Testing Of New Therapeutic Agents Research Articles

A Novel External Auditory Canal Squamous Cell Carcinoma Cell Line Sensitive to CDK4/6 Inhibition.

To characterize cell line CAE606 derived from a squamous cell carcinoma (SCC) of the external auditory canal (EAC) and to show its usefulness as a model for testing candidate therapeutic agents. Preclinical translational research. Biomedical research institute. The cell line was initiated from a moderately differentiated T2N0M0 EAC SCC. We studied its histologic and genetic features as well as growth and invasion parameters. Sensitivity to cell CDK4/6 cell cycle inhibitor palbociclib was analyzed. CAE606 cells expressed heavy molecular weight cytokeratin, p63, and vimentin. The population doubling time was 25.8 hours, and the cells showed fast collective cell migration in a wound-healing assay. Short tandem repeat analysis confirmed it to be derived from the primary tumor of the patient. Next-generation sequencing revealed alterations in cell cycle regulation genes, including inactivating mutations in CDKN2A and TP53 and high-level amplification of CCND1 and EGFR. CAE606 showed a strong decrease of phospo-Rb expression upon exposure to the CDK4/6 inhibitor palbociclib, causing significant growth inhibition with an IC50 of 0.46 µM. This is the first report of a stable EAC SCC cell line. Its genetic features make it a useful tool for preclinical testing of new therapeutic agents for EAC SCC, particularly those targeting cell cycle regulation in combination with radio- and chemotherapy or other specific signaling pathway inhibitors.

Efficacy of High-Dose Chemotherapy and Three-Dimensional Conformal Radiation for Atypical Teratoid/Rhabdoid Tumor: A Report From the Children's Oncology Group Trial ACNS0333.

Atypical teratoid/rhabdoid tumor (AT/RT) is an aggressive, early-childhood brain tumor without standard effective treatment. To our knowledge, we conducted the first AT/RT-specific cooperative group trial, ACNS0333, to examine the efficacy and safety of intensive postoperative chemotherapy and focal radiation to treat AT/RT. Patients from birth to 22 years of age with AT/RT were eligible. After surgery, they received 2 courses of multiagent chemotherapy, followed by 3 courses of high-dose chemotherapy with peripheral blood stem cell rescue and involved-field radiation therapy. Timing of radiation was based on patient age and disease location and extent. Central testing of tumor and blood for SMARCB1 status was mandated. Tumor molecular subclassification was performed retrospectively. The primary analysis was event-free survival (EFS) for patients < 36 months of age compared with a cooperative groups' historical cohort. Although accrual was based on the therapeutic question, potential prognostic factors, including age, tumor location, M stage, surgical resection, order of therapy, germline status, and molecular subtype, were explored. Of 65 evaluable patients, 54 were < 36 months of age. ACNS0333 therapy significantly reduced the risk of EFS events in patients < 36 months of age compared with the historical cohort (P < .0005; hazard rate, 0.43; 95% CI, 0.28 to 0.66). Four-year EFS and overall survival for the entire cohort were 37% (95% CI, 25% to 49%) and 43% (95% CI, 31% to 55%), respectively. Timing of radiation did not affect survival, and 91% of relapses occurred by 2 years from enrollment. Treatment-related deaths occurred in 4 patients. The ACNS0333 regimen dramatically improved survival compared with historical therapies for patients with AT/RT. Clinical characteristics and molecular subgrouping suggest prognostic differences. ACNS0333 results lay a foundation on which to build future studies and incorporate testing of new therapeutic agents.

Abstract A17: The role of post-translational activation of MYC in pancreatic cancer development and progression

Abstract Pancreatic cancer is the 4th leading cause of cancer-related deaths in the United States, with 46,420 new cases and 39,590 deaths estimated in 2014. Pancreatic cancer is a disease with a high rate of mortality, as it is generally diagnosed in the advanced stages when no curative treatments are available. Despite increased understanding of the morphology, biology, and molecular aberrations in pancreatic cancer, the 5-year survival rate has not improved significantly in the past 30 years. The c-MYC (MYC) oncoprotein is a potent regulator of cell fate decisions that drives nearly all aspects of tumorigenesis, including tumor cell proliferation and tumor microenvironment remodeling. High levels of MYC expression occur in a wide variety of human tumors, and animal models demonstrate that MYC overexpression can drive tumorigenesis in many tissues. The MYC gene is frequently amplified in human pancreatic cancer and MYC mRNA and/or protein are often overexpressed. However, the role of MYC in pancreatic cancer development and progression is not well understood. Our laboratory has demonstrated that MYC is post-translationally activated by KRAS signaling. Specifically, the RAS-RAF-MEK-ERK signaling pathway leads to phosphorylation of MYC at Serine 62 (pS62-MYC), which increases MYC’s protein stability as well as its activation of oncogenic target gene expression. Furthermore, we have observed high expression of pS62-MYC in primary pancreatic tumors, starting as early as PanIN 1, compared to normal adjacent tissue. Since activating mutations in KRAS are an early and nearly universal event in the development of pancreatic ductal adenocarcinoma (PDAC), these data suggest that MYC may be a critical downstream effector of KRAS-driven pancreatic cancer. We have generated unique c-MYC knock-in mice in which MYC is expressed from the ROSA26 locus following tissue-specific expression of Cre recombinase (ROSA-LSL-MYC). These mice express transcriptionally deregulated, but physiological levels of MYC. At these levels, however, MYCWT knock-in is not sufficient to drive tumors in any tested tissue. Since KRAS can post-translationally activate MYC, we hypothesized that low-level expression of deregulated MYC would increase KRAS driven pancreatic tumors in mice that co-express KRASG12D and MYCWT . We crossed ROSA-LSL-MYC mice with LSL-KRASG12D;Pdx1-Cre (KC) mice to generate the LSL-KRASG12D;ROSA-LSL-MYC;Pdx1-Cre (KMC) model. We found that co-expression of MYC and activated KRAS induces acinar to ductal metaplasia (ADM) and accelerates both PanIN precursor lesion formation and its conversion to PDAC relative to KRAS only. Tumors from KMC mice histologically resemble human PDAC, with a 20% metastatic rate to the liver. Together, these results suggest that MYC is important for both the initiation and progression of pancreatic tumors. In addition to ductal adenocarcinoma, the MYC/KRAS expressing mice also developed metastatic, poorly differentiated carcinomas with neuroendocrine features. While activation of KRAS is thought to be one of the earliest oncogenic changes in PDAC-precursor cells, studies suggest that several other genetic events occur during the evolution of PanIN lesions to PDAC, including loss of p16, mutation/loss of p53, and loss of SMAD4. Importantly, tumors arising in our KMC model spontaneously exhibit these stage-appropriate molecular aberrations, suggesting that this model accurately recapitulates the genetic evolution of human pancreatic cancers. We also observe a strong desmoplastic reaction with prominent stroma and immune cell infiltration in these spontaneous pancreatic tumors. Thus, our novel KMC mice may provide a physiologically relevant model of human pancreatic cancer for the testing of new therapeutic agents. Citation Format: Amy S. Farrell, Colin J. Daniel, Zina Jenny, Brett Sheppard, Jody Hooper, Rosalie C. Sears. The role of post-translational activation of MYC in pancreatic cancer development and progression. [abstract]. In: Proceedings of the AACR Special Conference on Pancreatic Cancer: Innovations in Research and Treatment; May 18-21, 2014; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2015;75(13 Suppl):Abstract nr A17.

Establishment and genetic characterization of six unique tumor cell lines as preclinical models for sinonasal squamous cell carcinoma.

Sinonasal squamous cell carcinomas (SCC) are rare tumors, etiologically related to occupational exposure to wood and leather dust. In spite of surgical and radiotherapeutic advances, the 5 year survival is still 30–50%. Therefore, alternative treatment options are needed. We report the establishment and characterization of six unique human sinonasal SCC cell lines, named SCCNC1, 2, 4, 5, 6 and 7. In vitro growth and invasion characteristics were evaluated and genetic profiles were compared to those of the original primary tumors. The population doubling times ranged from 21 to 34 hours. Cell lines SCCNC2 and 7 were highly invasive in matrigel. Five cell lines carried a high number of copy number alterations, including amplifications and homozygous deletions, while one showed only three abnormalities. Sequence analysis revealed three cell lines with TP53 mutation and none with KRAS or BRAF. Overexpression of p53 was observed in five, and of EGFR in four cell lines. None of the cell lines showed strong immunopositivity of p16 or presence of human papilloma virus. In conclusion, we have created six new cell lines that are clinically and genetically representative of sinonasal SCC and that will be a useful tool for the preclinical testing of new therapeutic agents.

Advances in imaging vascular inflammation

Inflammation is central to the pathogenesis of many vascular diseases. Despite an understanding of the natural history, predicting progression and acute complications remains an important clinical challenge in both atherosclerosis and aortic aneurysms. Current means of monitoring rely mainly on structural information, such as the extent of luminal stenosis on coronary angiography or maximum aneurysm diameter on ultrasonography. Although often useful in making clinical decisions about intervention, such measures do not always correlate well with the risk of acute complications. As an example, the degree of luminal obstruction correlates poorly with the risk of future coronary plaque rupture and, similarly, aneurysm size does not account entirely for the subsequent rate of expansion and risk of rupture. It might be possible to improve risk prediction using imaging modalities that focus on the underlying disease processes rather than their consequences. This article reviews non-invasive imaging techniques that track inflammation within the arterial wall as applied to atherosclerosis and abdominal aneurysms. These all aim to improve risk prediction, to highlight the underlying pathology non-invasively and to permit the testing of new therapeutic agents against vascular disease.

NIA Commentary: Translational Issues in Alzheimer’s Disease Drug Development

Alzheimer's disease (AD) mainly affects elderly individuals, and because of the aging of populations worldwide, this disorder is reaching epidemic proportions, with an enormous human and economic burden. Effective treatments are urgently needed to treat the cognitive and behavioral symptoms of AD and to slow its progression. The National Institute on Aging (NIA) has developed a number of grant and contract mechanisms to support this effort. The NIA has an exploratory R21 grant program (http://grants.nih.gov/grants/guide/pa-files/PAS-10-151.html) for early AD drug discovery, in collaboration with the Alzheimer's Drug Discovery Foundation, and a program for preclinical drug development of small molecules, biologics, or other compounds for the treatment of AD, mild cognitive impairment (MCI), and age-related cognitive decline, using the U01 cooperative agreement mechanism (http://grants.nih.gov/grants/guide/pa-files/PAR-08-266.html). Examples of funded grants include studies of small molecule neurotrophin mimetics, novel anti-inflammatory compounds, and τ-aggregation inhibitors. The NIA provides investigational new drug-enabling toxicology services for novel AD therapeutic drugs through a contract, which is open to individual investigators or small companies. The NIA also participates in many trans-NIH programs including program announcements for drug discovery for nervous system diseases utilizing the R21 and R01 grant mechanisms, Small Business Innovation Research/Small Business Technology Transfer Research grant programs, and translational programs through the NIH Neuroscience Blueprint (http://neuroscienceblueprint.nih.gov/). The overall goal of these translational research initiatives is to provide support to investigators from academia and the biotechnology sector to increase the number of drug candidates that can be clinically tested. Clinical development can be done through partnership with industry or through NIA-supported clinical trial programs. These include a program announcement for pilot clinical trials (http://grants.nih.gov/grants/guide/pa-files/PAR-11-100.html), large R01 grant applications, and the Alzheimer's Disease Cooperative Study, an NIA-supported clinical trials consortium. Examples of pilot trials include intranasal insulin in people with AD/MCI, and carvedilol in AD, which is an anti-hypertensive drug that was re-purposed for AD, with support from the U01 program. Through these programs, the NIA hopes to facilitate the discovery, development, and testing of new therapeutic agents for AD, MCI, and age-related cognitive impairment. There are very few drugs that have been approved by the Food and Drug Administration for the treatment of AD. There have been many recent clinical trials of potential new therapeutic agents, but none has been shown to be effective in providing symptomatic benefit or slowing disease progression. This indicates the critical nature of the NIA/NIH programs in trying to facilitate getting new therapies out to the patients and families who desperately need them.

Cell culture model that mimics drusen formation and triggers complement activation associated with age-related macular degeneration

We introduce a human retinal pigmented epithelial (RPE) cell-culture model that mimics several key aspects of early stage age-related macular degeneration (AMD). These include accumulation of sub-RPE deposits that contain molecular constituents of human drusen, and activation of complement leading to formation of deposit-associated terminal complement complexes. Abundant sub-RPE deposits that are rich in apolipoprotein E (APOE), a prominent drusen constituent, are formed by RPE cells grown on porous supports. Exposure to human serum results in selective, deposit-associated accumulation of additional known drusen components, including vitronectin, clusterin, and serum amyloid P, thus suggesting that specific protein-protein interactions contribute to the accretion of plasma proteins during drusen formation. Serum exposure also leads to complement activation, as evidenced by the generation of C5b-9 immunoreactive terminal complement complexes in association with APOE-containing deposits. Ultrastructural analyses reveal two morphologically distinct forms of deposits: One consisting of membrane-bounded multivesicular material, and the other of nonmembrane-bounded particle conglomerates. Collectively, these results suggest that drusen formation involves the accumulation of sub-RPE material rich in APOE, a prominent biosynthetic product of the RPE, which interacts with a select group of drusen-associated plasma proteins. Activation of the complement cascade appears to be mediated via the classical pathway by the binding of C1q to ligands in APOE-rich deposits, triggering direct activation of complement by C1q, deposition of terminal complement complexes and inflammatory sequelae. This model system will facilitate the analysis of molecular and cellular aspects of AMD pathogenesis, and the testing of new therapeutic agents for its treatment.

Establishment and genetic characterization of an immortal tumor cell line derived from intestinal-type sinonasal adenocarcinoma

Intestinal-type sinonasal adenocarcinoma (ITAC) is a rare tumor etiologically related to professional exposure to wood dust. The overall prognosis is poor, mainly due to the difficulty to resect the tumor completely in this anatomically complex region. Therefore, there is great need for alternative treatments. However, the lack of a good tumor model system for ITAC has hampered the development and testing of new therapeutic agents. Here, we report the establishment and characterization of the first human ITAC cell line named ITAC-3. The cell line was initiated from small explants of a T4bN0M0 colonic type ITAC from the ethmoid sinus. Growth and invasion parameters as well as genetic characteristics were analyzed. The population doubling time was 18 h and the cell line was capable of invasion in matrigel. Chromosomal analysis showed a tetraploid karyotype with both numerical and structural aberrations. High resolution microarray CGH analysis identified many copy number alterations, including homozygous deletions. TP53 carried a mutation c.818G>T in exon eight concurring with a strong nuclear protein overexpression. Immunohistochemical analysis showed protein overexpression of EGFR and normal expression of β-catenin and p16. This is the first report of the establishment of a cell line derived from a primary ITAC. The genomic profile of the cell line was the same as the primary tumor from which it was derived. This new cell line will be a useful tool for the development and testing of new therapeutic agents for this tumor type.

Observance médicamenteuse et rechutes dans la schizophrénie : des neuroleptiques classiques aux APAP

In recent years, the goals of treatment in schizophrenia have evolved from objective improvements in psychotic symptoms to encompass patient-related factors such as subjective response and quality of life. Patient satisfaction with antipsychotic therapy is influenced by multiple factors. The most frequently reported reasons for dissatisfaction include drug side effects, lack of involvement in treatment planning or decision-making and lack of involvement of family members in the care plan. Conventional clinical trials have been largely focused on establishing efficacy of drugs, leaving the study of compliance to a loosely organized postmarketing surveillance system. There has been renewed interest in studying compliance behaviour in recent years propelled by two forces: an exponential growth in the development and testing of new therapeutic agents, and the growing burden of chronic diseases that require reliance on life-long medical treatments. Both the pharmaceutical industry and clinicians have come to appreciate the old adage “much falls between the cup and the lip” that aptly conveys the barriers between the development of new drugs in a laboratory and improving the quality of life of people in the community. Novel antipsychotic drugs are perhaps unduly criticized for not improving patients’ compliance, while the reasons for noncompliance in schizophrenia may lie elsewhere, warranting a reexamination of the prevailing concepts, constructs and measures of compliance. Noncompliance and treatment dropouts are inherent aspects of the natural history of schizophrenia, irrespective of the type of treatment and the quality of care. This does not preclude, however, the continued search for newer and better anti-schizophrenic drugs, and more reliable alternative treatment strategies. The majority of studies have demonstrated that the APAP (long-Acting atyPical AntiPsychotic) are associated with significant improvements in quality of life, functional status and patient satisfaction compared with conventional agents. The therapeutic alliance is the key to achieving optimal outcomes, by providing information and education to meet patients’ needs, while facilitating compliance with drug therapy to ensure better clinical outcomes. A long-acting atypical antipsychotic that can ensure medication delivery will provide a platform for psychosocial interventions, and thus may further increase patient satisfaction and, ultimately, improve long-term outcomes in schizophrenia. Attitudes towards APAP play an important part in the treatment for schizophrenia and related disorders. The effectiveness of APAP is evident in acute and maintenance treatment of these disorders, and most mental health professionals recognize APAP as a cornerstone in treating affected people.

Psoriasis: evolution of pathogenic concepts and new therapies through phases of translational research

Psoriasis is perhaps unique for a disease studied through translational science in that there is not an accepted animal model, yet many rounds of bidirectional translation have taken place that have helped to define disease pathogenesis and to advance therapy. In this review, we illustrate the evolution of new pathogenic concepts and the testing of new therapeutic agents through translational research in humans. We present a current view of disease pathogenesis that stems from research in patients and animal models, but with the perspectives (i) that disease models can advance or hinder the overall translational enterprise and (ii) that the research process must be firmly grounded in the pathophysiology of the actual human condition.

290. Alpha-Tocopherol Succinate (VES) Enhances Ad-mda7-Mediated Killing of Ovarian Cancer Cells

Ovarian cancer is the second most common gynecological malignancy in the United States and is the fifth leading cause of death among women. An estimated 25,000 women will be diagnosed with ovarian cancer and 50% of these patients will die of the disease. Despite advances made in the treatment of ovarian cancer the overall long-term survival of patients have not significantly improved. Hence developing and testing of new therapeutic agents and strategies are warranted.

Magnetic resonance imaging predictors of disability in primary progressive multiple sclerosis: a 5-year study.

Magnetic resonance imaging (MRI) has become an accepted tool for monitoring therapeutic trials in relapsing-remitting and secondary progressive multiple sclerosis (MS); it is however unclear whether such MRI markers are equally applicable to primary progressive MS (PPMS). Forty-two patients with PPMS were reviewed five years after commencing a two-year MRI and clinical study. Clinical measures recorded at baseline and five years included both the Expanded Disability Status Scale and the MS functional composite. MRI data collected at baseline and two years included T1 and T2 lesion loads, the number of new brain and cord lesions, and measures of both brain and cord atrophy. The study demonstrated that both the number of new T2 lesions and rate of increase in ventricular volume over two years were modestly predictive of subsequent disease progression and therefore may be useful tools in the testing of new therapeutic agents in PPMS.

Lessons from animal models of vasculitis.

Vasculitis can occur as a primary disease or as a secondary manifestation of either another illness or a type-III hypersensitivity response to a foreign antigen. Over the past four decades, a number of animal models of vasculitis have been described. These models have served as important tools for enhancing our understanding of the basic mechanisms underlying the pathogenesis of vasculitis. In addition, animal models have made possible the preclinical testing of new therapeutic agents. Animal models of vasculitis can be broadly classified into two types--those that are experimentally induced and those that occur spontaneously. Vasculitis can be experimentally induced in animals through the stimulation of a type-III hypersensitivity response to a variety of foreign antigens, by viral or bacterial infection of vascular cells and the immune response to that infection, or by the in-vivo administration of antineutrophil cytoplasmic antibodies, estrogen, or mercuric chloride (HgCl(2)). Systemic vasculitis spontaneously develops in several strains of mice and rats. This paper reviews the current state of knowledge of several animal models of vasculitis and the lessons that have been learned from them.

Current trends in antiglaucoma therapy

No abstract.

Construction of human-SCID chimeric mice.

Until recently, testing of new therapeutic agents has relied extensively upon the use of mice and nonhuman primates for in vivo preclinical studies. Unfortunately, these animal models do not always mimic the physiological and pathophysiological processes that occur in humans. The finding that C.B-17 severe combined immunodeficiency (SCID) mice lack a competent immune system, and therefore are unable to mount effective cellular and humoral responses to foreign antigens, has led to their use as recipients for xenografts of human tissues. This unit is focused on the construction of human-SCID chimeric through the surgical implantation of human fetal hematolymphoid tissues into SCID mice (SCID-hu mice). The Basic Protocol describes the surgical implantation of human fetal thymus and liver under the kidney capsules of SCID mice (SCID-hu Thy/Liv model). Subcutaneous transplantation of human fetal bone marrow and thymus (SCID-hu Bm/Thy mice) is described in the Alternate Protocol. Additional support protocols provide procedures to analyze human lymphocyte populations in the peripheral blood and grafted organs of SCID-hu mice. The advantages and disadvantages of each protocol and potential applications are discussed in the Commentary.

Genetically engineered models of inflammatory bowel disease

Recent advances in genetic engineering techniques have made possible new and exciting approaches to generating models of inflammatory bowel disease in mice and rats. Several recent studies have demonstrated that targeted deletions of genes encoding for interleukin-2, interleukin-10, or certain T-cell receptor proteins produce mice that spontaneously develop chronic intestinal inflammation similar to human inflammatory bowel disease. In addition, it has been demonstrated that the introduction of HLA-B27 and human β2-microglobulin genes into rats produces transgenic animals that spontaneously develop chronic gastrointestinal inflammation. Data derived from these studies suggest that T cells play an important role in regulating B-cell activity, such that an unrestrained or unregulated B-cell response to normal luminal antigens or bacterial products promotes chronic gut inflammation. In addition, these studies demonstrate that endogenous luminal bacteria are important in promoting or exacerbating chronic gut inflammation. Indeed, these studies indicate that the production of very different defects in the immune system leads, in many cases, only to gut mucosal inflammation, suggesting that the gut mucosa is subjected to substantially more antigenic “stress” than are other tissues. Thus, these new models offer new and exciting avenues of research for the study of the pathogenesis of inflammatory bowel disease as well as providing new and possibly more appropriate models for the development and testing of new therapeutic agents for the treatment of these diseases.

Advances in the understanding and treatment of multiple sclerosis: Boston, Massachusetts, USA, 28–29 October 1992

Basic and applied research on multiple sclerosis (MS) has become increasingly focused on the development and testing of new therapeutic agents for this devastating disease. After a generation or more of exposing patients to non-specific immunoregulatory and other agents that have been, at best, of minimum benefit and significant potential or actual toxicity, attention has turned to attempts to identify treatment modalities that might be used in a more specific, effective and safe fashion. This trend has not been lost on the emerging biotechnology industry, which tends to view MS as both a disease to be explored on its own and as a disease whose solution may also open doors into other chronic diseases of purported autoimmune etiology, such as rheumatoid arthritis, type I diabetes, and others. These trends toward an increasing biotechnological approach to MS, coupled with an academic and industrial focus on the disease, were the clear motivations behind a recent two-day conference entitled “Advances in the Understanding and Treatment of Multiple Sclerosis” held in Boston and sponsored by a commercial meeting planner, International Business Communications USA Conferences, Inc.