Deconstructing Sonic Hedgehog Medulloblastoma: Molecular Subtypes, Drivers, and Beyond

TIGS 1760 No. of Pages 16

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Genetics Feature Review

Deconstructing Sonic Hedgehog Medulloblastoma: Molecular Subtypes, Drivers, and Beyond Jesus Garcia-Lopez,1,2 Rahul Kumar,1,2 Kyle S. Smith,1 and Paul A. Northcott1,* Highlights

Medulloblastoma (MB) is a highly malignant cerebellar tumor predominantly diagnosed during childhood. Driven by pathogenic activation of sonic hedgehog (SHH) signaling, SHH subgroup MB (SHH-MB) accounts for nearly one-third of diagnoses. Extensive molecular analyses have identified biologically and clinically relevant intertumoral heterogeneity among SHH-MB tumors, prompting the recognition of novel subtypes. Beyond germline and somatic mutations promoting constitutive SHH signaling, driver alterations affect a multitude of pathways and molecular processes, including TP53 signaling, chromatin modulation, and post-transcriptional gene regulation. Here, we review recent advances in the underpinnings of SHH-MB in the context of molecular subtypes, clarify novel somatic and germline drivers, highlight cellular origins and developmental hierarchies, and describe the composition of the tumor microenvironment and its putative role in tumorigenesis.

SHH-MBs are characterized by pathogenic activation of SHH signaling. Continued exploration of the somatic and germline molecular landscapes have revealed alterations in a variety of functional processes, including chromatin modulation and posttranscriptional gene regulation. Intertumoral heterogeneity amongst SHH-MB subgroup tumors has been described in terms of molecular subtypes (α, β, γ, and δ) with distinctive patient demographics, genetic lesions, and clinical outcomes. Developmental origins of SHH-MB have been pinpointed to the GN lineage with age-associated distinctions in cellular and differentiation hierarchies.

Clinicomolecular Framework MB (see Glossary) comprises four consensus molecular subgroups: WNT, SHH, Group 3, and Group 4. Such molecular heterogeneity has been illuminated through genetic, genomic, transcriptional, epigenomic, and proteomic approaches and carries relevant clinical implications. Constitutive activation of the SHH signaling pathway defines approximately 30% of MBs, with the namesake subgroup designation SHH-MB. Affected patients exhibit a bimodal age distribution, with tumors occurring in infants or adults and less frequently in older children. Notably, infant and adult SHH-MBs are characterized by distinct survival outcomes and molecular alterations. With early appreciation of transcriptional and epigenetic distinctions between infant and adult patients, recent advances in molecular classification and further subtyping of SHH-MB have disclosed additional heterogeneity within this subgroup. Such heterogeneity prompts deeper exploration into tumor biology and cerebellar development through contextualizing the contributions of recurrent genetic events seen in patients. Various subtypes of SHH-MB have been proposed through detailed molecular studies leveraging transcriptomics and/or DNA methylation profiling [1–4]. Four main subtypes have been defined – SHH-α, -β, -γ, and -δ – and these will be formally recognized in the forthcoming edition of the WHO Classification of Central Nervous System Tumors and redesignated SHH-1 (-β), SHH-2 (γ), SHH-3 (-α), and SHH-4 (-δ) (D.W. Ellison, personal communication). While defined by orthogonal molecular features, each subtype is characterized by relatively specific patient demographics, genetic lesions, and outcomes (Figure 1, Key Figure). SHH-α tumors predominantly affect non-infant (>3 years old) children and are characterized by germline or somatic TP53 mutations and amplifications of MYCN or GLI2, as well as pathogenic germline variants in ELP1. SHH-β (also known as infant SHH-I) and SHH-γ (also known as infant SHH-II) occur in infants Trends in Genetics, Month 2020, Vol. xx, No. xx

Over 40% of pediatric SHH-MBs exhibit damaging germline mutations, while such events are much rarer among adults. Quiescent, therapy-resistant cells, such as SOX2+ and OLIG2+ progenitor cells, may serve as reservoirs for tumor regrowth in relapsed SHH-MB. Divergent clonal selection may drive recurrence through selective pressures imposed by therapy.

1

Department of Developmental Neurobiology, St. Jude Children’s Research Hospital, Memphis, TN, USA 2 Equal contributions

*Correspondence: [email protected] (P.A. Northcott).

https://doi.org/10.1016/j.tig.2020.11.001 © 2020 Elsevier Ltd. All rights reserved.

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Key Figure

Glossary

Summary of Demographic, Clinical, and Molecular Features of Sonic Hedgehog Medulloblastoma (SHH-MB) Subtypes

Central nervous system (CNS): comprises the brain, spinal cord, optic nerves, and retina. The CNS receives information and feedback from the sensory organs and from nerves throughout the body, processes the information, and controls the response to those stimuli. Chromothripsis: a mutational phenomenon characterized by massive, clustered genomic rearrangements. Chromosomes that undergo chromothripsis first fragment into many pieces and are then stitched back together in a random order by DNA repair processes, most likely nonhomologous end joining. External germinal layer (EGL): a transient zone of granule cell precursors that is formed from cells that migrate tangentially from the uRL to cover the pial surface of the developing cerebellum. Genetically engineered models (GEMs): animals that harbor genetic alterations artificially introduced by humans and have been broadly used to study genetic disorders. Granule neuron progenitor cells (GNPs): a highly proliferative population that gives rise to GN. GNPs remain mitotically active in the EGL during the first postnatal week in mice and then start to migrate and differentiate into GNs once they reach the IGL. Granule neurons (GNs): glutamatergic excitatory neurons located in the IGL of the cerebellar cortex. Granule cells extend their axons into the molecular layer where they synapse with Purkinje cells. Histone acetyltransferases (HATs): modify histones by means of catalyzing the transfer of an acetyl group from acetyl-CoA to the ε-amino group of a histone lysine residue. Epigenetic activity of HATs facilitates transcriptional access to DNA by either neutralizing the positive histone charge or serving as a binding site for chromatin remodeling complexes. Internal granule layer (IGL): final position in the cerebellum where cells from the EGL migrate and differentiate into mature GNs. Locked nucleic acids (LNAs): modified RNA nucleotides in which the ribose moiety is modified with an extra bridge connecting the 2′ oxygen and 4′ carbon. LNAs have been used for the detection of low-abundance nucleic

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Figure 1. Age (in years), sex distribution, histology, frequency of metastasis, 5-year overall survival (OS), cytogenetics, and prominent driver gene alterations are summarized according to SHH-MB subtype.

(≤3 years), with the former having high rates of metastasis at diagnosis and variable prognosis depending on treatment [1,3,5,6]. By contrast, SHH-δ largely occurs among older adolescents/ adults and carries a more favorable prognosis [1]. The subtype-specific landscapes of cytogenetic and driver gene alterations suggest particular vulnerabilities in spatiotemporally distinct developmental populations. With obligatory derangement of SHH signaling as a defining feature of SHH-MB, the distinct subtypes are likely to manifest through a diverse combination of tumorigenic insults in distinct cerebellar subpopulations at discrete developmental stages. Furthermore, the identification of subtypes through unsupervised DNA methylation analysis alone highlights the orthogonality of this approach from the driver gene landscape and associated clinicodemographic features. While facilitating refined risk stratification and serving as a framework for the dissection of distinct tumor biology, novel subtyping of 2

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SHH-MB will enable the exploration of novel therapeutic approaches that exploit inherent pathophysiological and developmental vulnerabilities. Informed by a deeper appreciation of intrinsic molecular heterogeneity in the form of novel subtypes, the pathogenesis of SHH-MB is reviewed herein with an emphasis on developmental origins, genetic drivers and predisposition, and recent insights into the tumor microenvironment.

Developmental Origins and SHH Signaling The essential role of SHH signaling in cerebellar development was first established more than two decades ago when independent groups reported that cerebellar granule neuron progenitor cell (GNP) proliferation is dependent on active SHH signaling [7–9]. Mutations altering the SHH signaling cascade lead to tumorigenic transformation of GNPs into MB [10–12]. These findings have provided crucial insights into the role of aberrant SHH signaling in MB and implicated GNPs as the probable lineage of origin for this particular subgroup. GNPs reside in the external germinal layer (EGL) of the cerebellar anlage and are responsible for producing cerebellar granule neurons (GN), the most abundant neuronal subtype in the central nervous system (CNS) [13–15]. Furthermore, the role of non-tumor-cell SHH signaling in the tumor and stromal microenvironment may play a pivotal role in tumor initiation and maintenance, as described in subsequent sections. During mammalian embryonic development, a highly orchestrated process controls the migration of glutamatergic neurons, GABAergic neurons, and glial populations into precise, spatially organized locations in the cerebellum. In mice (e13.5) and humans (after ten post-conceptional weeks), GNPs are born from the upper rhombic lip (uRL), a primary germinal zone that produces the majority of glutamatergic populations in the developing cerebellum [13,16]. GNPs exit the uRL and then migrate tangentially towards the EGL. During the first two to three post-natal weeks in mice, GNPs are exposed to SHH secreted by Purkinje neurons and undergo massive proliferation within the EGL (peaking between P5 and P7) before migrating towards the internal granule layer (IGL), culminating as mature GNs (Figure 2A). The specification and proliferation of GNPs is driven in large part by key neuronal transcription factors, including Meis1, Pax6, and Atoh1, along with activation of the SHH pathway [17]. Atoh1 expression sensitizes GNPs to SHH signaling by maintaining the primary cilium on their surface and therefore enabling the binding of the mitogen Shh to Ptch1 [18]. By contrast, the Meis1–Pax6 pathway promotes GNP differentiation by means of Atoh1 degradation via bone morphogenic protein (BMP) signaling [17].

acids and chromosomal DNA due to their increased sensitivity compared with DNA and RNA oligos/probes. Loss of function (LOF): mutations that result in reduced or total ablation of normal protein function. Medulloblastoma (MB): an embryonal cerebellar tumor that is among the most common malignant pediatric brain tumors and a leading cause of cancerrelated death among children. MB is divided into biologically and clinically distinct molecular subgroups – WNT, SHH, Group 3, and Group 4. Sonic hedgehog (SHH): SHH signaling is a signal transduction cascade that regulates key events during developmental processes such as growth and the patterning of multicellular embryos. The regulatory action of SHH signaling is precisely linked to the secretion, uptake, and translocation of SHH ligand. Tumor-associated macrophages (TAMs): represent a major component of the tumor microenvironment in SHH-MBs. TAMs have been proposed to play a role in tumor growth, metastatic dissemination, and treatment failure. TAMs release cytokines and induce antiapoptotic programs in transformed GNPs. Unfolded protein response (UPR): an adaptive response triggered by endoplasmic reticulum (ER) stress that reduces the unfolded protein load to maintain cell viability and function. Upper rhombic lip (uRL): a primary germinal zone of the developing cerebellum. The uRL gives rise to numerous glutamatergic neuronal populations during development, including GNPs, cerebellar nuclei, and unipolar brush cells.

Recently, single-cell RNA-seq of the developing mouse cerebellum demonstrated that human SHH-MB tumors mirror the cerebellar granule cell hierarchy in mice [19,20]. Cross-species comparisons between human MB tumors and mouse cerebellar single-cell reference datasets have provided a clearer definition of the lineage hierarchy underlying bulk SHH-MB tumors, revealing a compositional trajectory of undifferentiated GNP-like and more-differentiated GN-like populations [19,20]. Both GNP-like and GN-like populations observed in SHH-MB exhibit expression signatures related to neuronal populations characteristic of cerebellar development. This suggests the derivation of tumors from discrete developmental populations, particularly the Atoh1+ lineage in the EGL. Rare subpopulations in the EGL, marked by neural stem cell markers including Nestin, Olig2, or Sox2, may be particularly vulnerable to transformation [21,22]. Paradoxically, pediatric tumors transcriptionally mirror more mature GN-like populations while adult tumors resemble less-differentiated GNP-like populations (Figure 2A). While PTCH1 mutations are highly prevalent in both pediatric and adult SHH-MBs, the repertoire of driver gene alterations in pediatric tumors is relatively sparse compared with adult tumors, the latter of which exhibit frequent co-operating lesions (e.g., TERT promoter, DDX3X, CREBBP, BRPF1 Trends in Genetics, Month 2020, Vol. xx, No. xx

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Figure 2. Dysregulation of Sonic Hedgehog (SHH) Signaling in the Developmental Origins of SHH Medulloblastoma (SHH-MB). (A) Cellular origins of SHH-MB. The figure depicts an embryonic mouse cerebellum highlighting progenitor populations proposed to be developmentally linked to infant and adult SHH-MB. (B) Primary cilium and aberrant activation of SHH signaling in MB. Inactivating mutations in PTCH1 or binding of SHH ligands to PTCH1 derepress SMO and allow its translocation into the primary cilium, leading to the activation of GLI transcription factors. Activation of GLI1 in MB promotes the expression of oncogenes such as MYCN along with genes that stimulate proliferation and survival. SUFU protein negatively regulates the pathway by sequestering GLI transcription factors in the cytoplasm and preventing the activation of GLI target genes. SUFU either maintains the GLI2/3 proteins in a repressive state (GLIR) or facilitates their degradation through cytoplasmic sequestration. An enzymatic cascade involving cAMP and protein kinase A (PKA) helps to maintain the GLIR state. The cascade is initiated by the GPR161 transmembrane protein that transmits signals through heterotrimeric G proteins, such as Gαs, increasing cellular cAMP levels, which ultimately increase PKA activity. (C) Chromosomal and chromatin dysregulation in SHH-MB. Chromothripsis, a phenomenon that involves massive chromosome rearrangements leading to pathogenic gene fusions such as DNAJB6-SHH, is frequently associated with TP53 mutations in medulloblastoma. TERT mutations directly impact telomere maintenance, also resulting in DNA damage. In SHH-MB, TERT somatic mutations are almost entirely restricted to adult patients and are associated with cell cycle and survival deregulation, which ultimately fuel tumor growth. Finally, epigenetic regulation of gene transcription by histone modification is frequently altered in SHH-MB tumors. The mutations affect methyltransferases (KMT2C and KMT2D), histone demethylases (KDM4C and KDM4B), acetyltransferases (CREBBP, EP300, KANSL1, MYST3, KAT6B, and BRPF1), and histone acetylases (BCOR, LBD1, and GPS2). Abbreviations: GN, granule neuron; EGL, external germinal layer; IGL, internal granule layer; GNP, granule neuron progenitor; GOF, gain of function; LOF, loss of function.

mutations) [2]. Given these differences, we speculate that pediatric SHH-MB tumors may have greater inherent differentiation capacity than adult SHH-MBs. Activation of SHH signaling can occur in a ‘canonical’ or ‘noncanonical’ manner to regulate the survival and proliferation of GNPs [23]. Canonical activation leads to transcriptional cascades mediated by the Gli family transcription factors on SHH ligand binding to Ptch1 [24]. Ptch1 4

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encodes the transmembrane protein Patched-1, which in the absence of SHH ligand acts as a suppressor of Smo (Figure 2B). Recent cryogenic electron microscopy studies have also demonstrated that mutations in sterol-binding sites of Ptch1 interfere with binding to SHH [25,26]. Given the lack of direct physical interaction between Ptch1 and Smo, SHH pathway regulation is likely to be mediated by intermediate molecules, such as sterols [25,27,28]. While various sterols may mediate signal transduction, cholesterol has been identified as necessary and sufficient to activate cellular SHH signaling via Smo [27,28]. In addition, the fatty acid modification of SHH plays an important role in its potency as a ligand and in its subcellular localization [29]. As a consequence, alterations of key metabolic components may represent cryptic drivers in SHH-MB tumorigenesis and serve as novel therapeutic targets. On exposure to SHH or constitutive activation of the signaling pathway via genetic lesions, Smo promotes the accumulation of Gli2/3 transcription factors in the primary cilium, shifting Gli to an active state (GliA) and overcoming negative regulation imposed by Sufu and protein kinase A (PKA). GliA translocates to the nucleus to activate the expression of Gli1, Mycn, and other transcription factors that promote cell proliferation. By contrast, noncanonical signaling involves the activation of Gli transcription factors independent of SHH ligand or Ptch1–Smo regulation [30]. Mechanistically, Gli1 is phosphorylated and activated by Mapk/Erk1/2 signaling [31]. In addition, PI3k–Akt–mTor signaling contributes to noncanonical SHH pathway activation by two different mechanisms: Akt prevents proteasomal degradation of Gli2 while PI3k–Akt signaling enhances Gli1 protein stability [32–34]. Aberrant SHH signaling can also promote the expression of prosurvival genes, such as Bcl-2 or Akt, through noncanonical mechanisms [33,35]. Moreover, growing evidence suggests that Ptch1 plays a role in programmed cell death, independent of its role in SHH signaling [36,37]. Thus, PTCH1 mutations observed in SHH-MBs might activate cell proliferation and survival through SHH-dependent and independent mechanisms.

Somatic Driver Alterations Somatic mutations affecting genes involved in SHH signaling have been extensively described in SHH-MB and occur across all molecular subtypes (Box 1). Large-scale human sequencing studies have revealed frequent loss-of-function (LOF) mutations and focal deletions in PTCH1 (40%) and SUFU (13%), activating mutations in SMO (9%), and amplifications of MYCN (13%) and GLI2 (6%) [2,3,38]. In humans, SHH-MB tumors also display an enrichment of catastrophic genomic rearrangements, referred to as chromothripsis, particularly in TP53mutant SHH-α, that affect SHH signaling genes (Figure 2C). For example, chromothripsisassociated fusion of the first exon of DNAJB6 with exons 2 and 3 of the SHH gene results in aberrant overexpression of SHH ligand [39]. The rapid proliferation of GNPs in a tight developmental window to yield the most numerous neurons in the CNS might also represent a stochastic vulnerability. SHH exposure causes S-phase acceleration in GNPs through increased replication origin firing and fork speed [40]. Since replicative stress may drive increased homologous recombination, an increase in somatic recombination rates under Shh-dependent replicative stress may explain the focal deletions and genomic instability in GNPs that promote SHH-MB tumor initiation [41,42]. Beyond mostly ubiquitous SHH pathway-associated alterations, recurrent alterations in the TP53 and PI3K/AKT/mTOR signaling pathways also act as key drivers of tumorigenesis in both human and mouse SHH-MB. Somatic TP53 mutations (21%) are enriched among children and adolescents (SHH-α) and confer an extremely poor prognosis [1,43]. In children, TP53 mutations co-occur frequently with MYCN and GLI2 amplifications but rarely with PTCH1 mutations [1,38,43], suggesting that downstream alterations of the SHH signaling pathway might trigger a semi-limiting apoptotic response that is overcome through either TP53 mutations or the Trends in Genetics, Month 2020, Vol. xx, No. xx

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Box 1. Summary of Biological Pathways and Molecular Processes Altered in SHH-MB Dysregulation of SHH signaling represents a unifying theme in SHH-MB. A subset of tumors in each subtype exhibits isolated alterations in the SHH pathway that are mutually exclusive of affecting other pathways and processes (Figure I), suggesting that aberrant activation of SHH signaling is sufficient to drive tumorigenesis irrespective of subtype. By contrast, in a large proportion of SHH-MB tumors, genetic deregulation of other functional pathways appears to cooperate with aberrant SHH signaling, which varies by molecular subtype. For example, in infants with SHH-β or SHH-γ tumors, dysregulation of chromatin modulation appears to be a key contributor to tumorigenesis, especially in the SHH-β subtype. TP53 pathway alterations are prominent in SHH-α tumors and either co-occur with alterations in the SHH pathway or occur in combination with events targeting transcription factors, cell cycle regulators, or protein translation. In older patients with SHH-δ tumors, alterations in the translational machinery and protein homeostasis appear to cooperate with alterations in chromatin modifiers and transcriptional regulators, on the backbone of aberrant SHH signaling.

β (SHH-1) n = 50

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Figure I. Genetically Altered Molecular Pathways and Processes in SHH-MB. Data extracted from [44]

induction of prosurvival genes, such as BCL2 [35]. Additionally, focal amplifications of negative regulators of the TP53 pathway, including PPM1D (6%) and MDM4 (2%), occur in a subset of SHH-α human tumors [44]. Similarly, PI3K/AKT/mTOR pathway genes are recurrently altered in SHH-MB (10%). Focal somatic copy number aberrations or mutations of PIK3CA, PTEN, and PIK3C2G have been described in more than 5% of human SHH-MBs [2], promoting GLI activation through a SMOindependent mechanism [45,46]. The vast majority of patients with PI3K/AKT/mTOR pathway mutations belong to the SHH-δ group, which almost universally harbors TERT promoter mutations (98%) (Figure 2C) and frequent somatic DDX3X (54%) helicase domain mutations [1].

Genetic Predisposition Current estimates suggest that 20–25% of all SHH-MB patients harbor pathogenic germline variants predisposing them to malignancy [47]. When considering only pediatric SHH-MB patients under the age of 18 years, the frequency of germline predisposition is much higher (>40%). Germline mutations are rarely found in adult SHH-MBs [47]. By contrast, germline mutations in PTCH1 and SUFU (4.5% and 7.4% of all SHH-MB cases) are enriched in infants. Similarly, germline LOF mutations in GPR161, a G protein-coupled receptor located in the cilium, and GNAS, which encodes the G protein alpha subunit (Gαs), have been reported in rare cases of infant SHH-MB [48–50]. Both mutations impair the production of cAMP, reducing PKA activity, and therefore represent an alternative mechanism to activate GLI transcription factors. 6

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Primary risk factors for the development of SHH-MB include rare hereditary syndromes such as Gorlin syndrome (GS) (mutations in PTCH1 and SUFU) and Li–Fraumeni syndrome (LFS) (mutations in TP53) [47]. GS, also known as nevoid basal cell carcinoma syndrome, is an inherited autosomal dominant disorder caused by germline inactivation of PTCH1 or SUFU that is associated with an increased risk of development of skin cancer and MB during adolescence or early adulthood. LFS is also an inherited autosomal dominant condition caused by germline mutations in TP53 that is associated with an increased risk for a spectrum of various cancers, including MB. In SHH-MB, both germline and somatic TP53 mutations and chromothripsis are strongly associated, suggesting that loss of p53 might potentiate catastrophic DNA rearrangements (Figure 2C) [51]. Similar to somatic events, hereditary TP53 mutations (6.4%) are more common in older children (median age 9 years) and largely restricted to SHH-α. By contrast, germline mutations affecting PALB2 (1%) or BRCA2 (2%) show no apparent association with age at diagnosis [47]. PALB2 is a component of the BRCA2 complex required for DNA repair [52]. Pathogenic germline variants of these genes in SHH-MB are associated with mutational signatures typical of homologous recombination (HR) repair deficiency [47,53,54]. Based on numerous genetically engineered models (GEMs) of SHH-MB, germline HR deficiency or genomic instability renders the rapidly expanding cerebellar GN lineage exquisitely vulnerable to transformation [53–57]. In an attempt to further explain the etiology of MB in patients lacking an obvious genetic predisposition, recent studies have broadened focus to look beyond prototypical cancer predisposition genes [44]. The discovery of frequent pathogenic germline variants targeting ELP1 (also known as IKBKAP) in 15% of pediatric SHH-MB patients substantiates this approach [44]. Deleterious ELP1 germline variants are restricted to children with SHH-α MB (median age of onset ~6 years) and twice as common as germline alterations affecting hallmark SHH-MB predisposition genes such as SUFU, PTCH1, or TP53. Although ELP1 mutations are heterozygous in the germline and almost never somatic, associated tumors display universal biallelic inactivation of ELP1 (chr9q31.3) by means of chromosome 9q deletion. Likewise, ~80% of ELP1-associated SHH-MBs exhibit somatic biallelic PTCH1 (chr9q22.32) inactivation (i.e., mutation of one allele plus chr9q deletion), suggesting that putative developmental defects resulting from ELP1 deficiency synergize with constitutive activation of SHH signaling to promote tumorigenesis. By contrast, ELP1 LOF mutations are mutually exclusive with germline and somatic TP53 mutations (Box 1). In addition to somatic PTCH1 alterations, approximately one-third of ELP1-associated SHH-MBs harbor somatic amplifications of PPM1D and MDM4, which act as negative regulators of the p53 response. ELP1 is a scaffolding subunit of a multimeric protein complex known as Elongator. The primary function of Elongator is to modulate translational elongation by catalyzing posttranscriptional modification of tRNAs in the uridine 34 (U34) wobble base position [44,58–60]. Deficiencies in U34 tRNA modifications lead to codon-specific ribosome pausing that can promote protein aggregation, imbalanced protein homeostasis, and induction of the unfolded protein response (UPR) (Figure 3A). As a consequence of ELP1 LOF, affected SHH-MBs are characterized by a pronounced reduction in Elongator subunit protein expression, a significant reduction in Elongator-dependent U34 tRNA modifications (namely, ncm5U, mcm5U, and mcm5s2U), codon-dependent translational reprogramming that leads to downregulation of proteins enriched in AA-ending codons and upregulation of proteins enriched in AG-ending codons, and the activation of UPR pathways. Further functional, biochemical, and mechanistic studies are required to deeply understand the interaction between ELP1-associated deregulation of translation and constitutive activation of SHH signaling in the etiology of these tumors.

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(A) Normal cerebellum

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Figure 3. Pathobiology of Post-transcriptional Deregulation in Sonic Hedgehog Medulloblastoma (SHH-MB). (A) ELP1 germline mutations destabilize the Elongator complex and cause loss of Elongator-dependent tRNA modifications, leading to stalled mRNA translation, protein misfolding, aggregation, and induction of the unfolded protein response (UPR). (B) Recurrent mutations in the U1 snRNA cause aberrant splicing of PTCH1, GLI2, and CCND2, generating cryptic transcript isoforms. (C) DDX3X mutations promote global failure of translational initiation due to the compromised binding between the 5′ untranslated regions (UTRs) of mRNAs and the catalytic domain of DDX3X.

Chromatin Modifiers and the Transcriptional Machinery Somatic mutations in chromatin modulators are prevalent in SHH-MB and occur across all subtypes, with some genes demonstrating notable subtype associations [2,38,44]. For example, inactivating KMT2D mutations are found in 15% of SHH-MBs overall but occur in approximately one-third of SHH-β tumors; namely, in infants. Likewise, deleterious CREBBP mutations occur in 8% of all SHH-MB tumors but are found almost exclusively among adults with the SHH-δ subtype. Rather than merely enhancing intrinsic SHH signaling, the KMT2D and CREBBP mutations found in infant and adult tumors, respectively, suggest that certain chromatin remodelers might have multifaceted roles in SHH-MB tumorigenesis. Although further functional and mechanistic studies are necessary, it is provocative to speculate that deregulation of important chromatin modifier complexes prevents progression through normal differentiation cascades, while aberrant 8

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SHH signaling provides a constitutive proliferative stimulus. Conversely, KMT2C mutations (6%) are found across all SHH subtypes, suggesting they may play a broader role in SHH-MB tumorigenesis. Thematically, genomic analyses have uncovered frequent somatic alterations targeting histone acetyltransferase (HAT) complexes (Figure 2C). In addition to CREBBP, other HAT genes, such as MYST3, KAT6B, EP300, and the HAT complex regulatory components BRPF1 and KANSL1, all exhibit recurrent alterations that are largely restricted to the SHH subgroup (19%) [38]. HATs play context-dependent roles in MB and GNP development. For example, embryonal loss of Crebbp impairs normal cerebellar development, promoting apoptosis and cerebellar hypoplasia in embryos, whereas postnatal Crebbp disruption in GNPs drives SHH-MB genesis [61]. Further functional and mechanistic studies are required to elucidate how somatic alterations targeting histone modifiers in GNPs drive SHH-MB development. SHH-MBs exhibit recurrent LOF mutations in genes encoding key components of epigenetic repressor complexes. For example, nonsense mutations in GPS2 and LDB1, subunits of the nuclear co-repressor (N-CoR) complex, have been reported [62]. However, the downstream consequences of these mutations remain poorly understood. The N-CoR complex recruits histone deacetylases to DNA promoter regions and acts as a repressor of the JNK/MAPK signaling pathway [63,64]. Aberrant MAPK activation drives invasion, promotes metastasis, and mediates resistance to SMO inhibitors in MB patients [65]. These findings suggest that dependencies for tumor growth may shift from SHH signaling to RAS/MAPK pathway activation through N-CoR complex deregulation. In addition, SHH-MBs exhibit mutations in the BCL6/BCOR/SIRT1 repressor complex, which has been suggested to negatively regulate SHH signaling. Mutations in BCOR, which acts as a transcriptional repressor of GLI transcription factors, might constitutively activate SHH signaling and promote the transformation of GNPs [66]. Disruption of Bcor in the GN lineage of transgenic mice combined with loss of Ptch1 results in fully penetrant SHH-MBs [67]. Molecular characterization of these Bcor;Ptch1 tumors identified marked overexpression of Igf2, implicating a BCOR–PRC1.1-dependent mechanism that might explain how BCOR mutations seen in SHH-MB may promote malignant transformation through compromised Igf2 repression in developing GNPs. Additional somatic alterations indirectly affect the epigenetic landscape of SHH-MB tumors. For example, hotspot IDH1 R132C mutations in SHH-δ (4%) tumors are associated with genome-wide CpG hypermethylation [38]. In addition to protein-coding genes, two recent human studies [68,69] reported highly recurrent hotspot mutations in the third nucleotide (r.3A>G) of U1 spliceosomal small nuclear RNAs (snRNAs). Such mutations were detected in multiple cancer types, including SHH-MB. SHH-α (25%) and -δ (97%) exhibit the vast majority of U1 mutations, which broadly disrupt RNA splicing to promote an excess of 5′ cryptic splicing events (Figure 3B). Such aberrant splicing was proposed to lead to inactivation of PTCH1 and activation of SHH driver genes such as GLI2 and CCND2 [69]. Whereas aberrant GLI2 splicing results in isoforms lacking the repressor domain, atypical splicing of CCND2 generates isoforms that might potentially escape protein degradation pathways. The observed U1 mutations co-occur with TERT and DDX3X mutations in adult patients (SHH-δ), but also with TP53 mutations in children (SHH-α) [69]. Although further confirmation in broader patient cohorts and experimental validation are needed to fully understand the role of U1 snRNA mutations in MB initiation and maintenance, these findings highlight the potential for drugging the spliceosome in affected SHH-MB subtypes [70,71].

Post-transcriptional Dysregulation While genomics, epigenomics, and transcriptomics have helped to decipher the molecular basis of the biological and clinical heterogeneity in SHH-MBs, recent proteomic profiling has provided new insights into the putative role of post-transcriptional dysregulation in MB pathogenesis [72–74]. Trends in Genetics, Month 2020, Vol. xx, No. xx

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Numerous pathogenic mechanisms have been suggested, including codon usage biases, deregulation of RNA-binding proteins, aberrant miRNA expression, failure of physiological protein turnover, and more. As described earlier, SHH-MBs harboring ELP1 LOF mutations exhibit codon usage biases and preferential upregulation of shorter proteins [44]. RNA-binding proteins regulate splicing, translation efficiency, and mRNA decay, accounting for as much as 30% of protein expression variation (reviewed in [75]). DDX3X, which encodes an RNA-helicase, is mutated in 19% of all SHH-MBs, contributing to 12% of SHH-α and 50% of SHH-δ tumors [38,39,62,76]. The DDX3X protein interacts with the ribosomal machinery and is involved in RNA processing through secondary structure remodeling [77,78]. DDX3X mutations found in MB impair catalytic activity, promoting a global deficiency in translation initiation [77,78] (Figure 3C). Besides their impact on translation, DDX3X mutations seen in MB enhance casein kinase 1 (CK1) activity [79]. CK1 plays a dual role in SHH signaling, positively by phosphorylating and activating SMO [80] and negatively by phosphorylating and destabilizing GLI transcription factors [81]. Hyperstimulation of CK1 driven by DDX3X-mutated isoforms leads to activation of the SHH signaling cascade downstream of SMO. In MB, DDX3X mutations are almost exclusively found in the WNT and SHH subgroups. In contrast to SHH-MBs that arise from GNPs in the EGL, WNT-MBs originate from the lower rhombic lip proximal to the brainstem [82]. Cre-mediated deletion of Ddx3x in GEMs representative of WNT-MB (Blbp-Cre+/-;Ctnnb1Flx/+;Tp53Flx/+ in combination with Ddx3xFlx/y or Ddx3xFlx/Flx) or SHH-MB (Blbp-Cre+/-;Ptch1Flx/+ in combination with Ddx3xFlx/y or Ddx3xFlx/Flx) shifted the typical anatomical boundaries of these models, yielding MBs in the brainstem and cerebellar hemispheres in both models [83]. Although these mouse models do not explicitly recapitulate the DDX3X point mutations observed in human SHH-MBs, the observation suggests that Ddx3x acts as a tumor suppressor to restrict the cell lineages that are capable of giving rise to WNT- and SHH-MB tumors [83]. As post-transcriptional regulators, miRNAs also play an important role in SHH-MB pathogenesis. Systematic miRNA expression profiling of patient tumors identified a number of aberrantly expressed miRNAs including the oncogenic miRNA cluster miR-17-92, which is specifically overexpressed in SHH-MB [84] and required for tumor formation in the Ptch1+/- GEM [85,86]. Although inhibition of miRNAs therapeutically remains challenging, the use of locked nucleic acid (LNA)-modified anti-miRs targeting miR-17-92 has exhibited promising preclinical results, prolonging survival in mice with intracranial MB allografts [87]. Alternatively, miR-125b, miR-324-5p, and miR-326, suppressors of SMO and GLI1 [88], are downregulated in SHH-MB, and their absence might contribute to the maintenance of constitutive SHH signaling.

Tumor Microenvironment The recent promise of immunotherapy as a novel and effective treatment option for some cancer patients has motivated an increased interest in understanding the composition of the tumor microenvironment and its role in tumorigenesis. In addition to malignant cells, the tumor microenvironment comprises immune cells, fibroblasts, smooth muscle cells, blood vessels, and extracellular matrix that support tumor growth [89]. The MB microenvironment is characterized by minimal immune infiltrate and is generally considered immunosuppressive [89–91]. By contrast, endothelial cells, pericytes, fibroblasts, microglia, astrocytes, and neurons are frequently identified as tissue residents in the MB tumor microenvironment [89–91] (Figure 4). Furthermore, paracrine SHH signaling between tumor cells and the stromal microenvironment highlights the critical role of cell-extrinsic pathways in tumorigenesis, motivating novel therapeutic strategies that target SHH signaling between tumor and stromal cells [92]. 10

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Figure 4. The Medulloblastoma (MB) Tumor Microenvironment. Sonic hedgehog MBs (SHH-MBs) contain multiple cell types, including granule neuron progenitor cell (GNP)-like tumor cells, rare SOX2+ cells capable of repopulating the tumor after therapy, and immune cells including macrophages. GNPs are able to trans-differentiate into tumor-associated astrocytes. Tumor-associated astrocytes produce interleukin (IL)-4, which hijacks microglial function for the benefit of the tumor. IL-4-stimulated microglia modify their secretome in a process defined as polarization and produce the cytokine insulin-like growth factor 1 (IGF-1), which signals back to malignant cells to promote MB outgrowth. Activated microglial populations recruit bone-marrow-derived monocytes through CCL2 release in response to tumor growth. Abbreviation: ECM, extracellular matrix.

Compared with other brain tumors, such as glioblastoma, ependymoma, or meningioma, MBs have been inferred to contain fewer immune cells based on the low expression of immune markers reported in transcriptional analyses [89,90,93]. Moreover, recent single-cell transcriptomic studies of SHH-MB patient tumors [19,20] revealed a paucity of nonmalignant cells, substantiating prior suggestions that MBs generally constitute ‘immune deserts’. Nonmalignant immune cell populations in these studies lacked CNVs and clustered with reference immune cell populations irrespective of tumor identity. However, further studies are underway to more fully and specifically characterize the gamut of immune cells in the MB tumor microenvironment. Despite their rarity, the contributions of different immune cell types appears to vary among MB subgroups. Tumor-associated macrophages (TAMs) have been suggested to be more abundant in SHH-MBs compared with other subgroups [90,93]. TAMs are particularly intriguing due to their controversial role in tumor growth [94]. Independent studies associate the presence of TAMs with both favorable and unfavorable prognoses [93,95,96]. In GEMs that resemble Trends in Genetics, Month 2020, Vol. xx, No. xx

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SHH-MBs driven by Smo activation (NeuroD2:SmoA1), the TAM population is enriched for bone- marrow-derived monocytes that are recruited to the niche via Ccl2 release by activated microglia [93] (Figure 4). Ccl2 interacts with Ccr2 to promote diapedesis of leukocytes, which allows their intravasation into the CNS [97]. SHH-MB cells may exploit the Ccl2–Ccr2 signaling axis to facilitate hematogenous dissemination and widespread leptomeningeal metastases [98]. In addition, SHH-MB cells produce Tgf-β, a well-known negative regulator of Cd8 T cell expansion and activation, thereby limiting intrinsic antitumor immune responses [95]. Recent studies have also highlighted an important role for astrocytes in SHH-MB tumor growth. Tumor-associated astrocytes can secrete Shh ligand and specialized extracellular matrix components that help to maintain tumor cell proliferation [99,100]. Moreover, a recent study implicated trans-differentiation of tumorigenic GNPs into tumor-associated astrocytes as a novel mechanism fueling tumor growth [101] (Figure 4). These tumor-derived astrocytes secrete interleukin-4 (IL-4), which drives the expression of insulin-like growth factor 1 (Igf1), which in turn promotes tumor growth. This study [101] opens a series of important questions, especially concerning the mechanism(s) regulating trans-differentiation. GNPs and astrocytes are characterized by contrasting expression profiles and originate from distinct cell lineages (glutamatergic progenitors and glial progenitors, respectively) with clearly defined developmental programs. Furthermore, production of Ccl2 by tumor-associated astrocytes may promote the maintenance of stem-like properties of tumor cells in the microenvironment [102]. Further studies are needed to understand how tumorigenic GNPs give rise to cells of a different lineage and thereby modulate the expression and behavior of the normal cells surrounding them. In addition to astrocytes, glial progenitors have been implicated in tumor maintenance and growth [103]. Similar to Sox2+ cells, which have been suggested to represent tumor-propagating cells in SHH-MB [104], Olig2+ progenitors are enriched in therapy-resistant and relapsed tumors and might be an additional source of MB-repopulating cells subsequent to therapy in SHH-MB. However, the role of Olig2+ cells in tumorigenesis remains to be determined. For example, Olig2 depletion in a SHH-MB GEM (Ptch1Flx/Flx;hGFAP-Cre) substantially reduced tumor growth but mice eventually developed MB, suggesting that Olig2+ progenitors might have a role in tumor maintenance rather than tumor initiation [103].

Therapeutic Resistance and Relapse Aberrant activation of SHH signaling in SHH-MB has led to focused translational efforts pharmacologically targeting SMO protein to prevent the downstream activation of GLI transcription factors. Among therapeutics developed to block SMO activation, sonidegib and vismodegib have been extensively evaluated in MB clinical trials (NCT00939484, NCT01239316, NCT03434262, and NCT01878617) [105–107]. Although some SHH-MB patients show a favorable transient response to targeted therapy, the long-term efficacy of SMO inhibitors has been limited, necessitating the development of novel therapeutics to overcome treatment-induced resistance [108,109]. Tumor heterogeneity may critically limit the efficacy of targeted therapies in SHH-MB patients. Single-cell transcriptomic analysis of a SHH-MB mouse model evaluated cellular composition in tumors treated with vismodegib versus vehicle control [110], demonstrating that vismodegib initially promoted differentiation and slowed tumor growth. However, certain populations remained resistant to SMO inhibition and managed to survive, representing candidate populations driving recurrence and treatment failure. Among them, Sox2+ cells have been recurrently detected following treatment [111]. Sox2+ cells produce highly proliferative progenitors expressing Doublecortin (Dcn+) that give rise to post-mitotic cells expressing Neuronal nuclei (NeuN), which form the predominant malignant population [104]. Thus, Sox2+ cells act as a reservoir for tumor regrowth and represent a therapeutic challenge for SMO inhibitors such 12

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as vismodegib. The presence of Sox2 + cells in MB tumors predicted a poor outcome in patients where relapse is almost uniformly fatal [21,104]. Unfortunately, there are very few studies disentangling the genomics of recurrent MB [112,113]. Conventional GEMs and patient-derived xenografts (PDXs) of SHH-MB have been shown to adequately maintain the molecular and phenotypic properties of the primary patient tumors from which they are derived [114–116], whereas models of recurrent disease are mostly lacking. Reported comparisons performed between primary and relapsed tumors have to date demonstrated only modest conservation of genetic and transcriptional landscapes between disease stages [112]. Such genetic divergence might explain the limited successes of targeted therapeutics at relapse [105,106,108,109]. For example, although oncogenic events observed in primary tumors are enriched in SHH signaling pathway genes, relapsed tumors have been reported to exhibit relapse-specific oncogenic events affecting the TP53 pathway and DNA damage response [112]. The emergence of ‘relapse-specific’ molecular signatures may thus result from clonal evolution in the face of selective pressures imposed by up-front therapies [112]. Notably, SHH-MBs often exhibit purely local anatomical patterns of relapse, in contrast to other molecular subgroups, which tend to recur distally [117]. In addition to biopsy at relapse to clearly guide the implementation of targeted agents, therapeutic efforts should continue to focus on the elimination of rare tumor reservoirs that may be difficult to target using conventional cytotoxic approaches.

Concluding Remarks Analysis of the genomes, epigenomes, and transcriptomes of SHH-MBs has enabled a deeper understanding of the molecular events converging on seemingly universal deregulation of the SHH signaling pathway in these tumors. The latter, combined with the enhanced delineation of SHH-MB into distinct molecular subtypes, affords new opportunities for improved patient risk stratification and the deployment of rational molecularly targeted therapies in the appropriate patient populations. The synergy of the multiomic explorations into SHH-MB tumor biology also scaffolds critical insights into cellular origins and evolutionary dynamics in these tumors. This can be exploited to expose lineage- or subtype-specific vulnerabilities, especially in high-risk SHH-MB subtypes that exhibit resistance to current therapeutic approaches and are prone to relapse. Paradoxically, relapsed SHH-MBs exhibit appreciable genetic divergence from their primary counterparts. This substantiates the need to search for therapeutic targets that effectively undermine the proliferative potential of quiescent cell populations, such as Sox2+ and Olig2+ progenitors, which act as a reservoir for tumor regrowth. In addition, the role of TAMs in the MB tumor microenvironment remains controversial, with both pro- and antitumoral contributions ascribed. The development of future therapeutic approaches that modulate TAMs in SHH-MB requires a more comprehensive understanding of the functional interactions between TAMs and other populations in the tumor microenvironment. During the past decade, we have substantially increased our knowledge of the molecular and genetic features of SHH-MB. Nevertheless, several hurdles remain (see Outstanding Questions). The next generation of clinical and biological studies must incorporate the evolving understanding of SHH-MB contextualized with advanced appreciation of intertumoral heterogeneity, developmental origins, and novel genetic drivers. By appreciating the interplay between the key developmental pathways and dysregulated molecular processes that directly or indirectly affect SHH signaling, efforts towards improved outcomes for patients with SHH-MB hinge on the development of faithful preclinical models, rational implementation of targeted therapeutics, and continued discovery of therapeutic vulnerabilities through exhaustive profiling of primary and relapsed tumors, combined with innovative functional genomics and diligent mechanistic studies.

Outstanding Questions How will evolving appreciation of the intertumoral heterogeneity in SHHMBs affect risk stratification and treatment paradigms? Given discriminatory genetic lesions, can subtype-specific therapeutic vulnerabilities be exploited? While cross-species comparisons have suggested correlates between developmental populations in the cerebellum and tumor cells, to what extent are these findings recapitulated using human developmental references? Do specific developmental hierarchies and cellular differentiation programs disclose population-specific dependencies for tumor initiation and maintenance? Given the array of genetic alterations affecting chromatin modifiers and remodelers in SHH-MB, what are the downstream epigenomic consequences? Do these alterations converge on specific developmental or signaling pathways? Are such alterations critical for tumor initiation, maintenance, or both? With new evidence suggesting dysregulation of post-transcriptional machinery as a common theme in SHH-MB, how does the altered proteome contribute to tumorigenesis? Will deep proteomic profiling of SHH-MBs disclose novel therapeutic avenues? What additional lesions can be identified in noncoding regions of the genome? Can models be developed to understand the molecular pathogenesis of lesions such as ELP1 or U1 snRNA? How does acquired or intrinsic quiescence among rare subpopulations of tumor cells contribute to therapeutic resistance and relapse? How do SOX2+ or OLIG2+ populations interact or promote changes in the tumor microenvironment? Despite the relative paucity of immune infiltrates in MB, can the tumor-associated microenvironment be leveraged to improve the efficacy of immunotherapy?

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