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. Author manuscript; available in PMC: 2022 Jun 20.
Published in final edited form as: Sci Transl Med. 2021 Aug 18;13(607):eabd5750. doi: 10.1126/scitranslmed.abd5750

ALT neuroblastoma chemoresistance due to telomere dysfunction-induced ATM activation is reversible with ATM inhibitor AZD0156

Balakrishna Koneru 1,2,3, Ahsan Farooqi 1, Thinh H Nguyen 1, Wan Hsi Chen 1, Ashly Hindle 1,3, Cody Eslinger 1, Monish Ram Makena 1, Trevor A Burrow 1, Joanne Wilson 5, Aaron Smith 5, Venkatesh Pilla Reddy 6, Elaine Cadogan 5, Stephen T Durant 5, C Patrick Reynolds 1,2,3,4,*
PMCID: PMC9208664  NIHMSID: NIHMS1812549  PMID: 34408079

Abstract

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 relapse or progression in patients with ALT neuroblastoma during or after front-line therapy is frequent and almost uniformly 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 (as SN-38 in vitro, P<0.05; in vivo mouse event-free survival (EFS), P<0.0001) relative to telomerase-positive neuroblastomas. We observed that ALT neuroblastoma cells manifested constitutive ATM kinase activation due to spontaneous telomere dysfunction whereas telomerase-positive tumors lacked constitutive ATM activation or spontaneous telomere DNA damage. 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 kinase knock-down (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 4 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. ATR inhibition using AZD6738 did not enhance temozolomide + SN-38 activity in ALT neuroblastoma cell lines. Thus, resistance to chemotherapy in ALT neuroblastoma occurs via ATM kinase activation and was reversed with the ATM inhibitor AZD0156. Combining AZD0156 with temozolomide + irinotecan warrants clinical testing in neuroblastoma.

One Sentence Summary

ATM activation at telomeres confers chemoresistance in ALT neuroblastoma and is reversible in preclinical models by ATM knockdown or inhibition.

Introduction

Unlimited proliferation of cancer cells requires that they maintain telomeres (1). Most cancers maintain their telomeres by activating telomerase (2). Alternatively, some cancers use the recombination-mediated alternative lengthening of telomeres (ALT) mechanism (3), which is more prevalent in tumors of mesenchymal and neuroepithelial origin, including osteosarcoma, pancreatic neuroendocrine tumors, gliomas, and neuroblastoma (4). ALT is characterized by a high frequency of telomere-sister chromatid exchanges (T-SCEs) (5, 6), heterogeneous telomere length (7), presence of ALT-associated promyelocytic leukemia (PML) nuclear bodies (APBs) (8), and presence of extrachromosomal telomeric DNA repeats in the form of partially double-stranded circles, termed C-circles (9). Although the molecular mechanism of how ALT telomere maintenance occurs is not yet clear, previous studies suggest that ALT cells extend telomeres via a complex break-induced replication pathway initiated by either DNA damage at telomeres or telomere replication stress (10–12). One study showed that ALT cells are hypersensitive to ATR inhibitors due to telomere replication stress (13). However, several subsequent studies refuted these findings, suggesting that ALT cancer cells do not show general hypersensitivity to ATR inhibitors, including neuroblastoma (14, 15). With recent advances in understanding ALT-mediated telomere maintenance, several other therapeutic strategies have been proposed to exploit the ALT-associated vulnerabilities of ALT tumors (16–19), but none of these ALT specific therapeutic strategies have yet translated into the clinic. Also, long-term growth of tumor cells with no apparent telomere maintenance has been reported, suggesting that direct targeting of telomere maintenance mechanisms as a therapeutic strategy may encounter resistance mechanisms (20, 21).

Neuroblastoma is a pediatric solid tumor of the peripheral sympathetic nervous system that is responsible for 11% of pediatric cancer deaths (22, 23). Neuroblastoma patients stratified as high-risk continue to have poor outcomes, with an overall survival rate of ~ 50% with current comprehensive therapy (24–26). Using next generation sequencing and molecular approaches, we and several other groups observed that most common genomic alterations in high-risk neuroblastoma (MYCN amplification, TERT rearrangements, and ATRX genomic alterations) converge on activating telomere maintenance mechanisms (20, 27–29). We have recently shown that telomere maintenance mechanisms (TMM) define clinical outcome in high-risk neuroblastoma, irrespective of currently employed risk factors, suggesting that TMM are pivotal for neuroblastoma pathogenesis and may potentially provide therapeutic targets (29).

Previous studies, using less-than-specific means of identifying ALT, suggest that ALT neuroblastomas have distinct clinical behavior such as older age at diagnosis, protracted disease progression, and poor response to chemotherapy (30–32). Using more specific ALT markers, especially the robust telomeric DNA C-circle assay, we showed that ALT activation occurs in ~25–30% of high-risk neuroblastomas with or without ATRX genomic alterations (29). Patients with ALT neuroblastoma have very poor long-term survival and are often non-salvageable following progression or relapse (29). Similar to ALT neuroblastoma, ALT pancreatic neuroendocrine tumors, gliomas, and soft tissue sarcomas are known to have distinct clinical behavior and outcome relative to non-ALT tumors (33–37), suggesting that ALT mediated telomere maintenance may impact cancer cell growth and the response to treatment.

Due to a lack of relevant tractable in vitro and in vivo ALT neuroblastoma models, development of therapeutic approaches against ALT neuroblastoma has remained challenging since the first report of ALT in neuroblastoma (38). In the present study, using patient-derived ALT neuroblastoma cell lines and patient derived xenografts (PDXs) established from relapsed neuroblastoma patients (29, 39), we determined if ALT neuroblastoma cell lines and xenografts manifested chemoresistance to DNA damaging agents due to the constitutive activation of ataxia-telangiectasia mutated (ATM) kinase signaling as a result of constitutive telomere dysfunction. ATM is known to play a critical role in detection and repair of double-strand breaks (DSBs) resulting from endogenous genomic stress or due to exogenous irradiation or DNA damaging chemotherapy (40). We also determined if the chemo-resistant phenotype could be reversed in ALT neuroblastoma by inhibiting ATM kinase using AZD0156, a clinical-stage small molecule ATM inhibitor, in vitro and in vivo.

Results

ALT neuroblastoma models are associated with resistance to DNA-damaging drugs in vitro and in vivo.

To investigate differences in sensitivity to chemotherapy among neuroblastoma cell lines based on ALT status, we compared cytotoxicity profiles of ALT (n=4) vs non-ALT (n=100) patient-derived neuroblastoma cell lines (data file S1) treated with drugs commonly used to treat patients with neuroblastoma: topoisomerase inhibitors 7-Ethyl-10-hydroxycamptothecin (SN-38; an active metabolite of irinotecan (IRN)), topotecan (TOPO), and etoposide, as well as alkylating agents cyclophosphamide (as 4-hydroperoxy cyclophosphamide (4-HC) in vitro), melphalan, and carboplatin. ALT neuroblastoma cell lines showed significantly higher resistance to these DNA damaging agents when compared to non-ALT neuroblastoma cell lines (Fig. 1A, Wilcoxon, P<0.05). Additionally, ALT cell lines (n=4) were also more resistant than non-ALT cell lines (n=36) to drug combinations (4-HC + TOPO, or temozolomide (TMZ) + SN-38) that are commonly used as an induction or salvage therapy for patients with relapsed neuroblastoma (Fig. 1B; Wilcoxon, P<0.05). All ALT cell lines were DNA C-circle positive, had ALT-associated PML bodies, and were TERT mRNA (enzymatic component of telomerase) non-expressing, whereas most non-ALT lines were TERT-expressing (data file S1).

Fig. 1. ALT neuroblastoma models manifest chemoresistance to DNA damaging agents in vitro and in vivo.

Fig. 1.

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(A) Box plot comparing survival fraction of ALT (n=4) versus non-ALT (n=100) neuroblastoma cell lines treated with clinically achievable doses of 7-ethyl-10-hydroxycamptothecin (SN-38; an active metabolite of irinotecan), topotecan (TOPO), etoposide (ETOPO), 4-hydroperoxy cyclophosphamide (4-HC; an active metabolite of cyclophosphamide), melphalan (L-PAM), and carboplatin (CARBO). (B) Box plot comparing survival of ALT (n=4) versus telomerase-positive (n=36) cell lines in response to combination treatment with either 4-HC (1 μM) + TOPO (100 nM) or temozolomide (TMZ; at 30 μM) + SN-38 (3 nM). Survival fraction was determined using the DIMSCAN cytotoxicity assay. ALT positive cell lines COG-N-512 and COG-N-515 that were established from the same patient are shown in red in A and B. (C) Kaplan–Meier event-free survival (EFS) curves for ALT (red lines; ALT CDXs: COG-N-515m, CHLA-90m, SK-N-FIm; ALT PDX: COG-N-625x) versus telomerase-positive (blue lines; telomerase-positive CDX: CHLA-79m, CHLA-119m, FELIXm, telomerase-positive PDXs: COG-N-452x, COG-N-519x, COG-N-632x, COG-N-421x, COG-N-470x, COG-N-561x, COG-N-564x) xenograft models in response to treatment with 2 cycles of TMZ (25 mg/kg) + irinotecan (IRN; 7.5 mg/kg) on days 1–5 in a 21 day cycle. Control mice were administered sterile carrier solutions. EFS was calculated as the time to reach the tumor volume of 1500 mm3 from initiation of treatment with vehicle or TMZ+IRN or death from any cause. Each xenograft model had n=3–5 mice in both control and treatment groups. (D and E) Tumor volume of (D) ALT and (E) telomerase-positive xenograft models treated with vehicle or TMZ+IRN, in the same mice as shown in C. Survival between ALT versus non-ALT cell lines was compared using Wilcoxon-rank sum test. Kaplan-Meier survival curves were compared using the log-rank test. *: P<0.05, **: P<0.01.

Loss of p53 function has been shown to be associated with chemoresistance in neuroblastoma (41). Our group previously reported TP53 mutations in 2 neuroblastoma cell lines (CHLA-90 and SK-N-FI) (39). We established ALT neuroblastoma cell lines COG-N-512 and COG-N-515 from different biopsies obtained from the same patient; both cell lines matched by short tandem repeat assay and were wild-type for TP53 using whole exome and Sanger sequencing and wild-type for p53-pathway genes (data file S2). However, COG-N-515 failed to induce p53 and p21 protein expression upon irradiation (fig. S1A & S1B), indicating that this cell line was non-functional for p53. As p53 loss-of-function can induce chemoresistance in neuroblastoma (41), we determined if the ALT cell lines had cytotoxicity profiles similar to that of p53 non-functional telomerase-positive cell lines. ALT neuroblastoma cell lines showed resistance to topoisomerase inhibitors (SN-38, etoposide, and topotecan; P<0.05), alkylating agents (4-HC, melphalan), and carboplatin (P<0.05) and to DNA-damaging drug combinations (TMZ+SN-38 and 4-HC+TOPO; P<0.05) when compared to p53 non-functional telomerase-positive cell lines (fig. S1C).

To determine if the chemoresistance observed in vitro could be recapitulated in vivo, we evaluated the in vivo response to TMZ + irinotecan (IRN) in an ALT PDX (COG-N-625x) and cell line-derived xenografts (CDX) (CHLA-90m, COG-N-515m, and SK-N-FIm) compared to 10 randomly chosen telomerase-positive PDXs (COG-N-519x, COG-N-421x, COG-N-470x, COG-N-561x, COG-N-564x COG-N-452x, COG-N-623x) and CDXs (CHLA-119m, CHLA-79m and Felix-m) (data file S1), all of which were established from patients with neuroblastoma after therapy at time of disease progression or at time of death from progressive disease. Mice with ALT xenografts treated with TMZ+IRN had a substantially lower event-free survival (EFS) when compared to telomerase-positive xenograft models (log-rank test: P<0.0001; Fig. 1C). The majority of mice in ALT xenograft models (3 of 4 ALT models; CHLA-90m, SK-N-FIm, COG-N-625x) treated with TMZ+IRN either progressed rapidly after responding or had minimal to no response (Fig. 1D) compared to complete responses in 8/10 telomerase-positive xenograft models (Fig. 1E) after treatment with TMZ+IRN (Fisher exact test, mice in complete response for ALT vs telomerase-positive models: P<0.0001). Telomerase-positive PDXs COG-N-564x and COG-N-519x were the only telomerase-positive models that did not achieve complete responses when treated with TMZ+SN38. Although complete responses were observed in most telomerase-positive xenograft models, tumors progressively grew after completion of treatment cycles.

ALT neuroblastoma is associated with constitutive DNA damage signaling and ATM activation.

To elucidate mechanisms of the high-level chemo-resistance observed in ALT neuroblastoma, we compared gene expression profiles of ALT (n=3) versus randomly selected telomerase-positive non-MYCN-amplified (n=8) neuroblastoma cell lines (all established from patients with progressive disease) using RNA sequencing (data file S1). MYCN-amplified cell lines were excluded from global gene expression analysis as ALT occurs mutually exclusive of MYCN amplification in neuroblastoma and MYCN amplified tumors have chromosomal alterations distinct from non-MYCN amplified tumors (29, 39). Gene set enrichment analysis (GSEA) of Biocarta gene sets indicated that ATM and ATR-BRCA signaling pathways were enriched in ALT neuroblastoma cell lines (Fig. 2A and 2B; data file S3). Both ATM and ATR-BRCA signaling pathways are known to be key players in DNA damage repair induced after genomic stress such as double strand breaks (DSBs). As DNA damage signaling appears to be constitutively active in ALT cell lines, we assessed basal DNA damage by immunofluorescent staining for 53BP1 (a DSBs marker) in ALT (n=3) versus telomerase-positive (n=8) neuroblastoma cell lines; telomerase-positive cell lines used for 53BP1 analysis included 4 MYCN non-amplified cell lines used for GSEA and 4 randomly selected MYCN-amplified cell lines (data file S1). ALT cell lines had substantially higher numbers of 53BP1 foci compared to telomerase-positive cell lines, irrespective of MYCN amplification (Fig. 2C; P<0.001). In line with the notion that ALT is associated with chronic endogenous DNA damage, higher tail moment by comet assay (fig. S2A & S2B; P < 0.001) and greater abundance of γ-H2AX (marker for DNA-damage; fig. S2C; P<0.01) were observed in ALT cell lines compared to 2 telomerase-positive p53 non-functional cell lines (MYCN-amplified SK-N-BE(2) and non-MYCN amplified CHLA-171).

Fig. 2. ALT neuroblastoma is associated with constitutive DNA-damage signaling and ATM activation.

Fig. 2.

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(A) Table listing upregulated Biocarta gene sets comparing ALT (n=3) versus telomerase-positive non-MYCN amplified (n=8) neuroblastoma patient-derived cell lines using Gene Set Enrichment Analysis GSEA. Biocarta gene sets with P-value < 0.05 and false discovery rate (FDR) < 5% are listed. (B) Enrichment plots showing positive enrichment for ATM and ATR-BRCA pathways are shown. Plots were derived from GSEA results in gene pattern environment. (C) IF staining for 53BP1, a double strand break marker, in ALT versus telomerase-positive neuroblastoma cell lines. Top panel displays representative images of IF staining for 53BP1 (red) in ALT cell lines CHLA-90 and SK-N-FI compared to telomerase-positive cell line SK-N-BE (2). Nuclei were stained with DAPI (blue). Scale bar, 10 μm. Bottom panel displays a violin plot for 53BP1 foci quantification in ALT (CHLA-90, SK-N-FI, and COG-N-515) versus telomerase-positive cell lines (n=8; non-MYCN amplified: CHLA-171, COG-N-618, COG-N-619 and COG-N-709; MYCN amplified COG-N-519, COG-N-564, SK-N-BE(2) and CHLA-136). A minimum of 100 nuclei per cell line were analyzed for quantification of 53BP1 foci. (D) Immunoblotting for pATM (S1981), ATM, pCHK2 (T68), CHK2, pATR (S345), ATR, pCHK1 (S345), CHK1 and β-actin in same cell lines as shown in C. CHLA-136 twelve hours post-irradiation with x-rays (10 Grays) served as a positive control. (E) Quantification of pATM (S1981) and pCHK2 (T68) expression for the immunoblot in D and its replicates. The bars represent means with SDs from three experimental replicates. (F) Representative images of immunohistochemistry for pATM (s1981) in ALT PDXs (COG-N-625x and COG-N-620x) versus telomerase-positive PDX (COG-N-623x). COG-N-623x mice irradiated with 10 grays of x-rays served as a positive control for ATM activation. Scale bar, 50 μm. Statistical analysis for C and E was performed by Wilcoxon-rank sum test. ***: P<0.001, **: P<0.01.

Immunoblotting for ATM and ATR activation demonstrated that ATM kinase and its downstream target CHK2 were constitutively phosphorylated in ALT cell lines but not in telomerase-positive cell lines (Fig. 2D & 2E; P<0.01). Consistent with immunoblotting, ALT neuroblastoma cell lines had numerous p-ATM (s1981) foci (by immunofluorescence) in the nucleus relative to two telomerase-positive cell lines (fig. S3). Consistent with the ATM activation observed in vitro, ALT PDXs, but not a telomerase-positive PDX, were also positive for p-ATM (s1981) by immunohistochemistry (IHC) (Fig. 2F). We did not observe selective ATR/CHK1 activation in ALT relative to telomerase-positive neuroblastoma cell lines (Fig. 2D and fig. S4; P >= 0.05). As we have observed that ALT neuroblastoma cell lines have a basal degree of DNA damage, we determined if ALT cells were defective for DNA repair by assessing for γ-H2AX at different time points after irradiation. We observed that both ALT cell lines and a telomerase-positive cell line robustly induced γ-H2AX at 1 hour after irradiation but returned to basal levels of γ-H2AX by 24 hours after irradiation (fig. S2D), suggesting that ALT and telomerase-positive cell lines have a similar ability to repair DNA damage after genotoxic stress.

ALT neuroblastoma cell lines manifest constitutive telomere damage signaling via ATM kinase.

As we did not observe a difference between ALT and telomerase-positive cell lines in DNA repair following exogenous genotoxic stress, we determined if constitutive DNA damage signaling occurred in ALT cells at telomeres. We assessed telomere dysfunction-induced foci (42, 43) as detected by immunofluorescence of the DNA damage repair marker 53BP1 at telomeres identified by fluorescence in situ hybridization (FISH). We observed that ~40% of 53BP1 foci localized to telomeres in ALT neuroblastoma cell lines (Fig. 3A and 3B). However, ALT cell lines often have heterogeneous telomere length with some chromosomal ends having undetectable telomere signals, making it difficult to determine if all of the DNA damaging signals emanate from telomeres or from other genomic loci. Further, we observed that ALT-positive cell lines had a substantially higher number of telomere dysfunction-induced foci (TIFs) compared to three TP53 mutant telomerase-positive cell lines (Fig. 3C), which is consistent with previous findings in fibroblast transformed ALT cell lines (44).

Fig. 3. ALT neuroblastoma cell lines manifest constitutive ATM dependent telomere damage signaling.

Fig. 3.

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(A) Representative images of immunofluorescence in combination with florescent in situ hybridization (IF-FISH) to detect telomere dysfunction-induced foci (TIFs) in ALT (n=3) versus telomerase-positive cell line. 53BP1 was detected by IF (red) and telomeres by FISH with a [TTAGGG]3 probe (green). (B) Percentage of 53BP1 foci that were co-localized to telomeres in 3 ALT cell lines. (C) Percentage of cells with ≥ 3 TIFs in ALT versus telomerase-positive (TEL+) cell lines. (D) Representative IF-FISH staining to detect TIFs in the ALT neuroblastoma cell line CHLA-90 treated with ATM inhibitor AZD0156 (100 nM) or ATR inhibitor AZD6738 (300 nM). Cells treated with DMSO were used as controls. 53BP1 was detected by IF (red) and telomeres by FISH with a [TTAGGG]3 probe (green). (E) Bar graph shows percentage of cells with ≥ 3 TIFs in ALT cell lines treated with ATM or ATR inhibitor relative to vehicle. Scale bar represents 2 μm in distance. A minimum of 50 cells for each experimental replicate was assessed using IF-FISH for B, C and E. Bars represent mean with SD from three experimental replicates. Statistical significance was calculated using Wilcoxon-rank sum test for C and two-tailed t-test for E. ***: P<0.001, **: P<0.01, *: P<0.05, ns: not significant.

As 53BP1 foci could be induced due to activation of either ATM or ATR kinase signaling (43), we assessed the effect of ATM or ATR inhibition on TIFs in ALT neuroblastoma cell lines (Fig. 3D). We observed that the frequency of TIFs were consistently diminished with the inhibition of ATM kinase (Fig. 3D and 3E; P<0.01) in ALT neuroblastoma cell lines, pointing towards an ATM-dependent DNA damage response at telomeres. However, ATR inhibition had only a modest effect on TIFs in 1 of 3 ALT neuroblastoma cell lines (Fig. 3E).

Induction of ATM-dependent TIFs in a p53 non-functional telomerase-positive cell line is associated with resistance to TMZ+SN-38.

It is well known that in a p53-deficient setting, ATM protects cells from DNA-damaging chemotherapy by inducing DNA repair (45). However, it is not known if constitutive activation of ATM has any effect on response to DNA-damaging chemotherapy. To mimic the ATM-dependent TIF response observed in ALT cells in a telomerase-positive p53 non-functional cell line, we overexpressed dominant-negative TRF2 (TRF2ΔBΔM; Fig. 4A) using a retroviral vector in a telomerase-positive TP53 mutant (SK-N-BE (2)) neuroblastoma cell line. The dominant-negative TRF2 is known to induce an ATM kinase-dependent TIF response at telomeres (44). Expression of TRF2ΔBΔM in SK-N-BE (2) (Fig. 4B) induced ATM activation (phospho-ATM) in both the TRF2ΔBΔM clones relative to the empty vector control (Fig. 4B and 4C). As expected, we were able to induce ATM-dependent TIFs in cells transduced with pLPC-nMyc-TRF2ΔBΔM and the TIFs were abrogated by treating with the ATM inhibitor AZD0156 (Fig. 4D).

Fig. 4. Induction of ATM dependent TIFs caused hyper-resistance to temozolomide (TMZ) + irinotecan (IRN) that is reversed with ATM inhibitor AZD0156.

Fig. 4.

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(A) Schematic of TRF2 and dominant-negative form of TRF2 (TRF2 ΔBΔM). (B) Immunoblotting for TRF2, pATM (S1981), ATM and β-actin in cells transduced with pLPC-nMyc or pLPC-nMyc-TRF2ΔBΔM. (C) Quantitative analysis of immunoblotting for pATM (S1981) in B and its replicates. (D) (Top panel) Representative images of telomere dysfunctional foci (TIF) analysis using IF-FISH in same cells as in B (Clone 1 only) +/−ATM inhibitor (AZD0156). 53BP1 was detected by IF (red) and telomeres by FISH with a [TTAGGG]3 probe (green). (Bottom panel) Bar graph showing meant TIF count in same cells as in B +/−ATM inhibitor (AZD0156). A minimum of 50 cells for each experimental replicate were assessed using IF-FISH. Scale bar, 2 μm. (E) DIMSCAN cytotoxicity assay curves in response to TMZ+SN-38 +/− AZD0156 in the same cells as shown in C. (F) Immunoblotting for PARP, cleaved-PARP, cleaved caspase-3, and β-actin in same cells as shown in B treated with TMZ+SN-38 +/− AZD0156, cells with no treatment were included for comparison. (G) Quantification of cleaved caspase 3 and (H) cleaved PARP for immunoblot in F and its replicates. Bars represent means with SDs from three experimental replicates. Statistical significance was calculated using two-tailed t-test for G and H.****: P<0.0001; ***: P<0.001, **: P<0.01, *: P<0.05, ns: not significant.

To evaluate the effect of constitutive ATM activation on DNA-damaging chemotherapy in neuroblastoma cell lines, we assessed the cytotoxic response to TMZ+SN-38 (a drug combination used to salvage patients with relapsed neuroblastoma) in SK-N-BE(2) transduced with pLPC-nMyc-TRF2ΔBΔM or pLPC-nMyc (empty vector) +/− the ATM inhibitor AZD0156. Cells transduced with pLPC-nMyc-TRF2ΔBΔM showed increased resistance to TMZ+SN-38 when compared to the empty vector control (Fig. 4E & fig. S5A; IC50: P<0.05; two-way ANOVA: P<0.001). Transduction of pLPC-nMyc-TRF2ΔBΔM into another p53 non-functional telomerase-positive cell line (CHLA-171) also increased resistance to TMZ+ SN-38 (Fig. 5B; IC50: P<0.05; two-way ANOVA: P<0.001). Consistent with the cytotoxicity assay, cells transduced with pLPC-nMyc-TRF2ΔBΔM showed lower induction by TMZ+ SN-38 of markers for apoptosis, caspase-3, and PARP cleavage. (Fig. 4F–H). Addition of the ATM inhibitor AZD0156 to TMZ+SN-38 reversed the chemoresistance in cells transduced with pLPC-nMyc-TRF2ΔBΔM (Fig. 4E–H, fig. S5A and S5B; IC50: P = ns; two-way ANOVA: P = ns).

Fig. 5. ATM inhibitor AZD0156 sensitizes ALT neuroblastoma cell lines to temozolomide (TMZ) + irinotecan (IRN).

Fig. 5.

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(A) DIMSCAN cytotoxicity assay curves in ALT (n=4) and chemoresistant telomerase-positive (n=4) cell lines treated with TMZ+SN-38 +/− AZD0156 (100 nM). Statistical significance was calculated using two-way ANOVA. (B) Fold-change in IC50 with the addition of AZD0156 to TMZ+SN-38 in ALT (n=4) versus telomerase-positive (n=12) cell lines in vitro. ALT cell lines COG-N-512 and COG-N-515 that were established from same patient are highlighted in red. Statistical significance was calculated using Wilcoxon-rank sum test. (C) Immunoblotting for PARP, cleaved-PARP, cleaved caspase-3, and β-actin in ALT cell lines (CHLA-90 and COG-N-515) and a telomerase-positive cell line (CHLA-146) treated with TMZ+SN-38 +/− AZD0156 (100 nM). (D) Tunnel assay to detect apoptosis using flow cytometry in same cells as shown in C. The bars represent means with SDs from three experimental replicates. Statistical significance was calculated using two-tailed t-test. ***: P<0.001, **: P<0.01, *: P<0.05, ns: not significant.

As overexpression of TRF2ΔBΔM can increase the frequency of non-homologous end joining (46), we employed an alternative approach to induce ATM dependent TIFs in the SK-N-BE (2) cell line by overexpressing the nuclease domain of FOKI fused to N-terminus domain of shelterin protein TRF1 (FokIWTTRF1) using a retroviral vector. A nuclease dead mutant (FokID450ATRF1) fused to TRF1 was used as a control (fig. S6A). As expected, fusion proteins expressed in these cells co-localized to telomeres in both FokIWTTRF1 and FokID450ATRF1 (fig. S6B). Cells expressing FokIWTTRF1 showed higher phosphorylation of ATM compared to a nuclease dead mutant or the empty vector control (fig. S6C and S6D). Consistent with previous work, (47) we were able to induce ATM-dependent TIFs in cells transduced with pLPC-FokIWTTRF1 and the TIFs were abrogated when treated with the ATM inhibitor AZD0156 (fig. S6E).

We assessed the cytotoxic response to TMZ+SN-38 in SK-N-BE (2) transduced with pLPC- FokIWTTRF1 or pLPC- FokID450ATRF1 (control vector) +/− the ATM inhibitor AZD0156. Cells transduced with pLPC-FokIWTTRF1 were relatively more resistant to TMZ+SN-38 when compared to cells transduced with the nuclease dead (pLPC- FokID450ATRF1) transduced cells (fig. S6F; IC50: P<0.05; two-way ANOVA: P<0.001). Addition of the ATM inhibitor AZD0156 to TMZ+SN-38 reversed chemoresistance in cells transduced with pLPC-FokIWTTRF1, with cytotoxicity equal to control vector transduced cells treated with AZD0156+TMZ+SN-38 (fig. S6F; IC50: P = ns; two-way ANOVA: P = ns).

ATM inhibition reverses resistance of ALT neuroblastoma cell lines to TMZ+SN-38 in vitro.

To evaluate the therapeutic potential for inhibition of ATM activation in ALT cells, we knocked down ATM using lentiviral shRNA in two ALT neuroblastoma cell lines (CHLA-90 and COG-N-515; fig. S7A). Knockdown or pharmacological inhibition of ATM (fig. S7A, S7C–D) reduced DNA C-circle content (fig. S7B and S7E) in ALT neuroblastoma cell lines, but cell viability was not altered following ATM knockdown or inhibition, indicating that inhibition of ATM activation by itself did not have an immediate effect on ALT cell survival.

As a small molecule ATM inhibitor (AZD0156) showed reversal of drug resistance in cells with ATM dependent TIFs (Fig. 4E, 4F and S6F), we determined if pharmacological inhibition of ATM was effective in sensitizing ALT cell lines to TMZ+SN-38. We compared the cytotoxic response to TMZ+SN-38 +/−AZD0156 in ALT (n=4) versus telomerase-positive (n=12) cell lines in vitro; the telomerase-positive in vitro models selected here were those least responsive to TMZ+SN38 in Fig. 1B. AZD0156 consistently caused a marked increase in sensitivity of ALT neuroblastoma cell lines to TMZ+SN-38 (Fig. 5A, fold-change in IC 50: P < 0.005; two-way ANOVA: P<0.001), whereas telomerase-positive cell lines showed a heterogeneous response to the combination (7/12 telomerase-positive cell lines showed enhanced cytotoxicity to TMZ+SN38 with AZD0156: fold-change in IC 50, P < 0.05; two-way ANOVA, P<0.01; Fig. 5A and fig. S8). The ATM kinase inhibitor KU60019 also sensitized ALT neuroblastoma cell lines to TMZ+SN-38 (fig. S9; fold-change in IC 50, P < 0.001; two-way ANOVA, P<0.001).

Across all cell lines, the fold decrease in IC50 for TMZ+SN-38 with the addition of AZD0156 was significantly greater in ALT relative to telomerase-positive cell lines (Fig. 5B; P = 0.007). AZD0156 at concentrations as low as 6.25 nM significantly sensitized ALT cells to TMZ+SN-38 (fig. S10; P<0.05). We assessed the combination index (CI) in ALT neuroblastoma cell lines using fixed-ratio dosing and demonstrated that AZD0156 synergized with TMZ+SN-38 in all 4 ALT cell lines in vitro (fig. S11; data file S4; CI < 1). Additionally, we observed an increase in PARP cleavage, caspase-3 cleavage, and TUNEL staining in ALT cells treated with TMZ+SN-38+AZD0156 relative to TMZ+SN-38 alone (Fig. 5C, 5D and fig. S12), indicating that synergy was driven by apoptotic cell death. A telomerase-positive cell line (CHLA-146) with no enhancement in cytotoxicity to TMZ+SN38 with AZD0156 was used for comparison. Consistent with the results obtained using pharmacological ATM inhibition, ATM knockdown sensitized ALT cell lines to TMZ+SN-38 (fig. S13; fold-change in IC 50 P<0.001; two-way ANOVA: P<0.001).

ALT cells have been reported to be to be hypersensitive to ATR inhibitors (13), but we did not see a marked difference in sensitivity to an ATR inhibitor (AZD6738) between ALT and telomerase-positive cell lines (fig. S14A; P >= 0.05). Additionally, we did not see consistently enhanced sensitization to TMZ+SN-38 with the ATR inhibitor AZD6738 in ALT neuroblastoma cell lines in vitro (fig. S15B).

Thus, ATM inhibitor AZD0156 enhanced TMZ+SN-38 activity in all ALT neuroblastoma cell lines, but to lesser degree in a subset of comparably chemoresistant telomerase-positive neuroblastoma cell lines in vitro. Reversal of drug resistance in ALT cell lines was not observed with an ATR inhibitor.

ATM inhibition enhances the activity of TMZ+IRN in ALT PDXs and CDXs.

We assessed the activity of the ATM inhibitor AZD0156 in combination with TMZ+IRN in 4 ALT neuroblastoma xenograft models (3 CDXs and 1 PDX) and 2 telomerase-positive neuroblastoma PDXs. We determined that AZD0156 at 20 mg/kg was well-tolerated when dosed on day 6–19 after treatment with TMZ+IRN on day 1–5 in nu/nu mice. Pharmacokinetic assessment of AZD0156 at 20 mg/kg showed that maximum unbound plasma concentration was 204 nM at 2 hours after dosing and dissipating over a 24-hour period (fig. S15; data file S5). AZD0156 alone had low activity against 3 of 4 ALT xenografts and intermediate activity against 1 of 4 ALT xenografts, compared to only low activity in 1 of 2 telomerase-positive xenografts (Fig. 6A and 6B; Table 1). AZD0156 alone failed to induce objective responses in any of the xenografts (Table 1). All mice in control and AZD0156-treated groups showed progressive disease (fig. S16; Table 1). Treatment of ALT xenografts with TMZ+IRN increased EFS relative to control and AZD0156 groups (Fig. 6A; Table 1). TMZ+IRN achieved partial responses in most of the mice in 2 of 4 ALT xenograft models (CHLA-90m and SK-N-FIm), progressive disease in an ALT PDX (COG-N-625x), and complete responses in ALT COG-N-515m (fig. S16; Table 1). Although objective responses to TMZ+ IRN were observed in 3 of 4 ALT xenograft models, the observed responses were maintained for < 60 days. Resistance of the ALT xenografts to TMZ+ IRN was high in COG-N-625x and substantial in CHLA-90m and SK-N-FI. (fig. S16A; Table 1).

Fig. 6. ATM inhibitor AZD0156 enhances temozolomide (TMZ) + irinotecan (IRN) activity in ALT PDX and CDXs in vivo.

Fig. 6.

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(A) Kaplan–Meier event-free survival (EFS) curves according to each treatment group for tumor-bearing either ALT PDX (COG-N-625x) or ALT CDXs (CHLA-90m, SK-N-FIm or COG-N-515m). EFS was defined as the time taken for tumors to reach 1500 mm3 in tumor volume, from initiation of treatment, or to death from any cause. All mice for each xenograft model were blindly randomized to control (n=5–7), AZD0156 (n=5–6), TMZ+IRN (n=5–8), TMZ+IRN+AZD0156 (n=6–8) groups. Mice treated with TMZ+IRN were given 2 cycles of TMZ (25 mg/kg) + IRN (7.5 mg/kg) on days 1–5 in a 21-day cycle. For the TMZ+IRN+AZD0156 group, AZD0156 (20 mg/kg) was administered following TMZ+IRN treatment on days 6–19. An additional TMZ+IRN+AZD0156 group with a lower dose of AZD0156 (10 mg/kg) for the ALT xenograft CHLA-90 is included. COG-N-515m mice treated with 2 cycles of TMZ (25 mg/kg + IRN (7.5 mg/kg) on days 1–5 in a 21-day cycle were repurposed in Fig. 1C and 1D. (B) Kaplan–Meier EFS curves according to each treatment group for tumor-bearing telomerase-positive PDXs (COG-N-564x and COG-N-519x). (C) Representative images of immunohistochemistry for Ki67 and immunofluorescence staining for apoptosis using tunnel assay (in green) on FFPE sections in each treatment group. KI-67 and tunnel staining are shown for 1 ALT PDX (COG-N-625x), 1 ALT CDX (CHLA-90m) and 2 telomerase-positive PDX (COG-N-519x and COG-N-564x) models. FFPE sections were obtained from resected tumor on day 9 from initiation of treatment. Scale bar, 50 μm. EFS was assessed for statistical significance using the log-rank test.

Table 1.

Summary of in vivo response to TMZ+IRN+AZD0156, TMZ+IRN, AZD0156, and no treatment control in 4 ALT and 2 telomerase-positive xenograft models.

Groups Name n CR (%) MCR (%) PR (%) SD (%) PD (%) Median EFS EFS T/C
ALT COG-N-625x
 Control 5 0 0 0 0 5 (100) 26 NA
 AZD0156 5 0 0 0 0 5 (100) 44d 1.7
 TMZ+IRN 7 0 0 0 0 7 (100) 71ab 2.7
 TMZ+IRN+AZD0156 8 8 (100) 0 0 0 0 >150abc >5.8
CHLA-90m
 Control 5 0 0 0 0 5 (100) 25 NA
 AZD0156 5 0 0 0 0 5 (100) 40d 1.6
 TMZ+IRN 5 1 (20) 0 4 (80) 0 0 63ab 2.5
 TMZ+IRN+AZD0156 6 6 (100) 0 0 0 0 124 a b c 5.0
COG-N-515m
 Control 5 0 0 0 0 5 (100) 22 NA
 AZD0156 5 0 0 0 0 5 (100) 57d 2.6
 TMZ+IRN 5 5 (100) 0 0 0 0 130ab 5.9
 TMZ+IRN+AZD0156 6 0 6 (100) 0 0 0 >150abc >6.8
SK-N-FIm
 Control 5 0 0 0 0 5 (100) 18 NA
 AZD0156 5 0 0 0 0 5 (100) 32d 1.8
 TMZ+IRN 7 2 (29) 0 5 (71) 0 0 76ab 4.2
 TMZ+IRN+AZD0156 8 8 (100) 0 0 0 0 110abc 6.1
TEL+ COG-N-564x
 Control 6 0 0 0 0 6 (100) 14 NA
 AZD0156 6 0 0 0 0 6 (100) 15 1.1
 TMZ+IRN 8 1 (13) 0 7 (88) 0 0 42ab 3.1
 TMZ+IRN+AZD0156 8 1 (13) 0 7 (88) 0 0 43.5ab 3.2
COG-N-519x
 Control 7 0 0 0 0 7 (100) 9 NA
 AZD0156 6 0 0 0 0 6 (100) 17 1.9
 TMZ+IRN 6 2 (33) 0 2 (33) 0 2 (33) 43ab 4.8
 TMZ+IRN+AZD0156 7 3 (43) 1 (14) 3 (43) 0 0 65abe 7.2
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Abbreviations: CR, complete response; EFS: event-free survival; EFS T/C: median EFS of treated group/median EFS of control group; IRN: irinotecan; MCR, maintained complete response; Median EFS: median days taken to reach end point (tumor volume ⩾1500 mm3); N, total number of mice in a group; PD: progressive disease; PR: partial response; SD: stable disease; TMZ: temozolomide; NA, not applicable.

a

Log-rank test relative to control: P<0.001

b

Log-rank test relative to AZD0156: P<0.001

c

Log-rank test relative to TMZ+IRN: P<0.001

d

Log-rank test relative to control: P<0.05

e

Log-rank test relative to TMZ+IRN: P<0.05

To assess the effect of ATM inhibition on TMZ+IRN activity, we treated mice first with TMZ+IRN (days 1 to 5) and then followed with AZD0156 given on days 6 to 19, to mimic clinical use and prior in vivo preclinical studies of AZD0156 in combination with IRN (48). All mice bearing ALT xenografts treated with TMZ+IRN+AZD0156 achieved complete responses (fig. S16A; Table 1) and EFS was significantly longer for mice treated with TMZ+IRN+AZD0156 relative to control, AZD0156 alone, or TMZ+IRN groups (fig. 6A; Table 1; log-rank test: P<0.0001). In the CHLA-90m xenograft model, mice treated with TMZ+IRN+AZD0156 (AZD0156 at 20 mg/kg) showed greater activity than mice treated with TMZ+IRN+AZD0156 (AZD0156 at 10 mg/kg) (Fig. 6A. log-rank test: P=0.005). The combination of AZD0156+TMZ+IRN was well-tolerated as indicated by mouse body weight over time (fig. S17). Histopathological examination at day 9 of therapy for xenografts treated with TMZ+IRN+AZD0156 showed a reduction in proliferative activity by Ki-67 immunohistochemistry and increased apoptosis by TUNEL staining relative to mice in the control, AZD0156, and TMZ+IRN treatment groups (Fig. 6C).

To assess the activity of AZD0156+TMZ+IRN in telomerase-positive neuroblastoma, we chose two telomerase-positive PDXs (COG-N-564x and COG-N-519x) that showed resistance to TMZ+IRN comparable to that seen in the ALT PDX (COG-N-625x). Mice treated with TMZ+IRN had increased EFS compared to mice in the control and AZD0156 groups in both the telomerase-positive PDXs (Fig. 6B and fig. S16B; Table 1). Mice treated with TMZ+IRN+AZD0156 showed an increase of EFS in COG-N-519x but no improvement of EFS in COG-N-564x relative to TMZ+IRN (Fig. 6B and fig. S16B; Table 1). Histopathological examination of COG-N-519x and COG-N-564x mice treated with TMZ+IRN+AZD0156 had substantial reduction in proliferative activity and showed increased apoptosis when compared to the TMZ+IRN group in COG-N-519x but not in COG-N-564x (Fig. 6C). The median EFS (Table 1) was > 100 days for all four ALT xenografts treated with TMZ+IRN+AZD0156 (> 150 days for 2 of 4), whereas median EFS for telomerase positive PDXs was 43.5 and 65 days.

Discussion

About 25–30% of high-risk neuroblastomas (age > 18 months) activate the ALT mechanism to maintain telomeres (28, 29), with a long-term overall survival under 25%, despite intensive multimodal treatment regimens (29). Unfortunately, relapse or progression in patients with ALT neuroblastoma after front-line therapy is almost uniformly fatal (29). Clinical observations have categorized presumptive ALT neuroblastoma tumors as chemo-resistant (32). Although potentially therapeutically actionable genomic alterations such as ATRX are being investigated in ALT neuroblastoma (49), ~50% of ALT neuroblastoma tumors lack any known genomic alterations (27, 29). Furthermore, due to a prior lack of robust in vitro and in vivo ALT neuroblastoma models, development of targeted therapy against ALT neuroblastoma has been challenging. We recently established and characterized a small panel of ALT neuroblastoma patient-derived cell lines and a PDX by screening a large number of neuroblastoma PDCLs and PDXs (29, 39). We observed that ALT neuroblastoma PDCLs and PDXs are p53 non-functional, in line with previous reports on ALT cancers (39, 50, 51). Although p53 non-functionality is known to induce chemotherapy resistance in neuroblastoma (41), ALT neuroblastoma PDCLs showed greater de novo resistance to DNA damaging agents than did p53 non-functional telomerase-positive lines. Thus, based on our data, the ALT phenotype is associated with hyper-resistance to DNA damaging agents in neuroblastoma.

To elucidate chemoresistant mechanisms operative in ALT neuroblastoma, we performed gene set enrichment analysis in comparing ALT to telomerase-positive non-MYCN-amplified PDCLs and we observed an enrichment of ATM and ATR-BRCA pathways in ALT cell lines. ATM, but not ATR, was specifically activated in ALT but not in telomerase-positive PDCLs in vitro and in PDXs in vivo. Additionally, ALT cells manifested evidence of constitutive DNA damage signaling. However, ALT PDCLs repaired DNA upon irradiation, indicating that endogenous DNA damage is not due to major defects in DNA repair. Previous studies on the ALT phenotype suggested that ALT cells show a high degree of spontaneous telomere DNA damage (44). We demonstrated in ALT neuroblastoma cell lines large amounts of DNA damage foci that were localized to telomeres, indicating that telomere dysfunction is partly responsible for constitutive DNA damage signaling in ALT neuroblastoma. We observed that the frequency of telomere dysfunctional foci (TIFs) markedly diminished with the inhibition of ATM kinase in ALT cell lines, pointing towards an ATM-dependent DNA damage response at telomeres. Our data indicate that constitutive ATM activation is at least partially due to telomere dysfunction in ALT neuroblastoma.

ATM kinase is known to act as a binary switch to control the contribution of p53 signaling to the DNA damage response (45). In the presence of functional p53, ATM kinase activation largely contributes to induction of apoptosis via p53. In cells with loss of p53 function, apoptosis is reduced and ATM signaling is redirected to promote homologous recombination mediated DSB repair. The net result of this switch in ATM signaling leads to increased cellular survival in response to genotoxic stress (45), but how endogenous ATM activation (such as we observed in ALT cells) affects the response to genotoxic stress in a p53 deficient setting is unclear. To investigate the role of constitutive ATM activation in chemo-resistance, we artificially induced telomere dysfunction by forced expression of a dominant-negative TRF2 or TRF1-FOKI in a p53 non-functional telomerase-positive cell line, as described previously (44). Forced generation of telomere dysfunction in p53-non-functional telomerase-positive cells induced hyper resistance to TMZ + irinotecan (IRN, as the active metabolite SN-38 in vitro), DNA damaging agents used commonly to salvage relapsed neuroblastoma patients. ATM inhibition reversed resistance to TMZ + SN-38, indicating constitutive ATM activation (in response to telomere dysfunction) in ALT cells is at least partly responsible for the chemoresistant phenotype.

We demonstrated that AZD0156, a clinical-stage ATM kinase inhibitor, sensitized ALT neuroblastoma cell lines and xenografts to TMZ + IRN (as SN-38 in vitro). By contrast, modest or no enhancement of TMZ + IRN activity was observed in telomerase-positive models in vitro and in vivo. Although AZD0156 consistently (and to a greater degree) enhanced the activity of TMZ + IRN in ALT cell lines and xenografts, our data suggest that a subset of telomerase-positive neuroblastomas showed an enhanced response to TMZ+IRN when combined with AZD0156, indicating that addition of ATM inhibitor to TMZ+IRN might be a useful approach in treating a subset of telomerase positive neuroblastomas. However, it is currently unclear how to identify telomerase-positive tumors for which AZD0156 may enhance the activity of TMZ + IRN.

There are limitations of our study. Although our data indicate that ATM kinase is central to ALT and the associated drug resistance in neuroblastoma, the role of ATM kinase in other cancer types remains to be defined. Also, although our mouse xenograft studies suggest that by sequencing AZD0156 after TMZ + IRN the combination therapy is well-tolerated, tolerability in children with neuroblastoma remains to be determined by clinical trials.

In summary, ALT neuroblastomas are a distinct subgroup of patients with a robust biomarker (DNA C-circles) that are in need of effective therapeutic options. We have demonstrated that telomere dysfunction induced ATM kinase in ALT neuroblastomas, and that constitutive activation of ATM kinase promoted resistance to DNA damaging therapy observed in ALT neuroblastoma. We also showed that the clinical-stage ATM inhibitor AZD0156 can reverse resistance to TMZ + IRN in ALT neuroblastoma cell lines and xenografts. These data support undertaking early-phase clinical trials of AZD0156 with TMZ + IRN in children with neuroblastoma.

Materials and Methods

Study Design.

The objectives of this study were to identify mechanism(s) of chemo-resistance in relapsed/refractory ALT neuroblastoma and to identify a means of reversing such resistance. To enable in vitro and in vivo experimentation, we used ALT and telomerase-positive neuroblastoma PDCLs, PDXs, and CDXs established from neuroblastoma patients with progressive disease, or postmortem from progressive disease. Using genetic and pharmacological experimentation, we determined if telomere dysfunction could drive chronic ATM activation, and if ATM activation in turn induced chemo-resistance to DNA damaging agents in ALT neuroblastoma. To determine if we could reverse the chemo-resistant phenotype in ALT PDXs and CDXs in vivo, we employed the clinical-stage orally available ATM kinase inhibitor AZD0156 given following treatment with TMZ + IRN. We used 4 ALT xenografts (3 CDX and 1 PDX) and 2 telomerase positive PDXs to assess the activity of AZD0156 alone or in combination with TMZ+IRN. Sample size was determined by previous experimental experience and based on the growth profile of individual models (n= 5 to 8), with 8 animals providing an 80% power (= 0.05 significance level) for a Wilcoxon rank-sum test to detect a significant difference between the treated and control mice, assuming that the standard deviation is 13.4 days, the increase in median survival is 40 days, and no censoring in the xenograft experiments. This was not a blinded study at the time of investigation. The endpoint was tumor volume ≥ 1500 mm3, if mouse weight was reduced by ≥ 20% of initial weight, if the mice exhibited signs of impaired health, or death from any cause. Mice were monitored until they reached endpoint of the study or until 150 days from the start of treatment.

In vivo Drug Testing.

The Texas Tech University Health Sciences Center Institutional Animal Care and Use Committee (IACUC) approved all animal protocols. We used 4 CDXs (CHLA-90m, SK-N-FIm, COG-N-515m, FELIXm) and 5 PDXs (COG-N-625x, COG-N-519x, COG-N-564x, COG-N-452x and COG-N-623x) in this study. All the CDXs and PDXs were established from high-risk neuroblastoma patients at progressive disease after therapy or at postmortem at progressive disease after therapy. H&E staining of formalin-fixed paraffin-embedded sections and expression of tyrosine hydroxylase mRNA were assessed to verify neuroblastoma origin of new CDXs or PDXs. PDXs and CDXs were verified to match the patient of origin by STR profiling and were passaged in mice (nu/nu) after establishment from the patient sample in NOD scid gamma mouse. Strained cells from a previous tumor were prepared for injection in RPMI-1640 and Matrigel (Corning). nu/nu mice were injected subcutaneously between the shoulder blades with 200 μl of cell preparation containing 10–20 million viable cells. Mice were randomized to treatment groups when progressively growing tumors reached 150–250 mm3, determined as previously described (52). CDX experiments were carried out similarly, as previously described (53).

In Vivo dosing of TMZ + IRN was designed to mimic clinical dosing (54). TMZ was prepared as a slurry in sterile water; 20 mg/ml of irinotecan hydrochloride was diluted 10x in 0.9% saline; AZD0156 was prepared in 10% DMSO and 30% captisol for oral gavage. In a 21-day cycle, on days 1 to 5 mice were treated with TMZ by oral gavage at 25 mg/kg, followed one hour later by IRN by tail vein injection at 7.5 mg/kg. Triple combination mice were additionally given AZD0156 by oral gavage at 20 mg/kg from days 6 to 19, sequencing of AZD0156 was designed to minimize system toxicities and to mimic clinical usage and previous in vivo testing (48).

In vivo responses were categorized based on National Cancer Institute Pediatric Preclinical Testing Program classification (55). For each mouse, complete response (CR) is defined as disappearance of measurable tumor mass (< 0.1cm3) at any time following treatment during the study period. A CR is considered as maintained (MCR) if the tumor volume was undetectable (<0.1 cm3) at the end of study period. Partial response (PR) is defined as tumor volume that reduces by > 50% of initial volume for at least one time point during the study but with a measurable tumor mass (≥ 0.1cm3). Progressive disease (PD) was defined as < 50% reduction in initial tumor volume at any point during the study and > 25% increase in tumor volume by the end of the study. Stable disease was defined as < 50% reduction in initial tumor volume until the end of the study and ≤ 25% increase in tumor volume by the end of the study. An event-free survival of tumor/control (EFS T/C) was calculated by the ratio of the median time to an event in the treatment group divided by median time to an event in the control group. For EFS T/C measure, a treatment is considered as highly active if EFS T/C is > 2, statistically significant (log rank test: P ≤ 0.05) difference in EFS compared to control, and there is a net reduction in median tumor volume by the end of treatment. Treatment groups with EFS T/C > 2 with statistically significant (log rank test: P ≤ 0.05) difference in EFS compared to control, but no net reduction in median tumor volume is considered to have intermediate activity. For EFS T/C < 2, the agents are considered to have low activity. If the treatment group does not have a median EFS, then EFS T/C is defined as greater than the ratio of last day of follow up for the treatment group divided by median time to an event in the control group.

Statistical Analysis.

Comparison of two sample sets was done using Mann-Whitney U test. Comparison of biological replicates assessed for normality and used the unpaired two-tailed t-test. Dose response curves were assessed using two-way-ANOVA test. IC50 concentration was calculated, as described previously (56); comparison of IC50 was based on unpaired two-tailed t-test with Welch’s correction. Survival analysis for xenograft studies was done by the Kaplan-Meier method, as assessed using a log-rank test. All statistical analysis was done using GraphPad prism v7.0 and were considered statistically significant if P ≤ 0.05.

Supplementary Material

Koneru et al Supplementary Material
Koneru et al Supplementary Tables

Acknowledgments

We thank Children’s Oncology Group repository for providing cell lines and PDXs for this study. We thank the patients and their families for donating samples to enable this research. We thank Titia de Lange for providing pLPC-NMYC TRF2 deltaB deltaM, pLPC-NMYC-hTRF1 and pLPC-NMYC vector. We thank Dr. David Wheeler of the Human Genome Sequencing Center at Baylor College of Medicine, Houston Texas for the RNA sequencing and whole exome sequencing. We thank Tito Woodburn, Heather L. Davidson, Kristyn E. McCoy, and Jonas A. Nance for their efforts in establishing neuroblastoma models.

Funding

This work was supported by grants from the Cancer Prevention & Research Institute of Texas RP170510 (to C.P.R.), the National Cancer Institute (NCI) CA217251 (to C.P.R.), NCI CA221957 (to C.P.R.), and Alex’s Lemonade Stand Foundation which supports the COG Childhood Cancer Repository (www.CCcells.org).

Footnotes

Supplementary Materials

Materials and Methods

Fig. S1 to S17

data files S1–S6

Competing interests

AZD0156 is covered by patent WO2015170081 “Imidazo[4,5-C] quinoline-2-one compounds and their use in treating cancer. The authors declare no other competing interests.

Data and materials availability

All data associated with this study are in the paper or supplementary materials. PDCLs and PDXs are freely available from the COG/ALSF Childhood Cancer Repository (www.CCcells.org). Any additional materials will be provided upon request. Requests for the materials should be submitted to C.P.R. Primary data from the paper are available in data file S6. Raw RNA sequencing data have been deposited to dbGaP under accession number phs002421.v1.p1.

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Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

Koneru et al Supplementary Material
NIHMS1812549-supplement-Koneru_et_al_Supplementary_Material.pdf (3.9MB, pdf)
Koneru et al Supplementary Tables
NIHMS1812549-supplement-Koneru_et_al_Supplementary_Tables.xlsx (350.9KB, xlsx)

Data Availability Statement

All data associated with this study are in the paper or supplementary materials. PDCLs and PDXs are freely available from the COG/ALSF Childhood Cancer Repository (www.CCcells.org). Any additional materials will be provided upon request. Requests for the materials should be submitted to C.P.R. Primary data from the paper are available in data file S6. Raw RNA sequencing data have been deposited to dbGaP under accession number phs002421.v1.p1.

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