Amy B. Hall

A paper-based invasion assay: assessing chemotaxis of cancer cells in gradients of oxygen.

This work describes a 3D, paper-based assay that can isolate sub-populations of cells based on their invasiveness (i.e., distance migrated in a hydrogel) in a gradient of concentration of oxygen (O2). Layers of paper impregnated with a cell-compatible hydrogel are stacked and placed in a plastic holder to form the invasion assay. In most assays, the stack comprises a single layer of paper containing mammalian cells suspended in a hydrogel, sandwiched between multiple layers of paper containing only hydrogel. Cells in the stack consume and produce small molecules; these molecules diffuse throughout the stack to generate gradients in the stack, and between the stack and the bulk culture medium. Placing the cell-containing layer in different positions of the stack, or modifying the permeability of the holder to oxygen or proteins, alters the profile of the gradients within the stack. Physically separating the layers after culture isolates sub-populations of cells that migrated different distances, and enables their subsequent analysis or culture. Using this system, three independent cell lines derived from A549 cancer cells are shown to produce distinguishable migration behavior in a gradient of oxygen. This result is the first experimental demonstration that oxygen acts as a chemoattractant for cancer cells.

Quantitative analysis of cell signaling and drug action via mass spectrometry-based systems level phosphoproteomics

Protein phosphorylation is a primary form of information transfer in cell signaling pathways and plays a crucial role in regulating biological responses. Aberrant phosphorylation has been implicated in a number of diseases, and kinases and phosphatases, the cellular enzymes that control dynamic phosphorylation events, present attractive therapeutic targets. However, the innate complexity of signaling networks has presented many challenges to therapeutic target selection and successful drug development. Approaches in phosphoproteomics can contribute functional, systems‐level datasets across signaling networks that can provide insight into suitable drug targets, more broadly profile compound activities, and identify key biomarkers to assess clinical outcomes. Advances in MS‐based phosphoproteomics efforts now provide the ability to quantitate phosphorylation with throughput and sensitivity to sample a significant portion of the phosphoproteome in clinically relevant systems. This review will discuss recent work and examples of application data that demonstrate the utility of MS, with a particular focus on the use of quantitative phosphoproteomics and phosphotyrosine‐directed signaling analyses to provide robust measurement for functional biological interpretation of drug action on signaling and phenotypic outcomes.

Profiling Protein Tyrosine Phosphorylation: A Quantitative 45-Plex Peptide-Based Immunoassay

Cellular homeostasis and responses to stimuli are mediated by complex signaling network events dominated by changes in protein phosphorylation states. Understanding information flow in the network is essential for correlating signaling changes to cell physiology. Tyrosine phosphorylation constitutes only a small portion of all protein phosphorylation, but its importance is manifested by the significant role it plays in diseases such as cancer. A peptide-based immunoassay microarray, designed to provide site specificity, quantification, broad coverage, and accessibility, is described that profiles 45 tyrosine phosphorylation sites across 34 proteins. Epidermal growth factor—stimulated A431 cells in the absence and presence of kinase inhibitors analyzed by microarrays showed biologically validated tyrosine phosphorylation changes and unanticipated activation of other targets. The approach is scalable for increasing the breadth of content as well as for interrogating other types of protein posttranslational modifications. ( Journal of Biomolecular Screening 2008:626-637)

Evaluating the immortal strand hypothesis in cancer stem cells: symmetric/self-renewal as the relevant surrogate marker of tumorigenicity.

Stem cells subserve repair functions for the lifetime of the organism but, as a consequence of this responsibility, are candidate cells for accumulating numerous genetic and/or epigenetic aberrations leading to malignant transformation. However, given the importance of this guardian role, stem cells likely harbor some process for maintaining their precious genetic code such as non-random segregation of chromatid strands as predicted by the Immortal Strand Hypothesis (ISH). Discerning such non-random chromosomal segregation and asymmetric cell division in normal or cancer stem cells has been complicated by methodological shortcomings but also by differing division kinetics amongst tissues and the likelihood that both asymmetric and symmetric cell divisions, dictated by local extrinsic factors, are operant in these cells. Recent data suggest that cancer stem cells demonstrate a higher incidence of symmetric versus asymmetric cell division with both daughter cells retaining self-renewal characteristics, a profile which may underlie poorly differentiated morphology and marked clonal diversity in tumors. Pathways and targets are beginning to emerge which may provide opportunities for preventing such a predilection in cancer stem cells and that will hopefully translate into new classes of chemotherapeutics in oncology. Thus, although the existence of the ISH remains controversial, the shift of cell division dynamics to symmetric random chromosome segregation/self-renewal, which would negate any likelihood of template strand retention, appears to be a surrogate marker for the presence of highly malignant tumorigenic cell populations.

Abstract B24: Differentiation screen identifies small molecules that target histologically divergent subtypes of patient-derived lung cancer stem cells

Non-small cell lung cancer (NSCLC) is a prevalent and deadly disease because of high incidence and relapse rates. One hypothesis for the high relapse rate is the existence of cancer stem cells (CSCs), a rare subpopulation of cells within these tumors, that are resistant to therapy and thought to be responsible for local and distal recurrence. CSCs are also able to self-renew and differentiate into multiple cell types forming the mass of new tumors. These differentiated progeny that constitute the bulk of the tumor are more sensitive to radiation and chemotherapeutic agents, express tissue of origin markers, and have an intrinsically limited lifespan. While most solid tumors have these properties, the degree of heterogeneity from patient to patient in the multi-lineage potential of the CSC population is not known. In particular, NSCLC CSC populations from primary patient tumors have not been well studied. The goal of this study was to characterize newly derived NSCLC CSC lines to determine the degree of heterogeneity between patient samples, and to provide a starting point for the discovery of novel differentiation therapeutic agents to target CSCs. We isolated six CSC lines from primary patient NSCLC tumors using an unbiased culture-based enrichment method, rather than a biased marker-based approach. Each of these six CSC lines was tumorigenic in immunodeficient mice at low cell numbers and the resulting tumors recapitulated the original tumor histology. All-trans retinoic acid (ATRA), a well-known differentiation agent for the treatment of acute promyelocytic leukemia (APL), maintains the normal growth and differentiation of human bronchial epithelial cells in culture. Each of the six CSC lines was treated with ATRA for two weeks to induce differentiation and then assessed for transcript and protein levels of candidate CSC and differentiation markers. Pre-treatment, the CSC lines all expressed CD44. Post-ATRA treatment, the majority of the CSC lines expressed Mucin-2, a marker of Goblet cell lineage. In addition, each cell line started to show evidence of the very early stages of differentiation into other lineages including Clara Cells, Neuroendocrine Cells, and Alveolar Type I and Type II cells. In one CSC line, ATRA treatment both in vitro and in vivo delayed tumor cell growth, induced the expression of Mucin-2 and decreased the expression of Nestin, a cancer stem cell marker. To further characterize CSC differentiation potential, we screened a pilot set of small molecules in two independent CSC lines to identify compounds that induce terminal differentiation, using Mucin-2 expression as readout. Surprisingly, despite the fact that these two CSC lines were from different histological subtypes (adenocarcinoma and squamous cell carcinoma) and displayed different transcriptional profiles post-differentiation, there was a good correlation between hit sets in the screens. Taken together, these data suggest that while the CSC of origin in tumors differs between patient samples in profile and function, there is promise to identify differentiation agents that are broadly active in different NSCLC subtypes. Further screening is currently being performed to identify these novel differentiation agents for therapeutic use. Citation Format: Dina Shlyakhter, Diane Boucher, Yong Gu, Amy B. Hall, Elaine Krueger, Anna Lindquist, Cheryl Murphy, Yuxin Wang, Mark Wood, Brenda Eustace. Differentiation screen identifies small molecules that target histologically divergent subtypes of patient-derived lung cancer stem cells. [abstract]. In: Proceedings of the AACR Special Conference: Developmental Biology and Cancer; Nov 30-Dec 3, 2015; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Res 2016;14(4_Suppl):Abstract nr B24.

Abstract 1644: VX-970, the first-in-class inhibitor of the DNA damage repair enzyme ATR

Proficient repair of DNA damage is important for cancer cell survival and is a leading cause for the poor response many patients experience when treated with DNA-damaging drugs or ionizing radiation. The protein kinase ataxia telangiectasia mutated and Rad3 related (ATR) regulates an important DNA damage response pathway that is most commonly activated by replication stress (RS). RS arises during S-phase when the cell9s DNA replication machinery attempts to copy through an unresolved damage lesion. Such events are common after cells are treated with DNA-damaging agents. Unresolved RS often leads to double strand breaks, which in turn may cause DNA mutations, chromosomal rearrangements or cell death. Pre-clinical data suggests a reliance on ATR for survival is a common feature in cancer cells. This may occur when there are defects in other DNA damage repair pathways or high levels of background RS. VX-970 is the first potent (Ki VX-970 is currently in Phase 1 clinical studies as monotherapy and in combination with gemcitabine, cisplatin and carboplatin. Note: This abstract was not presented at the meeting. Citation Format: John Pollard, Philip Reaper, Julie Jones, Christopher Barnes, Scott Gladwell, Stuart Hughes, Adele Peak, Hakim Djeha, Amy Hall, David Newsome, Yuxin Wang, Diane Boucher, Brenda Eustace, Yong Gu, Brian Hare, Mac Johnson, Sean Milton, Cheryl Murphy, Darin Takemoto, Crystal Tolman, Mark Wood, Brinley Furey, Marina Penney, Howard Li, Christopher Defranco, Mohammed Asmal, Scott Fields. VX-970, the first-in-class inhibitor of the DNA damage repair enzyme ATR. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 1644. doi:10.1158/1538-7445.AM2015-1644

Abstract LB-299: Comprehensive preclinical evaluation of VE-822, the first ATR-targeted drug candidate: a novel approach to transforming the efficacy of DNA damaging agents.

DNA damaging agents have been the cornerstone of cancer therapy for decades yet they provide only modest benefit for most patients. For example, standard of care for patients with non-small cell lung cancer (NSCLC) is dominated by the use of platinating drugs and ionizing radiation (IR), however outcome remains very poor with 5-year survival rates of VE-822 potently inhibits ATR in biochemical assays with Ki 90% of lines showed >3-fold shifts in IC50 for cisplatin in the presence of VE-822, with ~50% of lines showing >10-fold increases in cisplatin cytotoxicity. In contrast normal cells tolerate inhibition of ATR. In a panel of mouse xenograft models, derived from various primary human NSCLC tumor tissues, oral or IV administration of VE-822 strongly sensitized tumors to cisplatin treatment. In many cases, combinations including VE-822 led to tumor regression or extensive tumor growth delay. Inhibition of ATR activity and accumulation of DNA damage by VE-822 was observed coincident with efficacy. When administered alone or in combination with cisplatin VE-822 was well tolerated in mice at doses that block ATR activity. These data support the potential for ATR inhibitors to substantially increase the efficacy of standard-of-care agents in diseases such as NSCLC. Citation Format: Diane Boucher, Peter Charlton, Jean-Damien Charrier, Brinley Furey, Yong Gu, Amy Hall, Brian Hare, Howard Li, Sean Milton, Cheryl Murphy, Philip Reaper, Darin Takemoto, Taturo Udagawa, Yuxin Wang, Mark Wood, John Pollard. Comprehensive preclinical evaluation of VE-822, the first ATR-targeted drug candidate: a novel approach to transforming the efficacy of DNA damaging agents. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr LB-299. doi:10.1158/1538-7445.AM2013-LB-299

Abstract 5491: Evaluation of the first potent and highly selective inhibitor of ATR kinase: An approach to selectively sensitize cancer cells to genotoxic drugs

Proceedings: AACR 102nd Annual Meeting 2011‐‐ Apr 2‐6, 2011; Orlando, FL DNA damaging agents have been the cornerstone of solid cancer therapy for decades yet they provide only modest benefit for patients with many tumor types. This reflects, in part, the efficient repair of DNA damage via a complex signaling and repair network known as the DNA damage response (DDR). Key regulators of the DDR are the phosphoinositol 3-kinase-like serine/threonine protein kinase (PIKK) family members ATR, ATM and DNA-PK. The DDR acts to detect DNA lesions, enforce checkpoints to halt cell cycle progression, and stimulate repair. Recent data have shown that elements of the DDR are commonly defective in cancer cells. It is widely believed that these cells become dependent on the remaining DDR pathways for survival from DNA damage. Inhibitors have been reported for a number of DDR enzymes, including ATM, DNA-PK, CHK1 and PARP, however there are no reports of drug-like ATR inhibitors. Here we disclose the in vitro characterization of a potent and highly selective ATR inhibitor (VE-821). This compound selectively blocks ATR signaling in cells (IC50 = 0.7 µM), but has little impact on ATM or DNA-PK signaling (IC50 >10 µM). Treatment with 10 µM VE-821 for 144 h causes little cell death in normal cell lines (5-11 %) but markedly higher death in cancer cell lines (28-46 %). VE-821 also dramatically sensitizes many cancer cells to multiple classes of genotoxic agents including antimetabolites, topoisomerase inhibitors and crosslinking agents; with over 10-fold increases genotoxic potency observed in some cases. In a panel of 36 lung cancer cell lines, VE-821 sensitized the cytotoxic effect of cisplatin to a far greater magnitude and over a broader subset of these lines than potent inhibitors of ATM, Chk1, or PARP. In over half of these cell lines, the IC50 of cisplatin was reduced by greater than 5 fold upon the addition of VE-821. We show that a basis for the cancer-selective effects of VE-821 is a synthetic lethal interaction between loss of ATM signaling (a frequent event in cancer resulting from loss of function of proteins such as ATM or p53) and ATR inhibition when cells encounter DNA damage. In keeping with this, ATR inhibition does not sensitize normal cells (with functional ATM) to the cytotoxic effects of genotoxic therapy. In this case a compensatory DDR is activated that is associated with marked activation of ATM, which in turn leads to reversible checkpoint arrest and a strong survival response. These studies show for the first time that a selective ATR inhibitor can preferentially sensitize cancer cells to genotoxic drugs by exploiting a synthetic lethal interaction between ATM and ATR signaling. This underpins the broad potential of ATR inhibition as a highly promising new strategy to improve the efficacy of genotoxic therapy. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 5491. doi:10.1158/1538-7445.AM2011-5491