Eden L. Sikorski, David Pontoriero, Evan K. Day, Bryan W. Berger, Matthew J. Lazzara, and Damien Thévenin It was determined via STR profiling that cell line UMSCC2 should have been labeled as HSC3 cells. Like UMSCC2 cells, HSC3 cells are squamous cell carcinoma cells that express wild type EGFR, the primary substrate of PTPRJ/DEP1 investigated in this publication. Therefore, this correction does not affect the results and conclusions of the paper.

Sunday, February 20, 2022 83a represents a novel allosteric and a possibly orthogonal way to target the activity of PTPRJ and RTKs phosphorylation. It could therefore be used not only as probes to tease out PTPRJ regulating mechanisms but also for therapeutic purposes via the attenuation of signaling by dysregulated RTKs in cancers. We also expect that the basic framework developed here can be extended to other RPTPs.411-Pos Cryo-EM structures of the near full-length HER2/HER3 heterodimer reveal a novel allosteric mechanism of activation Devan C. Diwanji1, 2, Raphael Trenker1, Tarjani M. Thaker3, Feng Wang1, David A. Agard1, Kliment A. Verba4, Natalia Jura5. 1University of California San Francisco, San Francisco, CA, USA, 2Medical Scientist Training Program, University of California San Francisco, San Francisco, CA, USA, 3The University of Arizona, Tuscon, AZ, USA, 4Department of …

Simple Summary CX-5461 is an RNA polymerase I inhibitor that is in clinical trials for both advanced hematological cancers and solid tumors. Experimentally, this drug has been shown to induce a p53-independent DNA damage response through ATM and ATR kinase, and has particular activity against chemoresistant tumors. The current study shows for the first time that CX-5461 treatment in ovarian cancer cells induces the release of cytoplasmic DNA that stimulates cGAS–STING signaling, leading to the production of IFN type I in both cancer cells and xenografts in vivo. Because the cGAS–STING pathway is a key mediator of the immune response against cancer cells, this novel finding may lead to utilization of RNA Pol I inhibitors in combination with checkpoint inhibition. Abstract Epithelial ovarian cancer (EOC) is the deadliest of the gynecologic malignancies, with an overall survival rate of <30%. Recent research has suggested that targeting RNA polymerase I (POL I) with small-molecule inhibitors may be a viable therapeutic approach to combating EOC, even when chemoresistance is present. CX-5461 is one of the most promising POL I inhibitors currently being investigated, and previous reports have shown that CX-5461 treatment induces DNA damage response (DDR) through ATM/ATR kinase. Investigation into downstream effects of CX-5461 led us to uncovering a previously unreported phenotype. Treatment with CX-5461 induces a rapid accumulation of cytosolic DNA. This accumulation leads to transcriptional upregulation of ‘STimulator of Interferon Genes’ (STING) in the same time frame …

Phosphorylation (activation) and dephosphorylation (deactivation) of the slit diaphragm proteins NEPHRIN and NEPH1 are critical for maintaining the kidney epithelial podocyte actin cytoskeleton and, therefore, proper glomerular filtration. However, the mechanisms underlying these events remain largely unknown. Here we show that NEPHRIN and NEPH1 are novel receptor proteins for hepatocyte growth factor (HGF) and can be phosphorylated independently of the mesenchymal epithelial transition receptor in a ligand-dependent fashion through engagement of their extracellular domains by HGF. Furthermore, we demonstrate SH2 domain–containing protein tyrosine phosphatase-2–dependent dephosphorylation of these proteins. To establish HGF as a ligand, purified baculovirus-expressed NEPHRIN and NEPH1 recombinant proteins were used in surface plasma resonance binding experiments. We report …

Dynamically activated differential adhesion within cell populations enables the emergence of unique patterns in heterogeneous multicellular systems. This process has previously been explored using synthetically engineered heterogenous multicell spheroid systems in which cell subpopulations engage in bidirectional intercellular signaling to regulate the expression of different cadherins. While engineered cell systems provide excellent experimental tools to observe pattern formation in cell populations, computational models may be leveraged to explore the key parameters that drive the emergence of different patterns more systematically. Here, we developed and validated two-and three-dimensional agent-based models (ABMs) of spheroid patterning for cells engineered with a bidirectional signaling circuit regulating N-and P-cadherin expression. The model was used to predict how varying initial cell seeding, cadherin induction probabilities, or homotypic adhesion strengths between cells leads to different spheroid patterns, and unsupervised machine learning techniques were used to map system parameters to unique spheroid patterns. The model was also used as a tool to design new synthetic cell signaling circuits based on a desired final multicell pattern.

IntroductionPharmacologic approaches for promoting angiogenesis have been utilized to accelerate healing of chronic wounds in diabetic patients with varying degrees of success. We hypothesize that the distribution of proangiogenic drugs in the wound area critically impacts the rate of closure of diabetic wounds. To evaluate this hypothesis, we developed a mathematical model that predicts how spatial distribution of VEGF-A produced by delivery of a modified mRNA (AZD8601) accelerates diabetic wound healing.MethodsWe modified a previously published model of cutaneous wound healing based on coupled partial differential equations that describe the density of sprouting capillary tips, chemoattractant concentration, and density of blood vessels in a circular wound. Key model parameters identified by a sensitivity analysis were fit to data obtained from an in vivo wound healing study performed in the dorsum …

Oncogenic protein tyrosine phosphatases have long been viewed as drug targets of interest, and recently developed allosteric inhibitors of SH2 domain–containing phosphatase-2 (SHP2) have entered clinical trials. However, the ability of phosphatases to regulate many targets directly or indirectly and to both promote and antagonize oncogenic signaling may make the efficacy of phosphatase inhibition challenging to predict. Here we explore the consequences of antagonizing SHP2 in glioblastoma, a recalcitrant cancer where SHP2 has been proposed as a useful drug target. Measuring protein phosphorylation and expression in glioblastoma cells across 40 signaling pathway nodes in response to different drugs and for different oxygen tensions revealed that SHP2 antagonism has network-level, context-dependent signaling consequences that affect cell phenotypes (e.g., cell death) in …

Suicide gene therapies provide a unique ability to target cancer cells selectively, often based on modification of viral tropism or transcriptional regulation of therapeutic gene expression. We designed a novel suicide gene therapy approach wherein the gene product (herpes simplex virus thymidine kinase or yeast cytosine deaminase) is phosphorylated and stabilized in expression by the extracellular signal-regulated kinase (ERK), which is overactive in numerous cancers with elevated expression or mutation of receptor tyrosine kinases or the GTPase RAS. In contrast to transcriptional strategies for selectivity, regulation of protein stability by ERK allows for high copy expression via constitutive viral promoters, while maintaining tumor selectivity in contexts of elevated ERK activity. Thus, our approach turns a signaling pathway often coopted by cancer cells for survival into a lethal disadvantage in the presence of a …

Triple-Negative Breast Cancers (TNBCs) constitute roughly 10-20% of breast cancers and are associated with poor clinical outcomes. Previous work from our laboratory and others has determined that the cytoplasmic adaptor protein Breast Cancer Antiestrogen Resistance 3 (BCAR3) is an important promoter of cell motility and invasion of breast cancer cells. In this study, we use both in vivo and in vitro approaches to extend our understanding of BCAR3 function in TNBC. We show that BCAR3 is upregulated in ductal carcinoma in situ (DCIS) and invasive carcinomas compared to normal mammary tissue, and that survival of TNBC patients whose tumors contained elevated BCAR3 mRNA is reduced relative to individuals whose tumors had less BCAR3 mRNA. Using mouse orthotopic tumor models, we further show that BCAR3 is required for efficient TNBC tumor growth. Analysis of publicly available RNA …