Characterization of a novel OTX2‐driven stem cell program in Group 3 and Group 4 medulloblastoma

Abstract

Medulloblastoma (MB) is the most common malignant primary pediatric brain cancer. Among the most aggressive subtypes, Group 3 and Group 4 originate from stem/progenitor cells, frequently metastasize, and often display the worst prognosis, yet we know the least about the molecular mechanisms driving their progression. Here, we show that the transcription factor orthodenticle homeobox 2 (OTX2) promotes self‐renewal while inhibiting differentiation in vitro and increases tumor initiation from MB stem/progenitor cells in vivo. To determine how OTX2 contributes to these processes, we employed complementary bioinformatic approaches to characterize the OTX2 regulatory network and identified novel relationships between OTX2 and genes associated with neuronal differentiation and axon guidance signaling in Group 3 and Group 4 MB stem/progenitor cells. In particular, OTX2 levels were negatively correlated with semaphorin (SEMA) signaling, as expression of 9 SEMA pathway genes is upregulated following OTX2 knockdown with some being potential direct OTX2 targets. Importantly, this negative correlation was also observed in patient samples, with lower expression of SEMA4D associated with poor outcome specifically in Group 4 tumors. Functional proof‐of‐principle studies demonstrated that increased levels of select SEMA pathway genes are associated with decreased self‐renewal and growth in vitro and in vivo and that RHO signaling, known to mediate the effects of SEMA genes, is contributing to the OTX2 KD phenotype. Our study provides mechanistic insight into the networks controlled by OTX2 in MB stem/progenitor cells and reveals novel roles for axon guidance genes and their downstream effectors as putative tumor suppressors in MB.

Modeling medulloblastoma in vivo and with human cerebellar organoids

“…2i-l), suggesting that our new Group 3 mouse models can recapitulate the malignant phenotype of human MB. Interestingly, OTX2 is overexpressed/amplified in human Group 3 MB and has been already found to be required for the tumorigenesis of MB cell lines [29][30][31] . Here, we have demonstrated that Otx2 is able to induce MB in vivo from mouse cerebellar progenitors that have not been manipulated outside the mouse brain.…”

An OTX2-PAX3 signaling axis regulates Group 3 medulloblastoma cell fate

“…Increased PAX3 expression has similar functional consequences to OTX2 silencing, including reduced self-renewal and proliferation 24 , 25 . Therefore, we predicted that OTX2 silencing would also decrease mTORC1 activity.…”

“…Based on this integrated data, genes containing diffReps identified H3K4me3 differentially enriched regions in the −5 kb to +2 kb regions around the TSS and H3K27me3-enriched regions over the gene body were generated, and the gene list submitted to Ingenuity Pathway Analysis ( https://www.qiagenbioinformatics.com/products/ingenuity-pathway-analysis ) to determine enriched pathways and Gene Ontology terms. In addition, OTX2 motifs were identified in OTX2 peaks 25 using the following motif patterns (TCTAATCCC, TAATCC, TAAGCC, TAATCT, TAACCC) available as a part of the publication UCSC track hub ( http://dropbox.ogic.ca/trackhubs/ogilvie/hub.txt ). For patient histone ChIP-sequencing data 22 , the GEO series GSE92585 provided wiggle files of mean read depth over 25 bp windows across the genome.…”

“…We have previously characterized a critical role for OTX2 in controlling cell fate decisions in Group 3 MB 24 , 25 . OTX2 silencing is accompanied by a robust increase in the expression of axon guidance genes, suggesting that OTX2 actively represses differentiation while maintaining Group 3 MB cells in a primitive, stem/progenitor cell state 25 . However, the majority of axon guidance genes identified were found to be indirect targets of OTX2 25 .…”

“…OTX2 silencing is accompanied by a robust increase in the expression of axon guidance genes, suggesting that OTX2 actively represses differentiation while maintaining Group 3 MB cells in a primitive, stem/progenitor cell state 25 . However, the majority of axon guidance genes identified were found to be indirect targets of OTX2 25 . The mechanisms by which OTX2 inhibits differentiation of Group 3 MB cells are largely unknown.…”