Reflection of neuroblastoma intratumor heterogeneity in the new OHC-NB1 disease model

Accurate modeling of intratumor heterogeneity presents a bottleneck against drug testing. Flexibility in a preclinical platform is also desirable to support assessment of different endpoints. We established the model system, OHC-NB1, from a bone marrow metastasis from a patient diagnosed with MYCN-amplified neuroblastoma and performed whole-exome sequencing on the source metastasis and the different models and passages during model development (monolayer cell line, 3D spheroid culture and subcutaneous xenograft tumors propagated in mice). OHC-NB1 harbors a MYCN amplification in double minutes, 1p deletion, 17q gain and diploid karyotype, which persisted in all models. A total of 80–540 single-nucleotide variants (SNVs) was detected in each sample, and comparisons between the source metastasis and models identified 34 of 80 somatic SNVs to be propagated in the models. Clonal reconstruction using the combined copy number and SNV data revealed marked clonal heterogeneity in the originating metastasis, with four clones being reflected in the model systems. The set of OHC-NB1 models represents 43% of somatic SNVs and 23% of the cellularity in the originating metastasis with varying clonal compositions, indicating that heterogeneity is partially preserved in our model system.     

儿童神经母细胞瘤染色体分析

目的总结55例神经母细胞瘤（NB）患儿的染色体结果,分析与临床特点及近期治疗效果的关系,提高对伴有染色体异常NB的认识。方法回顾性分析55例NB患儿的临床资料,包括分期分组、肿瘤标记物、染色体结果、治疗方案及近期预后情况。结果55例NB患儿中有4例存在17q获得（7％）,1例患儿同时存在17q获得及1 p缺失（2％）,余50例（91％）患儿染色体检查均正常。伴有染色体异常的5例患儿肿瘤标记物在病初均有不同程度的增高,而且血神经元特异性烯醇化酶（NSE）高于染色体正常组;5例染色体异常患儿均为Ⅳ期、高危组,均伴有MYCN基因获得,其中1例在治疗过程中失访,余4例中有2例肿瘤进展,1例死亡,1例经化疗联合手术切除及自体造血干细胞移植,门诊随访33个月疾病稳定。结论结果提示染色体1 p缺失和17q获得可能是NB的预后不良因素,染色体异常在NB的诊断及预后评估中具有一定临床指导意义。但本研究中的异常染色体检出率偏低,考虑与常规检测的误差有关,方法学有待进一步改进。     

MiR‐34a deficiency accelerates medulloblastoma formation in vivo

Previous studies have evaluated the role of miRNAs in cancer initiation and progression. MiR‐34a was found to be downregulated in several tumors, including medulloblastomas. Here we employed targeted transgenesis to analyze the function of miR‐34a in vivo. We generated mice with a constitutive deletion of the miR‐34a gene. These mice were devoid of mir‐34a expression in all analyzed tissues, but were viable and fertile. A comprehensive standardized phenotypic analysis including more than 300 single parameters revealed no apparent phenotype. Analysis of miR‐34a expression in human medulloblastomas and medulloblastoma cell lines revealed significantly lower levels than in normal human cerebellum. Re‐expression of miR‐34a in human medulloblastoma cells reduced cell viability and proliferation, induced apoptosis and downregulated the miR‐34a target genes, MYCN and SIRT1. Activation of the Shh pathway by targeting SmoA1 transgene overexpression causes medulloblastoma in mice, which is dependent on the presence and upregulation of Mycn. Analysis of miR‐34a in medulloblastomas derived from ND2:SmoA1(tg) mice revealed significant suppression of miR‐34a compared to normal cerebellum. Tumor incidence was significantly increased and tumor formation was significantly accelerated in mice transgenic for SmoA1 and lacking miR‐34a. Interestingly, Mycn and Sirt1 were strongly expressed in medulloblastomas derived from these mice. We here demonstrate that miR‐34a is dispensable for normal development, but that its loss accelerates medulloblastomagenesis. Strategies aiming to re‐express miR‐34a in tumors could, therefore, represent an efficient therapeutic option.     

From fish to FISH: the comparative pathology of neuroblastomas in humans, mice and fish

Neuroblastoma is a malignant cancer of the sympathetic nervous system and is one of the most frequent solid cancers in young children. Only a few of the many advances in our understanding of basic genetic and cellular mechanisms leading to neuroblastoma development have translated to clinical practice, and the prognosis for children with neuroblastoma, particularly at advanced stages, has remained poor. Major directions for neuroblastoma management and control include the application of prognostic parameters, particularly amplified MYCN, which can be readily visualized by chromosomal fluorescence in situ hybridization (FISH), for individual therapy design, as well as the initiation of a presymptomatic screening program for early tumor detection to reduce the fraction of advanced-stage tumors. In addition, new and innovative therapeutic approaches are being sought. The understanding of molecular and cellular pathways resulting in spontaneous regression in up to 10% of neuroblastoma patients, possibly by apoptosis, could provide the basis for new biologically based therapeutic interventions. Unlike most other pediatric cancers, neuroblastoma can be studied in two experimental animal systems. One is the fish system Xiphophorus, where neuroblastomas can be induced in specific strains by exposure to mutagens/carcinogens; the second is mice that carry MYCN as a transgene. These animal systems demonstrate that neuroblastomas are evolutionarily conserved tumors. Their study could well result in a better understanding of neuroblastoma development. At the same time they represent systems in which experimental therapies can be preclinically tested. Received: 31 October 1998 / Accepted: 12 November 1998     

Catastrophic ATP loss underlies a metabolic combination therapy tailored for MYCN-amplified neuroblastoma

MYCN-amplified neuroblastoma is a lethal subset of pediatric cancer. MYCN drives numerous effects in the cell, including metabolic changes that are critical for oncogenesis. The understanding that both compensatory pathways and intrinsic redundancy in cell systems exists implies that the use of combination therapies for effective and durable responses is necessary. Additionally, the most effective targeted therapies exploit an “Achilles’ heel” and are tailored to the genetics of the cancer under study. We performed an unbiased screen on select metabolic targeted therapy combinations and correlated sensitivity with over 20 subsets of cancer. We found that MYCN-amplified neuroblastoma is hypersensitive to the combination of an inhibitor of the lactate transporter MCT1, AZD3965, and complex I of the mitochondrion, phenformin. Our data demonstrate that MCT4 is highly correlated with resistance to the combination in the screen and lowly expressed in MYCN-amplified neuroblastoma. Low MCT4 combines with high expression of the MCT2 and MCT1 chaperone CD147 in MYCN-amplified neuroblastoma, altogether conferring sensitivity to the AZD3965 and phenformin combination. The result is simultaneous disruption of glycolysis and oxidative phosphorylation, resulting in dramatic disruption of adenosine triphosphate (ATP) production, endoplasmic reticulum stress, and cell death. In mouse models of MYCN-amplified neuroblastoma, the combination was tolerable at concentrations where it shrank tumors and did not increase white-blood-cell toxicity compared to single drugs. Therefore, we demonstrate that a metabolic combination screen can identify vulnerabilities in subsets of cancer and put forth a metabolic combination therapy tailored for MYCN-amplified neuroblastoma that demonstrates efficacy and tolerability in vivo.

Despite their relative rarity compared to blood cancers, solid-tumor pediatric cancers are now the leading cause of pediatric cancer-related deaths. Among the most deadly is high-risk neuroblastoma (NB): amplification of MYCN confers high risk and is the clear driver of NB in these cancers (1). As such, MYCN remains the most important drug target in NB and one of the most important in pediatric cancer. Unfortunately, direct chemical targeting of MYCN has not yet been successful, and despite advancements in anti-GD2 immunotherapy (2), alternate ways of targeting MYCN-amplified NB may be needed to successfully treat this cancer.One approach is to find tumor-specific vulnerabilities, which are exploitable pharmacologically. Many efforts, including ours (3), have exhaustively looked for kinase inhibitors with particular efficacy in MYCN-amplified NBs. However, the emerging picture is a lack of kinase inhibitor efficacy in MYCN-amplified NB. Other vulnerabilities may be classified under the broad category of drugs targeting epigenetic modifiers. For example, using a CRISPR/Cas9 screen, Stegmaier and colleagues demonstrated that MYCN-amplified NB may be susceptible to targeting the H3K27me methylase EZH2 (4); in a different study, they demonstrated the susceptibility of MYCN-amplified NB to the combination of BRD4 inhibitors with CDK7 inhibitors (5). In addition, Thiele and colleagues (6) demonstrated high-risk NBs were susceptible to inhibition of the lysine methyltransferase SETD8. As promising as these data are, it remains unknown whether tolerability and/or clinical activity in MYCN-amplified NB will occur and SETD8, BRD4, and CDK7 inhibitors so far are not in the pediatric clinic. Cell death inducers constitute a third category. To this point, we recently uncovered a susceptibility of MYCN-amplified NB to the BCL-2 inhibitor venetoclax (3), confirmed by others (7). There, MYCN-driven NOXA expression sensitizes cells to venetoclax (3). Venetoclax is now in early phase trials in pediatric patients including those with NB ({"type":"clinical-trial","attrs":{"text":"NCT03236857","term_id":"NCT03236857"}}NCT03236857). It remains to be seen whether or not it will elicit responses in NB patients as a single agent.A fourth distinct category of therapeutic strategies to indirectly target oncogenes is through metabolism targeting, involving the growing coterie of drugs targeting the pathways fulfilling the high-energy demands of cancer cells. A major energy currency in cells is adenosine triphosphate (ATP). The Warburg effect describes the propensity of cancer cells (and highly proliferating normal cells) to produce ATP in the presence of oxygen with the less efficient, extramitochondrial glycolysis, as opposed to the more efficient mitochondria-based oxidative phosphorylation occurring in most noncancerous cells (8). The mechanistic explanation of the Warburg effect and how it might benefit cancer cells has been revised dramatically over the years. It was originally proposed that mitochondria from cancer cells were defective and lacked oxidative phosphorylation capabilities (9); on the contrary, emerging data show that many cancers rely on oxidative phosphorylation to facilitate the generation of ATP (8, 10). Interestingly, while amplified MYCN directly regulates the expression of many of the key glycolytic enzymes and as such contributes to the Warburg effect (11, 12), a study utilizing a Seahorse respirator demonstrated that a MYCN-amplified NB cell line favored oxidative phosphorylation over glycolysis for the metabolic needs, while the reverse was true for a MYCN wild-type NB cell line (13). In an independent study, MYCN was associated with higher glycolytic flux and oxidative phosphorylation and conferred sensitivity to fatty acid oxidation disruption (12). Overall, since c-MYC, which shares ∼40% binding homology to DNA-binding sites throughout the genome with MYCN, has been extensively characterized as a metabolic master regulator (14, 15), it is likely there are other MYCN-driven metabolic processes that may represent significant drug targets.Monocarboxylate transporters (MCTs) consist of four members (MCT1–4) in mammalian cells. Among their most critical substrates are lactate and pyruvate; MCT1 and MCT4 are responsible for lactate export across the plasma membrane to the extracellular space (16). AZD3965 (17) (AstraZeneca) is the first in-class–specific MCT1/2 dual inhibitor and is currently in early phase trials for diverse cancers; however, other inhibitors from different companies have recently been developed as well (18). Of note, AZD3965 has demonstrated good tolerability in diverse patients (clinical trial number {"type":"clinical-trial","attrs":{"text":"NCT01791595","term_id":"NCT01791595"}}NCT01791595). Although rare (65 cases/100,000 person-years), lactic acidosis led to the market retrieval of phenformin in America (19), yet phenformin remains in use as a type II antidiabetic drug in Europe, functioning centrally as a mitochondrial complex I electron transport chain (ETC) inhibitor. Phenformin reduces both glycolytic intermediates and pyruvate, increases shunting of glucose-derived carbon (increasing total lactate production), and markedly reduces tricarboxylic acid cycle intermediates (20). Indeed, there has been a recent resurgence in interest in the use of phenformin to treat cancer. For example, in BRAF mutant melanoma, phenformin sensitized cells to BRAF inhibitor through cooperative suppression of the metabolic sensor pathway mTORC1 (21). These preclinical data have led to a clinical trial of phenformin in combination with BRAF inhibitor in BRAF mutant melanoma ({"type":"clinical-trial","attrs":{"text":"NCT03026517","term_id":"NCT03026517"}}NCT03026517). Overall, while targeting individual metabolic pathways has demonstrated some preclinical success in different cancer models, it is limited with significant redundancy in pathways to generate ATP and regenerate NAD+ (22). We therefore assessed potential combination therapies involving metabolic targeting drugs to identify a strategy for MYCN-amplified NB.     

Effects of small molecule inhibitors of PI3K/Akt/mTOR signaling on neuroblastoma growth in vitro and in vivo

Activation of the PI3K/Akt signaling pathway is correlated with poor prognosis in neuroblastoma, the most common and deadly extracranial tumor of childhood. In this study, we show that the small-molecule inhibitors of phosphoinositide-dependent protein kinase-1 (PDK1) OSU03012 and the dual class I PI3K/mTOR inhibitor PI103 have profound effects on neuroblastoma survival in vitro and in vivo. Both OSU03012 and PI103 inhibited neuroblastoma growth in vitro. In treated cells, OSU03012 induced apoptosis and an S phase cell cycle arrest, whereas only minor apoptosis was detected in PI103 treated cells together with a G1 arrest. Both OSU03012 and PI103 downregulated phosphorylation of Akt and inhibited the downstream targets glycogen synthase kinase-3β (GSK3β) and p70 S6 kinase-1 (S6K1), as well as downregulated the expression of cyclin D1 and Mycn protein. Neuroblastoma cells expressing high levels of Mycn were more sensitive to OSU03012 or PI103 compared with cells expressing low Mycn levels. Both compounds significantly inhibited the growth of established, subcutaneous MYCN-amplified neuroblastoma xenografts in nude NMRI nu/nu mice. These results suggest that inhibition of the PI3K/Akt signaling pathway represent a clinical relevant target for the treatment of patients with high-risk MYCN-amplified neuroblastoma.