Recurrent somatic driver events
Group 3 and group 4 MBs are genetically heterogeneous and, unlike WNT and SHH-activated MBs, are not driven by well-defined, constitutively activated signaling pathways. Tetraploidy is a recurrent early genetic event in both group 3 and group 4 MBs, leading to an increased number of large-scale copy number gains [
38]. A meta-analysis based on 550 samples identified a gain of 17q (in 58% of samples) and loss of 17p (55%) along with a
loss of 16q (42%),
10q (43%), and
9q (21%) and
gain of 7 (39%) and
1q (41%) as most recurrent structural aberrations in Group 3 MBs [
10] (Table
1). Tetraploidy also occurs early in approximately 40% of group 4 tumors [
38], but its prognostic significance is yet unclear.
Isochromosome 17q (a chromosome with two 17q arms) is present in about 80% of all group 4 samples but is not predictive of outcome. Chromosome
7 gain (47%),
8p loss (41%),
10q loss (15%), and
11p and
18q aberrations are also regular events (Table
2). Approximately 80% of females have a complete loss of one
X chromosome [
10,
12,
18,
39]. Both group 3 and group 4 MBs harbor frequent chromosomal aberrations although somatic mutations are relatively infrequent. In fact, more than half of group 3 samples are thought to be devoided of mutations; based on deep sequencing of 92 samples, none of the 12 most significantly mutated genes were altered in group 3 and group 4 tumors [
21,
40].
Table 1
Frequent genetic alterations in group 3 MBs according to [
6,
12,
28,
38,
40,
113]
58 | 17q | Mainly gain | – | – | – |
55 | 17p | Mainly loss | – | – | – |
55 | 8q | Gain or loss | – | – | – |
51 | 8p | Gain or loss | – | – | – |
48 | 7q | Mainly gain | – | – | – |
43 | 10q | Mainly loss | – | – | – |
42 | 16q | Mainly loss | – | – | – |
41 | 1q | Mainly gain | – | – | – |
39 | 7p | Mainly gain | – | – | – |
38 | 13q | Gain or loss | – | – | – |
34 | 11q | Mainly loss | – | – | – |
32 | 11p | Mainly loss | – | – | – |
31 | 5q | Mainly gain | – | – | – |
30 | 5p | Mainly gain | – | – | – |
21 | X | Loss | – | – | – |
17 | MYC | Amplification, overexpression | MYC proto-oncogene, bHLH transcription factor | 8q24.21 | Transcriptional regulation |
12 | PVT1 | Amplification | Pvt1 oncogene (non-protein coding) | 8q24.21 | Oncogenic lncRNA |
11 | GFI1B | overexpression, amplification, deletion | Growth factor independent 1B transcriptional repressor | 9q34.13 | Transcriptional regulation |
9 | SMARCA4 | Mutation | SWI/SNF-related, matrix-associated, actin-dependent regulator of chromatin, subfamily a, member 4 | 19p13.2 | Chromatin modulation, SWI/SNF Nucleosome-remodeling complex |
6 | KBTBD4 | Mutation | Kelch repeat and BTB domain containing 4 | 11p11.2 | Ubiquitination of target substrates |
6 | SHPRH | Low level amplification | SNF2 histone linker PHD RING helicase | 6q24.3 | Genome maintenance |
5 | CD109 | Deletion | CD109 molecule | 6q13 | TGF-β signaling |
5 | CTDNEP1 | Mutation | CTD nuclear envelope phosphatase 1 | 17p13.1 | Metabolism of fatty acids |
5 | KMT2D | Mutation | Lysine methyltransferase 2D | 12q13.12 | Chromatin modulation |
5 | KDM7A | Mutation | Lysine demethylase 7A | 7q34 | Chromatin modulation |
5 | CHD7 | Mutation | Chromodomain helicase DNA binding protein 7 | 8q12.2 | Chromatin modulation |
5 | DDX3X | Mutation | DEAD-box helicase 3, X-linked | Xp11.4 | RNA metabolism |
5 | KDM3A | Mutation | Lysine demethylase 3A | 2p11.2 | Chromatin modulation |
5 | KDM4C | Mutation | Lysine demethylase 4C | 9p24.1 | Chromatin modulation |
5 | KDM5B | Mutation | Lysine demethylase 5B | 1q32.1 | Chromatin modulation |
5 | KDM6A | Mutation | Lysine demethylase 6A | Xp11.3 | Chromatin modulation |
5 | MYCN | Amplification | MYCN proto-oncogene, bHLH transcription factor | 2p24.3 | Transcriptional regulation |
5 | CREBBP | Amplification | CREB binding protein | 16p13.3 | Chromatin modulation, transcription initiation |
5 | DDX31 | Amplification | DEAD-box helicase 31 | 9q34.13 | RNA metabolism |
4 | ESRRG | Low level amplification | Estrogen-related receptor gamma | 1q41 | Transcriptional regulation, estrogen signaling |
4 | SNX6 | Deletion | Sorting nexin 6 | 14q13.1 | TGF-β signaling |
4 | GFI1 | Overexpression, amplification | Growth factor independent 1 transcriptional repressor | 1p22.1 | Transcriptional regulation |
3 | OTX2 | Amplification, overexpression | Orthodenticle homeobox 2 | 14q22.3 | Transcriptional regulation |
3 | FKBP1A | Deletion | FK506 binding protein 1A | 20p13 | TGF-β signaling |
3 | CDK6 | Amplification | Cyclin-dependent kinase 6 | 7q21.2 | Cell cycle |
2 | ACVR2A | Amplification | Activin A receptor type 2A | 2q22.3-q23.1 | TGF-β signaling |
2 | TGFBR1 | Amplification | Transforming growth factor beta receptor 1 | 9q22.33 | TGF-β signaling |
2 | BRCA2 | Mutation | BRCA2, DNA repair associated | 13q13.1 | Genome maintenance |
1 | ACVR2B | Amplification | Activin A receptor type 2B | 3p22.2 | TGF-β signaling |
1 | E2F5 | Amplification | E2F transcription factor 5 | 8q21.2 | Transcriptional regulation |
– | FOXG1 | Overexpression | Forkhead box G1 | 14q12 | Transcriptional regulation |
– | IMPG2 | Overexpression | Interphotoreceptor matrix proteoglycan 2 | 3q12.3 | Proteoglycan |
– | GABRA5 | Overexpression | Gamma-aminobutyric acid type A receptor alpha5 subunit | 15q12 | Neurotransmission |
– | EGFL11 | Overexpression | Eyes shut homolog (Drosophila) | 6q12 | Cell signaling |
– | NRL | Overexpression | Neural retina leucine zipper | 14q11.2-q12 | Transcriptional regulation |
– | MAB21L2 | Overexpression | Mab-21 like 2 | 4q31.3 | TGF-β signaling, neural development |
– | NPR3 | Overexpression | Natriuretic peptide receptor 3 | 5p13.3 | Natriuretic peptide metabolism |
Table 2
Frequent genetic alterations in group 4 MBs according to [
6,
12,
28,
38,
40,
113]
86 | 17q | Mainly gain | – | – | – |
79 | 17p | Mainly loss | – | – | – |
54 | 7q | Mainly gain | – | – | – |
50 | 8p | Loss | – | – | – |
43 | 7p | Mainly gain | – | – | – |
43 | 8q | Loss | – | – | – |
32 | 11p | Loss | – | – | – |
28 | 11q | Mainly loss | – | – | – |
21 | X | Loss | – | – | – |
17 | PRDM6 | Amplification, overexpression | PR/SET domain 6 | 5q23.2 | Chromatin modulation |
10 | SNCAIP | Tandem duplication | Synuclein alpha interacting protein | 5q23.2 | Chromatin modulation |
9 | GFI1B | Amplification, overexpression, deletion | Growth factor independent 1B transcriptional repressor | 9q34.13 | Transcriptional regulation |
8 | DDX31 | Deletion | DEAD-box helicase 31 | 9q34.13 | RNA metabolism |
8 | MYC | Amplification | MYC proto-oncogene, bHLH transcription factor | 8q24.21 | Transcriptional regulation |
8 | CHD7 | Mutation | Chromodomain helicase DNA binding protein 7 | 8q12.2 | Chromatin modulation |
8 | DDX31 | Mutation | DEAD-box helicase 31 | 9q34.13 | RNA metabolism |
7 | KDM6A | Mutation | Lysine demethylase 6A | Xp11.3 | Chromatin modulation |
6 | KBTBD4 | Mutation | Kelch repeat and BTB domain containing 4 | 11p11.2 | Ubiquitination of target substrates |
6 | KMT2C | Mutation | Lysine methyltransferase 2C | 7q36.1 | Chromatin modulation |
6 | ZMYM3 | Mutation | Zinc finger MYM-type containing 3 | Xq13.1 | Chromatin modulation |
6 | OTX2 | Amplification | Orthodenticle homeobox 2 | 14q22.3 | Transcriptional regulation |
6 | MYCN | Amplification | MYCN proto-oncogene, bHLH transcription factor | 2p24.3 | Transcriptional regulation |
5 | KDM4C | Mutation | Lysine demethylase 4C | 9p24.1 | Chromatin modulation |
4 | ZIC1 | Mutation | Zic family member 1 | 3q24 | Transcriptional regulation |
4 | CDK6 | Amplification | Cyclin-dependent kinase 6 | 7q21.2 | Cell cycle |
3 | FLG | Mutation | Filaggrin | 1q21.3 | Matrix protein |
3 | KMT2D | Mutation | Lysine methyltransferase 2D | 12q13.12 | Chromatin modulation |
3 | TBR1 | Mutation | T-box, brain 1 | 2q24.2 | Transcriptional regulation |
3 | TERT | Mutation | Telomerase reverse transcriptase | 5p15.33 | Genome maintenance |
3 | GFI1 | Amplification, overexpression | Growth factor independent 1 transcriptional repressor | 1p22.1 | Transcriptional regulation |
3 | CCND2 | Amplification | Cyclin D2 | 12p13.32 | Cell cycle |
3 | CTNNB1 | Low level amplification | Catenin beta 1 | 3p22.1 | Wingless signaling |
3 | CTDNEP1 | Mutation | CTD nuclear envelope phosphatase 1 | 17p13.1 | Metabolism of fatty acids |
3 | KDM1A | Mutation | Lysine demethylase 1A | 1p36.12 | Chromatin modulation |
3 | KDM5A | Mutation | Lysine demethylase 5A | 12p13.33 | Chromatin modulation |
3 | PIK3CA | Mutation | Phosphatidylinositol-4,5-bisphosphate 3-kinase catalytic subunit alpha | 3q26.32 | Cell signaling |
2 | ATM | Mutation | ATM serine/threonine kinase | 11q22.3 | Genome maintenance |
2 | BRCA2 | Mutation | BRCA2, DNA repair associated | 13q13.1 | Genome maintenance |
2 | FAT1 | Mutation | FAT atypical cadherin 1 | 4q35.2 | Cell signaling |
2 | MED12 | Mutation | Mediator complex subunit 12 | Xq13.1 | Chromatin modulation |
2 | SMARCA4 | Mutation | SWI/SNF-related, matrix-associated, actin-dependent regulator of chromatin, subfamily a, member 4 | 19p13.2 | Chromatin modulation, SWI/SNF nucleosome-remodeling complex |
2 | ACVR2B | Amplification | Activin A receptor type 2B | 3p22.2 | Cell signaling |
2 | SEMA3D | Amplification | Semaphorin 3D | 7q21.11 | Axon guidance during development |
– | FOXG1 | Overexpression | Forkhead box G1 | 14q12 | Transcriptional regulation |
– | KCNA1 | Overexpression | Potassium voltage-gated channel subfamily A member 1 | 12p13.32 | Voltage-gated potassium (K+) channel |
– | EOMES | Overexpression | Eomesodermin | 3p24.1 | Transcriptional regulation |
– | KHDRBS2 | Overexpression | KH RNA binding domain containing, signal transduction associated 2 | 6q11.1 | RNA metabolism |
– | RBM24 | Overexpression | RNA binding motif protein 24 | 6p22.3 | RNA metabolism |
– | UNC5D | Overexpression | Unc-5 netrin receptor D | 8p12 | Cell adhesion, axon guidance |
– | OAS1 | Overexpression | 2′-5′-Oligoadenylate synthetase 1 | 12q24.2 | Cellular innate antiviral response |
Somatic
MYC (17% in group 3) and
MYCN (6% in group 4) amplifications are the most frequently observed driver events [
28]. The link between
MYC and group 3 MB outcome is well established, and high
MYC levels are associated with significantly reduced survival [
18,
41].
MYC activation develops because of amplification at the
MYC loci, genomic rearrangement of
PVT1–MYC, or other yet-unknown mechanisms [
22,
28,
42‐
44].
Recently, a study with a large sample size identified at least one potential driver events in 76% of group 3 and 82% of group 4 MBs, with an almost equal occurrence of
MYCN amplifications across group 3 (5%) and group 4 (6%), with
MYC amplifications restricted to group 3 tumors (17%) [
6]. Activation of the mutually exclusive
GFI1/GFI1B was identified as the most prevalent driver event through “enhancer hijacking”, by depositing them near active regulatory elements. Hotspot insertions targeting a novel potential oncogene,
KBTBD4, were also frequent both in group 3 and group 4 samples [
6,
38]. The prognostic significance of
GFI1/GFI1B activation is not yet clear [
45], although a large-scale integrative analysis of gene expression and methylation data indicated the presence of
GFI1/GFI1B activations mainly within a particular subtype of group 3 tumors [
31].
A single copy gain of the
SNCAIP gene is present in over 10% of group 4 tumors and represents the most distinctly upregulated gene within the group 4 signature.
SNCAIP is involved in the development of Parkinson’s disease, and its tandem duplications in group 4 MBs are mutually exclusive with
MYCN and
CDK6 amplifications, the latter present in 5–10% of all group 4 tumors [
18,
28]. In group 4 MBs,
PRDM6, an epigenetic regulator of gene activity, is the probable target of
SNCAIP-associated enhancer hijacking and is activated in about 17% of tumors [
6].
SMARCA4 encoding subunits of the
SWI/SNF-like chromatin-remodeling complex is among the most recurrently (~ 9%) mutated genes in group 3 tumors [
6,
38]. Network analysis of group 4 somatic copy number aberrations revealed the enrichment of genes responsible for
chromatin modification and identified a novel homologous deletion of a histone-lysine demethylase,
KDM6A [
28], that preferentially demethylates the H3K27 trimethyl mark (H3K27me3) [
46]. Somatic mutations of the
KDM6A gene are exclusively present in approximately 12% of group 4 tumors, along with frequent mutations of other 6 KDM family members (
KDM1A,
KDM3A,
KDM4A,
KDM5A,
KDM5B, and
KDM7A) [
21,
38,
40,
47] (Table
2).
EZH2 is also amplified or overexpressed in group 3 and 4 tumors, contributing to the inscription of H3K27me3, and is mutually exclusive with
KDM6A mutations. About 50% of tumors with
KDM6A and
KDM1A mutations also harbor
ZMYM3 mutations, suggesting a cooperation between these two genes [
47]. The relatively numerous
CHD7 or
ZMYM3 mutations partake in the regulation of the H3K4me3 mark [
6]. Inactivating mutations in
MLL2 and
MLL3 genes also participate in the reduction of H3K4me3 levels, promoting the deactivation of prodifferentiation genes [
38,
48].
TBR1 and
EOMES expression is significantly higher in group 3 and 4 tumors compared to other subgroups and strongly correlates with gene methylation [
38]. These observations suggest that by preserving methylation marks, both group 3 and group 4 MBs retain a stem-like epigenetic state and their pattern of gene expression is more consistent with progenitor and undifferentiated cells than cells with SHH- and WNT-activated MBs [
49]. Genes participating in chromatin remodeling, such as
KDM6A and
ZMYM3, are located on the X chromosome, explaining the higher prevalence of group 3 and group 4 MBs in males [
47]. The mutual theme of altered epigenetic regulation in tumorigenesis across group 3 and group 4 tumors (Fig.
2b and
3b) emphasizes the potential utility of drugs targeting dysregulated epigenetic modifiers, with promising in vitro results [
50].
Another hallmark of non-WNT/non-SHH MBs is the elevated expression of
OTX2, a target of TGFβ signaling
. OTX2 amplification in group 3 MBs is mutually exclusive to
MYC amplification and is also routinely found in group 4 MBs [
6,
28].
OTX2 regulates cell cycle, drives proliferation, inhibits cellular differentiation, and has been associated with MB development [
51]. Overexpression and knockdown of
OTX2 are associated with altered expression levels of several polycomb genes (
EED,
SUZ12, and
RBBP4) and genes encoding H3K27 demethylases (
KDM6A,
KDM6B,
JARID2, and
KDM7A) [
52]. Additionally, OTX2 targets
EZH2 that could be pharmacologically manipulated and is a potential target especially for patients with hematological malignancies [
53]. Transcriptional profiling identified an elevated expression of a photoreceptor program in Group 3 MBs, well characterized in the retina [
32]. OTX2 transactivation contributes to the regulation of transcription factors
NRL and
CRX, acting as master regulators of the photoreceptor-specific program. Both genes are required for tumor maintenance while the target of
NRL, the protein BCL-XL, is necessary for tumor cell survival. Anti-BCL therapy may serve as a rational therapeutic target in this subset of group 3 MBs [
54].
Approximately 20% of group 3 cases involve copy number alterations in
TGFβ pathway genes, including the deletion of pathway inhibitors (
CD109,
FKBP1A,
SNX6) and amplification of regulators (
ACVR2A,
ACVR2B,
TGFBR1); thus, TGFβ signaling may represent a rational target for personalized therapy [
6,
28].
Notch-mediated signaling pathway plays a critical role in CNS development, stem cell maintenance, and differentiation of cerebellar granule neuron precursors; modulates epithelial-to-mesenchymal transition; and has been implicated in MB disease etiology [
55]. Mutations in Notch signaling genes have been described in group 3 MBs [
6], with especially elevated expression of NOTCH1 in spinal metastases [
56]. Somatic copy number variations in group 4 MBs affect regulators of the
NF-κB signaling pathway, such as deletions of
NFKBIA and
USP4, providing an opportunity for a rational targeted treatment [
28].
We summarize the most frequent genetic aberrations of group 3 MBs in Table
1 and group 4 MBs in Table
2.
Tumor proteome analysis defines novel potentially targetable signaling pathways
Both group 3 and group 4 MBs are characterized by abundant within-subgroup genetic heterogeneity. The low rate of recurrent lesions sets a challenge for successful therapy development. Moreover, it is difficult to infer phenotypes based on genomic data only; thus, global proteome and phosphoproteome profiles may uncover yet unknown subgroup-specific biological processes [
43,
44,
57]. A recent phosphoproteomic comparison revealed profound divergence in post-transcriptional regulation and differential kinase activity between group 3 and group 4 samples: in group 3, the PDHK, CLK, and CK2 kinase families, while in group 4 MBs, the kinases downstream of the RTK-GPCR axis were primarily enriched. The study identified aberrant RTK signaling as a unifying feature of group 4, with a potentially pivotal role of
ERBB4 and
SRC signaling in MB development [
44]. Another tumor proteome analysis underlies the limited number of potentially targetable pathways; different transcriptional patterns from untreated SHH, group 3, and group 4 MB samples converged into only two protein-signaling profiles. The first profile resembled MYC-like signaling, encompassing all of the SHH-activated and majority of group 3 samples. The other protein profile consisted of the rest of group 3 and the bulk of group 4 tumors, displaying DNA damage/apoptosis/neuronal signaling [
58].
Elevated MYC-expression is a discriminatory feature of a subset of group 3 tumors. Some group 3 MBs are characterized with an increased post-translational activation of MYC even in the absence of MYC amplification and are linked to the elevated expression of kinases, such as
PRKDC, providing targets for future therapies [
43].
HMGA1 is a stem cell phenotype regulator that targets MYC and is also targeted by MYC, and plays a role in cell growth and invasion in cancer. In a proteomic analysis, HMGA1 isoforms a and b showed elevated expression in Group 3 MBs associated with poor outcome [
57].
In summary, proteomic platforms complement cytogenetic, transcriptomic, and mutation-based data and expand translational opportunities. Data integration on multiple levels yields a more complete understanding of cancer biology for the sake of novel therapeutic strategies.