Lineage specific transcription factor waves reprogram neuroblastoma from self-renewal to differentiation.

Temporal regulation of super-enhancer (SE) driven transcription factors (TFs) underlies normal developmental programs. Neuroblastoma (NB) arises from an inability of sympathoadrenal progenitors to exit a self-renewal program and terminally differentiate. To identify SEs driving TF regulators, we use all-trans retinoic acid (ATRA) to induce NB growth arrest and differentiation. Time-course H3K27ac ChIP-seq and RNA-seq reveal ATRA coordinated SE waves. SEs that decrease with ATRA link to stem cell development (MYCN, GATA3, SOX11). CRISPR-Cas9 and siRNA verify SOX11 dependency, in vitro and in vivo. Silencing the SOX11 SE using dCAS9-KRAB decreases SOX11 mRNA and inhibits cell growth. Other TFs activate in sequential waves at 2, 4 and 8 days of ATRA treatment that regulate neural development (GATA2 and SOX4). Silencing the gained SOX4 SE using dCAS9-KRAB decreases SOX4 expression and attenuates ATRA-induced differentiation genes. Our study identifies oncogenic lineage drivers of NB self-renewal and TFs critical for implementing a differentiation program.

MYOD-SKP2 axis boosts tumorigenesis in fusion negative rhabdomyosarcoma by preventing differentiation through p57Kip2 targeting.

Rhabdomyosarcomas (RMS) are pediatric mesenchymal-derived malignancies encompassing PAX3/7-FOXO1 Fusion Positive (FP)-RMS, and Fusion Negative (FN)-RMS with frequent RAS pathway mutations. RMS express the master myogenic transcription factor MYOD that, whilst essential for survival, cannot support differentiation. Here we discover SKP2, an oncogenic E3-ubiquitin ligase, as a critical pro-tumorigenic driver in FN-RMS. We show that SKP2 is overexpressed in RMS through the binding of MYOD to an intronic enhancer. SKP2 in FN-RMS promotes cell cycle progression and prevents differentiation by directly targeting p27Kip1 and p57Kip2, respectively. SKP2 depletion unlocks a partly MYOD-dependent myogenic transcriptional program and strongly affects stemness and tumorigenic features and prevents in vivo tumor growth. These effects are mirrored by the investigational NEDDylation inhibitor MLN4924. Results demonstrate a crucial crosstalk between transcriptional and post-translational mechanisms through the MYOD-SKP2 axis that contributes to tumorigenesis in FN-RMS. Finally, NEDDylation inhibition is identified as a potential therapeutic vulnerability in FN-RMS.

MYCN Amplification, along with Wild-Type RB1 Expression, Enhances CDK4/6 Inhibitors' Efficacy in Neuroblastoma Cells.

Neuroblastoma (NB) is one of the primary causes of death for pediatric malignancies. Given the high heterogeneity in NB's mutation landscape, optimizing individualized therapies is still challenging. In the context of genomic alterations, MYCN amplification is the most correlated event with poor outcomes. MYCN is involved in the regulation of several cellular mechanisms, including cell cycle. Thus, studying the influence of MYCN overexpression in the G1/S transition checkpoint of the cell cycle may unveil novel druggable targets for the development of personalized therapeutical approaches. Here, we show that high expression of E2F3 and MYCN correlate with poor prognosis in NB despite the RB1 mRNA levels. Moreover, we demonstrate through luciferase reporter assays that MYCN bypasses RB function by incrementing E2F3-responsive promoter activity. We showed that MYCN overexpression leads to RB inactivation by inducing RB hyperphosphorylation during the G1 phase through cell cycle synchronization experiments. Moreover, we generated two MYCN-amplified NB cell lines conditionally knockdown (cKD) for the RB1 gene through a CRISPRi approach. Indeed, RB KD did not affect cell proliferation, whereas cell proliferation was strongly influenced when a non-phosphorylatable RB mutant was expressed. This finding revealed the dispensable role of RB in regulating MYCN-amplified NB's cell cycle. The described genetic interaction between MYCN and RB1 provides the rationale for using cyclin/CDK complexes inhibitors in NBs carrying MYCN amplification and relatively high levels of RB1 expression.

MYCN-driven fatty acid uptake is a metabolic vulnerability in neuroblastoma.

Neuroblastoma (NB) is a childhood cancer arising from sympatho-adrenal neural crest cells. MYCN amplification is found in half of high-risk NB patients; however, no available therapies directly target MYCN. Using multi-dimensional metabolic profiling in MYCN expression systems and primary patient tumors, we comprehensively characterized the metabolic landscape driven by MYCN in NB. MYCN amplification leads to glycerolipid accumulation by promoting fatty acid (FA) uptake and biosynthesis. We found that cells expressing amplified MYCN depend highly on FA uptake for survival. Mechanistically, MYCN directly upregulates FA transport protein 2 (FATP2), encoded by SLC27A2. Genetic depletion of SLC27A2 impairs NB survival, and pharmacological SLC27A2 inhibition selectively suppresses tumor growth, prolongs animal survival, and exerts synergistic anti-tumor effects when combined with conventional chemotherapies in multiple preclinical NB models. This study identifies FA uptake as a critical metabolic dependency for MYCN-amplified tumors. Inhibiting FA uptake is an effective approach for improving current treatment regimens.

Targeting Oncogenic Transcriptional Networks in Neuroblastoma: From N-Myc to Epigenetic Drugs.

Neuroblastoma (NB) is one of the most frequently occurring neurogenic extracranial solid cancers in childhood and infancy. Over the years, many pieces of evidence suggested that NB development is controlled by gene expression dysregulation. These unleashed programs that outline NB cancer cells make them highly dependent on specific tuning of gene expression, which can act co-operatively to define the differentiation state, cell identity, and specialized functions. The peculiar regulation is mainly caused by genetic and epigenetic alterations, resulting in the dependency on a small set of key master transcriptional regulators as the convergence point of multiple signalling pathways. In this review, we provide a comprehensive blueprint of transcriptional regulation bearing NB initiation and progression, unveiling the complexity of novel oncogenic and tumour suppressive regulatory networks of this pathology. Furthermore, we underline the significance of multi-target therapies against these hallmarks, showing how novel approaches, together with chemotherapy, surgery, or radiotherapy, can have substantial antineoplastic effects, disrupting a wide variety of tumorigenic pathways through combinations of different treatments.