◥Neuroblastoma is a childhood cancer with heterogeneous clinical outcomes. To comprehensively assess the impact of telomere maintenance mechanism (TMM) on clinical outcomes in highrisk neuroblastoma, we integrated the C-circle assay [a marker for alternative lengthening of telomeres (ALT)], TERT mRNA expression by RNA-sequencing, whole-genome/exome sequencing, and clinical covariates in 134 neuroblastoma patient samples at diagnosis. In addition, we assessed TMM in neuroblastoma cell lines (n ¼ 104) and patient-derived xenografts (n ¼ 28). ALT was identified in 23.4% of high-risk neuroblastoma tumors and genomic alterations in ATRX were detected in 60% of ALT tumors; 40% of ALT tumors lacked genomic alterations in known ALT-associated genes. Patients with high-risk neuroblastoma were classified into three subgroups (TERT-high, ALT þ , and TERT-low/non-ALT) based on presence of C-circles and TERT mRNA expression (above or below median TERT expression). Event-free survival was similar among TERT-high, ALT þ , or TERT-low/non-ALT patients. However, overall survival (OS) for TERT-low/non-ALT patients was significantly higher relative to TERT-high or ALT patients (log-rank test; P < 0.01) independent of current clinical and molecular prognostic markers. Consistent with the observed higher OS in patients with TERT-low/non-ALT tumors, continuous shortening of telomeres and decreasing viability occurred in low TERT-expressing, non-ALT patient-derived high-risk neuroblastoma cell lines. These findings demonstrate that assaying TMM with TERT mRNA expression and C-circles provides precise stratification of high-risk neuroblastoma into three subgroups with substantially different OS: a previously undescribed TERT-low/non-ALT cohort with superior OS (even after relapse) and two cohorts of patients with poor survival that have distinct molecular therapeutic targets.Significance: These findings assess telomere maintenance mechanisms with TERT mRNA and the ALT DNA biomarker C-circles to stratify neuroblastoma into three groups, with distinct overall survival independent of currently used clinical risk classifiers.

Purpose: To determine the pharmacokinetics and the antitumor activity in pediatric cancer models of MM-398, a nanoliposomal irinotecan (nal-IRI).Experimental Design: Mouse plasma and tissue pharmacokinetics of nal-IRI and the current clinical formulation of irinotecan were characterized. In vivo activity of irinotecan and nal-IRI was compared in xenograft models (3 each in nu/nu mice) of Ewing's sarcoma family of tumors (EFT), neuroblastoma (NB), and rhabdomyosarcoma (RMS). SLFN11 expression was assessed by Affymetrix HuEx arrays, Taqman RT-PCR, and immunoblotting.Results: Plasma and tumor concentrations of irinotecan and SN-38 (active metabolite) were approximately 10-fold higher for nal-IRI than for irinotecan. Two doses of NAL-IRI (10 mg/kg/ dose) achieved complete responses maintained for >100 days in 24 of 27 EFT-xenografted mice. Event-free survival for mice with RMS and NB was significantly shorter than for EFT. High SLFN11 expression has been reported to correlate with sensitivity to DNA damaging agents; median SLFN11 mRNA expression was >100-fold greater in both EFT cell lines and primary tumors compared with NB or RMS cell lines or primary tumors. Cytotoxicity of SN-38 inversely correlated with SLFN11 mRNA expression in 20 EFT cell lines.Conclusions: In pediatric solid tumor xenografts, nal-IRI demonstrated higher systemic and tumor exposures to SN-38 and improved antitumor activity compared with the current clinical formulation of irinotecan. Clinical studies of nal-IRI in pediatric solid tumors (especially EFT) and correlative studies to determine if SLFN11 expression can serve as a biomarker to predict nal-IRI clinical activity are warranted.

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.

RLIP76 (RAL-binding protein-1, Rlip) is a stress-protective mercapturic-acid-pathway transporter protein that also plays a key role in regulating clathrin-dependent endocytosis as a Ral effector. Targeted inhibition or depletion of Rlip causes regression of xenografts of many cancers and is capable of abrogating tumor formation in p53-null mice. This is associated with the reversion of the abnormal methylomic profile of p53-null mice to wild-type. In a query of The Cancer Genome Atlas (TCGA) databases, we found that Rlip expression was associated with poor survival and with significant differences in the frequencies of PIK3CA mutation, MYC amplification, and CDKN2A/B deletion, which were the most commonly mutated, amplified, and deleted genes, respectively, among TCGA breast cancer patients. We conducted the present study to further examine the effects of Rlip inhibition and to evaluate the in vitro and in vivo efficacy in breast cancer. Using immunogold electron microscopy, we found that plasma-membrane Rlip was accessible to cell-surface antibodies in the MCF7 (ER+) breast cancer cell line. Rlip depletion resulted in decreased survival of MCF7 and MDA-MB-231 cells and increased terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) positivity and DNA laddering, indicating apoptotic cell death. Additionally, in vitro knockdown of Rlip inhibited EGF endocytosis and WNT/MAPK signaling. Xenograft studies in nude mice showed regression of breast cancer via antisense-mediated depletion of Rlip mRNA as well as by anti-Rlip antibody. Finally, knockdown of Rlip by antisense locked nucleic acid oligonucleotides increased markers for apoptotic signaling and decreased markers for proliferation, angiogenesis, and cell cycling in MCF7 and MDA-MB-231luc xenografts. Our findings validate Rlip as an attractive target in breast cancer.

Tumor invasion and metastasis remain a major cause of mortality in breast cancer patients. It was reported that BP1, a homeobox isoform of DLX4, is overexpressed in 80% of breast cancer patients and in 100% of estrogen receptor negative (ER-) tumors. The prevalence of BP1 positive cells and the intensity of BP1 immunoreactivity increased with the extent of ductal proliferation and tumorigenesis. These findings imply that BP1 may play an important role in ER- breast cancer. We sought to determine the effects and mechanisms of BP1 on cell proliferation and metastasis using ER- Hs578T cells as a model. Cells were transfected with either pcDNA3.2 plasmid containing BP1 gene, or pcDNA3.2 vector, then selected and cloned. Overexpression of BP1 increased cell proliferation rate by 2-5 fold (p<0.005), and enhanced the in vitro invasive activity by 25-65 fold (p<0.001). Microarray experiments were performed to identify differentially expressed genes when BP1 is overexpressed. The gene expression profile of the transfected cell lines were compared, resulting in 71 differentially expressed genes with a fold-change of >=2.0. Of those genes, 49 were up-regulated and 22 were down-regulated. Significant pathways were identified involving cell proliferation and metastasis. These data demonstrated that overexpression of BP1 significantly enhanced cell proliferation and metastatic potential in ER- Hs578T cells. Further analysis with more ER- cell lines and patient samples is warranted to establish BP1 as a therapeutic target for ER- breast cancer.

2′-hydroxyflavanone (2HF) is a dietary flavonoid with anticancer activity towards multiple cancers. Here, we report that topically applied 2HF inhibits the growth of intradermal implants of melanoma in immunocompetent mice. 2HF induced apoptosis and inhibited the growth of the human SK-MEL-24 as well as murine B16-F0 and B16-F10 melanoma cell lines in vitro. Apoptosis was associated with depletion of caspase-3, caspase-9, and PARP1 in B16-F0 and SK-MEL-24 cells. Caspase-9 and MEKK-15 were undetected even in untreated B16-F10 cells. Signaling proteins TNFα, and phospho-PDGFR-β were depleted in all three cell lines; MEKK-15 was depleted by 2HF in SK-MEL-24 cells. 2HF enhanced sunitinib (an MEK and PDGFR-β inhibitor) and AZD 2461 (a PARP1 inhibitor) cytotoxicity. 2HF also depleted the Ral-regulated, stress-responsive, antiapoptotic endocytic protein RLIP76 (RALBP1), the inhibition of which has previously been shown to inhibit B16-F0 melanoma growth in vivo. Functional inhibition of RLIP76 was evident from inhibition of epidermal growth factor (EGF) endocytosis by 2HF. We found that topically applied 2HF–Pluronic Lecithin Organogel (PLO) gel inhibited B16-F0 and B16-F10 tumors implanted in mice and caused no overt toxicity despite significant systemic absorption. 2HF treatment reduced phospho-AKT, vimentin, fibronectin, CDK4, cyclinB1, and BCL2, whereas it increased BIM and phospho-AMPK in excised tumors. Several cancer signals are controlled by endocytosis, a process strongly inhibited by RLIP76 depletion. We conclude that 2HF–PLO gel may be useful for topical therapy of cutaneous metastases of melanoma and could enhance the antineoplastic effects of sunitinib and PARP1 inhibitors. The mechanism of action of 2HF in melanoma overlaps with RLI76 inhibitors.

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Despite the improvement in clinical outcome with 13-cis-retinoic acid (13-cisRA) + anti-GD2 antibody + cytokine immunotherapy given in first response ~40% of high-risk neuroblastoma patients die of recurrent disease. MYCN genomic amplification is a biomarker of aggressive tumors in the childhood cancer neuroblastoma. MYCN expression is downregulated by 13-cisRA, a differentiating agent that is a component of neuroblastoma therapy. Although MYC amplification is rare in neuroblastoma at diagnosis, we report transcriptional activation of MYC medicated by the transcription factor OCT4, functionally replacing MYCN in 13-cisRA-resistant progressive disease neuroblastoma in large panels of patient-derived cell lines and xenograft models. We identified novel OCT4-binding sites in the MYC promoter/enhancer region that regulated MYC expression via phosphorylation by MAPKAPK2 (MK2). OCT4 phosphorylation at the S111 residue by MK2 was upstream of MYC transcriptional activation. Expression of OCT4, MK2, and c-MYC was higher in progressive disease relative to pre-therapy neuroblastomas and was associated with inferior patient survival. OCT4 or MK2 knockdown decreased c-MYC expression and restored the sensitivity to 13-cisRA. In conclusion, we demonstrated that high c-MYC expression independent of genomic amplification is associated with disease progression in neuroblastoma. MK2-mediated OCT4 transcriptional activation is a novel mechanism for activating the MYC oncogene in progressive disease neuroblastoma that provides a therapeutic target.