Search

Purpose: Rhabdomyosarcomas are a major cause of cancer death in children, described with MYCN amplification and, in the alveolar subtype, transcription driven by the PAX3-FOXO1 fusion protein. Our aim was to determine the prevalence of N-Myc protein expression and the potential therapeutic effects of reducing expression in rhabdomyosarcomas, including use of an antigene strategy that inhibits transcription.Experimental Design: Protein expression was assessed by immunohistochemistry. MYCN expression was reduced in representative cell lines by RNA interference and an antigene peptide nucleic acid (PNA) oligonucleotide conjugated to a nuclear localization signal peptide. Associated gene expression changes, cell viability, and apoptosis were analyzed in vitro. As a paradigm for antigene therapy, the effects of systemic treatment of mice with rhabdomyosarcoma cell line xenografts were determined.Results: High N-Myc levels were significantly associated with genomic amplification, presence of the PAX3/7-FOXO1 fusion genes, and proliferative capacity. Sustained reduction of N-Myc levels in all rhabdomyosarcoma cell lines that express the protein decreased cell proliferation and increased apoptosis. Positive feedback was shown to regulate PAX3-FOXO1 and N-Myc levels in the alveolar subtype that critically decrease PAX3-FOXO1 levels on reducing N-Myc. Pharmacologic systemic administration of the antigene PNA can eliminate alveolar rhabdomyosarcoma xenografts in mice, without relapse or toxicity.Conclusion: N-Myc, with its restricted expression in non-fetal tissues, is a therapeutic target to treat rhabdomyosarcomas, and blocking gene transcription using antigene oligonucleotide strategies has therapeutic potential in the treatment of cancer and other diseases that has not been previously realized in vivo. Clin Cancer Res; 18(3); 796–807. ©2011 AACR.

PDF file - 72K

PDF file - 135K

PDF file - 1.1MB

PDF file - 287K

supplementary tables Supplementary Table 1: List of cell lines used in this study. Supplementary Table 2: List of the primers used in this study for quantitative Real Time PCR. Supplementary Table 3: Table showing the EC50 effect for BGA001 and BGA002 (MYCN mRNA, decrease in cell viability, N-myc protein and apoptosis induction in Kelly and SK-N-BE(2)c cell lines). Supplementary Table 4: List of genes present in the Mitochondrial-related signature in alphabetical order. Supplementary Table 5: Table summarizing principal patient features and their cluster attribution. More extensive information can be seen at http://www.ebi.ac.uk/arrayexpress; accession: E-MTAB-1781. Supplementary Table 6: Table summarizing the mitoscore and its components for each gene of the mitochondrial signature; genes are order based on decreasing mitoscore. Supplementary Table 7: List of the top 200 differentially expressed genes between Cluster 1 (left) and Cluster 2 (right) Supplementary Table 8: List of Cluster 1 and Cluster 2 associated genes used for the pathway network analysis. Supplementary Table 9: List of the ClueGo pathways obtained from the pathway network analysis.

CCR Translation for This Article from Antitumor Activity of Sustained N-Myc Reduction in Rhabdomyosarcomas and Transcriptional Block by Antigene Therapy

PDF file - 750K

PDF file - 62K

Approximately half of high-risk neuroblastoma is characterized by MYCN amplification. N-Myc promotes tumor progression by inducing cell growth and inhibiting differentiation. MYCN has also been shown to play an active role in mitochondrial metabolism, but this relationship is not well understood. Although N-Myc is a known driver of the disease, it remains a target for which no therapeutic drug exists. Here, we evaluated a novel MYCN-specific antigene PNA oligonucleotide (BGA002) in MYCN-amplified (MNA) or MYCN-expressing neuroblastoma and investigated the mechanism of its antitumor activity. MYCN mRNA and cell viability were reduced in a broad set of neuroblastoma cell lines following BGA002 treatment. Furthermore, BGA002 decreased N-Myc protein levels and apoptosis in MNA neuroblastoma. Analysis of gene expression data from patients with neuroblastoma revealed that MYCN was associated with increased reactive oxygen species (ROS), downregulated mitophagy, and poor prognosis. Inhibition of MYCN caused profound mitochondrial damage in MNA neuroblastoma cells through downregulation of the mitochondrial molecular chaperone TRAP1, which subsequently increased ROS. Correspondingly, inhibition of MYCN reactivated mitophagy. Systemic administration of BGA002 downregulated N-Myc and TRAP1, with a concomitant decrease in MNA neuroblastoma xenograft tumor weight. In conclusion, this study highlights the role of N-Myc in blocking mitophagy in neuroblastoma and in conferring protection to ROS in mitochondria through upregulation of TRAP1. BGA002 is a potently improved MYCN-specific antigene oligonucleotide that reverts N-Myc–dysregulated mitochondrial pathways, leading to loss of the protective effect of N-Myc against mitochondrial ROS.Significance:A second generation antigene peptide oligonucleotide targeting MYCN induces mitochondrial damage and inhibits growth of MYCN-amplified neuroblastoma cells.

PDF file - 242K

PDF file - 399K

Figure S1: BGA002 induces apoptosis Figure S2: BGA002 inhibits MYCN expression in a broad neuroblastoma cell-line panel Figure S3: BGA002 is a specific MYCN inhibitor Figure S4: Mitochondria structural alteration are MYCN specific inhibition. Figure S5: BGA002 leads to MYCN-specific structural alteration in mitochondria. Figure S6: BGA001 does not lead to significant MYCN-specific structural alteration in mitochondria. Figure S7: MYCN inhibition leads to mitochondrial damage Figure S8: Alterations in mitochondrial pathways identify Neuroblastoma patients with poor prognosis. Figure S9: MYCN amplified patient are enriched for the mitochondrial gene signature. Figure S10: MYCN blocking leads to MYCN specific gene expression signature inhibition in NB cells and to mitophagy reactivation. Figure S11: OPTN controls mitophagy induction after MYCN silencing Figure S12: BGA002 reverts the MYCN control of ROS generation by TRAP1 down-regulation in MNA-NB cells. Figure S13: BGA002 causes elimination of MYCN-NB in mice

PDF file - 347K

PDF file - 104K

PDF file - 45K, Xenografts tumor growth is proportional to CHAF1A protein levels.

XLSX file - 17K, Gene Ontology (GO) analysis of gene sets regulated by retinoic acid treatment.

PDF file - 57K, In vitro effects of CHAF1A silencing on proliferation.

Supplementary Figure 1 from Activity of the Novel Dual Phosphatidylinositol 3-Kinase/Mammalian Target of Rapamycin Inhibitor NVP-BEZ235 against T-Cell Acute Lymphoblastic Leukemia

Neuroblastoma arises from the embryonal neural crest secondary to a block in differentiation. Long-term patient survival correlates inversely with the extent of differentiation, and treatment with retinoic acid or other prodifferentiation agents improves survival modestly. In this study, we show the histone chaperone and epigenetic regulator CHAF1A functions in maintaining the highly dedifferentiated state of this aggressive malignancy. CHAF1A is a subunit of the chromatin modifier chromatin assembly factor 1 and it regulates H3K9 trimethylation of key target genes regulating proliferation, survival, and differentiation. Elevated CHAF1A expression strongly correlated with poor prognosis. Conversely, CHAF1A loss-of-function was sufficient to drive neuronal differentiation in vitro and in vivo. Transcriptome analysis of cells lacking CHAF1A revealed repression of oncogenic signaling pathways and a normalization of glycolytic metabolism. Our findings demonstrate that CHAF1A restricts neural crest differentiation and contributes to the pathogenesis of high-risk neuroblastoma. Cancer Res; 74(3); 765–74. ©2013 AACR.

Supplementary Figure 2 from Activity of the Novel Dual Phosphatidylinositol 3-Kinase/Mammalian Target of Rapamycin Inhibitor NVP-BEZ235 against T-Cell Acute Lymphoblastic Leukemia

Recent investigations have documented that constitutively activated phosphatidylinositol 3-kinase (PI3K)/Akt/mammalian target of rapamycin (mTOR) signaling is a common feature of T-cell acute lymphoblastic leukemia (T-ALL), where it strongly influences growth and survival. These findings lend compelling weight for the application of PI3K/Akt/mTOR inhibitors in T-ALL. However, our knowledge of PI3K/Akt/mTOR signaling in T-ALL is limited and it is not clear whether it could be an effective target for innovative therapeutic strategies. Here, we have analyzed the therapeutic potential of the dual PI3K/mTOR inhibitor PI-103, a small synthetic molecule of the pyridofuropyrimidine class, on both T-ALL cell lines and patient samples, which displayed constitutive activation of PI3K/Akt/mTOR signaling. PI-103 inhibited the growth of T-ALL cells, including 170-kDa P-glycoprotein overexpressing cells. PI-103 cytotoxicity was independent of p53 gene status. PI-103 was more potent than inhibitors that are selective only for PI3K (Wortmannin, LY294002) or for mTOR (rapamycin). PI-103 induced G0-G1 phase cell cycle arrest and apoptosis, which was characterized by activation of caspase-3 and caspase-9. PI-103 caused Akt dephosphorylation, accompanied by dephosphorylation of the Akt downstream target, glycogen synthase kinase-3β. Also, mTOR downstream targets were dephosphorylated in response to PI-103, including p70S6 kinase, ribosomal S6 protein, and 4E-BP1. PI-103 strongly synergized with vincristine. These findings indicate that multitargeted therapy toward PI3K and mTOR alone or with existing drugs may serve as an efficient treatment toward T-ALL cells, which require up-regulation of PI3K/Akt/mTOR signaling for their survival and growth. [Cancer Res 2009;69(8):3520–28]

Supplementary Figure 1 from Dual Inhibition of Class IA Phosphatidylinositol 3-Kinase and Mammalian Target of Rapamycin as a New Therapeutic Option for T-Cell Acute Lymphoblastic Leukemia

PDF file - 53K, Effect of retinoic acid (RA) treatment on neuroblastoma cells.

PDF file - 29K

Supplementary Figure 2 from Dual Inhibition of Class IA Phosphatidylinositol 3-Kinase and Mammalian Target of Rapamycin as a New Therapeutic Option for T-Cell Acute Lymphoblastic Leukemia

Supplementary Figure 3 from Dual Inhibition of Class IA Phosphatidylinositol 3-Kinase and Mammalian Target of Rapamycin as a New Therapeutic Option for T-Cell Acute Lymphoblastic Leukemia

XLSX file - 14K, Kegg, Biocarta and Oncogenic Pathways GSEA analysis.

XLSX file - 17K, Gene Ontology (GO) analysis of gene sets regulated by Cyclin D1 and CDK4 silencing.

Supplementary Table 1 from Dual Inhibition of Class IA Phosphatidylinositol 3-Kinase and Mammalian Target of Rapamycin as a New Therapeutic Option for T-Cell Acute Lymphoblastic Leukemia

PDF file - 36K

PDF file - 77K, CHAF1A expression correlates with stage of disease.

Follicular lymphoma (FL) still remains an incurable disease, with most of the patients undergoing subsequent phases of remission and relapse. In the era of immunotherapy, understanding the immunobiology of FL patients who experience progression of disease within 24 months (POD24) and show chemotherapy resistance remains a priority and an unmet clinical need. Tumor-associated macrophages (TAM) are multifaceted cellular components of the tumor microenvironment (TME). In concert with lymphoid-lineage B and T cells at various developmental stages, TAM can mediate enhanced tumor progression, often leading to poor clinical prognosis and impact the clinical response to chemotherapy. Recently, Joshua and colleagues demonstrated that a subset of FL patients with low immune infiltration was enriched in POD24 events. In our work, we characterized the immune repertoire of lymph node biopsies collected retrospectively from 30 patients with histological diagnosis of FL according WHO criteria. Median follow up was 8 years. The expression levels of TAM (CD68, CD163, MS4a4a) and tumor-infiltrating T-lymphocytes (CD8, PD-1 positive subsets) were assessed by immunohistochemistry and summarized using descriptive statistics. Our data highlighted that the subset of patients with a high extrafollicular CD163/CD8 ratio was enriched in POD24 events (p = 0.01). In addition, patients who showed an higher number of intrafollicular CD68+ macrophages, showed a longer disease free survival (p= 0.04). Another difference was related to the number of CD68, CD163, MS4A4A-positive polarized macrophages that resulted higher in bcl-2 negative than bcl-2 positive cases (p< 0.05 for all markers). In the evaluation of T lymphocytes, most relevant associations were found between the content and distribution of PD1+ cells and the treatment response: the higher the number of intrafollicular PD1+ lymphocytes the lower chemotherapy response rates (p= 0.04). No association was found between the number of positive elements in extrafollicular areas, where instead the PD1/CD8 ratio seems to be related with therapy response: the higher the ratio the lower response rate (p= 0.01). This finding could be explained assuming that there may be different types of cellular interactions inside and outside the neoplastic follicles. No other statistically significant difference in terms of expression of these markers was observed in relation to clinical pathological features such as staging, grade and FLIPI score. In conclusion, extrafollicular high CD163/CD8 ratio is associated with POD24 in FL patients, this finding underlines the pathological significance of CD163-expresing macrophages in TME, suggesting this biomarker as a potential therapeutic target in this disease. Citation Format: Clara Bertuzzi, Maria Maddalena Tumedei, Sara Ravaioli, Claudio Agostinelli, Maurizio Puccetti, Sara Bravaccini, Simona Righi, Beatrice Casadei, Andrea Pession, Giovanni Martinelli, Giorgia Simonetti, Elena Sabattini, Serena De Matteis. Extrafollicular high CD163/CD8 ratio is associated with progression of disease within 24 months in follicular lymphoma patients [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 2682.

Introduction and aim: Childhood cytogenetically normal acute myeloid leukemia (CN-AML) is a subgroup of pediatric AML lacking any known cytogenetic and abnormality. CN-AML accounts for 20% of pediatric AML and exhibits a very heterogeneous response to treatment and, consequently, variable outcome. With the aim of providing new insights into the molecular lesions of this subset of AML, we analyzed, through RNA-seq, 13 cases of pediatric CN-AML, corroborating our findings in an independent cohort of 152 AML patients enrolled in the AIEOP AML 2002/01 clinical trial. Methods: Paired-end RNA-seq (75×2) was performed on PoliA(+) RNA extracted from blasts at diagnosis. ChimeraScan, deFuse and FusionMap algorithms were used for chimeric transcript detection. Results: An alteration that was present in 2 out 13 cases was a chimeric transcript involving the genes nucleoporin 98kDa (NUP98) and PHD finger protein 23 (PHF23), resulting from a cryptic translocation t(11;17)(p15;p13). Both patients showed an in-frame fusion between exon 13 of NUP98 and exon 4 of PHF23 that was confirmed by RT-PCR analysis and Sanger sequencing. To date, the cryptic translocation t(11;17)(p15;p13) was never described before in pediatric AML and has been reported only once in an adult AML patient (Reader, et al. Leukemia 2007). In both our patients the same breakpoint in PHF23 gene at the beginning of exon 4 was present, which was different from that present in the adult patient. To assess the incidence of NUP98-PHF23 fusion in pediatric CN-AML, then we studied by RT-PCR and Sanger sequencing a validation cohort of 152 CN-AML children negative for the most common recurrent cytogenetic and molecular lesions, i.e. those involving MLL, CBFB, NPM1 and FLT3. Overall, 2 out of the 152 CN-AML cases were found positive for NUP98-PHF23, demonstrating that this genomic aberrancy is not rare in pediatric CN-AML (tentative frequency 2.4%). Fluorescence in situ hybridization analysis confirmed the cryptic chromosomal translocation t(7;11)(p15;p13) leading to the fusion between NUP98 and PHF23 in all cases. Conclusions: Lately, genome-wide approaches have been widely used to investigate the mutational landscape of CN-AML in adults. However, the genetic profile of childhood CN-AML still needs to be defined. Here, for the first time, we report the identification of a NUP98-PHF23 chimeric transcript in pediatric CN-AML. Together with recently published data demonstrating that NUP98-PHF23 promoted leukemogenesis and the observation that treatment with inhibitors of PHD-domain-mediated H3K4me3 binding, such as disulfiram (an FDA-approved drug), could selectively killed cells expressing NUP98-PHF23 (Gough, et al Cancer Discovery 2014), our findings enforce the role of epigenetic regulators in pediatric AML and suggest screening for this fusion gene at diagnosis in CN-AML pediatric patients. Citation Format: Marco Togni, Riccardo Masetti, Martina Pigazzi, Annalisa Astolfi, Daniele Zama, Valentina Indio, Salvatore Serravalle, Elena Manara, Valeria Bisio, Sergio Rutella, Franca Fagioli, Giuseppe Basso, Andrea Pession, Franco Locatelli. NUP98-PHF23 is a novel fusion gene in pediatric cytogenetically normal acute myeloid leukemia. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr LB-211. doi:10.1158/1538-7445.AM2015-LB-211

Wilms Tumor (WT), originating through perturbation of developmental processes in embryonic kidney, is the most frequent pediatric genitourinary malignancy. Despite its overall survival rate has reached 90%, several patients develop relapses and, among them, approximately 50% will suffer of fatal disease. Hence, reduction of the relapse burden is key to improve WT outcome, and incorporation of novel prognostic markers is currently explored to achieve this goal. Chromosomal alterations and gene expression signatures in WT cohorts are thus examined to identify patients with risk disease. By evaluating gene expression profiles of 58 untreated primary tumor tissues from patients enrolled in the AIEOP multicenter protocol, we could identify four WT subtypes displaying enrichment for genes that were differentially expressed, and divergent clinical outcome. These patterns were validated against a previously published large WT cohort, confirming the accuracy of the prediction. A correlation analysis based on the expression of WT1, which deregulation is a crucial event in this disease, allowed us to verify known WT1 targets and to identify novel potential effectors implicated in this disease. However, although the majority or primary tumors from relapsing patients (7 out of 11) features lower WT1 level and increased 11p13 copy number (CN) losses, its expression did not significantly correlate with specific pathological parameters, confirming its poor prognostic impact as an independent disease predictor. Therefore, in order to reliably associate the risk of recurrence to a specific gene signature, we performed class comparison analysis of primary tumors between relapsing and non-relapsing patients. Unexpectedly, the pattern of genes up-regulated in relapsing tumors showed a negative enrichment of genes expressed in early embryonic kidney structures, such as the metanephric and cap mesenchyme, and a positive enrichment for genes expressed in nephrogenic structures developed upon mesenchymal-to-epithelial transition. These findings underscore the clinical heterogeneity of the disease and indicate that disruption of post-inductive renal processes occurs in high-risk WTs. Moreover, a few transcripts mapping at chromosome 1q21-44, with CN gain or allelic imbalance in 5/11 relapsing vs. 6/47 non-relapsing tumors, were found up-regulated in these patients. Remarkably, a few genes known to be implicated in metastatic disease in adult cancers, including SPP1, MUC1, MYC, CLDN1, and MAOA, showed up-regulation in relapsing WTs. Overall, our data suggests a gene signature associated to naive primary recidivant tumors, to be exploited as a potential predictor of disease at higher risk of relapse. Citation Format: Antonio Fiorino, Loris De Cecco, Beatrice Gamba, Edoardo Marchesi, Paola Collini, Andrea Pession, Marilina Nantron, Maurizio Bianchi, Filippo Spreafico, Silvana Canevari, Paolo Radice, Daniela Perotti. Gene expression associated to relapsing disease in Wilms tumor indicates a more differentiated phenotype unveiling a distinct transformation process for patients with a higher risk of relapse. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 3268. doi:10.1158/1538-7445.AM2015-3268