Neuroblastoma (NB) is a highly aggressive extracranial solid tumor in children. Due to its heterogeneity, NB remains a therapeutic challenge. Several oncogenic factors, including the Hippo effectors YAP/TAZ, are associated with NB tumorigenesis. Verteporfin (VPF) is an FDA-approved drug shown to directly inhibit YAP/TAZ activity. Our study aimed to investigate VPF’s potential as a therapeutic agent in NB. We show that VPF selectively and efficiently impairs the viability of YAP/TAZ-expressing NB GI-ME-N and SK-N-AS cells, but not of non-malignant fibroblasts. To investigate whether VPF-mediated NB cell killing is YAP-dependent, we tested VPF potency in CRISPR-mediated YAP/TAZ knock-out GI-ME-N cells, and BE(2)-M17 NB cells (a MYCN-amplified, predominantly YAP-negative NB subtype). Our data shows that VPF-mediated NB cell killing is not dependent on YAP expression. Moreover, we determined that the formation of higher molecular weight (HMW) complexes is an early and shared VPF-induced cytotoxic mechanism in both YAP-positive and YAP-negative NB models. The accumulation of HMW complexes, involving STAT3, GM130 and COX IV proteins, impaired cell homeostasis and triggered cell stress and cell death mechanisms. Altogether, our study shows significant in vitro and in vivo VPF-induced suppression of NB growth, making VPF a potential therapeutic candidate against NB.

AIM: Pediatric low-grade gliomas (pLGGs) are a heterogenous group of tumors, diverse in their localization, histology, mutational landscape, clinical behavior, and treatment response. Genomic alterations impacting the MYB family of transcription factors were identified in two distinct pLGG subtypes: Angiocentric Gliomas (AG) and Diffuse Astrocytomas (DA). The molecular profiles and therapeutic vulnerabilities associated with these genomic alterations remain unexplored. In this study we highlight the use of genome-wide CRISPR/Cas9 knock-out screens for an unbiased identification of translatable therapeutic targets. METHODOLOGY: Given the lack of patient-derived pLGG cell lines, we engineered in vitro pLGG mouse and human neural stem cell (NSC) models to harbor pLGG-relevant genomic alterations. We performed single cell RNA sequencing to investigate the transcriptional profiles driven by these mutations and to dissect the central regulatory networks enabling tumorigenesis. Specific genetic dependencies associated with MYB/MYBL1 mutations were screened using the Brie genome-wide mouse CRISPR lentiviral knock-out pooled library, consisting of 78,637 single guide RNAs (sgRNAs) targeting 19,674 mouse genes. RESULTS: We have successfully generated in vitro NSC-based pLGG models crucial to deepening our knowledge on pLGG biology and the identification of translatable therapeutic targets. Genome-scale CRISPR/Cas9 knock-out screens in isogenic NSCs models, expressing distinct MYB/MYBL1 alterations or a control transgene, revealed several differential genetic dependencies. Among the top identified dependencies are regulators of cell-stress response, cell-cycle progression, and modulators of the ubiquitin-proteasome degradation pathway. CONCLUSION: Genome-wide CRISPR knock-out screens are a powerful tool for the unbiased identification of mutation-specific genetic dependencies that can be explored as candidates for precision medicine approaches.

Neuroblastoma (NB) is a highly aggressive extracranial solid tumor in children. Due to its heterogeneity, NB remains a therapeutic challenge. Several oncogenic factors, including the Hippo effectors YAP/TAZ, are associated with NB tumorigenesis. Verteporfin (VPF) is an FDA-approved drug shown to directly inhibit YAP/TAZ activity. Our study aimed to investigate VPF’s potential as a therapeutic agent in NB. We show that VPF selectively and efficiently impairs the viability of YAP/ TAZ-expressing NB GI-ME-N and SK-N-AS cells, but not of normal fibroblasts. To investigate whether VPF-mediated NB cell killing is YAP-dependent, we tested VPF potency in CRISPR-mediated YAP/TAZ knock-out GI-ME-N cells, as well as YAP-negative BE(2)-M17 NB cells. Our data shows that VPF-mediated NB cell killing is not dependent on YAP expression. Moreover, we determined that the formation of high molecular weight (HMW) complexes is an early and shared VPF-induced cytotoxic mechanism in both YAP-positive and YAP-negative NB models. The accumulation of HMW complexes, involving STAT3, GM130 and COX IV proteins, impaired cell homeostasis and triggered cell stress and cell death mechanisms. Altogether, our study shows significant in vitro and in vivo VPF-induced suppression of NB growth, making VPF a potential therapeutic candidate against NB.

AIM Recurrent structural variants involving MYB and MYBL1 transcription factors were recently identified in pediatric low-grade gliomas (pLGGs), such as the MYB-QKI rearrangement in Angiocentric Gliomas and truncations of MYBL1 (MYBL1tr) in Diffuse Astrocytomas. However, therapeutic dependencies induced by these alterations remain unexplored. METHODOLOGY We have generated in vitro pLGG mouse neural stem cell (NSCs) models engineered to harbor distinct MYB/MYBL1 genomic alterations. We used single cell RNA sequencing approaches to determine the transcriptional profile and dissect the central regulatory networks of our in vitro pLGG models over time. To identify specific genetic dependencies associated with MYB/MYBL1 mutations, we employed the Brie genome-wide mouse CRISPR lentiviral knockout pooled library, consisting of 78,637 single guide RNAs (sgRNAs) targeting 19,674 mouse genes. RESULTS MYB/MYBL1 expression in neural stem cells induced activation of cell-cycle related, glioma-related and senescence-related pathways that are involved in normal development, including activation of MAPK and mTOR signaling which are also activated in human pLGG samples. Genome-scale CRISPR-cas9 screens in isogenic NSCs expressing MYB-QKI or MYBL1tr identified differential genetic dependencies relative to GFP controls. These included regulators of cell-cycle progression and several modulators of the ubiquitin-proteasome degradation pathway. Analysis of RNA-sequencing data from human tumors revealed several of these dependencies identified in the cell line model to be differentially expressed in MYB-altered pLGG tumors relative to normal brain. CONCLUSION Expression of MYB family alterations induces expression of key developmental and oncogenic pathways and genetic dependencies that represent potential therapeutic targets for MYB or MYBL1 rearranged pLGGs.