Background Inhibitors of poly-ADP ribose polymerase (PARP), an enzyme involved in base excision repair (BER) have demonstrated single agent activity against tumors deficient in homologous repair processes. Ewing sarcoma cells are also sensitive to PARP inhibitors, although the mechanism is not understood. Here we evaluated the stereo-selective PARP inhibitor, talazoparib (BMN 673), combined with temozolomide or topotecan. Procedures Talazoparib was tested in vitro in combination with temozolomide (0.3–1,000 μmol/L) or topotecan (0.03-100 nmol/L) and in vivo at a dose of 0.1 mg/kg administered twice daily for 5 days combined with temozolomide (30 mg/kg/daily x 5; combination A) or 0.25 mg/kg administered twice daily for 5 days combined with temozolomide (12 mg/kg/daily x 5; combination B). Results In vitro talazoparib potentiated the toxicity of temozolomide up to 85-fold, with marked potentiation in Ewing sarcoma and leukemia lines (30–50-fold). There was less potentiation for topotecan. In vivo, talazoparib potentiated the toxicity of temozolomide, and Combination A and Combination B represent the maximum tolerated doses when combined with low dose or high dose talazoparib, respectively. Both combinations demonstrated significant synergism against 5 of 10 Ewing sarcoma xenografts. The combination demonstrated modest activity against most other xenograft models. Pharmacodynamic studies showed a treatment-induced complete loss of PARP only in tumor models sensitive to either talazoparib alone or talazoparib plus temozolomide. Conclusions The high level of activity observed for talazoparib plus temozolomide in Ewing sarcoma xenografts makes this an interesting combination to consider for pediatric evaluation.

Acquisition of replicative immortality is currently regarded as essential for malignant transformation. This is achieved by activating a telomere lengthening mechanism (TLM), either telomerase or alternative lengthening of telomeres, to counter normal telomere attrition. However, a substantial proportion of some cancer types, including glioblastomas, liposarcomas, retinoblastomas, and osteosarcomas, are reportedly TLM-negative. As serial samples of human tumors cannot usually be obtained to monitor telomere length changes, it has previously been impossible to determine whether tumors are truly TLM-deficient, there is a previously unrecognized TLM, or the assay results are false-negative. Here, we show that a subset of high-risk neuroblastomas (with ∼50% 5-year mortality) lacked significant TLM activity. Cancer cells derived from these highly aggressive tumors initially had long telomeres and proliferated for >200 population doublings with ever-shorter telomeres. This indicates that prevention of telomere shortening is not always required for oncogenesis, which has implications for inhibiting TLMs for cancer therapy.

High-risk neuroblastoma is often distinguished by amplification of MYCN and loss of differentiation potential. We performed high-throughput drug screening of epigenetic-targeted therapies across a large and diverse tumor cell line panel and uncovered the hypersensitivity of neuroblastoma cells to GSK-J4, a small-molecule dual inhibitor of lysine 27 of histone 3 (H3K27) demethylases ubiquitously transcribed tetratricopeptide repeat, X chromosome (UTX), and histone demethylase Jumonji D3 (JMJD3). Mechanistically, GSK-J4 induced neuroblastoma differentiation and endoplasmic reticulum (ER) stress, with accompanying up-regulation of p53 up-regulated modulator of apoptosis (PUMA) and induction of cell death. Retinoic acid (RA)–resistant neuroblastoma cells were sensitive to GSK-J4. In addition, GSK-J4 was effective at blocking the growth of chemorefractory and patient-derived xenograft models of high-risk neuroblastoma in vivo. Furthermore, GSK-J4 and RA combination increased differentiation and ER stress over GSK-J4 effects and limited the growth of neuroblastomas resistant to either drug alone. In MYCN-amplified neuroblastoma, PUMA induction by GSK-J4 sensitized tumors to the B cell lymphoma 2 (BCL-2) inhibitor venetoclax, demonstrating that epigenetic-targeted therapies and BCL-2 homology domain 3 mimetics can be rationally combined to treat this high-risk subset of neuroblastoma. Therefore, H3K27 demethylation inhibition is a promising therapeutic target to treat high-risk neuroblastoma, and H3K27 demethylation can be part of rational combination therapies to induce robust antineuroblastoma activity.

Psychological stress has been shown to affect immune system status and function, but most studies of this relationship have focused on acute stress and/or laboratory situations. The present study compared total numbers of leukocytes and lymphocyte subpopulations (determined by flow cytometry) and antibody titers to latent and nonlatent viruses among a group of chronically stressed individuals living near the damaged Three Mile Island (TMI) nuclear power plant with those of a demographically comparable control group. Urinary catecholamine and cortisol levels were also examined. Residents of the TMI area exhibited greater numbers of neutrophils, which were positively correlated with epinephrine levels. The TMI group also exhibited fewer B lymphocytes, T-suppressor/cytotoxic lymphocytes, and natural killer cells. Antibody titers to herpes simplex were significantly different across groups as well, whereas titers to nonlatent rubella virus as well as IgG and IgM levels were comparable.

Neuroblastoma (NB) is the most common extracranial solid malignancy in children. Despite current aggressive treatment regimens, the prognosis for high‐risk NB patients remains poor, with the survival of less than 40%. Amplification/stabilization of MYCN oncogene, in NB is associated with a high risk of recurrence. Thus, there is an urgent need for novel therapeutics. The deregulated expression of microRNA (miR) is reported in NB; nonetheless, its effect on MYCN regulation is poorly understood. First, we identified that miR‐15a‐5p, miR‐15b‐5p, and miR‐16‐5p (hereafter miR‐15a, miR‐15b or miR‐16) were down‐regulated in patient‐derived xenografts (PDX) with high MYCN expression. MiR targeting sequences on MYCN mRNA were predicted using online databases such as TargetScan and miR database. The R2 database, containing 105 NB patients, showed an inverse correlation between MYCN mRNA and deleted in lymphocytic leukemia (DLEU) 2, a host gene of miR‐15. Moreover, overexpression of miR‐15a, miR‐15b or miR‐16 significantly reduced the levels of MYCN mRNA and N‐Myc protein. Conversely, inhibiting miR dramatically enhanced MYCN mRNA and N‐Myc protein levels, as well as increasing mRNA half‐life in NB cells. By performing immunoprecipitation assays of argonaute‐2 (Ago2), a core component of the RNA‐induced silencing complex, we showed that miR‐15a, miR‐15b and miR‐16 interact with MYCN mRNA. Luciferase reporter assays showed that miR‐15a, miR‐15b and miR‐16 bind with 3’UTR of MYCN mRNA, resulting in MYCN suppression. Moreover, induced expression of miR‐15a, miR‐15b and miR‐16 significantly reduced the proliferation, migration, and invasion of NB cells. Finally, transplanting miR‐15a‐, miR‐15b‐ and miR‐16‐expressing NB cells into NSG mice repressed tumor formation and MYCN expression. These data suggest that miR‐15a, miR‐15b and miR‐16 exert a tumor‐suppressive function in NB by targeting MYCN. Therefore, these miRs could be considered as potential targets for NB treatment.

Cancers overcome replicative immortality by activating either telomerase or an alternative lengthening of telomeres (ALT) mechanism. ALT occurs in ~25% of high-risk neuroblastomas, and progression in patients with ALT neuroblastoma during or after front-line therapy is frequent and often fatal. Temozolomide + irinotecan is commonly used as salvage therapy for neuroblastoma. Patient-derived cell lines and xenografts established from patients with relapsed ALT neuroblastoma demonstrated de novo resistance to temozolomide + irinotecan [SN-38 in vitro, P < 0.05; in vivo mouse event-free survival (EFS), P < 0.0001] vs. telomerase-positive neuroblastomas. We observed that ALT neuroblastoma cells manifested constitutive ataxia-telangiectasia mutated (ATM) activation due to spontaneous telomere dysfunction which was not observed in telomerase-positive neuroblastoma cells. We demonstrated that induction of telomere dysfunction resulted in ATM activation that, in turn, conferred resistance to temozolomide + SN-38 (4.2-fold change in IC50, P < 0.001). ATM knockdown (shRNA) or inhibition using a clinical-stage small-molecule inhibitor (AZD0156) reversed resistance to temozolomide + irinotecan in ALT neuroblastoma cell lines in vitro (P < 0.001) and in four ALT xenografts in vivo (EFS, P < 0.0001). AZD0156 showed modest to no enhancement of temozolomide + irinotecan activity in telomerase-positive neuroblastoma cell lines and xenografts. Ataxia telangiectasia and Rad3 related (ATR) inhibition using AZD6738 did not enhance temozolomide + SN-38 activity in ALT neuroblastoma cells. Thus, ALT neuroblastoma chemotherapy resistance occurs via ATM activation and is reversible with ATM inhibitor AZD0156. Combining AZD0156 with temozolomide + irinotecan warrants clinical testing for neuroblastoma.

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We used magnetic resonance spectroscopy to determine whether orthotopic mouse brain tumors grown as xenografts in immunocompromised mice either from human brain tumor cells implanted immediately after surgery or from cultured human tumor lines show metabolic profiles comparable to those of the original tumors. Using a 7 T scanner, spectra were acquired from mice with a human atypical teratoid/rhabdoid tumor (AT/RT) either implanted directly from the surgical specimen or first grown in culture, directly implanted choroid plexus carcinoma (CPC), and two medulloblastoma cell lines. The results were compared with spectra from these same tumors or tumor types in patients and with controls. Metabolic variability of tumors from a single cell line was also evaluated using the medulloblastoma lines. The main metabolic features of human tumors were qualitatively replicated in xenografts. AT/RTs in mice exhibited choline, creatine, and myo-inositol levels comparable to those observed in the patient. As in patients, choline was prominent in experimental CPC. Tumors from a single cell line were comparable. Significant correlations were found with key metabolites in humans and mice; however, differences including lower lipids in the implanted AT/RTs than in patient spectra and taurine observed in all animal spectra were also noted. The causes of these dissimilarities warrant further investigation.