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Prostate cancer is the leading cause of cancer-related death in men in the United States. A major cause of drug resistance in prostate and other epithelial tumors may be due to the presence of a fraction of tumor cells that retain the ability to initiate tumors and hence are termed tumor-initiating cells (TIC) or cancer stem cells. Here, we report that darinaparsin, an organic derivative of arsenic trioxide, is cytotoxic to prostate cancer cell lines as well as fresh prostate cancer cells from patients at low micromolar concentrations, and importantly inhibits the TIC subpopulations. It also inhibits growth of the castrate-resistant Du145 prostate tumor propagated as xenograft in mice and inhibits the tumor-initiating potential of prostate cancer cells. Although the mechanism by which darinaparsin acts is not completely known, we show that it kills prostate cancer cells by blocking cells in the G2–M phase of the cell cycle and inhibits Hedgehog signaling by downregulating Gli-2 transcriptional activity. These data provide a rationale for evaluating darinaparsin in patients with castrate-resistant prostate cancer. Mol Cancer Ther; 14(1); 23–30. ©2014 AACR.

Cyclin-dependent kinases (CDK), and their regulatory cyclin partners, play a central role in eukaryotic cell growth, division, and death. This key role in cell cycle progression, as well as their deregulation in several human cancers, makes them attractive therapeutic targets in oncology. A series of CDK inhibitors was developed using Astex's fragment-based medicinal chemistry approach, linked to high-throughput X-ray crystallography. A compound from this series, designated AT7519, is currently in early-phase clinical development. We describe here the biological characterization of AT7519, a potent inhibitor of several CDK family members. AT7519 showed potent antiproliferative activity (40-940 nmol/L) in a panel of human tumor cell lines, and the mechanism of action was shown here to be consistent with the inhibition of CDK1 and CDK2 in solid tumor cell lines. AT7519 caused cell cycle arrest followed by apoptosis in human tumor cells and inhibited tumor growth in human tumor xenograft models. Tumor regression was observed following twice daily dosing of AT7519 in the HCT116 and HT29 colon cancer xenograft models. We show that these biological effects are linked to inhibition of CDKs in vivo and that AT7519 induces tumor cell apoptosis in these xenograft models. AT7519 has an attractive biological profile for development as a clinical candidate, and the tolerability and efficacy in animal models compare favorably with other CDK inhibitors in clinical development. Studies described here formed the biological rationale for investigating the potential therapeutic benefit of AT7519 in cancer patients. [Mol Cancer Ther 2009;8(2):324–32]

AT7519 is a potent inhibitor of several cyclin-dependent kinases and is currently in early phase clinical development. Recently, cyclin-dependent kinases 7, 8, and 9 have been shown to regulate transcription through phosphorylation of RNA polymerase II. B-cell lymphoproliferative disorders, including chronic lymphocytic leukemia, rely on the expression of transcripts with a short half-life, such as Mcl-1, Bcl-2, and XIAP, for survival. Here, we describe the characterization of AT7519 in leukemia cell lines, and compare and contrast the response in cell lines derived from solid tumors. Finally, we use these mechanistic insights to show activity in peripheral blood mononuclear cells isolated from 16 chronic lymphocytic leukemia patients. AT7519 induced apoptosis at concentrations of 100 to 700 nmol/L and was equally effective regardless of Rai stage or known prognostic markers. Short-term treatments (4-6 hours) resulted in inhibition of phosphorylation of the transcriptional marker RNA polymerase II and downregulation of the antiapoptotic protein Mcl-1, with no effect on either XIAP or Bcl-2 levels. The reduction in Mcl-1 protein level was associated with an increase in cleaved poly(ADP-ribose) polymerase. Together the data suggest AT7519 offers a promising treatment for patients with advanced B-cell leukemia. Mol Cancer Ther; 9(4); 920–8. ©2010 AACR.

While monoclonal antibodies remain highly effective anticancer agents, their complex post-translational processing preclude their use in multi-effector gene therapy platforms. Breast and ovarian cancers often overexpress HER family receptors and consequently the receptors are targets for effective therapeutics such as the monoclonal antibodies trastuzumab and cetuximab. To address the need for new immune modulating formats that can be encoded in a single therapy, we have designed a platform for rapid conversion and assay of monoclonal antibodies into functional single chain variable fragment-Fc fusion proteins. Here we demonstrate results with trastuzumab and cetuximab as examples of the platform scFv-Fc conversion. The scFv-Fc trastuzumab displays efficient expression from transfected cell lines and high-affinity, high specificity binding to HER2 in plate-based screens with measured KD of 0.15nM compared to commercial trastuzumab's KD of 0.08nM in the same assay. scFv-Fc trastuzumab was shown to bind the HER2+ breast cancer cell lines BT-474 and SKBR3 in FACS-based assays while showing no affinity towards HER2- control cells SHP-77. Antibody-dependent cell-mediated cytotoxicity assays (ADCC) were performed to evaluate ADCC potency of the scFv-Fc trastuzumab. Using NK92/CD16A(158V/V) cells as effectors and BT474 cells as targets, the EC50 of the scFv-Fc trastuzumab was 3.6ng/ml, compared to the EC50 of monoclonal format trastuzumab at 0.94ng/ml. The data provide a direct demonstration of the scFv-Fc format as a viable alternative for achieving molecular targeting in a single chain entity at a level commensurate with KD values of full-length monoclonal antibodies while maintaining ADCC activity. In addition, functional scFv-Fc trastuzumab was expressed in combination with scFv-Fc cetuximab from a single vector. scFv-Fc molecules generated using this modular protein engineering platform were cloned into plasmid DNA vectors controlled by the RheoSwitch Therapeutic System®. Transient transfections into differentiated C2C12 myocytes demonstrated the production of scFv-Fc proteins from muscle cells under control of the RheoSwitch® Activator Ligand. The integrated scFv-Fc optimization platform and inducible multigenic systems described here provides support for future work for the treatment of cancer using multigenic therapeutic antibodies. Citation Information: Mol Cancer Ther 2013;12(11 Suppl):B247. Citation Format: Charles Reed, Vernon Dailey, Anissa Elayadi, Lindsay Williams, Dina Schneider, Thomas Reed, Jonathan Lewis, Richard Einstein, Samuel Broder. Integration of a modularized protein engineering technology and the RheoSwitch Therapeutic System® platform to develop high affinity trastuzumab single chain variable fragment-Fc proteins for gene therapy applications. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2013 Oct 19-23; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2013;12(11 Suppl):Abstract nr B247.

The localization capability of human mesenchymal stem cell (hMSCs) to tumors offers an attractive possibility as a cellular vehicle delivery route of immunotherapeutic genes. The RheoSwitch Therapeutic System® (RTS®) platform allows for regulated expression following exposure and withdrawal of activator ligand (AL) veledimex (also known as INXN-1001). In this study, we first evaluated RTS® regulated IL-12 expression from adenovirally transduced hMSC to generate a genetically modified hMSC (GM-hMSC) that could be used as a vehicle for gene delivery with the added benefit of migration potential to the tumor for therapy. Following establishment of the cultures in vitro, hMSCs were phentoypically similar following adenoviral vector transduction to mock transduced hMSCs based upon marker expression, characterized by flow cytometry. Levels of human and mouse IL-12 secreted from the hMSCs transduced with Ad-RTS-hIL-12 and Ad-RTS-mIL-12 respectively, directly correlated with the viral vector doses (1K-20K vp/cell). hMSC transduction efficiency was ∼90% with an MOI of 20K/cell. In addition, sustained cell levels of IL-12 expression were observed up to 53 days following transduction when maintained in the presence of veledimex. Furthermore, cycling of in vitro exposure periods between veledimex and excipient demonstrated the ability for on/off/on and off/on/off kinetics of IL-12 expression by transduced hMSCs. We then expanded on the concept of RTS® regulated gene expression in GM-hMSCs using multigenic plasmid constructs that simultaneously expressed three immunomodulators - human IL-12, human IFNα, and a CTLA4 decoy - in single, dual or triple combinations. Transient transfection of the multieffector plasmid using the AMAXA nucleofector system resulted in RTS® regulated concomitant expression of all three effectors. hIL-12 and hIFNα were found to be fully bio-functional in their respective cell based functional assays; - hIL-12 increased IFNγ secretion from NK92 cells, hIFNα enhanced STAT1 reporter activity, and the CTLA4 decoy functional assay is in progress. Taken together, these in vitro studies highlight the potential use of MSCs for tumor-targeted delivery of single or multiple RTS® regulated cancer immunotherapies. Altogether, use of these novel regulated immunotherapeutic approaches could potentially be translated into an effective clinical regimen for a variety of cancers. Citation Information: Mol Cancer Ther 2013;12(11 Suppl):C234. Citation Format: Tim Chan, Anjali Prabhu, Anissa Elayadi, Lindsay Williams, Vernon Dailey, Kristi Elliot, Tracey Snipas, Jonathan Carson, Jonathan Lewis, Stephen Schauer, Daniel Bednarik, Jayson M. Rieger, Laurent M. Humeau, Thomas R. Reed. Regulated immunomodulators expression using the RheoSwitch Therapeutic System® platform in human mesenchymal stem cells. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2013 Oct 19-23; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2013;12(11 Suppl):Abstract nr C234.

Palifosfamide (Pa) is a novel DNA cross linker molecule that consists of the active anti-tumor metabolite of ifosfamide (I). Potential advantages over I include the abrogation of I-metabolite related toxicities, increased activity by virtue of increased dose delivery over time, and overcoming resistance mediated by aldehyde dehydrogenase (ALDH) overexpression. ALDH overexpression is associated with cancer cell stem-like potential in several tumor types. Pa has broad activity in vitro and in vivo preclinical models and has shown early activity in humans. A previous randomized study evaluating the addition of I to cisplatin and etoposide (E) in small cell lung cancer (SCLC) demonstrated improved survival, but with disabling increase in toxicity with the three-drug combination. We hypothesized that the substitution of Pa for I could increase the therapeutic advantage of a similar three-drug regimen. We initiated a multicenter phase I, open-label, dose-escalation study assessing the safety and efficacy of Pa in combination with carboplatin and E (PaCE regimen) in SCLC and in other cancers in which C+E is considered an appropriate therapeutic option. Tumor responses were assessed by RECIST 1.1 and relevant tumor markers. A total of 15 patients (8 females and 7 males) have been treated to date: 5 with SCLC, 2 with NSCLC, 2 with ovarian, 1 with testicular and 5 with other cancers. Serious adverse events have been reported in six patients, including, thrombocytopenia (4), leukopenia (2), and neutropenia (2). The maximum tolerated dose of Pa was determined to be 130 mg/m2 when administered in combination with E 90 mg/m2 and C AUC4. The dose limiting toxicity was neutropenic fever. To date, two radiological partial responses in five patients with SCLC and one complete response assessed by tumor markers in a patient with testicular cancer have been noted. Conclusion: This study is ongoing, and an additional cohort evaluating Pa 130 mg/m2 in combination with E 100 mg/m2 and C AUC 4 is being evaluated. Further clinical data will be presented. The PaCE combination appears to be tolerable and has demonstrated clinical activity. Based on these data, a confirmatory study in SCLC is planned. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2011 Nov 12-16; San Francisco, CA. Philadelphia (PA): AACR; Mol Cancer Ther 2011;10(11 Suppl):Abstract nr B53.

Heat Shock Protein 90 (HSP90) is a member of a family of molecular chaperone proteins which directs the folding of polypeptides into functional configurations affecting stabilisation and activation. Many of these proteins are oncogenes regulating tumor cell growth, survival and apoptosis. This poster will focus on the screening and medicinal chemistry work that led to the identification of AT13387, a high affinity HSP90 inhibitor that is currently in clinical trials for the treatment of cancer. A fragment screening campaign was conducted against the N-terminal domain of HSP90 to detect very low molecular weight compounds (Molecular Weight Subsequent lead optimisation focussed on the improvement of in vivo distribution properties via the addition of basic moieties to the lead molecule. These compounds showed encouraging in vivo pharmacology and biological profiles, and further medicinal chemistry work led to the discovery of AT13387, an inhibitor with sub-nanomolar affinity, prolonged duration of action and excellent in vivo anti-tumor efficacy. This poster represents first disclosure of the structure of AT13387 and illustrates how a fragment-based drug discovery approach can be efficiently used to discover compounds suitable for clinical testing in oncology. Citation Information: Mol Cancer Ther 2009;8(12 Suppl):A211.