Andrea Odersky

Mutational dynamics between primary and relapse neuroblastomas

Neuroblastoma is a malignancy of the developing sympathetic nervous system that is often lethal when relapse occurs. We here used whole-exome sequencing, mRNA expression profiling, array CGH and DNA methylation analysis to characterize 16 paired samples at diagnosis and relapse from individuals with neuroblastoma. The mutational burden significantly increased in relapsing tumors, accompanied by altered mutational signatures and reduced subclonal heterogeneity. Global allele frequencies at relapse indicated clonal mutation selection during disease progression. Promoter methylation patterns were consistent over disease course and were patient specific. Recurrent alterations at relapse included mutations in the putative CHD5 neuroblastoma tumor suppressor, chromosome 9p losses, DOCK8 mutations, inactivating mutations in PTPN14 and a relapse-specific activity pattern for the PTPN14 target YAP. Recurrent new mutations in HRAS, KRAS and genes mediating cell-cell interaction in 13 of 16 relapse tumors indicate disturbances in signaling pathways mediating mesenchymal transition. Our data shed light on genetic alteration frequency, identity and evolution in neuroblastoma.

Targeted Expression of Mutated ALK Induces Neuroblastoma in Transgenic Mice

ALK inhibitors induce complete tumor regression in a mouse model of ALK-driven neuroblastoma. Driving Neuroblastoma: A wALK in the Park Correlation doesn’t prove causation. For example, even though you may always see your neighbors walking their dog right before you find that odiferous pile of unscooped pooh, unless you directly witness a walkaway or use DNA testing to trace the culprit, you can’t prove that they did it. Demonstrating causation is even more important in cancer biology—just finding a prevalent mutation in people with a particular type of cancer isn’t enough to show that mutation is actually relevant to disease. Heukamp et al. now address the potential causative role of anaplastic lymphoma kinase (ALK) mutations in neuroblastoma. ALK mutations are found in most familial and some sporadic cases of neuroblastoma, a malignant tumor that affects children. To determine whether ALK mutations can drive the development of neuroblastoma, the authors introduced the most common ALK mutation into neural crest stem cells in mice. Tumors driven by this mutation resembled human neuroblastomas physiologically and mimicked the genetic structure of the disease. Mutated ALK and MYCN, another driver mutation for neuroblastoma, combined synergistically for tumor development. Heukamp et al. then used their new model to demonstrate that an ALK inhibitor currently in preclinical testing induced complete tumor regression in these mice; however, it remains to be seen whether these inhibitors will be useful in treating neuroblastoma in people. 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, ALKF1174L, 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 ALKF1174L 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 ALKF1174L transgenic mice with the ALK inhibitor TAE-684 induced complete tumor regression, indicating that tumor cells were addicted to ALKF1174L 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.

MiR‐137 functions as a tumor suppressor in neuroblastoma by downregulating KDM1A

Neuroblastoma is the most common extracranial solid tumor of childhood, and accounts for ∼15% of all childhood cancer deaths. The histone demethylase, lysine‐specific demethylase 1 (KDM1A, previously known as LSD1), is strongly expressed in neuroblastomas, and overexpression correlates with poor patient prognosis. Inducing differentiation in neuroblastoma cells has previously been shown to down regulate KDM1A, and siRNA‐mediated KDM1A knockdown inhibited neuroblastoma cell viability. The microRNA, miR‐137, has been reported to be downregulated in several human cancers, and KDM1A mRNA was reported as a putative target of miR‐137 in colon cancer. We hypothesized that miR‐137 might have a tumor‐suppressive role in neuroblastoma mediated via downregulation of KDM1A. Indeed, low levels of miR‐137 expression in primary neuroblastomas correlated with poor patient prognosis. Re‐expressing miR‐137 in neuroblastoma cell lines increased apoptosis and decreased cell viability and proliferation. KDM1A mRNA was repressed by miR‐137 in neuroblastoma cells, and was validated as a direct target of miR‐137 using reporter assays in SHEP and HEK293 cells. Furthermore, siRNA‐mediated KDM1A knockdown phenocopied the miR‐137 re‐expression phenotype in neuroblastoma cells. We conclude that miR‐137 directly targets KDM1A mRNA in neuroblastoma cells, and activates cell properties consistent with tumor suppression. Therapeutic strategies to re‐express miR‐137 in neuroblastomas could be useful to reduce tumor aggressiveness.

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. Clin Cancer Res; 22(10); 2470–81. ©2015 AACR.

Analysis of double-strand break repair by nonhomologous DNA end joining in cell-free extracts from mammalian cells.

Double-strand breaks (DSB) in genomic DNA are induced by ionizing radiation or radiomimetic drugs but also occur spontaneously during the cell cycle at quite significant frequencies. In vertebrate cells, nonhomologous DNA end joining (NHEJ) is considered the major pathway of DSB repair which is able to rejoin two broken DNA termini directly end-to-end irrespective of sequence and structure. Genetic studies in various radiosensitive and DSB repair-deficient cell lines yielded insight into the factors involved in NHEJ. Studies in cell-free systems derived from Xenopus eggs and mammalian cells allowed the dissection of the underlying mechanisms. In the present chapter, we describe a protocol for the preparation of whole cell extracts from mammalian cells and a plasmid-based in vitro assay which permits the easy analysis of the efficiency and fidelity of DSB repair via NHEJ in different cell types.

Abstract 3967: BET protein inhibitor OTX015 has selective anti-tumoral activity in preclinical models of MYCN- amplified neuroblastoma

Neuroblastomas harboring MYCN amplifications are highly lethal tumors. They are often resistant to standard chemotherapy, yet the development of targeted therapies has been hampered by a lack of compounds targeting MYCN. We and others have recently discovered that targeting BET bromodomain proteins, especially BRD4, disrupts epigenetic regulation of MYCN and its targets in neuroblastoma. OTX015, a new BET protein inhibitor, is the first lead into clinical phase I/II trials and has shown promising pharmacological properties in adults. Here, we investigate the preclinical efficacy of OTX015 in MYCN-amplified neuroblastoma. We tested in vitro OTX015 efficacy in 6 established neuroblastoma (NB) cell lines. We performed cell cycle profiling and analyzed markers for apoptosis and proliferation after 72h-treatment at 500 nM OTX015. The effect of OTX015 on MYCN expression and global MYCN-associated transcriptional activity was assessed by quantitative real time PCR and gene expression microarray profiling, respectively. In vivo efficacy of orally OTX015 was assessed in IMR5 xenografts, a N-MYC driven NB model, using diffent treatment schedules (50mg/kg/day, 100mg/kg/day and 50mg/kg/bidaily). Treatment of MYCN-amplified neuroblastoma cells with OTX015 resulted in decreased cell viability, induction of apoptosis and reduced proliferation. Concentrations of 50% inhibition (IC50) ranged between 50nM and 500nM. OTX015 treatment also resulted in an increase in the percentage of cells in G1 phase. This corresponded with the downregulation of MYCN mRNA and protein levels and MYCN-associated transcriptional activity. Interestigly, MYCN amplified cell lines were most sensitive to OTX015 treatment. In contrast, no effect was observed with OTX15 on normal cells. In vivo treatment with OTX015, significantly decreased tumor burden after 4 weeks and prolonged survival as compared to vehicle-treated mice. These preclinical findings highlight the promise of BET bromodomain inhibitors as novel agents for MYCN-driven neuroblastomas and serve as rationale to move forward with early phase clinical trials for children with these highly lethal tumors. Citation Format: Johannes H. Schulte, Kristina Althoff, Emma Bell, Andrea Odersky, Anneleen Beckers, Frank Speleman, Simon Schafers, Alexander Schramm, Angelika Eggert, Frank Westermann, Eugenia Riveiro, Esteban Cvitkovic, Anton Henssen. BET protein inhibitor OTX015 has selective anti-tumoral activity in preclinical models of MYCN- amplified neuroblastoma. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 3967. doi:10.1158/1538-7445.AM2014-3967

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 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.

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.

Abstract 4731: Targeting super-enhancer induced gene expression with the novel BRD4 inhibitor OTX015 in preclinical models of MYCN-amplified neuroblastoma

Bromodomain-containing protein 4 (BRD4) functions as an epigenetic reader and binds to promoter super-enhancer regions driving oncogenes such as MYC. Neuroblastomas (NB) harboring MYCN amplifications are highly lethal tumors often resistant to standard chemotherapy. OTX015 is a novel BRD2/3/4 inhibitor currently in clinical Phase Ib studies in hematologic malignancies and solid tumors. We have previously reported that OTX015 displayed in vitro and in vivo antitumor effects, together with MYCN transcription attenuation in NB models (Henssen et al; AACR 2014). Here, we investigated OTX015 targeting of super-enhancer regulated genes in MYCN-amplified NB in vitro and in vivo models. Protein-DNA interactions were analyzed using ChipSeq in IMR 5 cells. We identified super-enhancers associated with a variety of genes of known importance in NB, including MYCN, as well as some previously undescribed genes. OTX015 inhibited cell proliferation in Chp-212, Chp-134, Gimen, IMR-32, NB69, SK-N-AS, SK-N-BE, and SK-N-BE2 NB cell lines after 72 h exposure. OTX015 reduced tumor burden in IMR 5 xenograft mice and in a genetically engineered model of MYCN-amplified NB LSL MYCN;Dbh-iCre, when administered by oral gavage at a dose of 25 mg/kg daily for 3 weeks. Antitumoral effects of OTX015 were coupled with decreased binding of BRD4 to chromatin and subsequent global transcriptional changes. Moreover, OTX015 exposure led to significant transcriptional downregulation of genes associated with super-enhancers, supporting the notion that BRD4 preferentially acts at these chromatin sites. Interestingly, BRD inhibition not only attenuated MYCN transcription but most significantly affected MYCN-regulated transcriptional programs. Ectopic expression of MYCN was not able to abrogate the antitumoral effects of BRD4 inhibition, indicating direct involvement of MYCN in super-enhancer regulated gene expression and possibly explaining the increased susceptibility of MYCN-amplified NB to OTX015 inhibition. We describe here for the first time that BRD inhibition by OTX015 selectively and preferentially targets global super-enhancer induced transcription in MYCN-driven NB. These new insights will serve as a rationale for a clinical trial in pediatric MYCN-amplified NB patients with OTX015. Citation Format: Anton Henssen, Kristina Althoff, Richard Koche, Andrea Odersky, Anneleen Beckers, Frank Speleman, Simon Schafers, Katleen De Preter, Alexandra Florin, Lukas Heukamp, Annika Spruessel, Kathy Astrahanseff, Natalie Sadowski, Alexander Schramm, Angelika Eggert, Lucile Astorgues-Xerri, Eugenia Riveiro, Esteban Cvitkovic, Johannes H. Schulte. Targeting super-enhancer induced gene expression with the novel BRD4 inhibitor OTX015 in preclinical models of MYCN-amplified neuroblastoma. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 4731. doi:10.1158/1538-7445.AM2015-4731