ABSTRACT
PURPOSE: We and others have demonstrated that MYC-amplified medulloblastoma (MB) cells are susceptible to class I histone deacetylase inhibitor (HDACi) treatment. However, single drug treatment with HDACi has shown limited clinical efficacy. We hypothesized that addition of a second compound acting synergistically with HDACi may enhance efficacy. METHODS: We used a gene expression dataset to identify PLK1 as a second target in MB cells and validated the relevance of PLK1 in MB. We measured cell metabolic activity, viability, and cycle progression in MB cells after treatment with PLK1-specific inhibitors (PLK1i). Chou-Talalay synergy calculations were used to determine the nature of class I HDACi entinostat and PLK1i interaction which was validated. Finally, the clinical potential of the combination was assessed in the in vivo experiment. RESULTS: MYC-amplified tumor cells are highly sensitive towards treatment with ATP-competitive PLK1i as a monotherapy. Entinostat and PLK1i in combination act synergistically in MYC-driven MB cells, exerting cytotoxic effects at clinically relevant concentrations. The downstream effect is exerted via MYC-related pathways, pointing out the potential of MYC amplification as a clinically feasible predictive biomarker for patient selection. While entinostat significantly extended survival of mice implanted with orthotopic MYC-amplified MB PDX, there was no evidence of the improvement of survival when treating the animals with the combination. CONCLUSION: The combination of entinostat and PLK1i showed synergistic interaction in vitro, but not in vivo. Therefore, further screening of blood-brain barrier penetrating PLK1i is warranted to determine the true potential of the combination as no on-target activity was observed after PLK1i volasertib treatment in vivo.
Subject(s)
Antineoplastic Agents , Cerebellar Neoplasms , Medulloblastoma , Mice , Animals , Histone Deacetylase Inhibitors/pharmacology , Histone Deacetylase Inhibitors/therapeutic use , Medulloblastoma/drug therapy , Medulloblastoma/metabolism , Antineoplastic Agents/therapeutic use , Cerebellar Neoplasms/drug therapy , Cell Line, TumorABSTRACT
Reliable preclinical drug testing models for cancer research are urgently needed with zebrafish embryo models emerging as a powerful vertebrate model for xenotransplantation studies. Here, we describe the evaluation of toxicity, efficacy, and on-target activity of histone deacetylase (HDAC) inhibitors in a zebrafish embryo yolk sac xenotransplantation model of medulloblastoma and neuroblastoma cells. For this, we performed toxicity assays with our zebrafish drug library consisting of 28 clinically relevant targeted as well as chemotherapeutic drugs with zebrafish embryos. We further engrafted zebrafish embryos with fluorescently labeled pediatric tumor cells (SK-N-BE(2)-C, HD-MB03, or MED8A) and monitored the progression after HDAC inhibitor treatment of xenotransplanted tumors through tumor volume measurements with high-content confocal microscopy in a multi-well format. The on-target activity of HDAC inhibitors was verified through immunohistochemistry staining on paraffin-embedded early larvae. Overall, the zebrafish embryo xenotransplantation model allows for fast and cost-efficient in vivo evaluation of targeted drug toxicity, efficacy, and on-target activity in the field of precision oncology.
Subject(s)
Neoplasms , Zebrafish , Animals , Humans , Histone Deacetylase Inhibitors/pharmacology , Histone Deacetylase Inhibitors/therapeutic use , Heterografts , Neoplasms/drug therapy , Precision Medicine , Disease Models, Animal , Histone Deacetylases , Xenograft Model Antitumor Assays , Cell Line, TumorABSTRACT
BACKGROUND: Pediatric low-grade gliomas (pLGG) are the most common pediatric central nervous system tumors, with driving alterations typically occurring in the MAPK pathway. The ERK1/2 inhibitor ulixertinib (BVD-523) has shown promising responses in adult patients with mitogen-activated protein kinase (MAPK)-driven solid tumors. METHODS: We investigated the antitumoral activity of ulixertinib monotherapy as well as in combination with MEK inhibitors (MEKi), BH3-mimetics, or chemotherapy in pLGG. Patient-derived pLGG models reflecting the two most common alterations in the disease, KIAA1549:BRAF-fusion and BRAFV600E mutation (DKFZ-BT66 and BT40, respectively) were used for in vitro and in vivo (zebrafish embryos and mice) efficacy testing. RESULTS: Ulixertinib inhibited MAPK pathway activity in both models, and reduced cell viability in BT40 with clinically achievable concentrations in the low nanomolar range. Combination treatment of ulixertinib with MEKi or BH3-mimetics showed strong evidence of antiproliferative synergy in vitro. Ulixertinib showed on-target activity in all tested combinations. In vivo, sufficient penetrance of the drug into brain tumor tissue in concentrations above the in vitro IC50 and reduction of MAPK pathway activity was achieved. In a preclinical mouse trial, ulixertinib mono- and combined therapies slowed tumor growth and increased survival. CONCLUSIONS: These data indicate a high clinical potential of ulixertinib for the treatment of pLGG and strongly support its first clinical evaluation in pLGG as single agent and in combination therapy in a currently planned international phase I/II umbrella trial.
Subject(s)
Glioma , Mitogen-Activated Protein Kinases , Animals , Mice , Zebrafish , Cell Line, Tumor , Glioma/genetics , Protein Kinase Inhibitors/pharmacology , Protein Kinase Inhibitors/therapeutic use , Proto-Oncogene Proteins B-raf/genetics , MutationABSTRACT
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.
ABSTRACT
APR-246 (Eprenetapopt/PRIMA-1Met) is a very potent anti-cancer drug in clinical trials and was initially developed as a p53 refolding agent. As an alternative mode of action, the elevation of reactive oxygen species (ROS) has been proposed. Through an in silico analysis, we investigated the responses of approximately 800 cancer cell lines (50 entities; Cancer Therapeutics Response Portal, CTRP) to APR-246 treatment. In particular, neuroblastoma, lymphoma and acute lymphocytic leukemia cells were highly responsive. With gene expression data from the Cancer Cell Line Encyclopedia (CCLE; n = 883) and patient samples (n = 1643) from the INFORM registry study, we confirmed that these entities express low levels of SLC7A11, a previously described predictive biomarker for APR-246 responsiveness. Combining the CTRP drug response data with the respective CCLE gene expression profiles, we defined a novel gene signature, predicting the effectiveness of APR-246 treatment with a sensitivity of 90% and a specificity of 94%. We confirmed the predicted APR-246 sensitivity in 8/10 cell lines and in ex vivo cultures of patient samples. Moreover, the combination of ROS detoxification-impeding APR-246 with approved HDAC-inhibitors, known to elevate ROS, substantially increased APR-246 sensitivity in cell cultures and in vivo in two zebrafish neuroblastoma xenograft models. These data provide evidence that APR-246, in combination with HDAC-inhibitors, displays a novel potent targeted treatment option for neuroblastoma patients.
ABSTRACT
The survival rate among children with relapsed neuroblastomas continues to be poor, and thus new therapeutic approaches identified by reliable preclinical drug testing models are urgently needed. Zebrafish are a powerful vertebrate model in preclinical cancer research. Here, we describe a zebrafish neuroblastoma yolk sac model to evaluate efficacy and toxicity of histone deacetylase (HDAC) inhibitor treatments. Larvae were engrafted with fluorescently labeled, genetically diverse, established cell lines and short-term cultures of patient-derived primary cells. Engrafted tumors progressed locally and disseminated remotely in an intact environment. Combination treatments involving the standard chemotherapy doxorubicin and HDAC inhibitors substantially reduced tumor volume, induced tumor cell death, and inhibited tumor cell dissemination to the tail region. Hence, this model allows for fast, cost-efficient, and reliable in vivo evaluation of toxicity and response of the primary and metastatic tumor sites to drug combinations.