Top Drug Candidates Research Articles

Abstract 4023: The SABRE platform: A novel, unbiased technology for drug discovery and prioritization

Abstract Targeted therapies designed to inhibit hyperactive oncogenic signaling have demonstrated some encouraging clinical responses. However, most of the time these responses are not durable as tumors “work around” pathway inhibition, often reactivating the inhibited pathway, leading to drug resistance and disease relapse. New technologies that enable more efficient discovery and development of “suites” of drugs that comprehensively inhibit multiple pathway nodes are needed. The SABRE (Splice Acceptor Brilliant Reporter) platform was developed to provide an unbiased method to screen large compound libraries and dramatically improve the speed and efficiency of which novel targeted therapeutics can be identified. SABRE is built off the premise that oncogenic output is exerted through changes in gene transcription and that such changes can be harnessed as powerful reporters of pathway activation status. SABRE utilizes the power of gene trap technology coupled with a drug selection process to isolate cells that generate a robust “off to on” signal in response to target or pathway inhibition. Via massively parallel comparative analysis, multiple traps let nature provide the best reporter for further analysis and drug discovery. In this proof-of-concept study, we employed the SABRE technology to identify insertion sites that are specifically regulated by the MAPK pathway. Experiments were performed using the human BRAFV600E mutant melanoma cell line A375. SABRE lentiviral transduced A375 cells were treated with trametinib, a MEK inhibitor, and clones were isolated that emitted a positive luciferase signal upon drug treatment. To determine if these reporters were MAPK pathway specific, the platform was miniaturized to a 1536 well format and used to screen a 6000+ compound library. Results from the screen found that 70% (28/40) of the top drug hits were known to directly modulate the MAPK pathway. Since drug resistance is a common occurrence in melanoma, we generated a “resistant” A375 SABRE reporter line to increasing concentrations of vemurafenib and interrogated the top drug candidates identified in the screen. As expected, the resistant SABRE line failed to respond to BRAFV600E specific inhibitors but continued to respond to downstream MEK inhibitors, which is helping to delineate the mechanism of resistance. We were also interested in the screening hits that were not previously known to influence the MAPK pathway including bafetinib and a Tie2 inhibitor. In follow-up testing, we found that these drugs induced robust and titratable reporter signals in both the SABRE A375 vemurafenib sensitive and resistant lines. In addition, treatment of either bafetinib or the Tie2 inhibitor reduced ERK phosphorylation in the vemurafenib resistant cells, suggesting that these drugs could be useful in the treatment of drug resistant melanomas. Studies are currently underway to determine the in vivo efficacy of these drugs in A375 SABRE reporter xenograft models. Citation Format: Shelli M. Morris, Andrew J. Mhyre, Savanna S. Carmack, Connor Burns, Marc Ferrer, Wenjuan Ye, James M. Olson, Richard A. Klinghoffer. The SABRE platform: A novel, unbiased technology for drug discovery and prioritization [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 4023. doi:10.1158/1538-7445.AM2017-4023

Preclinical drug screen reveals topotecan, actinomycin D, and volasertib as potential new therapeutic candidates for ETMR brain tumor patients.

Embryonal tumor with multilayered rosettes (ETMR) is a rare and aggressive embryonal brain tumor that solely occurs in infants and young children and has only recently been recognized as a separate brain tumor entity in the World Health Organization classification for CNS tumors. Patients have a very dismal prognosis with a median survival of 12 months upon diagnosis despite aggressive treatment. The aim of this study was to develop novel treatment regimens in a preclinical drug screen in order to inform potentially more active clinical trial protocols. We have carried out an in vitro and in vivo drug screen using the ETMR cell line BT183 and its xenograft model. Furthermore, we have generated the first patient-derived xenograft (PDX) model for ETMR and evaluated our top drug candidates in an in vitro drug screen using this model. BT183 cells are very sensitive to the topoisomerase inhibitors topotecan and doxorubicin, to the epigenetic agents decitabine and panobinostat, to actinomycin D, and to targeted drugs such as the polo-like kinase 1 (PLK1) inhibitor volasertib, the aurora kinase A inhibitor alisertib, and the mammalian target of rapamycin (mTOR) inhibitor MLN0128. In xenograft mice, monotherapy with topotecan, volasertib, and actinomycin D led to a temporary response in tumor growth and a significant increase in survival. Finally, using multi-agent treatment regimens of topotecan or doxorubicin combined with methotrexate and vincristine, the response in tumor growth and survival was further increased compared with mice receiving single treatments. We have identified several promising candidates for combination therapies in future clinical trials for ETMR patients.

Gametocytocidal screen identifies novel chemical classes with Plasmodium falciparum transmission blocking activity.

Discovery of transmission blocking compounds is an important intervention strategy necessary to eliminate and eradicate malaria. To date only a small number of drugs that inhibit gametocyte development and thereby transmission from the mosquito to the human host exist. This limitation is largely due to a lack of screening assays easily adaptable to high throughput because of multiple incubation steps or the requirement for high gametocytemia. Here we report the discovery of new compounds with gametocytocidal activity using a simple and robust SYBR Green I- based DNA assay. Our assay utilizes the exflagellation step in male gametocytes and a background suppressor, which masks the staining of dead cells to achieve healthy signal to noise ratio by increasing signal of viable parasites and subtracting signal from dead parasites. By determining the contribution of exflagellation to fluorescent signal and using appropriate cutoff values, we were able to screen for gametocytocidal compounds. After assay validation and optimization, we screened an FDA approved drug library of approximately 1500 compounds, as well as the 400 compound MMV malaria box and identified 44 gametocytocidal compounds with sub to low micromolar IC50s. Major classes of compounds with gametocytocidal activity included quaternary ammonium compounds with structural similarity to choline, acridine-like compounds similar to quinacrine and pyronaridine, as well as antidepressant, antineoplastic, and anthelminthic compounds. Top drug candidates showed near complete transmission blocking in membrane feeding assays. This assay is simple, reproducible and demonstrated robust Z-factor values at low gametocytemia levels, making it amenable to HTS for identification of novel and potent gametocytocidal compounds.

Filamin A mutations cause periventricular heterotopia with Ehlers-Danlos syndrome

To define the clinical, radiologic, and genetic features of periventricular heterotopia (PH) with Ehlers-Danlos syndrome (EDS). Exonic sequencing and single stranded conformational polymorphism (SSCP) analysis was performed on affected individuals. Linkage analysis using microsatellite markers on the X-chromosome was performed on a single pedigree. Western blotting evaluated for loss of filamin A (FLNA) protein and Southern blotting assessed for any potential chromosome rearrangement in this region. The authors report two familial cases and nine additional sporadic cases of the EDS-variant form of PH, which is characterized by nodular brain heterotopia, joint hypermobility, and development of aortic dilatation in early adulthood. MRI typically demonstrated bilateral nodular PH, indistinguishable from PH due to FLNA mutations. Exonic sequencing or SSCP analyses of FLNA revealed a 2762 delG single base pair deletion in one affected female. Another affected female harbored a C116 single point mutation, resulting in an A39G change. A third affected female had a 4147 delG single base pair deletion. One pedigree with no detectable exonic mutation demonstrated positive linkage to the FLNA locus Xq28, an affected individual in this family also had no detectable FLNA protein, but no chromosomal rearrangement was detected. These results suggest that the Ehlers-Danlos variant of periventricular heterotopia (PH), in part, represents an overlapping syndrome with X-linked dominant PH due to filamin A mutations.