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Supplementary Figure Legends. Supplementary Figure 1: Targeted disruption of AMPKα1/2 does not affect arsenicinducible phosphorylation of AKT at Ser473. Supplementary Figure 2: As- Biotin binds to AMPKα as measured by mass spectroscopy

The urokinase system is overexpressed in epithelial ovarian cancer cells and is expressed at low levels in normal cells. To develop a platform for intracellular and targeted delivery of therapeutics in ovarian cancer, we conjugated urokinase plasminogen activator (uPA) antibodies to liposomal nanobins. The arsenic trioxide–loaded nanobins had favorable physicochemical properties and the ability to bind specifically to uPA. Confocal microscopy showed that the uPA-targeted nanobins were internalized by ovarian cancer cells, whereas both inductively coupled plasma optical mass spectrometry (ICP-MS) and fluorescence-activated cell sorting (FACS) analyses confirmed more than four-fold higher uptake of targeted nanobins when compared with untargeted nanobins. In a coculture assay, the targeted nanobins showed efficient uptake in ovarian cancer cells but not in the normal primary omental mesothelial cells. Moreover, this uptake could be blocked by either downregulating uPA receptor expression in the ovarian cancer cells using short-hairpin RNA (shRNA) or by competition with free uPA or uPA antibody. In proof-of-concept experiments, mice bearing orthotopic ovarian tumors showed a greater reduction in tumor burden when treated with targeted nanobins than with untargeted nanobins (47% vs. 27%; P < 0.001). The targeted nanobins more effectively inhibited tumor cell growth both in vitro and in vivo compared with untargeted nanobins, inducing caspase-mediated apoptosis and impairing stem cell marker, aldehyde dehydrogenase-1A1 (ALDH1A1), expression. Ex vivo fluorescence imaging of tumors and organs corroborated these results, showing preferential localization of the targeted nanobins to the tumor. These findings suggest that uPA-targeted nanobins capable of specifically and efficiently delivering payloads to cancer cells could serve as the foundation for a new targeted cancer therapy using protease receptors. Mol Cancer Ther; 12(12); 2628–39. ©2013 AACR.

PDF - 469KB, Supplementary Materials & Methods; Supplementary Figure S1: Specific uptake of ATN-291-NB(Ni,As) in ovarian cancer cells; Supplementary Figure S2: Confocal studies of early apoptosis in HeyA8 cells treated with nanobins; Supplementary Figure S3: Targeted delivery using a urokinase antibody (ATN-291) does not inhibit uPA enzymatic activity in vitro; Supplementary Figure S4: Immunohistochemical analysis of the HeyA8 xenograft tumors upon the treatments of nanobins in vivo; Supplementary Figure S5: Organ function of mice treated by ATN-291-NB(Ni,As).

Supplementary Data from A Novel Nanoparticulate Formulation of Arsenic Trioxide with Enhanced Therapeutic Efficacy in a Murine Model of Breast Cancer

Purpose: The clinical success of arsenic trioxide (As2O3) in hematologic malignancies has not been replicated in solid tumors due to poor pharmacokinetics and dose-limiting toxicity. We have developed a novel nanoparticulate formulation of As2O3 encapsulated in liposomal vesicles or “nanobins” [(NB(Ni,As)] to overcome these hurdles. We postulated that nanobin encapsulation of As2O3 would improve its therapeutic index against clinically aggressive solid tumors, such as triple-negative breast carcinomas.Experimental Design: The cytotoxicity of NB(Ni,As), the empty nanobin, and free As2O3 was evaluated against a panel of human breast cancer cell lines. The plasma pharmacokinetics of NB(Ni,As) and free As2O3 were compared in rats to measure drug exposure. In addition, the antitumor activity of these agents was evaluated in an orthotopic model of human triple-negative breast cancer.Results: The NB(Ni,As) agent was much less cytotoxic in vitro than free As2O3 against a panel of human breast cancer cell lines. In contrast, NB(Ni,As) dramatically potentiated the therapeutic efficacy of As2O3 in vivo in an orthotopic model of triple-negative breast cancer. Reduced plasma clearance, enhanced tumor uptake, and induction of tumor cell apoptosis were observed for NB(Ni,As).Conclusions: Nanobin encapsulation of As2O3 improves the pharmacokinetics and antitumor efficacy of this cytotoxic agent in vivo. Our findings demonstrate the therapeutic potential of this nanoscale agent and provide a foundation for future clinical studies in breast cancer and other solid tumors. Clin Cancer Res; 16(14); 3607–17. ©2010 AACR.

Arsenoplatins are adducts of two chemically important anticancer drugs, cisplatin and arsenic trioxide, that have a Pt(II) bond to an As(III) hydroxide center. Screens of the NCI-60 human tumor cell lines reveal that arsenoplatin-1 (AP1), [Pt(μ-NHC(CH(3))O)(2)ClAs(OH)(2)], the first representative of this novel class of anti-cancer agents, displays a superior activity profile relative to the parent drugs As(2)O(3) or cisplatin in majority of cancer cell lines tested. These activity profiles are important because the success of arsenic trioxide in blood cancers (such as APL) has not been seen in solid tumors due to the rapid clearance of arsenous acid from the body. To understand the biological chemistry of these compounds, we evaluated interactions of AP-1 with the two important classes of biomolecules – proteins and DNA. The first structural studies of AP-1 bound to model proteins reveal that platinum(II) binds the Nε of His in a manner that preserves the Pt-As bond. We find that AP-1 readily enters cells and binds to DNA with an intact Pt-As bond (Pt:As ratio of 1). At longer incubation times, however, the Pt:As ratio in DNA samples increases, suggesting that the Pt-As bond breaks and releases the As(OH)(2) moiety. We conclude that arsenoplatin-1 has the potential to deliver both Pt and As species to a variety of hematological and solid cancers.

Ephrin receptor A2 (EphA2) is a member of the Ephrin/Eph receptor cell-to-cell signaling family of molecules, and it plays a key role in cell proliferation, differentiation, and migration. EphA2 is overexpressed in a broad range of cancers, and its expression is in many cases associated with poor prognosis. We recently developed a novel EphA2-targeting antibody-directed nanotherapeutic encapsulating a labile prodrug of docetaxel (EphA2-ILs-DTXp) for the treatment of EphA2-expressing malignancies. Here, we characterized the expression of EphA2 in bladder cancer using immunohistochemistry in 177 human bladder cancer samples and determined the preclinical efficacy of EphA2-ILs-DTXp in four EphA2-positive patient-derived xenograft (PDX) models of the disease, either as a monotherapy, or in combination with gemcitabine. EphA2 expression was detected in 80&ndash, 100% of bladder cancer samples and correlated with shorter patient survival. EphA2 was found to be expressed in tumor cells and/or tumor-associated blood vessels in both primary and metastatic lesions with a concordance rate of approximately 90%. The EphA2-targeted antibody-directed nanotherapeutic EphA2-ILs-DTXp controlled tumor growth, mediated greater regression, and was more active than free docetaxel at equitoxic dosing in all four EphA2-positive bladder cancer PDX models. Combination of EphA2-ILs-DTXp and gemcitabine in one PDX model led to improved tumor growth control compared to monotherapies or the combination of free docetaxel and gemcitabine. These data demonstrating the prevalence of EphA2 in bladder cancers and efficacy of EphA2-ILs-DTXp in PDX models support the clinical exploration of EphA2 targeting in bladder cancer.

// Meenakshi Upreti 1, 3 , Amar Jyoti 1, 3 , Sara E. Johnson 2 , Elden P. Swindell 4 , Dana Napier 3 , Pallavi Sethi 1, 3 , Ryan Chan 1 , Jonathan M. Feddock 5 , Heidi L. Weiss 3 , Thomas V. O’Halloran 4 , B. Mark Evers 3, 6 1 Department of Pharmaceutical Sciences, College of Pharmacy, University of Kentucky, Lexington, KY, USA 2 Markey Cancer Center, University of Kentucky, Lexington, KY, USA 3 Department of Pathology, University of Kentucky, Lexington,KY, USA 4 Department of Chemistry, Chemistry of Life Processes Institute, Northwestern University, Evanston, IL, USA 5 Department of Radiation Medicine, University of Kentucky Chandler Hospital, Lexington, KY, USA 6 Department of Surgery, University of Kentucky, Lexington, KY, USA Correspondence to: Meenakshi Upreti, email: m.upreti@uky.edu Keywords: galectin-1, triple negative breast cancer, stromal-targeting, TNBC tumor model, tumor tissue analog (TTA) Received: February 18, 2016 Accepted: May 04, 2016 Published: May 19, 2016 ABSTRACT Currently there are no FDA approved targeted therapies for Triple Negative Breast Cancer (TNBC). Ongoing clinical trials for TNBC have focused primarily on targeting the epithelial cancer cells. However, targeted delivery of cytotoxic payloads to the non-transformed tumor associated-endothelium can prove to be an alternate approach that is currently unexplored. The present study is supported by recent findings on elevated expression of stromal galectin-1 in clinical samples of TNBC and our ongoing findings on stromal targeting of radiation induced galectin-1 by the anginex-conjugated arsenic-cisplatin loaded liposomes using a novel murine tumor model. We demonstrate inhibition of tumor growth and metastasis in response to the multimodal nanotherapeutic strategy using a TNBC model with orthotopic tumors originating from 3D tumor tissue analogs (TTA) comprised of tumor cells, endothelial cells and fibroblasts. The ‘rigorous’ combined treatment regimen of radiation and targeted liposomes is also shown to be well tolerated. More importantly, the results presented provide a means to exploit clinically relevant radiation dose for concurrent receptor mediated enhanced delivery of chemotherapy while limiting overall toxicity. The proposed study is significant as it falls in line with developing combinatorial therapeutic approaches for stroma-directed tumor targeting using tumor models that have an appropriate representation of the TNBC microenvironment.

Platinum drugs (cisplatin, oxaliplatin, and carboplatin) and arsenic trioxide are the only commercial inorganic non-radioactive anticancer drugs approved by the US Food and Drug Administration. Numerous efforts are underway to take advantage of the synergy between the anticancer activity of cisplatin and arsenic trioxide - two drugs with strikingly different mechanisms of action. These include co-encapsulation of the two drugs in novel nanoscale delivery systems as well as the development of small molecule agents that combine the activity of these two inorganic materials. Several of these new molecular entities containing Pt-As bonds have broad anticancer activity, are robust in physiological buffer solutions, and form stable complexes with biopolymers. This review summarizes results from a number of preclinical studies involving the combination of cisplatin and As(2)O(3), co-encapsulation and nanoformulation efforts, and the chemistry and cytotoxicity of the first member of platinum anticancer agents with an arsenous acid moiety bound to the platinum(II) center: arsenoplatins.

The urokinase system is overexpressed in epithelial ovarian cancer cells and is expressed at low levels in normal cells. To develop a platform for intracellular and targeted delivery of therapeutics in ovarian cancer, we conjugated urokinase plasminogen activator (uPA) antibodies to liposomal nanobins. The arsenic trioxide–loaded nanobins had favorable physicochemical properties and the ability to bind specifically to uPA. Confocal microscopy showed that the uPA-targeted nanobins were internalized by ovarian cancer cells, whereas both inductively coupled plasma optical mass spectrometry (ICP-MS) and fluorescence-activated cell sorting (FACS) analyses confirmed more than four-fold higher uptake of targeted nanobins when compared with untargeted nanobins. In a coculture assay, the targeted nanobins showed efficient uptake in ovarian cancer cells but not in the normal primary omental mesothelial cells. Moreover, this uptake could be blocked by either downregulating uPA receptor expression in the ovarian cancer cells using short-hairpin RNA (shRNA) or by competition with free uPA or uPA antibody. In proof-of-concept experiments, mice bearing orthotopic ovarian tumors showed a greater reduction in tumor burden when treated with targeted nanobins than with untargeted nanobins (47% vs. 27%; P < 0.001). The targeted nanobins more effectively inhibited tumor cell growth both in vitro and in vivo compared with untargeted nanobins, inducing caspase-mediated apoptosis and impairing stem cell marker, aldehyde dehydrogenase-1A1 (ALDH1A1), expression. Ex vivo fluorescence imaging of tumors and organs corroborated these results, showing preferential localization of the targeted nanobins to the tumor. These findings suggest that uPA-targeted nanobins capable of specifically and efficiently delivering payloads to cancer cells could serve as the foundation for a new targeted cancer therapy using protease receptors. Mol Cancer Ther; 12(12); 2628–39. ©2013 AACR.

Recent advances in nanotechnology, materials science, and biotechnology have enabled the design of theranostic nanoparticles that co-deliver therapeutic compounds and imaging probes in a single platform to heterogeneous disease sites such as tumors, simultaneously treating and enabling non-invasive monitoring of the response to treatment.[1] This can allow the clinician to detect previously unseen tumor sites and immediately adjust the treatment regimen to improve the margin for success. Magnetic resonance imaging (MRI) has emerged as an ideal imaging modality for this application due to its high-resolution, real-time 3D tomography capability, excellent penetration depth, and independence from ionizing radiation (CT) or radionuclides (PET-CT/SPECT).[2] In contrast, optical imaging techniques utilizing two-photon fluorescence or intravital microscopy/endoscopy as well as bioluminescence are generally low resolution, only provide limited (or no) tomographic information, have insufficient penetration depth, and, requires multiple labels to assess the surrounding cells or tissues, thus limiting the amount of salient information that can be obtained on the tumor and its neighboring environment.[3] The aforementioned attributes make MRI the ideal imaging modality for evaluating the treatment of solid tumors.[4] However, to maximize the full potential of MRI for the evaluation of solid tumors, theranostic platforms have to overcome a number of limitations before they can be clinically successful. For in vivo imaging, the intrinsically low probe sensitivity, especially of T1 contrast agents, require efficient delivery and maximum cellular accumulation of contrast agents like gadolinium (Gd) chelates.[5] However, these contrast agents have poor cellular uptake, leading to insignificant contrast enhancements.[2] For therapy, the lack of specificity of chemotherapeutics to only the tumor tissue necessitates a high administered dose and reduces the efficacy of the drug.[6] This high-dosage requirement increases the prevalence of adverse side effects for patients and shrinks the therapeutic window.[7] The conjugation of GdIII chelates to a variety of nanostructures, including inorganic nanoparticles,[8] dendrimers,[9] viral capsids,[10] lipid nanoparticles,[11] liposomes,[12] and hydrogels,[13] has been shown to increase T1 MRI contrast compared to unconjugated GdIII chelates. Many of these platforms, however, have limited therapeutic efficacy due to insufficient drug-loading capabilities and/or lack of triggered drug-release ability under the specific conditions encountered at the tumor site. Additionally, when nanoparticles accumulate passively at the tumor site through the enhanced permeability and retention (EPR) effect[1a], their poor cellular internalization[14] and inability[15] to release the payload further limit their therapeutic efficacy. These major obstacles must be overcome for nanoparticle-based theranostic platforms to reach their full potential and become clinically viable.[1a, 16] Herein, we report the first multifunctional polymer-caged nanobin (PCN) theranostic platform that is capable of selectively targeting Her2-expressing tumor cells with high concentrations of T1 MRI contrast agents followed by the triggered release of a high payload of chemotherapeutics. We extended the PCN core-shell nanocarrier concept,[17] which comprises a doxorubicin (DXR)-loaded biocompatible liposomal core and an acid-sensitive, biodegradable polymer shell,[18] to include surface functionalization with Herceptin targeting groups and GdIII-based contrast agents (Figure 1a). In vitro testing of the Her2-targeted theranostic platform (Her-GdIII-PCNDXR) against Her2-overexpressing SK-BR-3 cells reveals an impressive 120-fold enhancement in cellular GdIII–uptake in comparison to free DOTA-GdIII, which significantly decreases T1 relaxation time and provides enhanced T1 MRI contrast for the targeted PCNs over the non-targeted nanocarriers. In addition, the 14-fold increase in cytotoxicity of the Her-GdIII-PCNDXR over the non-targeted analogue makes it a highly promising theranostic agent. Figure 1 (a) Schematic presentation of the preparation of DXR-loaded, Herceptin- and GdIII-conjugated PCN (Her-GdIII-PCNDXR). (b) Plots of the hydrodynamic diameters (DH) of BLDXR (black rectangle), PCNDXR (red circle), and Her-GdIII-PCNDXR (blue triangle) and ... Herceptin (Trastuzumab) is a monoclonal antibody that specifically binds to human epidermal growth factor receptor 2 (Her2), known to be overexpressed in ~25% of human breast cancers.[19] Recently, a promising clinical study[20] has reported that the Herceptin-conjugated chemotherapy agent Trastuzumab emtansine (T-DM1) significantly extends survival rate while reducing side effects in patients with aggressive Her2-overexpressing breast cancers, which suggests that targeting toxic chemotherapeutic agents to specific cell-surface receptors can indeed improve their therapeutic efficacies. We hypothesize that this strategy can be extended to our PCN-based theranostic nanoparticle platform[21] where the Her2-targeting functionality is expected to result in more efficient internalization of both drugs and imaging agents by Her2-overexpressing cancer cells through a receptor-mediated endocytosis pathway.[7, 22] Together with the pH-triggered drug-releasing capability[23] and high stability in physiological conditions,[17] the resulting enhanced cellular uptake would lead to major improvements in both cytotoxicity and MRI contrast enhancement at the target sites and would significantly advance the theranostic concept. While high specificity to Her2-overexpressing tumor cells has spurred usage of Herceptin as a targeting ligand for delivery nanocarriers of either drugs or imaging agents,[14, 24] only a few studies[25] have reported a Her2-targeted theranostic platform that includes both chemotherapeutics and imaging probes in a single delivery nanocarrier. Most of these platforms, however, have limited theranostic efficacy due to a lack of triggered drug release and limited ability for in vivo imaging. While a Her2-targeting, iron-oxide nanoparticle-based theranostic platform demonstrated acid-triggered drug release and MR imaing abilities[25c], this system allows T2 MR imaging and exhibits premature drug release under physiological conditions (pH 7.4 and 37 °C). To the best of our knowledge, there have been no reports of Her2-targeting theranostic platforms that combine a chemotherapeutic with a T1 MRI contrast agent and show significant enhancements in both therapy and diagnosis through triggered-release and Her2-targeting. In contrast to the iron oxide nanoparticle for T2 MR imaging, T1 MRI contrast agensts such as GdIII chelates must be further conjugated to a nanocarrier to enhance their intrinsic poor cellular internalization. The work described herein demonstrates the first smart multifunctional theranostic platform that presents facile acid-triggered drug release, high stability under physiological conditions, specific targeting of Her2-overexpresssing cancers, and enhanced T1-weighted MR imaging. Together, these four characteristics lead to outstanding in vitro theranostic performance against Her2-overexpressing breast cancer cells.

The urokinase plasminogen activator (uPA) system is a proteolytic system comprised of uPA, a cell surface receptor for uPA (uPAR), and an inhibitor of uPA (PAI-1) and is implicated in many aspects of tumor growth and metastasis. The uPA system has been identified in nearly all solid tumors examined to date as well as several hematological malignancies. In adults, transient expression of the uPA system is observed during wound healing and inflammatory processes while only limited expression is identified in healthy, quiescent tissue. Members of the uPA system are expressed not only on cancer cells but also on tumor-associated stromal cells. These factors make the uPA system an ideal therapeutic target for cancer therapies. To date most therapeutics targeted at the uPA system have been inhibitors of either the uPA-uPAR interaction or uPA proteolysis but have not shown robust anti-tumor activity. There is now mounting evidence that uPAR participates in a complex signaling network central to its role in cancer progression, which provides a basis for the hypothesis that uPAR may be a marker for cancer stem cells. Several new uPAR-directed therapies have recently been developed based on this new information. A monoclonal antibody has been developed that disrupts the interactions of uPAR with signaling partners and is poised to enter the clinic. In addition, nanoscale drug delivery vehicles targeted to the uPA system using monoclonal antibodies, without disrupting the normal functioning of the system, are also in development. This review will highlight some of these new discoveries and the new uPA system-based therapeutic approaches that have arisen from them.

Ephrin receptor A2 (EphA2) is a member of the Ephrin-Ephrin receptors cell to cell signaling family of molecules and plays a key role in proliferation, differentiation, and migration. EphA2 is overexpressed in a broad range of cancers, including bladder cancer, and is associated with increased metastasis and poor prognosis. Several potential EphA2-targeted therapies were developed and showed promising preclinical activity which failed to translate clinically due to narrow therapeutic window. we propose to use a novel antibody-directed nanotherapeutic (ADN) approach to target EphA2 for the treatment of bladder cancer. This work characterizes the expression of EphA2 in bladder cancer patients, evaluates its prognostic power, and tests an EphA2-targeted ADN, MM-310, in patient-derived xenograft (PDX) models. EphA2 expression in tumors was investigated using a validated immunohistochemistry assay performed in 147 bladder cancer samples, enabling analysis of prevalence and prognostic power. Whole sections of primary and metastatic tumor resections were used to assess EphA2 expression in tumor-associated vessels and tumor cells. Four EphA2+ PDX models were used to evaluate the activity of MM-310 compared to free docetaxel, alone or in combination with gemcitabine. EphA2 was expressed in tumor cells and tumor-associated blood vessels in primary and metastatic lesions. EphA2 overexpression was seen in 80%-100% of tumors and correlated with shorter survival. In the PDX models, MM-310 controlled tumor growth, mediated greater regression, and was more active than free docetaxel at equitoxic dosing (Table). Additionally, the combination of MM-310 and gemcitabine controlled tumor growth better than each drug alone, and outperformed the combination of free docetaxel and gemcitabine in the single PDX model where it was compared. Thus, in bladder cancer models, a docetaxel-based ADN as a monotherapy and in combination with gemcitabine targeted EphA2 and led to significant tumor regression. Maximum tumor regression (%) & Time to regrowth (days)Model NameControlDocetaxelMM-310p valueBL0293-2% & 3 days-58% & 55 days-100% & 120 days Citation Format: Walid Kamoun, Elden Swindell, Christine Pien, Lia Luus, Jason Cain, Irawati Kandela, Richard Huang, Suresh Tipparaju, Dmitri Kirpotin, Wiam Bshara, Vasileios Askoxylakis, Carl Morrison, Daryl Drummond. Targeting EphA2 in bladder cancer using a novel antibody-directed nanotherapeutic [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 5771.

An appropriate representation of the tumor microenvironment in tumor models can have a pronounced impact on directing combinatorial treatment strategies and cancer nanotherapeutics. The present study develops a novel 3D co-culture spheroid model (3D TNBC) incorporating tumor cells, endothelial cells and fibroblasts as color-coded murine tumor tissue analogs (TTA) to better represent the tumor milieu of triple negative breast cancer in vitro . Implantation of TTA orthotopically in nude mice, resulted in enhanced growth and aggressive metastasis to ectopic sites. Subsequently, the utility of the model is demonstrated for preferential targeting of irradiated tumor endothelial cells via radiation-induced stromal enrichment of galectin-1 using anginex conjugated nanoparticles (nanobins) carrying arsenic trioxide and cisplatin. Demonstration of a multimodal nanotherapeutic system and inclusion of the biological response to radiation using an in vitro / in vivo tumor model incorporating characteristics of tumor microenvironment presents an advance in preclinical evaluation of existing and novel cancer nanotherapies. From the Clinical Editor Existing in-vivo tumor models are established by implanting tumor cells into nude mice. Here, the authors described their approach 3D spheres containing tumor cells, enodothelial cells and fibroblasts. This would mimic tumor micro-environment more realistically. This interesting 3D model should reflect more accurately tumor response to various drugs and would enable the design of new treatment modalities.

Structural, thermodynamic, and gene expression studies provide a comprehensive picture of how the bacterial metalloregulatory transcriptional repressor Zur achieves its exquisite sensitivity to zinc concentrations., Commensal microbes, whether they are beneficial or pathogenic, are sensitive to host processes that starve or swamp the prokaryote with large fluctuations in local zinc concentration. To understand how microorganisms coordinate a dynamic response to changes in zinc availability at the molecular level, we evaluated the molecular mechanism of the zinc-sensing zinc uptake regulator (Zur) protein at each of the known Zur-regulated genes in Escherichia coli. We solved the structure of zinc-loaded Zur bound to the PznuABC promoter and show that this metalloregulatory protein represses gene expression by a highly cooperative binding of two adjacent dimers to essentially encircle the core element of each of the Zur-regulated promoters. Cooperativity in these protein-DNA interactions requires a pair of asymmetric salt bridges between Arg52 and Asp49′ that connect otherwise independent dimers. Analysis of the protein-DNA interface led to the discovery of a new member of the Zur-regulon: pliG. We demonstrate this gene is directly regulated by Zur in a zinc responsive manner. The pliG promoter forms stable complexes with either one or two Zur dimers with significantly less protein-DNA cooperativity than observed at other Zur regulon promoters. Comparison of the in vitro Zur-DNA binding affinity at each of four Zur-regulon promoters reveals ca. 10,000-fold variation Zur-DNA binding constants. The degree of Zur repression observed in vivo by comparison of transcript copy number in wild-type and Δzur strains parallels this trend spanning a 100-fold difference. We conclude that the number of ferric uptake regulator (Fur)-family dimers that bind within any given promoter varies significantly and that the thermodynamic profile of the Zur-DNA interactions directly correlates with the physiological response at different promoters., Author Summary Zinc is an essential nutrient for most organisms, with the Zn2+ ion performing numerous structural, regulatory, and catalytic roles in a range of proteins. However, this nutrient can neither be synthesized nor degraded and individual cells need to be able to maintain steady levels of zinc in the face of near-zero or excessively high environmental concentrations. Here we look at how the bacterium E. coli does this, by examining the structure and function of Zur, a transcriptional repressor that is exquisitely sensitive to Zn2+ concentration. Although the structures of related Zur proteins on their own are known, here we show how E. coli protein binds to DNA and explain its extreme sensitivity and specificity (it responds to Zn2+ concentrations in the femtomolar range). Our results reveal how the Zur protein switches on and off a bank of bacterial genes that control zinc physiology. Extensive analysis of protein-DNA interactions revealed both a surprising degree of cooperativity and an extremely large range of Zur-DNA binding affinities across the set of genes known as the Zur regulon. The results provide strong support for a controversial idea that the thermodynamics of an ensemble of protein-DNA interactions play a dominant role in the physiological control of gene regulation networks. In addition, we have used our structural and thermodynamic analysis to identify a novel gene target of Zur regulation.

Arsenic trioxide (As2O3) exhibits potent antineoplastic effects and is used extensively in clinical oncology for the treatment of a subset of patients with acute myeloid leukemia (AML). Although As2O3 is known to regulate activation of several signaling cascades, the key events, accounting for its antileukemic properties, remain to be defined. We provide evidence that arsenic can directly bind to cysteine 299 in AMPKα and inhibit its activity. This inhibition of AMPK by arsenic is required in part for its cytotoxic effects on primitive leukemic progenitors from patients with AML, while concomitant treatment with an AMPK activator antagonizes in vivo the arsenic-induced antileukemic effects in a xenograft AML mouse model. A consequence of AMPK inhibition is activation of the mTOR pathway as a negative regulatory feedback loop. However, when AMPK expression is lost, arsenic-dependent activation of the kinase RSK downstream of MAPK activity compensates the generation of regulatory feedback signals through phosphorylation of downstream mTOR targets. Thus, therapeutic regimens with As2O3 will need to include inhibitors of both the mTOR and RSK pathways in combination to prevent engagement of negative feedback loops and maximize antineoplastic responses. Mol Cancer Ther; 14(1); 202–12. ©2014 AACR.

A PCN theranostic platform comprises a doxorubicin (DXR)-loaded liposomal core and an acid-sensitive polymer shell that is functionalized with Herceptin and GdIII-based MRI contrast agents. In vitro testing reveals a 14-fold increase in DXR-based cytotoxicity versus a non-targeted analogue and an 120-fold improvement in cellular GdIII–uptake in comparison with clinically approved DOTA-GdIII, leading to significant T1 MRI contrast enhancement.

Starting in ancient China and Greece, arsenic-containing compounds have been used in the treatment of disease for over 3000 years. They were used for a variety of diseases in the 20th century, including parasitic and sexually transmitted illnesses. A resurgence of interest in the therapeutic application of arsenicals has been driven by the discovery that low doses of a 1% aqueous solution of arsenic trioxide (i.e., arsenous acid) lead to complete remission of certain types of leukemia. Since Food and Drug Administration (FDA) approval of arsenic trioxide (As2O3) for treatment of acute promyelocytic leukemia in 2000, it has become a front-line therapy in this indication. There are currently over 100 active clinical trials involving inorganic arsenic or organoarsenic compounds registered with the FDA for the treatment of cancers. New generations of inorganic and organometallic arsenic compounds with enhanced activity or targeted cytotoxicity are being developed to overcome some of the shortcomings of arsenic therapeutics, namely, short plasma half-lives and a narrow therapeutic window.

Starting in ancient China and Greece, arsenic-containing compounds have been used in the treatment of disease for over 3000 years. They were used for a variety of diseases in the 20th century, including parasitic and sexually transmitted illnesses. A resurgence of interest in the therapeutic application of arsenicals has been driven by the discovery that low doses of a 1% aqueous solution of arsenic trioxide (i.e., arsenous acid) lead to complete remission of certain types of leukemia. Since Food and Drug Administration (FDA) approval of arsenic trioxide (As2O3) for treatment of acute promyelocytic leukemia in 2000, it has become a front-line therapy in this indication. There are currently over 100 active clinical trials involving inorganic arsenic or organoarsenic compounds registered with the FDA for the treatment of cancers. New generations of inorganic and organometallic arsenic compounds with enhanced activity or targeted cytotoxicity are being developed to overcome some of the shortcomings of arsenic therapeutics, namely, short plasma half-lives and a narrow therapeutic window.

Polymer nanoparticles (PNPs) possessing a high density of drug payload have been successfully stabilized against aggregation in biological buffers after amine modification, which renders these PNPs positively charged. The resulting charge-stabilized PNPs retain their original narrow particle size distributions and well-defined spherical morphologies. This stabilization allows these PNPs to have an improved anti-proliferative effect on MDA-MB-231-Br human breast cancer cells compared to non-functionalized PNPs. As a non-cytotoxic control, similar surface-modified PNPs containing cholesterol in place of doxorubicin did not inhibit cell proliferation, indicating that the induced cytotoxic response was solely due to the doxorubicin release from the PNPs.

Purpose: The clinical success of arsenic trioxide (As2O3) in hematologic malignancies has not been replicated in solid tumors due to poor pharmacokinetics and dose-limiting toxicity. We have developed a novel nanoparticulate formulation of As2O3 encapsulated in liposomal vesicles or “nanobins” [(NB(Ni,As)] to overcome these hurdles. We postulated that nanobin encapsulation of As2O3 would improve its therapeutic index against clinically aggressive solid tumors, such as triple-negative breast carcinomas. Experimental Design: The cytotoxicity of NB(Ni,As), the empty nanobin, and free As2O3 was evaluated against a panel of human breast cancer cell lines. The plasma pharmacokinetics of NB(Ni,As) and free As2O3 were compared in rats to measure drug exposure. In addition, the antitumor activity of these agents was evaluated in an orthotopic model of human triple-negative breast cancer. Results: The NB(Ni,As) agent was much less cytotoxic in vitro than free As2O3 against a panel of human breast cancer cell lines. In contrast, NB(Ni,As) dramatically potentiated the therapeutic efficacy of As2O3 in vivo in an orthotopic model of triple-negative breast cancer. Reduced plasma clearance, enhanced tumor uptake, and induction of tumor cell apoptosis were observed for NB(Ni,As). Conclusions: Nanobin encapsulation of As2O3 improves the pharmacokinetics and antitumor efficacy of this cytotoxic agent in vivo. Our findings demonstrate the therapeutic potential of this nanoscale agent and provide a foundation for future clinical studies in breast cancer and other solid tumors. Clin Cancer Res; 16(14); 3607–17. ©2010 AACR.

The therapeutic efficacy of antitumor drugs is often limited by non-specific, systematic delivery. Here we report a novel targeted delivery platform designed by coupling liposomal nanobins (NB) with the urokinase plasminogen activator receptor (u-PAR) antibody (ATN-658). The urokinase system is overexpressed in epithelial ovarian cancer (OvCa) cells and is expressed only at low levels in normal cells. Doxorubicin (Dox), a FDA approved antitumor drug, had been loaded into the nanobins as a payload. The size and surface charge of the nanobins were optimized to facilitate specific binding to u-PAR expressing OvCa cells. Confocal and transmission electron microscopy showed that ATN-658-NB(Dox) was internalized in OvCa cells in a receptor-dependent manner and was released from endosomes as a function of time. This uptake could be blocked by stably down-regulating u-PAR expression in the OvCa cells using shRNA. In an orthotopic ovarian cancer model, athymic mice treated with ATN-658-NB(Dox) had a significantly greater reduction in tumor burden (0.06±0.01g versus 0.1±0.01g; p Citation Format: Yilin Zhang, Elden P. Swindell, Patrick L. Hankins, Hilary A. Kenny, Thomas V. O’Halloran, Andrew P. Mazar, Ernst Lengyel. Targeted delivery of doxorubicin loaded nanobins to ovarian cancer cells through the urokinase plasminogen activator system [abstract]. In: Proceedings of the 10th Biennial Ovarian Cancer Research Symposium; Sep 8-9, 2014; Seattle, WA. Philadelphia (PA): AACR; Clin Cancer Res 2015;21(16 Suppl):Abstract nr POSTER-THER-1440.

Glioblastoma multiforme (GBM) is fatal in nearly all instances, and responds poorly to current standards of care. GBMs exhibit resistance to chemotherapy which leads to tumor recurrence following initial surgical debulking. We have recently developed an 80 nm liposome containing a novel hydrophobic microtubule disrupting piperazine. This novel nanostructure, called IM-1 nanobin, contains 500-1000 piperazine drug molecules encapsulated in the hydrophobic inner leaflet of the lipid bilayer in each liposome. This nanoparticle inhibited the proliferation of GBM cells in vitro, although its cytotoxicity was reduced (3-5 fold) compared to free drug dissolved in DMSO. Results from FACS analysis showed that sub-micromolar concentrations of IM-1 caused G2/M phase cell cycle arrest and apoptosis of U87 GBM cells, as well as early passage GBM cells derived from a patient-derived xenograft (PDX). In collaboration with the Northwestern Brain Tumor Institute, we have established a panel of PDX. Since these tumors are not propagated in culture, they preserve the mixture of tumor cells and stroma critical for conducting meaningful therapy response experiments. IM-1 was evaluated in GBM PDX propagated in subcutaneous and intracranial compartments. In mice bearing subcutaneous PDX tumors, IM-1 treatment delayed the growth of the tumors compared to free drug alone; orthotopic GBM PDX studies are ongoing. Our results show that a novel liposome encapsulated piperazine, IM-1, is a promising new nanostructure for development as a therapeutic for treating patients with GBM. Citation Format: Elden P. Swindell, Andrey Ugolkov, Christian Freguia, Oleksii Dubrovskyi, Patrick L. Hankins, Jeong Yang, Jeffrey J. Raizer, James P. Chandler, Charles David James, Andrew P. Mazar, Thomas V. O'Halloran. Liposomes containing piperazine compounds inhibit tumor growth in a patient-derived xenograft model of glioblastoma multiforme. [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 4389. doi:10.1158/1538-7445.AM2015-4389

Several studies including our own have made it increasingly clear that genetic alterations of tumor cells alone are not the sole driving force behind tumor development but that tumor initiation, growth, progression and response to combinatorial treatments are rather intimately controlled by the microenvironment. Improved knowledge of cancer biology and investigation of the complex functional interrelation between the cellular and noncellular compartments of the tumor microenvironment have provided an ideal platform for the evolution of novel cancer nanotherapies. In the present study we have developed an in-vitro method to co-culture color coded triple negative breast carcinoma cells and cell types that exist in the tumor/tumor microenvironment in a “hanging drop” of medium to the size of an avascular microtissue (∼800 µm). Changes in the fluorescence intensity of different cell types and spheroid/microtissue sizing is being utilized as a method for high throughput therapeutic evaluation of the combination therapy using nanoparticles. When implanted orthotopically and in the dorsal skin fold window chambers in nude mice they facilitate the imaging of neovascularization and understanding response to therapy. We have also observed that radiation (3Gy) can significantly enhances expression of Galectin-1 in tumor vasculature associated endothelial cells which can serve as a specific ligand for the 33 amino acid antiangiogenic, anginex peptide. Thus Galectin-1 is a promising candidate for actively targeting irradiated tumors with anginex conjugated nanoparticles. The objective of this study is to establish a novel strategy for radiation therapy-amplified delivery of dual drugs for targeting the microenvironment-responsive endothelial component of the tumor vasculature. Use of radiation-guided anginex bound nanoparticles for delivery and controlled release of dual drugs (Arsenic-cisplatin) are expected to simulate metronomic therapy and preferentially target the tumor endothelial cells at the irradiated tumor site via tumor stroma enriched Galectin-1 protein. Our molecular studies indicate differential vulnerability for arsenic and cisplatin in cell types constituting the tumor/tumor microenvironment. This is the first study to understand a novel combinatorial nanotherapeutic system in an in-vitro/ in-vivo breast tumor model which incorporates characteristics of the tumor microenvironment and is thus expected to better predict treatment response in patients. Supported by NCI grant CA173609 and (CNPP) U01CA151461 Citation Format: Pallavi Sethi, Amar Jyoti, Elden Swindell, Ulrich W. Langner, William H. St. Clair, Ronald C. McGarry, Thomas V. O'Halloran, Meenakshi Upreti. Impact of tumor microenvironment on tumor growth, metastasis and response to combination therapy via microenvironment-responsive dual drug-loaded nanoparticles and radiation. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 4589. doi:10.1158/1538-7445.AM2014-4589

One of the primary goals of a successful cancer treatment regimen is to deliver an effective combination of radiation and/or drugs to tumors while minimizing damage to normal tissues. Many anti-angiogenic agents, while not able to control tumor growth, possess the ability to selectively target the location and process of tumor blood vessel formation. Conversely many chemotherapy agents are highly cytotoxic and lack selective targeting ability thus decreasing the therapeutic ratio. We are investigating a new drug delivery strategy exploiting the tumor endothelium “stimulated” by ionizing radiation to preferentially target and deliver a nanoparticulate formulation of arsenic trioxide (ATO) encapsulated in liposomal vesicles or “nanobins” to the irradiated tumor tissue. The targeting is via the 33 amino acid anti-angiogenic peptide, anginex. The identification of galectin-1 as the receptor for anginex expressed on activated endothelial cells involved in tumor angiogenesis has revealed a solid basis for this therapeutic rationale. Unlike the antiangiogenic agent Avastin, which is an antibody that targets the vascular endothelial growth factor (VEGF) released by the tumor cells into the microenvironment, this peptide binds to galectin-1, a tumor endothelial cell specific antigen that is expressed in solid tumors. We have made the novel discovery that galectin-1 expression is further upregulated in the tumor after radiation exposure, particularly on the endothelial cell surface. Exposure of murine SCK breast tumors to a clinical radiation dose of 2Gy induced a substantial average increase of 141 +/− 49% in anginex uptake as assessed by [18]-F-labeled anginex biodistribution. These results suggest that anginex may be an effective targeting molecule for image and radiation-guided therapy of solid tumors. To assist in delineating the exact mechanisms for our radiation-guided drug delivery strategy, we have developed a system to grow 3D tumor cell-endothelial cell spheroids and have observed increased galectin-1 expression upon radiation exposure. We are using these spheroidal cultures to implant tumors in dorsal skin fold window chambers for intravital wavelength imaging of drug delivery to tumor tissue before and after radiation exposure. Understanding how anginex uptake selectively increases in endothelial cells after irradiation and its nexus to radiation-sensitivity and drug delivery is our current focus. Further studies are underway to characterize the in vivo targeting of arsenic trioxide chemotherapy via anginex-conjugated ‘nanobins’ in combination with radiation exposure and assess the therapeutic potential in our solid tumor model. Supported by NCI grant CA107160 and the Central Arkansas Radiation Therapy Institute. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 4644. doi:1538-7445.AM2012-4644

That the urokinase plasminogen activator receptor (uPAR) system is constitutively expressed in primary and metastatic ovarian cancer regardless of grade or stage, while it is only weakly expressed in normal tissues, identifies it as a possible therapeutic target. Taking advantage of the tumor-specific expression of uPAR, a nanoscale liposome (nanobin, NB) was coupled with an antibody (ATN 291) against the uPAR ligand, uPA, to facilitate receptor-mediated, targeted drug delivery. The nanobin was loaded with arsenic trioxide (As2O3, in short As), since our previous results had demonstrated effective tumor uptake and induction of tumor cell apoptosis using As loaded nanobins in an orthotropic model of human triple negative breast cancer. We hypothesized that the ATN 291-conjugated nanobin (NB-uPA Ab) would allow specific cellular internalization and increase the therapeutic index of As in ovarian cancer cells. Using flow cytometry and fluorescence microscopy, we measured the cellular uptake of NB(Calcein)-uPA antibody (Ab) in HeyA8, ovarian cancer cells. The data showed a dose-dependent pattern of internalization with maximum internalization at 24 hours. Furthermore, NB-uPA Ab showed elevated cellular uptake when compared to non-targeted NB. More importantly, delivery of the targeted nanobins is uPA/uPAR dependent. High doses of ATN-291 or scuPA inhibited the cellular binding of NB-uPA Ab to HeyA8 cells. Moreover, down-regulation of uPAR expression via RNA interference in ovarian cancer cells inhibited the cellular uptake of the NB-uPA Ab. The cell viability of HeyA8 cells was significantly reduced following treatment of both non-targeted NB(As) and NB(As)-uPA Ab when compared to empty NB treatment as determined by a cytotoxicity assay. However, the cytotoxicity of NB(As)-uPA Ab is 2.1-fold more potent than the non-targeted NB(As). Taken together, these data indicate that the uPA Ab conjugated nanobins improved drug delivery and enhanced the anti-cancer effects of As, demonstrating the utility of targeting the uPA/uPAR system in ovarian cancer chemotherapeutics. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 2885. doi:1538-7445.AM2012-2885

We have developed a novel strategy to encapsulate a combination of cisplatin and arsenic trioxide as a precipitate inside 100 nm liposomal nanobins, NB(Pt, As). Cisplatin is a widely used chemotherapeutic but has significant dose-limiting toxicity. Arsenic trioxide (ATO) is a potent FDA-approved therapy for acute promyelocytic leukemia; however, ATO has not been effective in clinical trials of solid tumors. Encapsulation in nanoliposomes can improve the anti-cancer efficacy of chemotherapeutic agents by extending serum half-life, increasing tumor drug delivery and attenuating toxicity. We have tested the therapeutic efficacy of NB(Pt, As) in orthotopic xenograft models of triple negative breast cancer using MDA-MB-231 and MDA-MB-435/LvBr1 cancer cell lines. Treatment with either NB(Pt, As) or cisplatin for three weeks inhibited growth of MDA-MB-231 mammary tumors in vivo. However, the NB(Pt, As) did not cause weight loss in treated animals and did not result in abnormal serum chemistries. NB(Pt, As) was also evaluated for its ability to inhibit lung metastases. Mice inoculated intraductally with highly metastatic MDA-MB-435/LvBr1 cancer cells develop mammary tumors that metastasize and form macroscopic lesions in the lung. Treatment with NB(Pt, As) was initiated when primary tumors reached∼250 mm3. Both NB(Pt, As) and cisplatin inhibited primary tumor growth in this model, whereas only NB(Pt, As) treatment significantly reduced lung metastatic burden. Our results suggest that nanobin encapsulated cisplatin and ATO is a promising nanoscale therapeutic agent for metastatic breast cancer. 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 LB-202. doi:10.1158/1538-7445.AM2011-LB-202