scholarly journals ATRT-22. HIGH-THROUGHPUT DRUG SCREENING OF FDA-APPROVED CANCER DRUGS REVEALS POTENTIAL THERAPEUTIC APPROACHES FOR ATYPICAL TERATOID RHABDOID TUMOUR

2020 ◽  
Vol 22 (Supplement_3) ◽  
pp. iii280-iii280
Author(s):  
Wai Chin Chong ◽  
Nataliya Zhukova ◽  
Paul Wood ◽  
Peter A Downie ◽  
Jason E Cain
Keyword(s):  
High Throughput ◽  
Drug Screening ◽  
Prolonged Treatment ◽  
Allelic Loss ◽  
Rhabdoid Tumour ◽  
High Throughput Drug Screening ◽  
Atypical Teratoid ◽  
Rhabdoid Tumors

Abstract Atypical teratoid/rhabdoid tumors (ATRT), are the most common brain tumor in children under the age of 1 year with an overall survival of ~17%. Like extracranial rhabdoid tumors, ATRT is exclusively characterized by bi-allelic loss of SMARCB1, a critical subunit of the SWI/SNF chromatin remodeling complex, implicating epigenetic deregulation in the pathogenesis of disease. We have previously shown the ability of the histone deacetylase inhibitor, panobinostat, to mimic SMARCB1-mediated SWI/SNF functions in extracranial rhabdoid tumors to inhibit tumor growth by driving multi-lineage differentiation in vitro and in vivo. Whether this also applies to ATRT is unknown. Using a panel of human-derived ATRT cell lines, representing defined molecular subgroups, we have shown that prolonged treatment with panobinostat at nanomolar concentrations results in markedly reduced clonogenicity, and increased senescence, preceded by increased H3K27 acetylation, decreased H3K27 trimethylation and EZH2 expression. To determine potentially synergistic therapies, we performed high-throughput drug screening of 622 compounds already in advanced clinical trials or FDA-approved for other indications, across our panel of ATRT models and identified 30 common compounds, which decrease cell viability by >50%, with no effect on neural stem cell controls and 12 compounds which demonstrated subgroup specificity, highlighting the necessity to consider therapies in the molecular context. In addition to HDACi, consistent with our panobinostat in vitro findings, inhibitors of CDK, survivin and PI3K were the top hits. In vitro and in vivo validation of these compounds alone, and in combination with panobinostat is ongoing.

scholarly journals DIPG-07. HIGH THROUGHPUT DRUG SCREENING IDENTIFIES POTENTIAL NEW THERAPIES FOR DIFFUSE INTRINSIC PONTINE GLIOMAS (DIPGs)

2020 ◽  
Vol 22 (Supplement_3) ◽  
pp. iii288-iii288
Author(s):  
Dannielle Upton ◽  
Santosh Valvi ◽  
Jie Liu ◽  
Nicole Yeung ◽  
Sandra George ◽  
...  
Keyword(s):  
High Throughput ◽  
Apoptotic Cell ◽  
Safety Data ◽  
Biologically Active ◽  
Orthotopic Model ◽  
High Throughput Drug Screening ◽  
Diffuse Intrinsic Pontine Gliomas ◽  
Almost All

Abstract DIPGs are the most devastating of all brain tumors. There are no effective treatments, hence almost all children will die of their tumor within 12 months. There is an urgent need for novel effective therapies for this aggressive tumor. We performed a high-throughput drug screen with over 3,500 biologically active, clinically approved compounds against a panel of neurosphere-forming DIPG cells. We identified 7 compounds- auranofin, fenretinide, ivermectin, lanatoside, parthenolide, SAHA and mefloquine- that were confirmed to have potent anti-tumor activity against a panel of DIPG-neurospheres, with minimal effect on normal cells. Using cytotoxicity and clonogenic assays, we found that these drugs were able to inhibit DIPG-neurosphere proliferation and colony formation in-vitro. To determine whether the in-vitro efficacy could be replicated in-vivo, we tested the activity of each of these compounds in an orthotopic DIPG model. Of the agents tested, fenretinide and SAHA were the most active anti-tumor agents, significantly enhancing the survival of tumor bearing animals. Mechanistic studies showed fenretinide enhancing apoptotic cell death of DIPG cells via inhibition of PDGFRa transcription and downregulation of the PI3K/AKT/MTOR pathway. We therefore examined the therapeutic efficacy of fenretinide using a second orthotopic model with PDGFRa amplification. We used two different Fenretinide formulations (LYM-X-Sorb and NanoMicelle) which were found to enhance survival. Fenretinide is clinically available with safety data in children. Validation of the activity of Fenretinide in PDGFRa-amplified or overexpressed DIPGs will lead to the development of a clinical trial, allowing the advancement of fenretinide as potentially the first active therapy for DIPG.

scholarly journals Proteasome inhibition as a therapeutic approach in atypical teratoid/rhabdoid tumors

2020 ◽  
Vol 2 (1) ◽  
Author(s):  
Andrew Morin ◽  
Caroline Soane ◽  
Angela Pierce ◽  
Bridget Sanford ◽  
Kenneth L Jones ◽  
...  
Keyword(s):  
Clinical Trials ◽  
Cell Lines ◽  
Proteasome Inhibitor ◽  
High Grade Glioma ◽  
Proteasome Inhibition ◽  
Atypical Teratoid ◽  
Rhabdoid Tumors ◽  
Approved Drugs

Abstract Background Atypical teratoid/thabdoid tumor (AT/RT) remains a difficult-to-treat tumor with a 5-year overall survival rate of 15%–45%. Proteasome inhibition has recently been opened as an avenue for cancer treatment with the FDA approval of bortezomib (BTZ) in 2003 and carfilzomib (CFZ) in 2012. The aim of this study was to identify and characterize a pre-approved targeted therapy with potential for clinical trials in AT/RT. Methods We performed a drug screen using a panel of 134 FDA-approved drugs in 3 AT/RT cell lines. Follow-on in vitro studies used 6 cell lines and patient-derived short-term cultures to characterize selected drug interactions with AT/RT. In vivo efficacy was evaluated using patient derived xenografts in an intracranial murine model. Results BTZ and CFZ are highly effective in vitro, producing some of the strongest growth-inhibition responses of the evaluated 134-drug panel. Marizomib (MRZ), a proteasome inhibitor known to pass the blood–brain barrier (BBB), also strongly inhibits AT/RT proteasomes and generates rapid cell death at clinically achievable doses in established cell lines and freshly patient-derived tumor lines. MRZ also significantly extends survival in an intracranial mouse model of AT/RT. Conclusions MRZ is a newer proteasome inhibitor that has been shown to cross the BBB and is already in phase II clinical trials for adult high-grade glioma (NCT NCT02330562 and NCT02903069). MRZ strongly inhibits AT/RT cell growth both in vitro and in vivo via a moderately well-characterized mechanism and has direct translational potential for patients with AT/RT.

Characterization Of FLT3/ITD Resistant Cells and Identification Of Novel Therapeutic Targets Utilizing High Throughput Drug Screening

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 2522-2522
Author(s):  
Katherine Tarlock ◽  
C. Anthony Blau ◽  
Timothy Martins ◽  
Soheil Meshinchi
Keyword(s):  
Cell Lines ◽  
High Throughput ◽  
Drug Screening ◽  
Therapeutic Options ◽  
High Throughput Drug Screening ◽  
Mutational Profiling ◽  
Flt3 Inhibitors ◽  
Resistant Cells

Abstract The overall survival (OS) of pediatric acute myeloid leukemia (AML) exceeds 60%, however for high risk (HR) patients, including high allelic ratio FLT3/ITD+, survival remains poor. FLT3/ITD is one of the first genomic alterations in AML to be exploited for therapeutic benefit as it has greater sensitivity to the pro-apoptotic effects of FLT3-inhibitors. Children’s Oncology Group (COG) phase III AML trial AAML1031 is investigating the role of sorafenib in combination with chemotherapy in HR FLT3/ITD+ patients. In vitro and in vivo studies indicate that resistance to FLT3-inhibitors can develop through varying mechanisms including up-regulation of FLT3 receptor, acquisition of secondary mutations, or activation of alternate survival mechanisms leading to apoptotic escape. For FLT3/ITD+ patients who relapse despite treatment with FLT3-inhibitors, there are often no therapeutic options and survival is very poor. In evaluation of therapeutic options for those who relapse on sorafenib, we developed an in vitro resistance model using the FLT3/ITD+ cell line MV4-11. Resistance was induced thru long-term exposure to incrementally increasing doses of sorafenib. Two distinct cell lines with resistance at 10 and 100 fold above the IC50 of naïve MV4-11 were generated for experimental evaluation. Genotypic and phenotypic characterization of the resistant cells was conducted by multidimensional flow cytometry (MDF), conventional karyotyping, and mutational profiling. MDF revealed an overall similar immunophenotype, however the resistant cells were significantly more homogeneous for expression of HLA-DR and had significantly higher CD11b expression compared to their naïve counterparts. CD135 expression was minimally increased in the resistant cells. In comparison of the karyotypes, the resistant cells were a more homogenous population with emergence of one dominant clone and disappearance of a number of pre-existing sub-clones. Mutational profiling by Sanger sequencing revealed a novel N841Y mutation in activation loop, an area implicated in TKI-resistance. Using a high throughput drug screening assay, we explored sensitivity profiles of the naïve and resistant MV4-11 cells to 163 oncology agents, including 45 FDA approved and 118 investigational agents that target a number of key pathways regulating cell growth, differentiation, and survival. The naïve MV4-11s had a sorafenib IC50 of 1.3 nM (published 1-5nM) and resistant cells had IC50 of approximately 2-log folds above the naïve, which was consistent to what we had seen in our lab-based validations. We initially assessed whether resistance to sorafenib induces cross-resistance to other TKIs. Agents in the panel with previously demonstrated efficacy for FLT3/ITD included quizartinib (AC-220), tandutinib, ponatinib, sunitinib, and midostaurin, and in all cases sorafenib-resistant cells were also more resistant to these agents. We then examined whether we could identify agents with efficacy in the resistant cells. We identified 5 novel agents to which the resistant cells retained sensitivity. Two bcl-2 inhibitors tested maintained sensitivity in the resistant cells with IC50s in the 20-100nM range. In addition, YM-155, a survivin inhibitor, also maintained sensitivity in the resistant cells with IC50s of approximately 25-50nM across the cell lines. Survivin over-expression is associated with AML stem progenitor cells and decreased OS in adults, and transcription regulation has been linked to the FLT3/STAT5 pathway. Two CRM inhibitors, a novel class of agents which inhibit nuclear export to restore tumor suppressor function, also maintained sensitivity in the resistant cell lines with an approximate 3-fold increase in IC50 from 12nM in the naïve to 32-40nM in the resistant cells. Experience with the use of directed therapy to target specific somatic events has provided evidence that leukemic evolution can continue under this selection pressure and therapeutic options for patients with emergent disease is often insufficient. Using the high throughput drug assay in a FLT3/ITD+ cell line as an in vitro model for sorafenib-resistant FLT3/ITD patients, we identified classes of targeted agents that maintain sensitivity in resistant cells. Further validation of the targets in specimens from those with resistance to such TKIs can inform on the class of agents that can be used to treat or prevent refractory disease FLT3/ITD+ patients. Disclosures: No relevant conflicts of interest to declare.

scholarly journals Mutation Profiles in Glioblastoma 3D Oncospheres Modulate Drug Efficacy

2018 ◽  
Vol 24 (1) ◽  
pp. 28-40 ◽  
Author(s):  
Kelli M. Wilson ◽  
Lesley A. Mathews-Griner ◽  
Tara Williamson ◽  
Rajarshi Guha ◽  
Lu Chen ◽  
...  
Keyword(s):  
Cell Lines ◽  
High Throughput ◽  
Drug Screening ◽  
Kinase Inhibitors ◽  
Proteasome Inhibitors ◽  
Drug Efficacy ◽  
Driver Genes ◽  
Human Gbm

Glioblastoma (GBM) is a lethal brain cancer with a median survival time of approximately 15 months following treatment. Common in vitro GBM models for drug screening are adherent and do not recapitulate the features of human GBM in vivo. Here we report the genomic characterization of nine patient-derived, spheroid GBM cell lines that recapitulate human GBM characteristics in orthotopic xenograft models. Genomic sequencing revealed that the spheroid lines contain alterations in GBM driver genes such as PTEN, CDKN2A, and NF1. Two spheroid cell lines, JHH-136 and JHH-520, were utilized in a high-throughput drug screen for cell viability using a 1912-member compound library. Drug mechanisms that were cytotoxic in both cell lines were Hsp90 and proteasome inhibitors. JHH-136 was uniquely sensitive to topoisomerase 1 inhibitors, while JHH-520 was uniquely sensitive to Mek inhibitors. Drug combination screening revealed that PI3 kinase inhibitors combined with Mek or proteasome inhibitors were synergistic. However, animal studies to test these drug combinations in vivo revealed that Mek inhibition alone was superior to the combination treatments. These data show that these GBM spheroid lines are amenable to high-throughput drug screening and that this dataset may deliver promising therapeutic leads for future GBM preclinical studies.

Patient-derived organoids for personalized drug screening in intrahepatic cholangiocarcinoma.

2020 ◽  
Vol 38 (4_suppl) ◽  
pp. 581-581
Author(s):  
Ricardo J. Antonia ◽  
Kan Toriguchi ◽  
Eveliina Karelehto ◽  
Dania Annuar ◽  
Luika Timmerman ◽  
...  
Keyword(s):  
High Throughput ◽  
Drug Screening ◽  
Intrahepatic Cholangiocarcinoma ◽  
Three Dimensional ◽  
Small Samples ◽  
Patient Specific ◽  
Mtor Inhibition ◽  
Drug Responses

581 Background: Despite standard treatment with gemcitabine and cisplatin, median survival for unresectable Intrahepatic Cholangiocarcinoma (ICC) is < 1 year. Clearly, novel therapeutic strategies are urgently needed. The paucity of targetable mutations in ICC and the as yet unproven benefit of genetically targeted drugs led us to ask whether a reliable clinical benefit may be revealed by patient-specific therapeutic testing in novel models of ICC. Here we describe our ability to establish patient-derived three-dimensional organoid cultures (PDO) that enable individualized identification of active single agents or drug combinations in surrogate models of ICC. Methods: To model patient-specific drug responses, we used the freshly resected ICCs from small samples of single patient tumors to generate PDXs and PDOs, small spheroidal clusters of tumor cells grown in vitro. We have employed a high-throughput drug screening platform using AI-enhanced robotics (Yamaha Motor Corporation) to identify and distribute single, uniformly sized PDOs into 384-well ultra-low adherent plates. This is coupled with a TECAN D300e drug dispenser that rapidly delivers nanoliter volumes of a 34-drug panel, thereby facilitating rapid, reliable drug response analyses. Results: Our data show that PDOs retain characteristic genomic and histological features of the patients’ tumors. Drug responses were specific to each patient tumor, but PDOs from all patients responded to a greater or lesser degree to mTOR inhibition, suggesting that this pathway is important in ICC. The responses of PDO to the mTOR inhibitor Sapanisertib (INK128), was recapitulated in the same patient’s PDX. Further, INK128 was synergistic with gemcitabine in patient 970 PDOs as well as in vivo in PDX also from patient 970. Conclusions: As it is believed that PDX can predict patient responses to drugs, our results suggest that PDO may also predict patient drug responses. The establishment of PDO may allow economical patient-specific, high throughput drug screens that could ultimately inform clinical practice. [Table: see text]

scholarly journals THER-02. EVALUATION OF THE ONCOLYTIC VIRUS DELTA24-RGD AS AN ANTI-TUMOR AGENT IN PRECLINICAL MODELS OF LOCALIZED AND DISSEMINATED AT/RT

2020 ◽  
Vol 22 (Supplement_3) ◽  
pp. iii471-iii471
Author(s):  
Marc Garcia-Moure ◽  
Marisol González-Huarriz ◽  
Daniel de la Nava ◽  
Lucía Marrodán ◽  
Cande Gomez-Manzano ◽  
...  
Keyword(s):  
Intraventricular Injection ◽  
Therapeutic Choice ◽  
Ic50 Values ◽  
Current Therapies ◽  
Atypical Teratoid ◽  
Atypical Teratoid Rhabdoid Tumors ◽  
Rhabdoid Tumors ◽  
Functional Analyses

Abstract Current therapies for atypical teratoid/rhabdoid tumors (AT/RTs) are suboptimal, resulting in a 2-year OS below 20% and the development of severe side effects. Therefore, we need to explore alternative therapeutic approaches for this disease. Since the virus Delta24-RGD has already demonstrated its efficacy and safety as a therapeutic agent for brain tumors, including pediatric patients, here we propose to evaluate the anti-tumor effect of Delta24-RGD in AT/RT. In vitro, Delta24-RGD infects and replicates in AT/RT cultures followed by oncolysis, obtaining IC50 values below 1 PFU/cell. In vivo, a single local injection of Delta-24-RGD in three infratentorial AT/RT models (BT-12, CHLA-06 and CHLA-266) extended significantly the median OS (50 to 78 days BT-12; 21 to 31 days CHLA-06; 64 to 110 days CHLA-266). Delta-24-RGD also increased the survival of mice bearing supratentorial CHLA-266 tumors (from 93 to 132 days). Next, we evaluated the efficacy of Delta24-RGD in a model mimicking metastatic disease through intraventricular injection of BT-12-luciferase cells. Administration of Delta24-RGD inhibited tumor growth and development of metastases, leading to an increased OS and nearly 70% of long-term survivors. The interaction between Delta24-RGD and the immune system was evaluated in humanized mice models bearing CHLA-06. In this model, Delta24-RGD treatment extended OS (from 23 to 34 days) and we characterized the anti-tumor immune landscape in control and Delta24-RGD treated mice by transcriptional and functional analyses. These results underscore the potential of Delta24-RGD as a promising therapeutic choice for patients affected by AT/RT.

scholarly journals A high-throughput 3D bioprinted cancer cell migration and invasion model with versatile and broad biological applicability

2021 ◽  
Author(s):  
MoonSun Jung ◽  
Joanna Skhinas ◽  
Eric Y Du ◽  
Maria Kristine Tolentino ◽  
Robert Utama ◽  
...  
Keyword(s):  
Cell Migration ◽  
High Throughput ◽  
Drug Screening ◽  
Cancer Biology ◽  
Cell Movement ◽  
Migration And Invasion ◽  
Cell Migration And Invasion ◽  
Drop On Demand

Understanding the underlying mechanisms of migration and metastasis is a key focus of cancer research. There is an urgent need to develop in vitro 3D tumor models that can mimic physiological cell-cell and cell-extracellular matrix interactions, with high reproducibility and that are suitable for high throughput (HTP) drug screening. Here, we developed a HTP 3D bioprinted migration model using a bespoke drop-on-demand bioprinting platform. This HTP platform coupled with tunable hydrogel systems enables (i) the rapid encapsulation of cancer cells within in vivo tumor mimicking matrices, (ii) in situ and real-time measurement of cell movement, (iii) detailed molecular analysis for the study of mechanisms underlying cell migration and invasion, and (iv) the identification of novel therapeutic options. This work demonstrates that this HTP 3D bioprinted cell migration platform has broad applications across quantitative cell and cancer biology as well as drug screening.

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