Abstract
Introduction
Pilocytic astrocytomas (PA) are the most common pediatric brain tumors. They are characterized by MAPK pathway alterations, leading to its constitutive activation and modulating ...the balance between cell proliferation and the oncogene-induced senescence (OIS) sustained by senescence-associated secretory phenotype (SASP) factors. This makes PA suitable for MAPK inhibitor (MAPKi) therapies, showing encouraging results in phase 1/2 clinical trials. Little is known about the molecular implications of MAPK downregulation in the proliferating and senescent compartments.
Methods
DKFZ-BT66 PA cells derived from a primary KIAA:BRAF-fusion positive PA cell line, were used as model system. Gene expression and phospho-proteomic datasets were generated from DKFZ-BT66 cells, in both the proliferative and senescent states, and treated with the MEKi trametinib for different time-spans. A time course analysis based on differentially expressed genes was performed, followed by a single-sample gene set enrichment analysis (ssGSEA). Analysis of the phospho-proteomic data is ongoing.
Results
Differential gene expression analysis revealed that MEK inhibition leads to the inhibition of the OIS-SASP gene program in senescent DKFZ-BT66. ssGSEA showed that most MAPK-related signatures were downregulated upon MEKi treatment, while pathways related to upstream MAPK activators (including FGFR, NTRK and TGFB pathways) were upregulated, in both proliferating and senescent DKFZ-BT66. Genes regulated by the MAPK pathway and involved in OIS-SASP were identified by analyzing genes differentially regulated between proliferating and senescent DKFZ-BT66, and modulated upon MEKi treatment.
Conclusion
This data suggests that MAPKi reverses OIS in senescent PA cells, while inducing the activation of MAPK upstream regulators in proliferating and senescent PA cells, identifying putative co-targets that could help prevent growth rebound upon MAPKi withdrawal. Furthermore, the identification of the MAPK-related OIS-SASP genes provide insight about the regulation of OIS-SASP by the MAPK pathway. Validation of this data with the ongoing phospho-proteomic analysis and in primary samples is needed.
Abstract
Subgroups of ependymoma, especially RELA fusion-positive and posterior fossa type A tumors, are associated with poor prognosis. Curative therapeutic strategies have not yet been identified. ...We set up a high-throughput drug screening (HTS) pipeline to evaluate clinically established compounds (n=196) in primary ependymoma cultures (n=12). As culturing ependymoma is challenging, assay miniaturization to 1536-well microplates emerged as a key feature to process HTS despite smallest cell numbers. DNA methylation profiling showed that entity and subgroup affiliation from primary diagnosis was maintained in primary cultures, as assessed through molecular neuropathology 2.0 based classification (MNP 2.0, Capper, D. et al., Nature, 2018). A comparison of HTS data of ependymoma and other pediatric brain tumor models (n=48) revealed a remarkable chemoresistance in vitro. However, we identified Neratinib, an irreversible ERBB2 inhibitor, as the most prominent candidate which was preferentially active in a subset of the investigated ependymoma cultures (n=5). Combinatory treatment with Copanlisib, a PI3K inhibitor, was able to overcome resistance to single agent treatment using Neratinib in established cell lines of ependymoma (n=3) and 2/4 primary cultures for which combinatory treatment could be tested. Finally, we validated efficacy of Neratinib combined with Copanlisib in mice bearing ependymoma xenografts which revealed significantly reduced tumor size compared to vehicle-treated animals. In summary, our study demonstrates that HTS may reveal targeted therapies for pediatric brain tumors. Specifically, we found a synergistic interaction of Neratinib and Copanlisib for treatment of ependymoma, thereby providing a novel therapeutic approach in an otherwise largely chemoresistant entity.
Ependymoblastoma (EBL) and embryonal tumor with abundant neuropil and true rosettes (ETANTR) are very aggressive embryonal neoplasms characterized by the presence of ependymoblastic multilayered ...rosettes typically occurring in children below 6 years of age. It has not been established whether these two tumors really comprise distinct entities. Earlier, using array-CGH, we identified a unique focal amplification at 19q13.42 in a case of ETANTR. In the present study, we investigated this locus by fluorescence in situ hybridization in 41 tumors, which had morphologically been diagnosed as EBL or ETANTR. Strikingly, FISH analysis revealed 19q13.42 amplifications in 37/40 samples (93%). Among tumors harboring the amplification, 19 samples were identified as ETANTR and 18 as EBL. The three remaining tumors showed a polysomy of chromosome 19. Analysis of recurrent/metastatic tumors (
n
= 7) showed that the proportion of nuclei carrying the amplification was increased (up to 80–100% of nuclei) in comparison to the corresponding primary tumors. In conclusion, we have identified a hallmark cytogenetic aberration occurring in virtually all embryonal brain tumors with ependymoblastic rosettes suggesting that ETANTR and EBL comprise a single biological entity. FISH analysis of the 19q13.42 locus is a very promising diagnostic tool to identify a subset of primitive neuroectodermal tumors with distinct morphology, biology, and clinical behavior.
Pilocytic astrocytoma (PA) is the most frequent pediatric brain tumor. Activation of the MAPK pathway is well established as the oncogenic driver of the disease. It is most frequently caused by ...KIAA1549:BRAF fusions, and leads to oncogene induced senescence (OIS). OIS is thought to be a major reason for growth arrest of PA cells in vitro and in vivo, preventing establishment of PA cultures. Hence, valid preclinical models are currently very limited, but preclinical testing of new compounds is urgently needed. We transduced the PA short-term culture DKFZ-BT66 derived from the PA of a 2-year old patient with a doxycycline-inducible system coding for Simian Vacuolating Virus 40 Large T Antigen (SV40-TAg). SV40-TAg inhibits TP53/CDKN1A and CDKN2A/RB1, two pathways critical for OIS induction and maintenance. DNA methylation array and KIAA1549:BRAF fusion analysis confirmed pilocytic astrocytoma identity of DKFZ-BT66 cells after establishment. Readouts were analyzed in proliferating as well as senescent states, including cell counts, viability, cell cycle analysis, expression of SV40-Tag, CDKN2A (p16), CDKN1A (p21), and TP53 (p53) protein, and gene-expression profiling. Selected MAPK inhibitors (MAPKi) including clinically available MEK inhibitors (MEKi) were tested in vitro. Expression of SV40-TAg enabled the cells to bypass OIS and to resume proliferation with a mean doubling time of 45h allowing for propagation and long-term culture. Withdrawal of doxycycline led to an immediate decrease of SV40-TAg expression, appearance of senescent morphology, upregulation of CDKI proteins and a subsequent G1 growth arrest in line with the re-induction of senescence. DKFZ-BT66 cells still underwent replicative senescence that was overcome by TERT expression. Testing of a set of MAPKi revealed differential responses in DKFZ-BT66. MEKi efficiently inhibited MAPK signaling at clinically achievable concentrations, while BRAF V600E- and RAF Type II inhibitors showed paradoxical activation. Taken together, we have established the first patient-derived long term expandable PA cell line expressing the KIAA1549:BRAF-fusion suitable for preclinical drug testing.
BACKGROUND:
Histone deacetylases (HDACs) are crucial regulators of epigenetic and posttranslational modifications and therefore represent a promising therapeutic target in cancer cells that harbor ...distinct epigenomes from normal cells. To exploit the therapeutic potential of HDAC inhibitors (HDACi) we synthesized a unique in-house library of more than 200 inhibitors. For the evaluation of the antitumor effects of the compound library in brain tumor cell lines, we successfully established an optimal screening workflow.
METHODS:
The screening procedure was streamlined by automated dispensing of cell lines, reagents and inhibitors using state of the art equipment. The drugs were evaluated for their effect on tumor cell viability in a panel of cell lines derived from different brain tumor entities (8 glioblastoma, 10 medulloblastoma and 6 atypical teratoid/rhabdoid tumor cell lines) and compared to 5 normal control tissues. Corresponding dose-response profiles were generated using an optimized bioinformatics workflow. In addition to our in-house library commercially available and clinically used HDACi (e.g. Vorinostat) were included.
RESULTS:
The semi-automated setup enabled the miniaturization of the assay format to 384-well plates. In combination with additional modifications, a remarkable increase of the overall throughput was realized, while generating accurate and reproducible results. Based on this workflow, we created a unique and comprehensive data set and could thereby identify various HDACi acting universally across brain tumors or being specifically active in distinct tumor entities. Promising candidates are currently being further characterized, e.g. Panobinostat showed activity in the majority of brain tumor models at low nanomolar concentrations.
CONCLUSION:
The growing importance of HDAC inhibitors is reflected by an increasing number of HDACi in clinical trials, with four HDACi already approved by the FDA for treating lymphomas and multiple myelomas. Our study supports the finding that HDACi are valid therapeutic agents and that selected inhibitors are promising candidates for future epigenetic therapy of primary brain tumors.
Abstract
Medulloblastoma is the most common malignant pediatric brain tumor and comprises at least four distinct biological subgroups. MYC-driven tumorigenesis constitutes a hallmark feature ...underlying Group 3 biology and metastatic dissemination at diagnosis or recurrence constitutes a major clinical problem in this highly aggressive subgroup. Employing our institutional drug screening platform, we evaluated an in-house library of over 200 histone deacetylase inhibitors (HDACi) in various brain tumor cell lines and patient-derived primary cultures including glioblastoma (n=8), medulloblastoma (n=10) and atypical teratoid/rhabdoid tumors (n=11). Thereby, we identified CI-994, a clinically established class I specific HDAC inhibitor, which selectively inhibited proliferation of MYC-driven medulloblastoma in our primary and secondary screen. We confirmed the MYC-dependent response in medulloblastoma cell lines with CRISPR/CAS9-based MYC overexpression compared to their isogenic controls with low MYC expression. Notably, inhibitor treatment resulted in significantly reduced MYC mRNA and protein expression levels, decreased cell viability and induction of apoptosis. Additionally, a screen for synergism with a clinical inhibitor library (clinical phase III/IV and approved chemotherapeutics) revealed favorable interaction with NFκB inhibition. Furthermore, integrated proteogenomics using RNA sequencing and proteomic profiling corroborated NFκB pathway activation upon CI-994 treatment. Finally, we demonstrated a significantly prolonged survival, a decrease in tumor growth and spinal metastasis in two orthotopic xenograft mouse models of MYC-driven medulloblastoma. In conclusion, our results suggest a MYC-dependent response to class I HDAC inhibition in medulloblastoma and provide compelling rationale for further development of a novel, potentially highly effective therapeutic strategy against the primary site and, importantly, the metastatic compartment.
Abstract
Pilocytic astrocytomas (PAs) and other pediatric low-grade gliomas (pLGGs) exhibit aberrant activation of the MAPK signaling pathway caused by genetic alterations, most commonly ...KIAA1549:BRAF fusions, BRAF V600E and NF1 mutations. In such a single-pathway disease, novel drugs targeting the MAPK pathway (MAPKi) are prime candidates for treatment. We developed an assay suitable for pre-clinical testing of MAPKi in pLGGs, aiming at the identification of novel MAPK pathway suppressing synergistic drug combinations. We generated a reporter plasmid (pDIPZ) expressing destabilized firefly luciferase driven by a MAPK-responsive ELK-1-binding element, packaged in a lentiviral vector system. We stably transfected pediatric glioma cell lines with a BRAF fusion (DKFZ-BT66) and a BRAFV600E mutation (BT-40) background, respectively. Measurement of MAPK pathway activity was performed using the luciferase reporter. pERK protein levels were detected for validation. We performed a screen of a MAPKi library and calculated Combination Indices of selected combinations. The MAPKi library screen revealed MEK inhibitors as the class inhibiting the pathway with the lowest IC50s, followed by ERK and second generation RAF inhibitors. Synergistic effects in both BRAF-fusion and BRAFV600E mutation backgrounds were observed following combination treatments with different MAPKi classes (RAFi/MEKi, > RAFi/ERKi > MEKi/ERKi). We have generated a novel reporter assay for medium- to high-throughput pre-clinical drug testing of MAPKi in pLGG cell lines. MEK, ERK and next-generation RAF inhibitors were confirmed as potential treatment approaches for KIAA1549:BRAF and BRAFV600E mutated pLGGs. Synergistic suppression of MAPK pathway activity upon combination treatments was revealed using our assay in addition.
Histone deacetylases (HDACs) have been identified as promising epigenetic drug targets for the treatment of neuroblastoma and glioblastoma. In this work, we have rationally designed a novel class of ...peptoid-based histone deacetylase inhibitors (HDACi). A mini library of β-peptoid-capped HDACi was synthesized using a four-step protocol. All compounds were screened in biochemical assays for their inhibition of HDAC1 and HDAC6 and docking studies were performed to rationalize the observed selectivity profile. The synthesized compounds were further examined for tumor cell-inhibitory activity against a panel of neuroblastoma and glioblastoma cell lines. In particular, non-selective compounds with potent activity against HDAC1 and HDAC6 showed strong antiproliferative effects. The most promising HDACi, compound
, displayed submicromolar tumor cell-inhibitory potential (IC
: 0.21-0.67 μM) against all five cancer cell lines investigated and exceeded the activity of the FDA-approved HDACi vorinostat.
Abstract
Medulloblastoma is the most common malignant brain tumor in childhood and comprises four distinct molecular subgroups with further layers of intertumoral heterogeneity. Amplification of the ...oncogene MYC drives tumorigenesis and constitutes a hallmark feature underlying Group 3 biology. Metastatic dissemination is frequently observed at diagnosis or recurrence and confers a dismal prognosis. Employing our in-house drug screening pipeline, we evaluated a library of epigenetic inhibitors (n=78) in various brain tumor cell lines including glioblastoma (n=14), medulloblastoma (n=14) and atypical teratoid/rhabdoid tumors (n=11). By this cross-entity approach we revealed preferential activity of histone deacetylase inhibitors (HDACi) in MYC-driven medulloblastoma. In a secondary HDACi (n=20) screen, we identified the clinically established, class I selective HDACi CI-994 as the compound with the most preferential antitumoral effect in MYC-driven medulloblastoma. We confirmed that the inhibitor response was in part MYC-dependent as our lentiviral based MYC-overexpression model showed higher sensitivity towards CI-994 treatment as compared to the isogenic control with low endogenous MYC expression. Moreover, we observed significantly reduced MYC mRNA and protein expression levels, decreased cell viability and induction of apoptosis following CI-994 treatment. Notably, CI-994 showed significant antitumoral effects at the primary site and the metastatic compartment in two orthotopic mouse models of MYC-driven medulloblastoma. Additionally, a screen for synergistic drug interaction with a clinical inhibitor library of approved chemotherapeutics and clinical phase III/IV agents (n=199) revealed a favorable interaction of the NFκB inhibitor bardoxolone methyl with CI-994. In line with our synergy data, RNA sequencing profiling confirmed the functional relevance of NFκB pathway induction upon CI-994 treatment. In all, our pharmacogenomic approach suggests that the combination with NFκB inhibitors has the potential to increase the anitumoral activity of HDACi and the pronounced activity in MYC amplified medulloblastoma, where therapeutic options are limited, is particular noteworthy and warrants further clinical investigation.
PDF
