Structural Optimization and Enhanced Prodrug-Mediated Delivery Overcomes Camptothecin Resistance in High-Risk Solid Tumors.

Camptothecins are potent topoisomerase I inhibitors used to treat high-risk pediatric solid tumors, but they often show poor efficacy due to intrinsic or acquired chemoresistance. Here, we developed a multivalent, polymer-based prodrug of a structurally optimized camptothecin (SN22) designed to overcome key chemoresistance mechanisms. The ability of SN22 vs. SN38 (the active form of irinotecan/CPT-11) to overcome efflux pump-driven drug resistance was tested. Tumor uptake and biodistribution of SN22 as a polymer-based prodrug (PEG-[SN22]4) compared with SN38 was determined. The therapeutic efficacy of PEG-[SN22]4 to CPT-11 was compared in: (i) spontaneous neuroblastomas (NB) in transgenic TH-MYCN mice; (ii) orthotopic xenografts of a drug-resistant NB line SK-N-BE(2)C (mutated TP53); (iii) flank xenografts of a drug-resistant NB-PDX; and (iv) xenografts of Ewing sarcoma and rhabdomyosarcoma. Unlike SN38, SN22 inhibited NB cell growth regardless of ABCG2 expression levels. SN22 prodrug delivery resulted in sustained intratumoral drug concentrations, dramatically higher than those of SN38 at all time points. CPT-11/SN38 treatment had only marginal effects on tumors in transgenic mice, but PEG-[SN22]4 treatment caused complete tumor regression lasting over 6 months (tumor free at necropsy). PEG-[SN22]4 also markedly extended survival of mice with drug-resistant, orthotopic NB and it caused long-term (6+ months) remissions in 80% to 100% of NB and sarcoma xenografts. SN22 administered as a multivalent polymeric prodrug resulted in increased and protracted tumor drug exposure compared with CPT-11, leading to long-term "cures" in NB models of intrinsic or acquired drug resistance, and models of high-risk sarcomas, warranting its further development for clinical trials. SIGNIFICANCE: SN22 is an effective and curative multivalent macromolecular agent in multiple solid tumor mouse models, overcoming common mechanisms of drug resistance with the potential to elicit fewer toxicities than most cancer therapeutics.

RET receptor expression and interaction with TRK receptors in neuroblastomas.

Neuroblastomas (NBs) have heterogeneous clinical behavior, from spontaneous regression or differentiation to relentless progression. Evidence from our laboratory and others suggests that neurotrophin receptors contribute to these disparate behaviors. Previously, the role of TRK receptors in NB pathogenesis was investigated. In the present study, the expression of RET and its co­receptors in a panel of NB cell lines was investigated and responses to cognate ligands GDNF, NRTN, and ARTN with GFRα1­3 co­receptor expression, respectively were found to be correlated. RET expression was high in NBLS, moderate in SY5Y, low/absent in NBEBc1 and NLF cells. All cell lines expressed at least one of GFRα co­receptors. In addition, NBLS, SY5Y, NBEBc1 and NLF cells showed different morphological changes in response to ligands. As expected, activation of RET/GFRα3 by ARTN resulted in RET phosphorylation. Interestingly, activation of TrkA by its cognate ligand NGF resulted in RET phosphorylation at Y905, Y1015, and Y1062, and this was inhibited in a dose­dependent manner by the TRK inhibitor (CEP­701). Conversely, RET activation by ARTN in NBLS cells led to phosphorylation of TrkA. This suggests a physical association between RET and TRK proteins, and cross­talk between these two receptor pathways. Finally, RET, GFR and TRK expression in primary tumors was investigated and a significant association between RET, its co­receptors and TRK expression was demonstrated. Thus, the present data support a complex model of interacting neurotrophin receptor pathways in the regulation of cell growth and differentiation in NBs.

Mechanisms of Entrectinib Resistance in a Neuroblastoma Xenograft Model.

TrkB with its ligand, brain-derived neurotrophic factor (BDNF), are overexpressed in the majority of high-risk neuroblastomas (NB). Entrectinib is a novel pan-TRK, ALK, and ROS1 inhibitor that has shown excellent preclinical efficacy in NB xenograft models, and recently it has entered phase 1 trials in pediatric relapsed/refractory solid tumors. We examined entrectinib-resistant NB cell lines to identify mechanisms of resistance. Entrectinib-resistant cell lines were established from five NB xenografts initially sensitive to entrectinib therapy. Clonal cell lines were established in increasing concentrations of entrectinib and had >10X increase in IC50 Cell lines underwent genomic and proteomic analysis using whole-exome sequencing, RNA-Seq, and proteomic expression profiling with confirmatory RT-PCR and Western blot analysis. There was no evidence of NTRK2 (TrkB) gene mutation in any resistant cell lines. Inhibition of TrkB was maintained in all cell lines at increasing concentrations of entrectinib (target independent). PTEN pathway downregulation and ERK/MAPK pathway upregulation were demonstrated in all resistant cell lines. One of these clones also had increased IGF1R signaling, and two additional clones had increased P75 expression, which likely increased TrkB sensitivity to ligand. In conclusion, NB lines overexpressing TrkB developed resistance to entrectinib by multiple mechanisms, including activation of ERK/MAPK and downregulation of PTEN signaling. Individual cell lines also had IGF1R activation and increased P75 expression, allowing preservation of downstream TrkB signaling in the presence of entrectinib. An understanding of changes in patterns of expression can be used to inform multimodal therapy planning in using entrectinib in phase II/III trial planning.

Role of microRNAs in epigenetic silencing of the CHD5 tumor suppressor gene in neuroblastomas.

Neuroblastoma (NB), a tumor of the sympathetic nervous system, is the most common extracranial solid tumor of childhood. We and others have identified distinct patterns of genomic change that underlie diverse clinical behaviors, from spontaneous regression to relentless progression. We first identified CHD5 as a tumor suppressor gene that is frequently deleted in NBs. Mutation of the remaining CHD5 allele is rare in these tumors, yet expression is very low or absent, so expression is likely regulated by epigenetic mechanisms. In order to understand the potential role of miRNA regulation of CHD5 protein expression in NBs, we examined all miRNAs that are predicted to target the 3'-UTR using miRanda, TargetScan and other algorithms. We identified 18 miRNAs that were predicted by 2 or more programs: miR-204, -211, -216b, -17, -19ab, -20ab, -93, -106ab, -130ab, -301ab, -454, -519d, -3666. We then performed transient transfections in two NB cell lines, NLF (MYCN amplified) and SY5Y (MYCN non-amplified), with the reporter plasmid and miRNA mimic, as well as appropriate controls. We found seven miRNAs that significantly downregulated CHD5 expression in NB: miR-211, 17, -93, -20b, -106b, -204, and -3666. Interestingly, MYCN upregulates several of the candidates we identified: miR-17, -93, -106b & -20b. This suggests that miRNAs driven by MYCN and other genes represent a potential epigenetic mechanism to regulate CHD5 expression.

Entrectinib is a potent inhibitor of Trk-driven neuroblastomas in a xenograft mouse model.

Neuroblastoma (NB) is one of the most common and deadly childhood solid tumors. These tumors are characterized by clinical heterogeneity, from spontaneous regression to relentless progression, and the Trk family of neurotrophin receptors plays an important role in this heterogeneous behavior. We wanted to determine if entrectinib (RXDX-101, Ignyta, Inc.), an oral Pan-Trk, Alk and Ros1 inhibitor, was effective in our NB model. In vitro effects of entrectinib, either as a single agent or in combination with the chemotherapeutic agents Irinotecan (Irino) and Temozolomide (TMZ), were studied on an SH-SY5Y cell line stably transfected with TrkB. In vivo growth inhibition activity was studied in NB xenografts, again as a single agent or in combination with Irino-TMZ. Entrectinib significantly inhibited the growth of TrkB-expressing NB cells in vitro, and it significantly enhanced the growth inhibition of Irino-TMZ when used in combination. Single agent therapy resulted in significant tumor growth inhibition in animals treated with entrectinib compared to control animals [p < 0.0001 for event-free survival (EFS)]. Addition of entrectinib to Irino-TMZ also significantly improved the EFS of animals compared to vehicle or Irino-TMZ treated animals [p < 0.0001 for combination vs. control, p = 0.0012 for combination vs. Irino-TMZ]. We show that entrectinib inhibits growth of TrkB expressing NB cells in vitro and in vivo, and that it enhances the efficacy of conventional chemotherapy in in vivo models. Our data suggest that entrectinib is a potent Trk inhibitor and should be tested in clinical trials for NBs and other Trk-expressing tumors.

Retinoic acid-induced CHD5 upregulation and neuronal differentiation of neuroblastoma.

BACKGROUND: Chromodomain-helicase DNA binding protein 5 (CHD5) is an important tumor suppressor gene deleted from 1p36.31 in neuroblastomas (NBs). High CHD5 expression is associated with a favorable prognosis, but deletion or low expression is frequent in high-risk tumors. We explored the role of CHD5 expression in the neuronal differentiation of NB cell lines. METHODS: NB cell lines SH-SY5Y (SY5Y), NGP, SK-N-DZ, IMR5, LAN5, SK-N-FI, NB69 and SH-EP were treated with 1-10 µM 13-cis-retinoic acid (13cRA) for 3-12 days. qRT-PCR and Western blot analyses were performed to measure mRNA and protein expression levels, respectively. Morphological differences were examined by both phase contrast and immunofluorescence studies. RESULTS: Treatment of SY5Y cells with 13cRA caused upregulation of CHD5 expression in a time- and dose-dependent manner (1, 5, or 10 µM for 7 or 12 days) and also induced neuronal differentiation. Furthermore, both NGP and SK-N-DZ cells showed CHD5 upregulation and neuronal differentiation after 13cRA treatment. In contrast, 13cRA treatment of IMR5, LAN5, or SK-N-FI induced neither CHD5 expression nor neuronal differentiation. NB69 cells showed two different morphologies (neuronal and substrate adherent) after 12 days treatment with 10 µM of 13cRA. CHD5 expression was high in the neuronal cells, but low/absent in the flat, substrate adherent cells. Finally, NGF treatment caused upregulation of CHD5 expression and neuronal differentiation in SY5Y cells transfected to express TrkA (SY5Y-TrkA) but not in TrkA-null parental SY5Y cells, and both changes were blocked by a pan-TRK inhibitor. CONCLUSIONS: Treatment with 13cRA induces neuronal differentiation only in NB cells that upregulate CHD5. In addition, NGF induced CHD5 upregulation and neuronal differentiation only in TrkA expressing cells. Together, these results suggest that CHD5 is downstream of TrkA, and CHD5 expression may be crucial for neuronal differentiation induced by either 13cRA or TrkA/NGF signaling.

The tumour suppressor CHD5 forms a NuRD-type chromatin remodelling complex.

Eukaryotic gene expression is developmentally regulated, in part by chromatin remodelling, and its dysregulation has been linked to cancer. CHD5 (chromodomain helicase DNA-binding protein 5) is a tumour suppressor gene (TSG) that maps to a region of consistent deletion on 1p36.31 in neuroblastomas (NBs) and other tumour types. CHD5 encodes a protein with chromatin remodelling, helicase and DNA-binding motifs that is preferentially expressed in neural and testicular tissues. CHD5 is highly homologous to CHD3 and CHD4, which are the core subunits of nucleosome remodelling and deacetylation (NuRD) complexes. To determine if CHD5 forms a similar complex, we performed studies on nuclear extracts from NBLS, SY5Y (both with endogenous CHD5 expression), NLF (CHD5 null) and NLF cells stably transfected with CHD5 cDNA (wild-type and V5-histidine-tagged). Immunoprecipitation (IP) was performed with either CHD5 antibody or antibody to V5/histidine-tagged protein. We identified NuRD components both by GST-FOG1 (Friend Of GATA1) pull-down and by IP. We also performed MS/MS analysis to confirm the presence of CHD5 or other protein components of the NuRD complex, as well as to identify other novel proteins. CHD5 was clearly associated with all canonical NuRD components, including metastasis-associated protein (MTA)1/2, GATA zinc finger domain containing 2A (GATAD2A), histone deacetylase (HDAC)1/2, retinoblastoma-binding protein (RBBP)4/7 and methyl DNA-binding domain protein (MBD)2/3, as determined by Western blotting and MS/MS. Our data suggest CHD5 forms a NuRD complex similar to CHD4. However, CHD5-NuRD may also have unique protein associations that confer functional specificity and may contribute to normal development and to tumour suppression in NB and other cancers.

Role of CHD5 in human cancers: 10 years later.

CHD5 was first identified because of its location on 1p36 in a region of frequent deletion in neuroblastomas. CHD5 (chromodomain-helicase-DNA-binding-5) is the fifth member of a family of chromatin remodeling proteins, and it probably functions by forming a nucleosome remodeling and deacetylation (NuRD) complex that regulates transcription of particular genes. CHD5 is preferentially expressed in the nervous system and testis. On the basis of its position, pattern of expression, and function in neuroblastoma cells and xenografts, CHD5 was identified as a tumor suppressor gene (TSG). Evidence soon emerged that CHD5 also functioned as a TSG in gliomas and a variety of other tumor types, including breast, colon, lung, ovary, and prostate cancers. Although one copy of CHD5 is deleted frequently, inactivating mutations of the remaining allele are rare. However, DNA methylation of the CHD5 promoter is found frequently, and this epigenetic mechanism leads to biallelic inactivation. Furthermore, low CHD5 expression is strongly associated with unfavorable clinical and biologic features as well as outcome in neuroblastomas and many other tumor types. Thus, based on its likely involvement as a TSG in neuroblastomas, gliomas, and many common adult tumors, CHD5 may play an important developmental role in many other tissues besides the nervous system and testis.