Zaowen Chen

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.

G-CSF promotes neuroblastoma tumorigenicity and metastasis via STAT3-dependent cancer stem cell activation

Increasing evidence suggests that inflammatory cytokines play a critical role in tumor initiation and progression. A cancer stem cell (CSC)-like subpopulation in neuroblastoma is known to be marked by expression of the G-CSF receptor (G-CSFR). Here, we report on the mechanistic contributions of the G-CSFR in neuroblastoma CSCs. Specifically, we demonstrate that the receptor ligand G-CSF selectively activates STAT3 within neuroblastoma CSC subpopulations, promoting their expansion in vitro and in vivo. Exogenous G-CSF enhances tumor growth and metastasis in human xenograft and murine neuroblastoma tumor models. In response to G-CSF, STAT3 acts in a feed-forward loop to transcriptionally activate the G-CSFR and sustain neuroblastoma CSCs. Blockade of this G-CSF-STAT3 signaling loop with either anti-G-CSF antibody or STAT3 inhibitor depleted the CSC subpopulation within tumors, driving correlated tumor growth inhibition, decreased metastasis, and increased chemosensitivity. Taken together, our results define G-CSF as a CSC-activating factor in neuroblastoma, suggest a comprehensive reevaluation of the clinical use of G-CSF in these patients to support white blood cell counts, and suggest that direct targeting of the G-CSF-STAT3 signaling represents a novel therapeutic approach for neuroblastoma.

MYCN-directed centrosome amplification requires MDM2-mediated suppression of p53 activity in neuroblastoma cells.

The MYC family oncogenes cause transformation and tumor progression by corrupting multiple cellular pathways, altering cell cycle progression, apoptosis, and genomic instability. Several recent studies show that MYCC (c-Myc) expression alters DNA repair mechanisms, cell cycle checkpoints, and karyotypic stability, and this is likely partially due to alterations in centrosome replication control. In neuroblastoma cell lines, MYCN (N-Myc) expression induces centrosome amplification in response to ionizing radiation. Centrosomes are cytoplasmic domains that critically regulate cytokinesis, and aberrations in their number or structure are linked to mitotic defects and karyotypic instability. Whereas centrosome replication is linked to p53 and Rb/E2F-mediated cell cycle progression, the mechanisms downstream of MYCN that generate centrosome amplification are incompletely characterized. We hypothesized that MDM2, a direct transcriptional target of MYCN with central inhibitory effects on p53, plays a role in MYC-mediated genomic instability by altering p53 responses to DNA damage, facilitating centrosome amplification. Herein we show that MYCN mediates centrosome amplification in a p53-dependent manner. Accordingly, inhibition of the p53-MDM2 interaction with Nutlin 3A (which activates p53) completely ablates the MYCN-dependent contribution to centrosome amplification after ionizing radiation. We further show that modulating MDM2 expression levels by overexpression or RNA interference-mediated posttranscriptional inhibition dramatically affects centrosome amplification in MYCN-induced cells, indicating that MDM2 is a necessary and sufficient mediator of MYCN-mediated centrosome amplification. Finally, we show a significant correlation between centrosome amplification and MYCN amplification in primary neuroblastoma tumors. These data support the hypothesis that elevated MDM2 levels contribute to MYCN-induced genomic instability through altered regulation of centrosome replication in neuroblastoma.

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.

Transmembrane adaptor protein PAG1 is a novel tumor suppressor in neuroblastoma

(NB) is the most common extracranial pediatric solid tumor with high mortality rates. The tyrosine kinase c-Src has been known to play an important role in differentiation of NB cells, but the mechanism of c-Src regulation has not been defined. Here, we characterize PAG1 (Cbp, Csk binding protein), a central inhibitor of c-Src and other Src family kinases, as a novel tumor suppressor in NB. Clinical cohort analysis demonstrate that low expression of PAG1 is a significant prognostic factor for high stage disease, increased relapse, and worse overall survival for children with NB. PAG1 knockdown in NB cells promotes proliferation and anchorage-independent colony formation with increased activation of AKT and ERK downstream of c-Src, while PAG1 overexpression significantly rescues these effects. In vivo, PAG1 overexpression significantly inhibits NB tumorigenicity in an orthotopic xenograft model. Our results establish PAG1 as a potent tumor suppressor in NB by inhibiting c-Src and downstream effector pathways. Thus, reactivation of PAG1 and inhibition of c-Src kinase activity represents an important novel therapeutic approach for high-risk NB.

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.

MYCN acts as a direct co-regulator of p53 in MYCN amplified neuroblastoma

The MYC oncogenes and p53 have opposing yet interrelated roles in normal development and tumorigenesis. How MYCN expression alters the biology and clinical responsiveness of pediatric neuroblastoma remains poorly defined. Neuroblastoma is p53 wild type at diagnosis and repression of p53 signaling is required for tumorigenesis. Here, we tested the hypothesis that MYCN amplification alters p53 transcriptional activity in neuroblastoma. Interestingly, we found that MYCN directly binds to the tetrameric form of p53 at its C-terminal domain, and this interaction is independent of MYCN/MAX heterodimer formation. Chromatin analysis of MYCN and p53 targets reveals dramatic changes in binding, as well as co-localization of the MYCN-p53 complex at p53-REs and E-boxes of genes critical to DNA damage responses and cell cycle progression. RNA sequencing studies show that MYCN-p53 co-localization significantly modulated the expression of p53 target genes. Furthermore, MYCN-p53 interaction leads to regulation of alternative p53 targets not regulated in the presence of low MYCN levels. These novel targets include a number of genes involved in lipid metabolism, DNA repair, and apoptosis. Taken together, our findings demonstrate a novel oncogenic role of MYCN as a transcriptional co-regulator of p53 in high-risk MYCN amplified neuroblastoma. Targeting this novel oncogenic function of MYCN may enhance p53-mediated responses and sensitize MYCN amplified tumors to chemotherapy.