Combination drug screen identifies synergistic drug interaction of BCL-XL and class I histone deacetylase inhibitors in MYC-amplified medulloblastoma cells.

PURPOSE: Patients with MYC-amplified Group 3 medulloblastoma (MB) (subtype II) show poor progression-free survival rates. Class I histone deacetylase inhibitors (HDACi) are highly effective for the treatment of MYC-amplified MB in vitro and in vivo. Drug combination regimens including class I HDACi may represent an urgently needed novel treatment approach for this high risk disease. METHODS: A medium-throughput in vitro combination drug screen was performed in three MYC-amplified and one non-MYC-amplified MB cell line testing 75 clinically relevant drugs alone and in combination with entinostat. The drug sensitivity score (DSS) was calculated based on metabolic inhibition quantified by CellTiter-Glo. The six top synergistic combination hits were evaluated in a 5 × 5 combination matrix and a seven-ray design. Synergy was validated and characterized by cell counts, caspase-3-like-activity and poly-(ADP-ribose)-polymerase-(PARP)-cleavage. On-target activity of drugs was validated by immunoprecipitation and western blot. BCL-XL dependency of the observed effect was explored with siRNA mediated knockdown of BCL2L1, and selective inhibition with targeted compounds (A-1331852, A-1155463). RESULTS: 20/75 drugs effectively reduced metabolic activity in combination with entinostat in all three MYC-amplified cell lines (DSS ≥ 10). The combination entinostat and navitoclax showed the strongest synergistic interaction across all MYC-amplified cell lines. siRNA mediated knockdown of BCL2L1, as well as targeted inhibition with selective inhibitors showed BCL-XL dependency of the observed effect. Increased cell death was associated with increased caspase-3-like-activity. CONCLUSION: Our study identifies the combination of class I HDACi and BCL-XL inhibitors as a potential new approach for the treatment of MYC-amplified MB cells.

Drug sensitivity profiling of 3D tumor tissue cultures in the pediatric precision oncology program INFORM.

The international precision oncology program INFORM enrolls relapsed/refractory pediatric cancer patients for comprehensive molecular analysis. We report a two-year pilot study implementing ex vivo drug sensitivity profiling (DSP) using a library of 75-78 clinically relevant drugs. We included 132 viable tumor samples from 35 pediatric oncology centers in seven countries. DSP was conducted on multicellular fresh tumor tissue spheroid cultures in 384-well plates with an overall mean processing time of three weeks. In 89 cases (67%), sufficient viable tissue was received; 69 (78%) passed internal quality controls. The DSP results matched the identified molecular targets, including BRAF, ALK, MET, and TP53 status. Drug vulnerabilities were identified in 80% of cases lacking actionable (very) high-evidence molecular events, adding value to the molecular data. Striking parallels between clinical courses and the DSP results were observed in selected patients. Overall, DSP in clinical real-time is feasible in international multicenter precision oncology programs.

Patient-by-Patient Deep Transfer Learning for Drug-Response Profiling Using Confocal Fluorescence Microscopy of Pediatric Patient-Derived Tumor-Cell Spheroids.

Image-based phenotypic drug profiling is receiving increasing attention in drug discovery and precision medicine. Compared to classical end-point measurements quantifying drug response, image-based profiling enables both the quantification of drug response and characterization of disease entities and drug-induced cell-death phenotypes. Here, we aim to quantify image-based drug responses in patient-derived 3D spheroid tumor cell cultures, tackling the challenges of a lack of single-cell-segmentation methods and limited patient-derived material. Therefore, we investigate deep transfer learning with patient-by-patient fine-tuning for cell-viability quantification. We fine-tune a convolutional neural network (pre-trained on ImageNet) with 210 control images specific to a single training cell line and 54 additional screen -specific assay control images. This method of image-based drug profiling is validated on 6 cell lines with known drug sensitivities, and further tested with primary patient-derived samples in a medium-throughput setting. Network outputs at different drug concentrations are used for drug-sensitivity scoring, and dense-layer activations are used in t-distributed stochastic neighbor embeddings of drugs to visualize groups of drugs with similar cell-death phenotypes. Image-based cell-line experiments show strong correlation to metabolic results ( R ≈ 0.7 ) and confirm expected hits, indicating the predictive power of deep learning to identify drug-hit candidates for individual patients. In patient-derived samples, combining drug sensitivity scoring with phenotypic analysis may provide opportunities for complementary combination treatments. Deep transfer learning with patient-by-patient fine-tuning is a promising, segmentation-free image-analysis approach for precision medicine and drug discovery.

Functional Therapeutic Target Validation Using Pediatric Zebrafish Xenograft Models.

The survival rate among children with relapsed tumors remains poor, due to tumor heterogeneity, lack of directly actionable tumor drivers and multidrug resistance. Novel personalized medicine approaches tailored to each tumor are urgently needed to improve cancer treatment. Current pediatric precision oncology platforms, such as the INFORM (INdividualized Therapy FOr Relapsed Malignancies in Childhood) study, reveal that molecular profiling of tumor tissue identifies targets associated with clinical benefit in a subgroup of patients only and should be complemented with functional drug testing. In such an approach, patient-derived tumor cells are exposed to a library of approved oncological drugs in a physiological setting, e.g., in the form of animal avatars injected with patient tumor cells. We used molecularly fully characterized tumor samples from the INFORM study to compare drug screen results of individual patient-derived cell models in functional assays: (i) patient-derived spheroid cultures within a few days after tumor dissociation; (ii) tumor cells reisolated from the corresponding mouse PDX; (iii) corresponding long-term organoid-like cultures and (iv) drug evaluation with the corresponding zebrafish PDX (zPDX) model. Each model had its advantage and complemented the others for drug hit and drug combination selection. Our results provide evidence that in vivo zPDX drug screening is a promising add-on to current functional drug screening in precision medicine platforms.

iTReX: Interactive exploration of mono- and combination therapy dose response profiling data.

High throughput screening methods, measuring the sensitivity and resistance of tumor cells to drug treatments have been rapidly evolving. Not only do these screens allow correlating response profiles to tumor genomic features for developing novel predictors of treatment response, but they can also add evidence for therapy decision making in precision oncology. Recent analysis methods developed for either assessing single agents or combination drug efficacies enable quantification of dose-response curves with restricted symmetric fit settings. Here, we introduce iTReX, a user-friendly and interactive Shiny/R application, for both the analysis of mono- and combination therapy responses. The application features an extended version of the drug sensitivity score (DSS) based on the integral of an advanced five-parameter dose-response curve model and a differential DSS for combination therapy profiling. Additionally, iTReX includes modules that visualize drug target interaction networks and support the detection of matches between top therapy hits and the sample omics features to enable the identification of druggable targets and biomarkers. iTReX enables the analysis of various quantitative drug or therapy response readouts (e.g. luminescence, fluorescence microscopy) and multiple treatment strategies (drug treatments, radiation). Using iTReX we validate a cost-effective drug combination screening approach and reveal the application's ability to identify potential sample-specific biomarkers based on drug target interaction networks. The iTReX web application is accessible at https://itrex.kitz-heidelberg.de.

Drug-perturbation-based stratification of blood cancer.

As new generations of targeted therapies emerge and tumor genome sequencing discovers increasingly comprehensive mutation repertoires, the functional relationships of mutations to tumor phenotypes remain largely unknown. Here, we measured ex vivo sensitivity of 246 blood cancers to 63 drugs alongside genome, transcriptome, and DNA methylome analysis to understand determinants of drug response. We assembled a primary blood cancer cell encyclopedia data set that revealed disease-specific sensitivities for each cancer. Within chronic lymphocytic leukemia (CLL), responses to 62% of drugs were associated with 2 or more mutations, and linked the B cell receptor (BCR) pathway to trisomy 12, an important driver of CLL. Based on drug responses, the disease could be organized into phenotypic subgroups characterized by exploitable dependencies on BCR, mTOR, or MEK signaling and associated with mutations, gene expression, and DNA methylation. Fourteen percent of CLLs were driven by mTOR signaling in a non-BCR-dependent manner. Multivariate modeling revealed immunoglobulin heavy chain variable gene (IGHV) mutation status and trisomy 12 as the most important modulators of response to kinase inhibitors in CLL. Ex vivo drug responses were associated with outcome. This study overcomes the perception that most mutations do not influence drug response of cancer, and points to an updated approach to understanding tumor biology, with implications for biomarker discovery and cancer care.

BID links ferroptosis to mitochondrial cell death pathways.

Ferroptosis has been defined as an oxidative and iron-dependent pathway of regulated cell death that is distinct from caspase-dependent apoptosis and established pathways of death receptor-mediated regulated necrosis. While emerging evidence linked features of ferroptosis induced e.g. by erastin-mediated inhibition of the Xc- system or inhibition of glutathione peroxidase 4 (Gpx4) to an increasing number of oxidative cell death paradigms in cancer cells, neurons or kidney cells, the biochemical pathways of oxidative cell death remained largely unclear. In particular, the role of mitochondrial damage in paradigms of ferroptosis needs further investigation. In the present study, we find that erastin-induced ferroptosis in neuronal cells was accompanied by BID transactivation to mitochondria, loss of mitochondrial membrane potential, enhanced mitochondrial fragmentation and reduced ATP levels. These hallmarks of mitochondrial demise are also established features of oxytosis, a paradigm of cell death induced by Xc- inhibition by millimolar concentrations of glutamate. Bid knockout using CRISPR/Cas9 approaches preserved mitochondrial integrity and function, and mediated neuroprotective effects against both, ferroptosis and oxytosis. Furthermore, the BID-inhibitor BI-6c9 inhibited erastin-induced ferroptosis, and, in turn, the ferroptosis inhibitors ferrostatin-1 and liproxstatin-1 prevented mitochondrial dysfunction and cell death in the paradigm of oxytosis. These findings show that mitochondrial transactivation of BID links ferroptosis to mitochondrial damage as the final execution step in this paradigm of oxidative cell death.

Janus and PI3-kinases mediate glucocorticoid resistance in activated chronic leukemia cells.

Glucorticoids (GCs) such as dexamethasone (DEX) remain important treatments for Chronic Lymphocytic Leukemia (CLL) but the mechanisms are poorly understood and resistance is inevitable. Proliferation centers (PC) in lymph nodes and bone marrow offer protection against many cytotoxic drugs and circulating CLL cells were found to acquire resistance to DEX-mediated killing in conditions encountered in PCs including stimulation by toll-like receptor agonists and interactions with stromal cells. The resistant state was associated with impaired glucocorticoid receptor-mediated gene expression, autocrine activation of STAT3 through Janus Kinases (JAKs), and increased glycolysis. The JAK1/2 inhibitor ruxolitinib blocked STAT3-phosphorylation and partially improved DEX-mediated killing of stimulated CLL cells in vitro but not in CLL patients in vivo. An automated microscopy-based screen of a kinase inhibitor library implicated an additional protective role for the PI3K/AKT/FOXO pathway. Blocking this pathway with the glycolysis inhibitor 2-deoxyglucose (2-DG) or the PI3K-inhibitors idelalisib and buparlisib increased DEX-mediated killing but did not block STAT3-phosphorylation. Combining idelalisib or buparlisib with ruxolitinib greatly increased killing by DEX. These observations suggest that glucocorticoid resistance in CLL cells may be overcome by combining JAK and PI3K inhibitors.

High-content screening identifies kinase inhibitors that overcome venetoclax resistance in activated CLL cells.

Novel agents such as the Bcl-2 inhibitor venetoclax (ABT-199) are changing treatment paradigms for chronic lymphocytic leukemia (CLL) but important problems remain. Although some patients exhibit deep and durable responses to venetoclax as a single agent, other patients harbor subpopulations of resistant leukemia cells that mediate disease recurrence. One hypothesis for the origin of resistance to venetoclax is by kinase-mediated survival signals encountered in proliferation centers that may be unique for individual patients. An in vitro microenvironment model was developed with primary CLL cells that could be incorporated into an automated high-content microscopy-based screen of kinase inhibitors (KIs) to identify agents that may improve venetoclax therapy in a personalized manner. Marked interpatient variability was noted for which KIs were effective; nevertheless, sunitinib was identified as the most common clinically available KI effective in overcoming venetoclax resistance. Examination of the underlying mechanisms indicated that venetoclax resistance may be induced by microenvironmental signals that upregulate antiapoptotic Bcl-xl, Mcl-1, and A1, which can be counteracted more efficiently by sunitinib than by ibrutinib or idelalisib. Although patient-specific drug responses are common, for many patients, combination therapy with sunitinib may significantly improve the therapeutic efficacy of venetoclax.

Molecular Pathways: Leveraging the BCL-2 Interactome to Kill Cancer Cells--Mitochondrial Outer Membrane Permeabilization and Beyond.

The inhibition of apoptosis enables the survival and proliferation of tumors and contributes to resistance to conventional chemotherapy agents and is therefore a very promising avenue for the development of new agents that will enhance current cancer therapies. The BCL-2 family proteins orchestrate apoptosis at the mitochondria and endoplasmic reticulum and are involved in other processes such as autophagy and unfolded protein response (UPR) that lead to different types of cell death. Over the past decade, significant efforts have been made to restore apoptosis using small molecules that modulate the activity of BCL-2 family proteins. The small molecule ABT-199, which antagonizes the activity of BCL-2, is currently the furthest in clinical trials and shows promising activity in many lymphoid malignancies as a single agent and in combination with conventional chemotherapy agents. Here, we discuss strategies to improve the specificity of pharmacologically modulating various antiapoptotic BCL-2 family proteins, review additional BCL-2 family protein interactions that can be exploited for the improvement of conventional anticancer therapies, and highlight important points of consideration for assessing the activity of small-molecule BCL-2 family protein modulators.

N-acyl derivatives of 4-phenoxyaniline as neuroprotective agents.

Neuronal cell death is the main cause behind the progressive loss of brain function in age-related neurodegenerative disorders such as Alzheimer's and Parkinson's diseases. Despite the differing etiologies of these neurological diseases, the underlying neuronal damage is triggered by common mechanisms such as oxidative stress, impaired calcium homeostasis, and disrupted mitochondrial integrity and function. In particular, mitochondrial fragmentation, mitochondrial membrane permeability, and the release of death-promoting factors into the cytosol have been revealed as the "point of no return" in programmed cell death in neurons. Recent studies revealed a pivotal role for the pro-apoptotic Bcl-2-family protein Bid in models of neuronal cell death, which confirmed Bid as a potential drug target. Herein, we present N-acyl-substituted derivatives of 4-phenoxyaniline that were screened for their potential to attenuate Bid-mediated neurotoxicity. These compounds provided significant protection against glutamate- and Bid-induced toxicity in cultured neurons. Substitution of the amino group in the 4-phenoxyaniline scaffold with 4-piperidine carboxylic acid and N-hydroxyethyl-4-piperidine carboxylic acid yielded compounds that displayed significant neuroprotective activity at concentrations as low as 1 µM. Furthermore, findings of a tBid-overexpression assay and real-time measurements of cell impedance support the hypothesis that these compounds indeed address the Bid protein.

Novel N-phenyl-substituted thiazolidinediones protect neural cells against glutamate- and tBid-induced toxicity.

Mitochondrial demise is a key feature of progressive neuronal death contributing to acute and chronic neurological disorders. Recent studies identified a pivotal role for the BH3-only protein B-cell lymphoma-2 interacting domain death antagonist (Bid) for such mitochondrial damage and delayed neuronal death after oxygen-glucose deprivation, glutamate-induced excitotoxicity, or oxidative stress in vitro and after cerebral ischemia in vivo. Therefore, we developed new N-phenyl-substituted thiazolidine-2,4-dione derivatives as potent inhibitors of Bid-dependent neurotoxicity. The new compounds 6, 7, and 16 were identified as highly protective by extensive screening in a model of glutamate toxicity in immortalized mouse hippocampal neurons (HT-22 cells). These compounds significantly prevent truncated Bid-induced toxicity in the neuronal cell line, providing strong evidence that inhibition of Bid was the underlying mechanism of the observed protective effects. Furthermore, Bid-dependent hallmarks of mitochondrial dysfunction, such as loss of mitochondrial membrane potential, ATP depletion, as well as impairments in mitochondrial respiration, are significantly prevented by compounds 6, 7, and 16. Therefore, the present study identifies a class of N-phenyl thiazolidinediones as novel Bid-inhibiting neuroprotective agents that provide promising therapeutic perspectives for neurodegenerative diseases, in which Bid-mediated mitochondrial damage and associated intrinsic death pathways contribute to the underlying progressive loss of neurons.