◥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.

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.

T-cell lymphoid malignancies (TCLM) are in need of novel and more effective therapies. The histone deacetylase (HDAC) inhibitor romidepsin and the synthetic cytotoxic retinoid fenretinide both have achieved durable clinical responses in T-cell lymphomas as single agents. We investigated the potential for using these two agents in combination in TCLMs. We demonstrated cytotoxic synergy between romidepsin and fenretinide in 15 TCLM cell lines at clinically achievable concentrations that lacked cytotoxicity for nonmalignant cells (fibroblasts and blood mononuclear cells). , romidepsin + fenretinide + ketoconazole (enhances fenretinide exposures by inhibiting fenretinide metabolism) showed greater activity in subcutaneous and disseminated TCLM xenograft models than single-agent romidepsin or fenretinide + ketoconazole. Fenretinide + romidepsin caused a reactive oxygen species (ROS)-dependent increase in proapoptotic proteins (Bim, tBid, Bax, and Bak), apoptosis, and inhibition of HDAC enzymatic activity, which achieved a synergistic increase in histone acetylation. The synergistic cytotoxicity, apoptosis, and histone acetylation of fenretinide + romidepsin were abrogated by antioxidants (vitamins C or E). Romidepsin + fenretinide activated p38 and JNK via ROS, and knockdown of p38 and JNK1 significantly decreased the synergistic cytotoxicity. Romidepsin + fenretinide also showed synergistic cytotoxicity for B-lymphoid malignancy cell lines, but did not increase ROS, acetylation of histones, activation of p38 + JNK, or cytotoxicity in nonmalignant cells. Romidepsin + fenretinide achieved synergistic activity in preclinical models of TCLMs, but not in nonmalignant cells, via a novel molecular mechanism. These data support conducting clinical trials of romidepsin + fenretinide in relapsed and refractory TCLMs..