BET bromodomain protein inhibition is a therapeutic option for medulloblastoma

Medulloblastoma is the most common malignant brain tumor of childhood, and represents a significant clinical challenge in pediatric oncology, since overall survival currently remains under 70%. Patients with tumors overexpressing MYC or harboring a MYC oncogene amplification have an extremely poor prognosis. Pharmacologically inhibiting MYC expression may, thus, have clinical utility given its pathogenetic role in medulloblastoma. Recent studies using the selective small molecule BET inhibitor, JQ1, have identified BET bromodomain proteins, especially BRD4, as epigenetic regulatory factors for MYC and its targets. Targeting MYC expression by BET inhibition resulted in antitumoral effects in various cancers. Our aim here was to evaluate the efficacy of JQ1 against preclinical models for high-risk MYC-driven medulloblastoma. Treatment of medulloblastoma cell lines with JQ1 significantly reduced cell proliferation and preferentially induced apoptosis in cells expressing high levels of MYC. JQ1 treatment of medulloblastoma cell lines downregulated MYC expression and resulted in a transcriptional deregulation of MYC targets, and also significantly altered expression of genes involved in cell cycle progression and p53 signalling. JQ1 treatment prolonged the survival of mice harboring medulloblastoma xenografts and reduced the tumor burden in these mice. Our preclinical data provide evidence to pursue testing BET inhibitors, such as JQ1, as molecular targeted therapeutic options for patients with high-risk medulloblastomas overexpressing MYC or harboring MYC amplifications

Targeting tachykinin receptors in neuroblastoma

Neuroblastoma is the most common extracranial tumor in children. Despite aggressive multimodal treatment, high-risk neuroblastoma remains a clinical challenge with survival rates below 50%. Adding targeted drugs to first-line therapy regimens is a promising approach to improve survival in these patients. TACR1 activation by substance P has been reported to be mitogenic in cancer cell lines. Tachykinin receptor (TACR1) antagonists are approved for clinical use as an antiemetic remedy since 2003. Tachykinin receptor inhibition has recently been shown to effectively reduce growth of several tumor types. Here, we report that neuroblastoma cell lines express TACR1, and that targeting TACR1 activity significantly reduced cell viability and induced apoptosis in neuroblastoma cell lines. Gene expression profiling revealed that TACR1 inhibition repressed E2F2 and induced TP53 signaling. Treating mice harboring established neuroblastoma xenograft tumors with Aprepitant also significantly reduced tumor burden. Thus, we provide evidence that the targeted inhibition of tachykinin receptor signaling shows therapeutic efficacy in preclinical models for high-risk neuroblastoma

Ca2+^{2+} functions as a molecular switch that controls the mutually exclusive complex formation of pyridoxal phosphatase with CIB1 or calmodulin

Pyridoxal 5′‐phosphate (PLP) is an essential cofactor for neurotransmitter metabolism. Pyridoxal phosphatase (PDXP) deficiency in mice increases PLP and γ‐aminobutyric acid levels in the brain, yet how PDXP is regulated is unclear. Here, we identify the Ca$^{2+}$‐ and integrin‐binding protein 1 (CIB1) as a PDXP interactor by yeast two‐hybrid screening and find a calmodulin (CaM)‐binding motif that overlaps with the PDXP‐CIB1 interaction site. Pulldown and crosslinking assays with purified proteins demonstrate that PDXP directly binds to CIB1 or CaM. CIB1 or CaM does not alter PDXP phosphatase activity. However, elevated Ca$^{2+}$ concentrations promote CaM binding and, thereby, diminish CIB1 binding to PDXP, as both interactors bind in a mutually exclusive way. Hence, the PDXP‐CIB1 complex may functionally differ from the PDXP‐Ca$^{2+}$‐CaM complex

Absence of telomerase reverse transcriptase promoter mutations in neuroblastoma

Maintenance of telomere length is a critical hallmark of malignant transformation. While silenced in somatic cells, telomerase reverse transcriptase (TERT), the catalytic subunit of telomerase, is frequently overexpressed in malignant cells thereby maintaining their telomere length. Specific point mutations in the TERT promoter region have recently been identified in melanoma and other tumor entities resulting in high TERT expression. Neuroblastoma is the most common extracranial tumor of childhood, arising from neural-crest progenitor cells. TERT overexpression has been observed in the majority of neuroblastoma. Taking into consideration that TERT promoter mutations are frequently described in neural-crest-derived tumors such as melanoma, as well as a variety of other neuronal tumors, the present study analyzed the frequency of TERT promoter mutations in primary neuroblastoma and neuroblastoma cell lines. In 131 neuroblastoma primary tumors representing the whole spectrum of neuroblastoma, no TERT promoter mutations were detected. However, in 3 out of 19 neuroblastoma cell lines the previously described C228T TERT promoter mutation was present. In conclusion, the TERT promoter mutations are not a frequent mechanism of TERT overexpression in neuroblastoma

RITA displays anti-tumor activity in medulloblastomas independent of TP53 status

Current therapy of medulloblastoma, the most common malignant brain tumor of childhood, achieves 40-70% survival. Secondary chemotherapy resistance contributes to treatment failure, where TP53 pathway dysfunction plays a key role. MDM2 interaction with TP53 leads to its degradation. Reactivating TP53 functionality using small-molecule inhibitors, such as RITA, to disrupt TP53-MDM2 binding may have therapeutic potential. We show here that RITA decreased viability of all 4 analyzed medulloblastoma cell lines, regardless of TP53 functional status. The decrease in cell viability was accompanied in 3 of the 4 medulloblastoma cell lines by accumulation of TP53 protein in the cells and increased CDKN1A expression. RITA treatment in mouse models inhibited medulloblastoma xenograft tumor growth. These data demonstrate that RITA treatment reduces medulloblastoma cell viability in both in vitro and in vivo models, and acts independently of cellular TP53 status, identifying RITA as a potential therapeutic agent to treat medulloblastoma

Superiority of focused ion beam‐scanning electron microscope tomography of cardiomyocytes over standard 2D analyses highlighted by unmasking mitochondrial heterogeneity

BACKGROUND: Cardioprotection by preventing or repairing mitochondrial damage is an unmet therapeutic need. To understand the role of cardiomyocyte mitochondria in physiopathology, the reliable characterization of the mitochondrial morphology and compartment is pivotal. Previous studies mostly relied on two‐dimensional (2D) routine transmission electron microscopy (TEM), thereby neglecting the real three‐dimensional (3D) mitochondrial organization. This study aimed to determine whether classical 2D TEM analysis of the cardiomyocyte ultrastructure is sufficient to comprehensively describe the mitochondrial compartment and to reflect mitochondrial number, size, dispersion, distribution, and morphology. METHODS: Spatial distribution of the complex mitochondrial network and morphology, number, and size heterogeneity of cardiac mitochondria in isolated adult mouse cardiomyocytes and adult wild‐type left ventricular tissues (C57BL/6) were assessed using a comparative 3D imaging system based on focused ion beam‐scanning electron microscopy (FIB‐SEM) nanotomography. For comparison of 2D vs. 3D data sets, analytical strategies and mathematical comparative approaches were performed. To confirm the value of 3D data for mitochondrial changes, we compared the obtained values for number, coverage area, size heterogeneity, and complexity of wild‐type cardiomyocyte mitochondria with data sets from mice lacking the cytosolic and mitochondrial protein BNIP3 (BCL‐2/adenovirus E1B 19‐kDa interacting protein 3; Bnip3 ( −/− )) using FIB‐SEM. Mitochondrial respiration was assessed on isolated mitochondria using the Seahorse XF analyser. A cardiac biopsy was obtained from a male patient (48 years) suffering from myocarditis. RESULTS: The FIB‐SEM nanotomographic analysis revealed that no linear relationship exists for mitochondrial number (r = 0.02; P = 0.9511), dispersion (r = −0.03; P = 0.9188), and shape (roundness: r = 0.15, P = 0.6397; elongation: r = −0.09, P = 0.7804) between 3D and 2D results. Cumulative frequency distribution analysis showed a diverse abundance of mitochondria with different sizes in 3D and 2D. Qualitatively, 2D data could not reflect mitochondrial distribution and dynamics existing in 3D tissue. 3D analyses enabled the discovery that BNIP3 deletion resulted in more smaller, less complex cardiomyocyte mitochondria (number: P < 0.01; heterogeneity: C.V. wild‐type 89% vs. Bnip3 ( −/− ) 68%; complexity: P < 0.001) forming large myofibril‐distorting clusters, as seen in human myocarditis with disturbed mitochondrial dynamics. Bnip3 (−/−) mice also show a higher respiration rate (P < 0.01). CONCLUSIONS: Here, we demonstrate the need of 3D analyses for the characterization of mitochondrial features in cardiac tissue samples. Hence, we observed that BNIP3 deletion physiologically acts as a molecular brake on mitochondrial number, suggesting a role in mitochondrial fusion/fission processes and thereby regulating the homeostasis of cardiac bioenergetics

The GSK461364 PLK1 inhibitor exhibits strong antitumoral activity in preclinical neuroblastoma models

Polo-like kinase 1 (PLK1) is a serine/threonine kinase that promotes G2/M-phase transition, is expressed in elevated levels in high-risk neuroblastomas and correlates with unfavorable patient outcome. Recently, we and others have presented PLK1 as a potential drug target for neuroblastoma, and reported that the BI2536 PLK1 inhibitor showed antitumoral actvity in preclinical neuroblastoma models. Here we analyzed the effects of GSK461364, a competitive inhibitor for ATP binding to PLK1, on typical tumorigenic properties of preclinical in vitro and in vivo neuroblastoma models. GSK461364 treatment of neuroblastoma cell lines reduced cell viability and proliferative capacity, caused cell cycle arrest and massively induced apoptosis. These phenotypic consequences were induced by treatment in the low-dose nanomolar range, and were independent of MYCN copy number status. GSK461364 treatment strongly delayed established xenograft tumor growth in nude mice, and significantly increased survival time in the treatment group. These preclinical findings indicate PLK1 inhibitors may be effective for patients with high-risk or relapsed neuroblastomas with upregulated PLK1 and might be considered for entry into early phase clinical trials in pediatric patients

Targeting MYCN-Driven Transcription By BET-Bromodomain Inhibition

Purpose: Targeting BET proteins was previously shown to have specific antitumoral efficacy against MYCN-amplified neuroblastoma. We here assess the therapeutic efficacy of the BET inhibitor, OTX015, in preclinical neuroblastoma models and extend the knowledge on the role of BRD4 in MYCN-driven neuroblastoma. Experimental Design: The efficacy of OTX015 was assessed in in vitro and in vivo models of human and murine MYCN-driven neuroblastoma. To study the effects of BET inhibition in the context of high MYCN levels, MYCN was ectopically expressed in human and murine cells. The effect of OTX015 on BRD4-regulated transcriptional pause release was analyzed using BRD4 and H3K27Ac chromatin immunoprecipitation coupled with DNA sequencing (ChIP-Seq) and gene expression analysis in neuroblastoma cells treated with OTX015 compared with vehicle control. Results: OTX015 showed therapeutic efficacy against preclinical MYCN-driven neuroblastoma models. Similar to previously described BET inhibitors, concurrent MYCN repression was observed in OTX015-treated samples. Ectopic MYCN expression, however, did not abrogate effects of OTX015, indicating that MYCN repression is not the only target of BET proteins in neuroblastoma. When MYCN was ectopically expressed, BET inhibition still disrupted MYCN target gene transcription without affecting MYCN expression. We found that BRD4 binds to super-enhancers and MYCN target genes, and that OTX015 specifically disrupts BRD4 binding and transcription of these genes. Conclusions: We show that OTX015 is effective against mouse and human MYCN-driven tumor models and that BRD4 not only targets MYCN, but specifically occupies MYCN target gene enhancers as well as other genes associated with super-enhancers. (C) 2015 AACR

Targeted expression of mutated ALK induces neuroblastoma in transgenic mice

Activating anaplastic lymphoma kinase (ALK) mutations were recently detected in most familial and 10% of sporadic neuroblastomas. However, the role of mutated ALK in tumorigenesis remains elusive. We demonstrate that targeted expression of the most frequent and aggressive variant, ALK(F1174L), is tumorigenic in mice. Tumors resembled human neuroblastomas in morphology, metastasis pattern, gene expression, and the presence of neurosecretory vesicles as well as synaptic structures. This ALK-driven neuroblastoma mouse model precisely recapitulated the genetic spectrum of the disease. Chromosomal aberrations were syntenic to those in human neuroblastoma, including 17q gain and MYCN oncogene amplification. Targeted ALK(F1174L) and MYCN coexpression revealed a strong synergism in inducing neuroblastoma with minimal chromosomal aberrations, suggesting that fewer secondary hits are required for tumor induction if both oncoproteins are targeted. Treatment of ALK(F1174L) transgenic mice with the ALK inhibitor TAE-684 induced complete tumor regression, indicating that tumor cells were addicted to ALK(F1174L) activity. We conclude that an activating mutation within the ALK kinase domain is sufficient to induce neuroblastoma development, and ALK inhibitors show promise for treating human neuroblastomas harboring ALK mutations