Long-term Viability Assays Research Articles

HGG-08. COMBINATORIAL ACTIVITY OF CDK4/6-INHIBITORS AND TRAMETINIB IN PEDIATRIC HIGH-GRADE GLIOMA WITH BRAFV600E MUTATION AND HOMOZYGOUS CDKN2A DELETION

Abstract BRAFV600E mutations occur in 10-15% and homozygous CDKN2A deletions in approximately 40% of adolescent and pediatric high-grad glioma (HGG) and is associated with significantly decreased overall survival. Moreover, a subset of pHGG is defined by combined BRAFV600E mutation and concomitant CDKN2A homozygous deletion. Standard therapy has remained unchanged and consists of maximal safe resection and focal radiotherapy still resulting in poor overall survival. Therefore, new therapeutic approaches to increase patient survival are of utmost importance. Targeted therapy of BRAFV600E mutant HGG has emerged as effective treatment but remains challenging due to therapy resistance. CDK4/6 inhibitors represent a promising target in CDKN2A altered HGG. However, palbociclib demonstrated only low efficacy in HGG patients. Therefore, combination approaches may be more effective, and we investigated CDK4/6-inhibitors alone and in combination with trametinib in HGG cell models with BRAFV600E mutation and homozygous CDKN2A deletion. Efficacy of CDK4/6-inhibitor monotherapy and combinatorial approach with trametinib was assessed by short- and long-term viability assays in four patient-derived HGG cell models with homozygous CDKN2A deletion and BRAFV600E mutation. Furthermore, effects of CDK4/6 inhibitor therapy alone or in combination with trametinib on downstream signaling pathways (MAPK, PI3K) were analyzed with Western blots. Abemaciclib showed the highest activity in all cell models with IC50-values ranging from 0,5 – 2µM. Combined treatment approaches with trametinib showed synergistic effects across all cell models. Long-term viability assays revealed distinct sensitivity in the nanomolar range for CDK4/6-inhibitors and combined treatment with trametinib in all cell models. Exposure to abemaciclib and trametinib significantly decreased pRB, pERK, pS6, and pAKT protein expression levels when compared to monotherapy alone. Summarizing, combined treatment with CDK4/6-inhibitors and trametinib showed promising therapeutic effects on HGG models with homozygous CDKN2A deletion and BRAFV600E mutation. Currently, effects on cell cycle distribution and downstream molecular mechanisms are investigated to identify potential predictive biomarkers.

866 Preclinical Study of Pamiparib, a CNS Penetrant PARP Inhibitor, in IDH1/2 Mutated Gliomas

INTRODUCTION: Our group demonstrated IDH1/2-mutated gliomas harbor intrinsic homologous recombination (HR) defects mediated by 2-HG, conferring sensitivity to PARP-inhibitors. We studied pamiparib, a CNS-penetrant PARP-inhibitor with potent PARP-trapping ability, in combination with TMZ and radiation in IDH1/2-mutated gliomas and its pharmacokinetics to demonstrate CNS penetrance. METHODS: We performed DNA-repair functional studies with in-vitro short- and long-term viability assays, and in-vivo studies utilizing an intracranial rat glioma model with bioluminescence imaging. Pharmacokinetics was measured with LCMS/MS from harvested tumor, normal brain, and blood. RESULTS: In-vitro viability assays in paired isogenic IDH1-wildtype and mutant U87 cells showed the IDH1 mutation conferred several-fold enhanced sensitivity to TMZ, as expected. Differential TMZ sensitivity was less pronounced in HCT116 cells, as expected given higher MGMT expression. ELISA confirmed PARylation inhibition by pamiparib at the nanomolar range in a dose-dependent manner. Enhanced pamiparib sensitivity was seen in IDH-mutant U87 and HCT116 cells compared to wildtype. Pamiparib and TMZ demonstrated synergistic interactions in U87 cells, dependent upon IDH1 mutation status. In HCT116 cells, TMZ resistance was overcome with pamiparib in wildtype and mutant cells but notably at lower doses in mutant cells. Synergy was observed with pamiparib and radiation in U87-mutant, compared to wildtype cells. Pharmacokinetics demonstrated favorable CNS penetration with tumor:plasma ratios (>0.20) with 3 mg/kg and 6 mg/kg doses of pamiparib, orally administered BID to rats harboring RG2-cell intracranial tumors. These levels persisted between 2- and 8-hours after last dosing. Pamiparib selectively penetrated tumors over normal brain with normal brain:plasma ratios <0.20. CONCLUSIONS: Pamiparib may selectively target IDH1/2-mutated gliomas and act synergistically with TMZ and radiation to exploit intrinsic HR defects. Pharmacokinetics suggest favorable CNS penetration after BID oral administration.

EXTH-60. PRECLINICAL STUDY OF PAMIPARIB, A CNS-PENETRANT PARP INHIBITOR, IN IDH1/2 MUTATED GLIOMAS

Abstract Our group has demonstrated that IDH1/2 mutated gliomas harbor intrinsic homologous recombination (HR) defects mediated by the oncometabolite, 2-HG, rendering them sensitive to PARP inhibitors. Here, we studied the efficacy of pamiparib, a CNS-penetrant PARP inhibitor with potent PARP trapping ability, in combination with temozolomide (TMZ) and radiation therapy (RT) in IDH1/2 mutated gliomas. We also studied the pharmacokinetics of BGB290 to demonstrate CNS penetrance. We performed a series of DNA repair functional studies with in vitro short- and long-term viability assays, as well as in vivo studies utilizing an orthotopically injected rat glioma model with bioluminescence. Pharmacokinetics was measured with a previously validated LCMS/MS technique. Short-term and long-term viability assays in paired isogenic IDH1 wildtype and mutant cell lines showed the IDH1 mutation conferred enhanced sensitivity to pamiparib with several-fold decreases in IC50, as well as TMZ and RT as expected. Combination treatment with pamiparib and TMZ or RT also demonstrated synergistic interactions in these same cell lines, dependent upon IDH1 mutation status. An ELISA assay confirmed PARylation inhibition by pamiparib at the nanomolar range in a dose-dependent manner. Pharmacokinetic analysis demonstrated favorable CNS penetration with tumor:plasma ratios ( >0.20) observed with low (3 mg/kg) and high (6 mg/kg) doses of pamiparib, orally administered BID to rats harboring intrinsic rat glioma intracranial tumors. These levels persisted between 2 and 8 hours after last dosing. Pamiparib also selectively penetrated tumors over normal brain with normal brain:plasma ratios under 0.20. These data suggest pamiparib may selectively target IDH1/2 mutated gliomas and act synergistically with TMZ and RT to exploit intrinsic HR defects associated with these tumors. Pharmacokinetic analysis suggest favorable CNS penetration after standard bid oral administration. As such, these results lay the groundwork for study of pamiparib with TMZ and/or RT in patients with IDH1/2 mutated gliomas.

Abstract 4152: Preclinical evaluation of PRKC fusions and GNA11/GNAQ mutations as genetic drivers of PKC activation in non-MUM indications to support a phase 1/2 basket trial of IDE196

Abstract Background: IDE196 is a potent and selective PKC inhibitor targeting the novel (δ, ϵ, η, θ) and classical (α, β) PKC isoforms that has demonstrated preliminary clinical activity in patients with metastatic uveal melanoma (MUM). IDE196 is being further evaluated for safety and efficacy as a monotherapy in a Phase1/2 basket trial in non-MUM patients whose tumors harbor GNAQ/GNA11 hotspot mutations (Clinicialtrials.gov: NCT03947385). Patients with solid tumors harboring PRKC fusions or GNAQ/11 non-hotspot mutations may be eligible after preclinical validation of the activity of these fusions and mutations in non-MUM indications. PRKC gene fusions exist at very low frequencies with various gene partners across multiple indications. Hotspot mutations in GNAQ/GNA11 (codons Q209 and R183) constitutively activate the PKC pathway in 90% of MUM patients and may also occur in non-MUM indications such as colorectal cancer (CRC) and cutaneous melanoma. Furthermore, there are additional non-hotspot mutations found in these GTPases across various non-MUM indications. To help identify patients that may benefit from IDE196 in this basket trial, we assessed the sensitivity of non-MUM cell lines that have PRKC gene fusions or GNAQ/11 hotspot/non-hotspot mutations to IDE196. Methods The most prevalent fusions in patient samples listed in TCGA are the HIF1A-PRKCH (n=20) and RP11-47I22.3-PRKCH (n=3) fusions, therefore cell lines containing PRKCH fusions listed in CCLE were prioritized for cell viability screening using short (3-day) and long-term (10-day) assays after IDE196 treatment. For PD analyses, cells were treated for 3hrs with IDE196. Expression of the fusions and pharmacodynamic modulation of phospho-MARCKS was assessed using western blotting (WB). RNASeq data was used to determine fusion structure, size and whether the fusion was in-frame. Results 8/10 PRKCH fusion lines were highly resistant to IDE196 (IC50>10μM, including HIF1A-PRKCH and RP11-47I22.3-PRKCH fusion lines), whereas 2/10 showed sensitivity at high doses in the long-term viability assay (IC50≥1μM, lines with PRKCH-SPTB or VWA2-PRKCH fusions). PD assessment showed that the PKC substrate, pMARCKS, was decreased in all cell lines by IDE196 at doses similar to those seen in the MUM cell line, 92.1 (≥0.3μM). Fusion protein expression was not detectable by WB consistent with literature suggesting that PKC fusions are unstable (1). In summary, cell lines with clinically prevalent PRKCH fusions were insensitive to IDE196 despite robust PD modulation. Assessment of cell lines harboring GNAQ/GNA11 hotspot/non-hotspot mutations for IDE196 sensitivity is ongoing. (1) An-Angela N. Van, Timothy R. Baffi, Maya T. Kunkel, Corina E. Antal and Alexandra C. Newton, FASEB J. (2018); vol 32, suppl.no. 1, abstract 687.6. Citation Format: Marie Wagle, Nandini Ravindran, Carol O'Brien, Christian Frey, Julie Hambleton, Mark R. Lackner, Zineb Mounir. Preclinical evaluation of PRKC fusions and GNA11/GNAQ mutations as genetic drivers of PKC activation in non-MUM indications to support a phase 1/2 basket trial of IDE196 [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 4152.

Nintedanib Is Active in Malignant Pleural Mesothelioma Cell Models and Inhibits Angiogenesis and Tumor Growth In Vivo.

Purpose: Malignant pleural mesothelioma (MPM) is an aggressive thoracic tumor type with limited treatment options and poor prognosis. The angiokinase inhibitor nintedanib has shown promising activity in the LUME-Meso phase II MPM trial and thus is currently being evaluated in the confirmatory LUME-Meso phase III trial. However, the anti-MPM potential of nintedanib has not been studied in the preclinical setting.Experimental Design: We have examined the antineoplastic activity of nintedanib in various in vitro and in vivo models of human MPM.Results: Nintedanib's target receptors were (co)expressed in all the 20 investigated human MPM cell lines. Nintedanib inhibited MPM cell growth in both short- and long-term viability assays. Reduced MPM cell proliferation and migration and the inhibition of Erk1/2 phosphorylation were also observed upon nintedanib treatment in vitro Additive effects on cell viability were detected when nintedanib was combined with cisplatin, a drug routinely used for systemic MPM therapy. In an orthotopic mouse model of human MPM, survival of animals receiving nintedanib per os showed a favorable trend, but no significant benefit. Nintedanib significantly reduced tumor burden and vascularization and prolonged the survival of mice when it was administered intraperitoneally. Importantly, unlike bevacizumab, nintedanib demonstrated significant in vivo antivascular and antitumor potential independently of baseline VEGF-A levels.Conclusions: Nintedanib exerts significant antitumor activity in MPM both in vitro and in vivo These data provide preclinical support for the concept of LUME-Meso trials evaluating nintedanib in patients with unresectable MPM. Clin Cancer Res; 24(15); 3729-40. ©2018 AACR.

Alginate–polyester comacromer based hydrogels as physiochemically and biologically favorable entities for cardiac tissue engineering

The physiochemical and biological responses of tissue engineering hydrogels are crucial in determining their desired performance. A hybrid comacromer was synthesized by copolymerizing alginate and poly(mannitol fumarate-co-sebacate) (pFMSA). Three bimodal hydrogels pFMSA-AA, pFMSA-MA and pFMSA-NMBA were synthesized by crosslinking with Ca2+ and vinyl monomers acrylic acid (AA), methacrylic acid (MA) and N,N′-methylene bisacrylamide (NMBA), respectively. Though all the hydrogels were cytocompatible and exhibited a normal cell cycle profile, pFMSA-AA exhibited superior physiochemical properties viz non-freezable water content (58.34%) and water absorption per unit mass (0.97gwater/ggel) and pore length (19.92±3.91μm) in comparing with other two hydrogels. The increased non-freezable water content and water absorption of pFMSA-AA hydrogels greatly influenced its biological performance, which was evident from long-term viability assay and cell cycle proliferation. The physiochemical and biological favorability of pFMSA-AA hydrogels signifies its suitability for cardiac tissue engineering.

Highly Fluorescent and Photostable Probe for Long‐Term Bacterial Viability Assay Based on Aggregation‐Induced Emission

Long-term tracking of bacterial viability is of great importance for monitoring the viability change of bacteria under storage, evaluating disinfection efficiency, as well as for studying the pharmacokinetic and pharmacodynamic properties of antibacterials. Most of the conventional viability dyes, however, suffer from high toxicity and/or poor photostability, making them unsuitable for long-term studies. In this work, an aggregation-induced emission molecule, TPE-2BA, which can differentiate dead and living bacteria and serve as a highly fluorescent and photostable probe for long-term viability assay. TPE-2BA is a cell-impermeable DNA stain that binds to the groove of double-stranded DNA. Bacteria with compromised membrane open the access for TPE-2BA to reach DNA, endowing it with strong emission. The feasibility of using TPE-2BA for screening effective bactericides is also demonstrated. Plate count experiment reveals that TPE-2BA poses negligible toxicity to bacteria, indicating that it is an excellent probe for long-term bacterial viability assay.

In vitro and in vivo trypanocidal effect of lipophilic extracts of medicinal plants from Mali and Burkina Faso

Aim of the study To determine the in vitro and in vivo antitrypanosomal activity of extracts of traditionally used plants. Materials and methods 47 dichloromethane extracts were tested in vitro in the Long-term Viability Assay (LtVA) on Trypanosoma brucei brucei. The most active ones were also tested in vivo using a standardised mouse test. Results 13 extracts (28%) were active in vitro with MIC-values ≤ 100 μg/ml, 6 extracts showed MIC-values ≤ 50 μg/ml. The root extract of Securidaca longepedunculata Fresen. (Polygalaceae) and the leaf extract of Guiera senegalensis J. F. Gmel. (Combretaceae) were able to reduce parasitaemia in mice, experimentally infected with Trypanosoma brucei brucei by 48 and 42% at the dose of 150 mg/kg b.w. intraperitoneally, two times daily for 3 days. The extract of Acacia nilotica Delile (Mimosaceae) stem bark showed immunosuppressive effect in vivo. Conclusion The results confirm an effect of the ethnobotanically used plants. Further investigation is needed to optimize the effectiveness of the extracts.

Drug‐resistance of Trypanosoma b. rhodesiense isolates from Tanzania

To determine the drug resistance of Trypanosoma brucei rhodesiense strains isolated from sleeping sickness patients in Tanzania. We first screened 35 T. b. rhodesiense strains in the mouse model, for sensitivity to melarsoprol (1.8, 3.6 and 7.2 mg/kg), diminazene aceturate (3.5, 7 and 14 mg/kg), suramin (5, 10 and 20 mg/kg) and isometamidium (0.1, 1.0 and 2 mg/kg). A 13 isolates suspected to be resistant were selected for further testing in vitro and in vivo. From the in vitro testing, IC(50) values were determined by short-term viability assay, and MIC values were calculated by long-term viability assay. For in vivo testing, doses higher than those in the initial screening test were used. Two T. b rhodesiense stocks expressed resistance in vivo to melarsoprol at 5 mg/kg and at 10 mg/kg. These strains had high IC(50) and MIC values consistent with those of the melarsoprol-resistant reference strain. Another isolate relapsed after treatment with 5 mg/kg of melarsoprol although it did not appear resistant in vitro. One isolate was resistant to diminazene at 14 mg/kg and another was resistant at both 14 and 28 mg/kg of diminazene. These two isolates had high IC(50) values consistent with the diminazene-resistant reference strain. Two isolates relapsed at a dose of 5 mg/kg of suramin, although no isolate appeared resistant in the in vitro tests. Two isolates were resistant to isometamidium at 1.0 mg/kg and had higher IC(50) values. Two isolates were cross-resistant to melarsoprol and diminazene and one isolate was cross-resistant to suramin and isometamidium. The reduced susceptibility of T. b. rhodesiense isolates to these drugs strongly indicates that drug resistance may be emerging in north-western Tanzania.

Evaluation of medicinal plants from Mali for their in vitro and in vivo trypanocidal activity

Water, methanol and dichloromethane extracts prepared from various parts of 40 medicinal plant species from Mali were investigated for their trypanocidal activity against Trypanosoma brucei brucei. Of a total of 165 extracts tested in vitro in the Low Inoculation Long Incubation Test (LILIT), 24 extracts showed a high trypanocidal activity. Using the Long-Term Viability Assay (LtVA) for corroboration of the results of the 24 extracts, it was found that 15 samples had minimum inhibitory concentration (MIC) values > 10μg/ml, eight MIC values of 100 μg/ml and one MIC values of 50–100 μg/ml. So far, four extracts with MIC values ≤ 100 μg/ml were tested for antitrypanosomal activity in mice, experimentally infected with Trypanosoma brucei brucei. Only, the aqueous extracts of the leaves of Terminalia avicennioides Guill. and Perr. (Combretaceae) and the stem bark of Ceiba pentandra (L.) Gaertn. (Bombacaceae) were able to reduce the parasitaemia in animals treated at the dose of 100 mg/kg b.w. (intraperitoneally, two times daily for 3 days) and of 150 mg/kg b.w. (per os, two times daily for 3 days), respectively. The reduction of parasitaemia was, however, statistically significant ( p = 0.002) only in case of treatment with Terminalia avicennioides.

Axenic in vitro cultivation of Trypanosoma simiae bloodstream trypomastigotes

In vitro test methods have been developed both for determining the sensitivity of isolates of pathogenic African trypanosomes and for evaluating new compounds for antitrypanosomal activity. The principles of the assays and their main advantages or drawbacks are presented and discussed. In vitro assays which do not require trypanosomes pre-adapted to culture conditions are the Drug Incubation Infectivity Test (DIIT) and the [3H]Hypoxanthine Incorporation Assay. Chemosensitivity tests which do require continuous growth of trypanosomes in vitro include photometric, fluorescence, growth inhibition, long-term viability and metacyclic incubation assays. Evidence is presented to use metacyclic or bloodstream forms in such assays but to avoid procyclic trypanosomes. The drug sensitivity of a homogeneous trypanosome population can be quantified by using photometric, fluorescence or growth inhibition assays lasting 1–3 days. Small numbers of resistant organisms hidden in a sensitive population can be detected employing long-term viability assays (7–10 days). Final selection of the assay to be employed will depend on the parameter to be investigated, and the equipment available.