Eveline Barbieri

MDM2 inhibition sensitizes neuroblastoma to chemotherapy-induced apoptotic cell death

Novel therapeutic approaches are urgently needed for high-stage neuroblastoma, a major therapeutic challenge in pediatric oncology. The majority of neuroblastoma tumors are p53 wild type with intact downstream p53 signaling pathways. We hypothesize that stabilization of p53 would sensitize this aggressive tumor to genotoxic chemotherapy via inhibition of MDM2, the primary negative upstream regulator of p53. We used pharmacologic inhibition of the MDM2-p53 interaction with the small-molecule inhibitor Nutlin and studied the subsequent response to chemotherapy in neuroblastoma cell lines. We did 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide and terminal deoxynucleotidyl transferase assays to measure proliferation and apoptosis in several cell lines (IMR32, MYCN3, and JF) treated with combinations of cisplatin, etoposide, and Nutlin. We found consistent and robust decreases in proliferation and increases in apoptosis with the addition of Nutlin 3a to etoposide or cisplatin in all cell lines tested and no response to the inactive Nutlin 3b enantiomer. We also show a rapid and robust accumulation of p53 protein by Western blot in these cells within 1 to 2 hours of treatment. We conclude that MDM2 inhibition dramatically enhances the activity of genotoxic drugs in neuroblastoma and should be considered as an adjuvant to chemotherapy for this aggressive pediatric cancer and for possibly other p53 wild-type solid tumors. [Mol Cancer Ther 2006;5(9):2358–65]

A Genome-Wide Search for Promoters That Respond to Increased MYCN Reveals Both New Oncogenic and Tumor Suppressor MicroRNAs Associated with Aggressive Neuroblastoma

MYCN is a major driver of neuroblastoma tumorigenesis and MYCN amplification is the worst prognostic indicator of aggressive NB. To identify potentially therapeutic tumor suppressor microRNAs for aggressive NB, we utilized a conditional MYCN system to simulate MYCN-amplified and nonamplified tumor types and performed a genome-wide search for MYCN target microRNA promoters differentially repressed under high MYCN conditions. We identified 20 gene promoters hosting 30 microRNAs that were directly bound and differentially regulated by MYCN. Eleven of these genes showed significant clinical correlations for neuroblastoma with 4 genes linked with better survival and 7 genes linked with poor survival. Surprisingly, expression analysis of host genes and microRNAs demonstrated that 8 of 11 pairs were repressed by high levels of MYCN regardless of the clinical correlation of the host gene. We therefore predicted these intronic microRNAs would be tumor suppressors. In fact, detailed gain of function studies for two miRs, miR-591 and miR-558, confirmed potent tumor suppressive effects for miR-591 in orthotopic neuroblastoma xenografts. However, miR-558 markedly increased colony formation, proliferation, and tumor growth in vivo. Our data reveal host-gene independent functions of MYCN-target microRNAs and demonstrate that MYCN represses both tumor suppressive and proproliferative microRNAs.

G-CSF Receptor Positive Neuroblastoma Subpopulations Are Enriched in Chemotherapy-Resistant or Relapsed Tumors and Are Highly Tumorigenic

Neuroblastoma is a neural crest-derived embryonal malignancy, which accounts for 13% of all pediatric cancer mortality, primarily due to tumor recurrence. Therapy-resistant cancer stem cells are implicated in tumor relapse, but definitive phenotypic evidence of the existence of these cells has been lacking. In this study, we define a highly tumorigenic subpopulation in neuroblastoma with stem cell characteristics, based on the expression of CSF3R, which encodes the receptor for granulocyte colony-stimulating factor (G-CSF). G-CSF receptor positive (aka G-CSFr(+) or CD114(+)) cells isolated from a primary tumor and the NGP cell line by flow cytometry were highly tumorigenic and capable of both self-renewal and differentiation to progeny cells. CD114(+) cells closely resembled embryonic and induced pluripotent stem cells with respect to their profiles of cell cycle, miRNA, and gene expression. In addition, they reflect a primitive undifferentiated neuroectodermal/neural crest phenotype revealing a developmental hierarchy within neuroblastoma tumors. We detected this dedifferentiated neural crest subpopulation in all established neuroblastoma cell lines, xenograft tumors, and primary tumor specimens analyzed. Ligand activation of CD114 by the addition of exogenous G-CSF to CD114(+) cells confirmed intact STAT3 upregulation, characteristic of G-CSF receptor signaling. Together, our data describe a novel distinct subpopulation within neuroblastoma with enhanced tumorigenicity and a stem cell-like phenotype, further elucidating the complex heterogeneity of solid tumors such as neuroblastoma. We propose that this subpopulation may represent an additional target for novel therapeutic approaches to this aggressive pediatric malignancy.

A p53 drug response signature identifies prognostic genes in high-risk neuroblastoma.

Chemotherapy induces apoptosis and tumor regression primarily through activation of p53-mediated transcription. Neuroblastoma is a p53 wild type malignancy at diagnosis and repression of p53 signaling plays an important role in its pathogenesis. Recently developed small molecule inhibitors of the MDM2-p53 interaction are able to overcome this repression and potently activate p53 dependent apoptosis in malignancies with intact p53 downstream signaling. We used the small molecule MDM2 inhibitor, Nutlin-3a, to determine the p53 drug response signature in neuroblastoma cells. In addition to p53 mediated apoptotic signatures, GSEA and pathway analysis identified a set of p53-repressed genes that were reciprocally over-expressed in neuroblastoma patients with the worst overall outcome in multiple clinical cohorts. Multifactorial regression analysis identified a subset of four genes (CHAF1A, RRM2, MCM3, and MCM6) whose expression together strongly predicted overall and event-free survival (p<0.0001). The expression of these four genes was then validated by quantitative PCR in a large independent clinical cohort. Our findings further support the concept that oncogene-driven transcriptional networks opposing p53 activation are essential for the aggressive behavior and poor response to therapy of high-risk neuroblastoma.

Histone Chaperone CHAF1A Inhibits Differentiation and Promotes Aggressive Neuroblastoma

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.

Dual targeting of MDM2 and BCL2 as a therapeutic strategy in neuroblastoma

Wild-type p53 tumor suppressor activity in neuroblastoma tumors is hampered by increased MDM2 activity, making selective MDM2 antagonists an attractive therapeutic strategy for this childhood malignancy. Since monotherapy in cancer is generally not providing long-lasting clinical responses, we here aimed to identify small molecule drugs that synergize with idasanutlin (RG7388). To this purpose we evaluated 15 targeted drugs in combination with idasanutlin in three p53 wild type neuroblastoma cell lines and identified the BCL2 inhibitor venetoclax (ABT-199) as a promising interaction partner. The venetoclax/idasanutlin combination was consistently found to be highly synergistic in a diverse panel of neuroblastoma cell lines, including cells with high MCL1 expression levels. A more pronounced induction of apoptosis was found to underlie the synergistic interaction, as evidenced by caspase-3/7 and cleaved PARP measurements. Mice carrying orthotopic xenografts of neuroblastoma cells treated with both idasanutlin and venetoclax had drastically lower tumor weights than mice treated with either treatment alone. In conclusion, these data strongly support the further evaluation of dual BCL2/MDM2 targeting as a therapeutic strategy in neuroblastoma.Wild-type p53 tumor suppressor activity in neuroblastoma tumors is hampered by increased MDM2 activity, making selective MDM2 antagonists an attractive therapeutic strategy for this childhood malignancy. Since monotherapy in cancer is generally not providing long-lasting clinical responses, we here aimed to identify small molecule drugs that synergize with idasanutlin (RG7388). To this purpose we evaluated 15 targeted drugs in combination with idasanutlin in three p53 wild type neuroblastoma cell lines and identified the BCL2 inhibitor venetoclax (ABT-199) as a promising interaction partner. The venetoclax/idasanutlin combination was consistently found to be highly synergistic in a diverse panel of neuroblastoma cell lines, including cells with high MCL1 expression levels. A more pronounced induction of apoptosis was found to underlie the synergistic interaction, as evidenced by caspase-3/7 and cleaved PARP measurements. Mice carrying orthotopic xenografts of neuroblastoma cells treated with both idasanutlin and venetoclax had drastically lower tumor weights than mice treated with either treatment alone. In conclusion, these data strongly support the further evaluation of dual BCL2/MDM2 targeting as a therapeutic strategy in neuroblastoma.

Depletion of tRNA-halves enables effective small RNA sequencing of low-input murine serum samples

The ongoing ascent of sequencing technologies has enabled researchers to gain unprecedented insights into the RNA content of biological samples. MiRNAs, a class of small non-coding RNAs, play a pivotal role in regulating gene expression. The discovery that miRNAs are stably present in circulation has spiked interest in their potential use as minimally-invasive biomarkers. However, sequencing of blood-derived samples (serum, plasma) is challenging due to the often low RNA concentration, poor RNA quality and the presence of highly abundant RNAs that dominate sequencing libraries. In murine serum for example, the high abundance of tRNA-derived small RNAs called 5′ tRNA halves hampers the detection of other small RNAs, like miRNAs. We therefore evaluated two complementary approaches for targeted depletion of 5′ tRNA halves in murine serum samples. Using a protocol based on biotinylated DNA probes and streptavidin coated magnetic beads we were able to selectively deplete 95% of the targeted 5′ tRNA half molecules. This allowed an unbiased enrichment of the miRNA fraction resulting in a 6-fold increase of mapped miRNA reads and 60% more unique miRNAs detected. Moreover, when comparing miRNA levels in tumor-carrying versus tumor-free mice, we observed a three-fold increase in differentially expressed miRNAs.

p53 Nongenotoxic Activation and mTORC1 Inhibition Lead to Effective Combination for Neuroblastoma Therapy

Purpose: mTORC1 inhibitors are promising agents for neuroblastoma therapy; however, they have shown limited clinical activity as monotherapy, thus rational drug combinations need to be explored to improve efficacy. Importantly, neuroblastoma maintains both an active p53 and an aberrant mTOR signaling. Experimental Design: Using an orthotopic xenograft model and modulating p53 levels, we investigated the antitumor effects of the mTORC1 inhibitor temsirolimus in neuroblastoma expressing normal, decreased, or mutant p53, both as single agent and in combination with first- and second-generation MDM2 inhibitors to reactivate p53. Results: Nongenotoxic p53 activation suppresses mTOR activity. Moreover, p53 reactivation via RG7388, a second-generation MDM2 inhibitor, strongly enhances the in vivo antitumor activity of temsirolimus. Single-agent temsirolimus does not elicit apoptosis, and tumors rapidly regrow after treatment suspension. In contrast, our combination therapy triggers a potent apoptotic response in wild-type p53 xenografts and efficiently blocks tumor regrowth after treatment completion. We also found that this combination uniquely led to p53-dependent suppression of survivin whose ectopic expression is sufficient to rescue the apoptosis induced by our combination. Conclusions: Our study supports a novel highly effective strategy that combines RG7388 and temsirolimus in wild-type p53 neuroblastoma, which warrants testing in early-phase clinical trials. Clin Cancer Res; 23(21); 6629–39. ©2017 AACR.

Circulating microRNA biomarkers for metastatic disease in neuroblastoma patients

In this study, the circulating miRNome from diagnostic neuroblastoma serum was assessed for identification of non-invasive biomarkers with potential in monitoring metastatic disease. After determining the circulating neuroblastoma miRNome, 743 miRNAs were screened in two independent cohorts of 131 and 54 patients. Evaluation of serum miRNA variance in a model testing for tumor stage, MYCN status, age at diagnosis and overall survival, revealed tumor stage as the most significant factor impacting miRNA abundance in neuroblastoma serum. Differential expression analysis between patients with metastatic and localized disease revealed 9 miRNAs strongly associated with metastatic stage 4 disease in both patient cohorts. Increasing levels of these miRNAs were also observed in serum from xenografted mice bearing human neuroblastoma tumors. Moreover, murine serum miRNA levels were strongly associated with tumor volume, suggesting this miRNA signature may be applied to monitor disease burden.

Abstract A59: CHAF1A blocks neuroblastoma differentiation and promotes tumor growth via metabolic reprogramming

Neuroblastoma (NB), an embryonal tumor arising in tissues of the sympathetic nervous system, accounts for 13% of all deaths due to childhood malignancies. NB arises from embryonal neural crest secondary to a block in differentiation and long-term survival inversely correlates with the degree of neuronal differentiation. Importantly, treatment with differentiation agents, such as retinoic acid (RA), has modestly improved survival. We have recently demonstrated a novel function for the histone chaperone CHAF1A in promoting tumor progression by blocking NB differentiation. CHAF1A is a MYCN target gene and a subunit of the Chromatin Assembly Factor-1 (CAF1) which regulates H3K9-trimethylation and DNA methylation. Here, in order to define the mechanism for CHAF1A-mediated inhibition of differentiation, we performed transcriptome analyses (Affymetrix U133+2.0 arrays) in NB cells upon CHAF1A silencing. Notably, gene set enrichment analysis (GSEA) revealed repression of oncogenic pathways and enrichment of cell metabolism pathways (valine, leucine, and isoleucine degradation, glutamate metabolism and insulin pathways). Furthermore, Q-PCR confirmed that CHAF1A significantly affects the expression of critical genes regulating glucose uptake. Based on these findings and the fact that cancer cells have defective mitochondria, which forces them to depend on aerobic glycolysis, we then examined the sensitivity of NB cells to different glycolytic inhibitors. We hypothesized that targeting glucose metabolism will decrease NB cell growth and restore sensitivity to RA. The effect of the glycolytic inhibitors metformin, dichloroacetate (DCA), and 2-Deoxy-D- glucose (2-DG) on cell proliferation was determined by MTT-Assay in several NB lines under normoxic and hypoxic conditions (1% O2). 2-DG treatment significantly blocked cell proliferation in all NB lines tested, with enhanced effect under hypoxic conditions. However, metformin and DCA9s anti-proliferative effects were dependent on MYCN status and levels of O2 deprivation. Notably, MYCN non-amplified lines showed greater sensitivity to metformin and DCA under normoxia than under hypoxic conditions (IC50 normoxia: 6.25 mM vs. IC50 hypoxia: > 50 mM). In contrast, MYCN-amplified cell lines maintained high proliferation under hypoxia and revealed high sensitivity to metformin and DCA both under normoxic and hypoxic conditions (IC50 normoxia: 25 mM vs. IC50 hypoxia: 22.5 mM). In conclusion, we have shown that: 1) the differentiation of NB cells upon CHAF1A silencing induces metabolic reprogramming by affecting genes controlling glucose uptake, and 2) NB cells are extremely sensitive to glycolytic inhibition, dependently on MYCN status and O2 levels. A complete characterization of the metabolic changes occurring in NB cells with high and low CHAF1A expression, as well as a definition of MYCN regulation of mitochondrial functions under hypoxic conditions is currently undergoing. This will help identify vulnerable metabolic points to block NB cell growth and guide the development of novel differentiating therapies. Citation Format: Myrthala Moreno-Smith, Zaowen Chen, Eugene S. Kim, Jason M. Shohet, Eveline Barbieri. CHAF1A blocks neuroblastoma differentiation and promotes tumor growth via metabolic reprogramming. [abstract]. In: Proceedings of the AACR Special Conference: Metabolism and Cancer; Jun 7-10, 2015; Bellevue, WA. Philadelphia (PA): AACR; Mol Cancer Res 2016;14(1_Suppl):Abstract nr A59.