**8. Biology**

third ventriculostomy may be necessary to relieve raised intracranial pressure secondary to hydrocephalus. Many patients benefit from steroids which help by reducing peritumoral

Conventional RT in a dose of 54–60Gy over a 6 week period is commonly utilized in the treatment of DIPG [9]. Temporary improvement or stabilization of symptoms is seen in 70% of patients, but almost all patients have progressive disease. The mean progression-free survival (PFS) is 5.8 months with radiotherapy and 5 months without it [4]. As RT is effective temporarily, non-conventional doses and delivery schedules were investigated. In hyperfractionated RT, the total dose is divided into smaller doses and given more than once a day. Hyperfractionated RT in the form of 1Gy twice a day, to the total dose of 72Gy failed to improve the outcome [37]. Hypofractionated RT is given over a smaller period of time than standard RT by dividing the total RT dose into larger doses and treatments given once a day or less often. Studies investigating the role of hypofractionated RT with 39Gy [55] or 45Gy [56] delivered over 3 weeks have revealed similar survival outcomes, but may be more acceptable

Various chemotherapy and targeted agents have been used to potentiate the beneficial effects of RT. These agents were combined before, with, and after RT without much success [9, 10].

Chemotherapy combinations used with RT in the setting of phase I–III clinical trials. These include lomustine, vincristine and prednisone [41], cisplatin, etoposide, vincristine, ifosfamide and oral valproic acid [57], myeloablative thiotepa, isotretinoin and vinorelbine [58] and multiple other agents at relapse [59]. One trial evaluated the role of preradiation chemo-

**i.** Temozolomide (TMZ): TMZ is an alkylating prodrug which is converted into its active metabolite monomethyl 5-triazeno imidazole carboxamide. TMZ causes DNA damage

cacy in high grade gliomas, low toxicity and radiosensitization potential, temozolomide was trialed to potentiate RT efficacy without much success [8, 62, 63]. Addition of lomustine to adjuvant temozolomide [64] was not beneficial. O6-methylguanine DNA meth-

nucleotides. But this does not appear to be the cause of TMZ resistance as MGMT is not expressed in DIPG [47]. However, 3-methylpurine-DNA glycosylase (MPG), enzyme




yltransferase (MGMT) contributes to TMZ resistance by repairing alkylated O6

edema.

42 Brain Tumors - An Update

**7.2. Radiation therapy (RT)**

**7.3. Chemotherapy**

*7.3.1. Intensive chemotherapy*

*7.3.2. Radiosensitizing agents*

by alkylating O6

to fami9lies due to the shorter delivery times.

therapy [60]. The outcome was uniformly poor.


More than 250 therapeutic clinical trials including several targeted agents have not improved the dismal prognosis of DIPG [10]. The reason for this, at least in part, has been attributed to our lack of understanding of the biology of this disease. More has been published on the biology and pathophysiology of DIPG in the past 10 years than in all prior years combined [5]. A more recent and significant achievement in DIPG research is sample collection at autopsy. This has provided invaluable insights into understanding of the biology [71, 72]. Both autopsy and biopsy samples have allowed development of *in-vitro* (neurospheres) and *in-vivo* models (allograft and xenograft) [73–75].

#### **8.1. Cell of origin**

Pontine precursor-like cells (PPC), found in the ventral pons region, which are positive for the markers for the primitive neural precursor cells, nestin and vimentin, are postulated as the candidate cell of origin for DIPG [35]. Approximately half of PPC also expressed Olig2, a transcription factor which is associated with oligodendroglial precursors. This cell type was morphologically distinct from the nestin positive cells seen in the dorsal brainstem. PPC are present in all ventral brainstem structures during infancy and wane by 2 years of age. The ventral pontine and medullary nestin<sup>+</sup> cells show a second peak at 6 years, corresponding to the age of presentation of DIPG. Thus, temporal and spatial distribution of these cells correlates closely with the incidence of DIPG suggesting that tumors arise secondary to dysregulation of a postnatal neurodevelopmental process [35]. Expression of SOX2, a transcription factor with activity during embryogenesis, and Olig2 in another model supports the disordered neurodevelopmental origin of DIPG [76].

#### **8.2. The genomic landscape**

DIPG biopsy and autopsy samples have undergone extensive genomic profiling and major breakthroughs have been achieved in identifying key oncogenic pathways [77]. The drivers for DIPG tumorigenesis include epigenetic changes, gene mutations, deletions or overexpression and chromosomal number changes.

#### **8.3. Epigenetic changes**

#### *8.3.1. Histone mutations*

The DNA is packaged by histone proteins into a chain of nucleosomes which are the basic building blocks of the chromatin fiber [78]. In a single nucleosome, 147 base pairs (bp) of DNA wrap around histone octamers containing two copies each of histones H2A, H2B, H3 and H4 [79]. The N-terminal ends of histones containing lysine (K) and arginine (R) residues are posttranslationally modified by acetylation or methylation and regulate DNA repair, replication and transcription. The histone H3 family consists of a number of related proteins. Histone H3 isoforms H3.1 and H3.2 (also called as canonical H3) help in packaging newly replicated DNA. While H3.3 can function much the same as canonical H3 as a core part of the nucleosome, it is also deposited into transcriptionally active regions to replace histones lost during processes disrupting nucleosomes [80].

*8.3.1.1.3. Other novel mutations*

*8.3.1.2.1. K27 M mutations*

K27 M mutation has been described [87].

*TP53, PPM1D, ACVR1 or PI3KR1* [83, 95, 96].

*8.3.1.3. Co-mutations associated with H3 mutations in DIPG*

H3K27me3 and development of DIPG.

*8.3.1.4. Mutations of chromatin modifiers*

*8.3.1.2.2. G34R/V mutations*

*8.3.1.2. Downstream effects of H3 mutations and gliomagenesis*

*H3F3A* mutation resulting in lysine-to isoleucine substitution at K27 has been rarely seen in DIPG [87]. A mutation in the gene encoding the H3.2 variant, HIST2H3C, resulting in a novel

Diffuse Intrinsic Pontine Glioma

45

http://dx.doi.org/10.5772/intechopen.78578

K27M (and to a lesser extent K27I) is the only amino acid substitution which can ablate trimethylation (H3K27me3) [90]. The mutant H3K27M binds to the enhancer of zeste homolog 2 (EZH2) component of PRC2 interfering with methyltransferase activity of EZH2 which results in generalized hypomethylation. The downstream effect is derepression of targets of PRC2, upregulation of gene expression and gliomagenesis [91]. In addition, K27M mutation contributes to altered cell cycle control, inhibition of autophagy pathways and potentially increased resistance to radiotherapy [92]. Even though the mutant histone forms only 3.6–17.6% of the total cellular H3 pool, there is a near-absolute loss of H3K27me3. This represents a *trans*dominant-negative effect across all three isoforms of the wild-type H3 protein [90, 91, 93, 94]. However, the exact role of H3K27M in DIPG tumorigenesis remains unknown as it does not induce the tumors on its own *in vivo* [95]. One of the postulates of gliomagenesis is H3.3 K27M and G34R/V acting as driver mutations followed by a second hit by another mutation like

In summary, H3K27 mutations have a great significance irrespective of whichever histone H3 variant (H3.1-HIST13B, H2-HIST2H3C and H3.3-H3F3A) is targeted and will result in loss of

The role of H3.3G34R/V in gliomagenesis is less clear. It may act by disrupting K36me3 levels and activating potential oncogenes [90, 93]; inducing *MYCN* upregulation [97, 98] and disrupting interaction between H3.3 and ATRX/DAXX leading to aberrant deposition of H3.3

Other mutations associated with H3K27M mutation include α thalassemia/mental retardation syndrome X-linked (*ATRX)* or death-domain associated protein *(DAXX)* (30%), *TP53* (60%) and *NF-1*, *PDGFRA*, *BRAF*, *KRAS*, and *FGFR1* at lower frequencies [88, 99, 100]. G35R/V

Chromatin writers or erasers are the enzymes which catalyze the post-translational modifications of histone tails like methylation, acetylation and ubiquitylation of lysine residues,

near telomeric regions and leading to alternate lengthening of telomerase [88, 89, 99].

mutations coexpress with mutations in *TP53*, *ATRX/DAXX* and *PDGFRA* [83].

#### *8.3.1.1. H3 mutations and DIPG*

#### *8.3.1.1.1. H3K27M mutations*

*H3F3A* and *H3F3B* produce identical H3.3 proteins whereas *HIST1H3B* is one of the many genes encoding H3.1 [81, 82]. Distinct and recurrent mutations in H3 have been implicated in 70–80% of pediatric gliomas [83]. Lysine to methionine missense mutation at position 27 (K27M) was present in 78% of DIPG patients, with most of these mutations in *H3F3A* and up to 25% in *HIST1H3B* [84, 85]. H3K27M mutations are restricted to the midline structures [86] and H3.1 and H3.3 mutations involve two different oncogenic pathways resulting in distinct clinicopathological variants. H3.1 mutated tumors are exclusively linked to DIPG and exhibit a mesenchymal/astrocytic phenotype, a pro-angiogenic/hypoxic signature and are co-segregated with *ACVR1* mutations. Clinically, these tumors are less aggressive when compared to H3.3 mutant tumors, metastasize less frequently and respond better to radiation therapy (RT) with a median overall survival (OS) of 15 months. H3.3 mutated tumors are located in the midline structures including the brainstem, thalamus and spinal cord. They have a proneural/oligodendroglial phenotype, a pro-metastatic gene expression signature with *PDGFRA* activation. They behave more aggressively, responding poorly to RT with a median OS of 9 months and metastasize more frequently [87]. The gain-of-function H3K27M alterations are exclusive to pediatric high-grade gliomas and any H3 mutation is associated with a dismal outcome but identification of a specific mutation may help in developing specific therapeutic targets.

#### *8.3.1.1.2. G34R/V mutations*

*H3F3A* mutations encoding a glycine 34 to arginine or valine G34R/V comprise a smaller proportion of H3.3 mutations [88]. G34R/V mutations are seen in cerebral hemispheres of slightly older patients (9–42 years) as compared to K27M mutations (5–29 years) [86, 88, 89].

K27M and G34R/V mutations are mutually exclusive and heterozygously expressed, with one wild-type *H3F3A* allele [89].

#### *8.3.1.1.3. Other novel mutations*

**8.3. Epigenetic changes**

44 Brain Tumors - An Update

*8.3.1. Histone mutations*

processes disrupting nucleosomes [80].

*8.3.1.1. H3 mutations and DIPG*

*8.3.1.1.1. H3K27M mutations*

targets.

*8.3.1.1.2. G34R/V mutations*

wild-type *H3F3A* allele [89].

The DNA is packaged by histone proteins into a chain of nucleosomes which are the basic building blocks of the chromatin fiber [78]. In a single nucleosome, 147 base pairs (bp) of DNA wrap around histone octamers containing two copies each of histones H2A, H2B, H3 and H4 [79]. The N-terminal ends of histones containing lysine (K) and arginine (R) residues are posttranslationally modified by acetylation or methylation and regulate DNA repair, replication and transcription. The histone H3 family consists of a number of related proteins. Histone H3 isoforms H3.1 and H3.2 (also called as canonical H3) help in packaging newly replicated DNA. While H3.3 can function much the same as canonical H3 as a core part of the nucleosome, it is also deposited into transcriptionally active regions to replace histones lost during

*H3F3A* and *H3F3B* produce identical H3.3 proteins whereas *HIST1H3B* is one of the many genes encoding H3.1 [81, 82]. Distinct and recurrent mutations in H3 have been implicated in 70–80% of pediatric gliomas [83]. Lysine to methionine missense mutation at position 27 (K27M) was present in 78% of DIPG patients, with most of these mutations in *H3F3A* and up to 25% in *HIST1H3B* [84, 85]. H3K27M mutations are restricted to the midline structures [86] and H3.1 and H3.3 mutations involve two different oncogenic pathways resulting in distinct clinicopathological variants. H3.1 mutated tumors are exclusively linked to DIPG and exhibit a mesenchymal/astrocytic phenotype, a pro-angiogenic/hypoxic signature and are co-segregated with *ACVR1* mutations. Clinically, these tumors are less aggressive when compared to H3.3 mutant tumors, metastasize less frequently and respond better to radiation therapy (RT) with a median overall survival (OS) of 15 months. H3.3 mutated tumors are located in the midline structures including the brainstem, thalamus and spinal cord. They have a proneural/oligodendroglial phenotype, a pro-metastatic gene expression signature with *PDGFRA* activation. They behave more aggressively, responding poorly to RT with a median OS of 9 months and metastasize more frequently [87]. The gain-of-function H3K27M alterations are exclusive to pediatric high-grade gliomas and any H3 mutation is associated with a dismal outcome but identification of a specific mutation may help in developing specific therapeutic

*H3F3A* mutations encoding a glycine 34 to arginine or valine G34R/V comprise a smaller proportion of H3.3 mutations [88]. G34R/V mutations are seen in cerebral hemispheres of slightly older patients (9–42 years) as compared to K27M mutations (5–29 years) [86, 88, 89]. K27M and G34R/V mutations are mutually exclusive and heterozygously expressed, with one *H3F3A* mutation resulting in lysine-to isoleucine substitution at K27 has been rarely seen in DIPG [87]. A mutation in the gene encoding the H3.2 variant, HIST2H3C, resulting in a novel K27 M mutation has been described [87].

#### *8.3.1.2. Downstream effects of H3 mutations and gliomagenesis*

#### *8.3.1.2.1. K27 M mutations*

K27M (and to a lesser extent K27I) is the only amino acid substitution which can ablate trimethylation (H3K27me3) [90]. The mutant H3K27M binds to the enhancer of zeste homolog 2 (EZH2) component of PRC2 interfering with methyltransferase activity of EZH2 which results in generalized hypomethylation. The downstream effect is derepression of targets of PRC2, upregulation of gene expression and gliomagenesis [91]. In addition, K27M mutation contributes to altered cell cycle control, inhibition of autophagy pathways and potentially increased resistance to radiotherapy [92]. Even though the mutant histone forms only 3.6–17.6% of the total cellular H3 pool, there is a near-absolute loss of H3K27me3. This represents a *trans*dominant-negative effect across all three isoforms of the wild-type H3 protein [90, 91, 93, 94]. However, the exact role of H3K27M in DIPG tumorigenesis remains unknown as it does not induce the tumors on its own *in vivo* [95]. One of the postulates of gliomagenesis is H3.3 K27M and G34R/V acting as driver mutations followed by a second hit by another mutation like *TP53, PPM1D, ACVR1 or PI3KR1* [83, 95, 96].

In summary, H3K27 mutations have a great significance irrespective of whichever histone H3 variant (H3.1-HIST13B, H2-HIST2H3C and H3.3-H3F3A) is targeted and will result in loss of H3K27me3 and development of DIPG.

#### *8.3.1.2.2. G34R/V mutations*

The role of H3.3G34R/V in gliomagenesis is less clear. It may act by disrupting K36me3 levels and activating potential oncogenes [90, 93]; inducing *MYCN* upregulation [97, 98] and disrupting interaction between H3.3 and ATRX/DAXX leading to aberrant deposition of H3.3 near telomeric regions and leading to alternate lengthening of telomerase [88, 89, 99].

#### *8.3.1.3. Co-mutations associated with H3 mutations in DIPG*

Other mutations associated with H3K27M mutation include α thalassemia/mental retardation syndrome X-linked (*ATRX)* or death-domain associated protein *(DAXX)* (30%), *TP53* (60%) and *NF-1*, *PDGFRA*, *BRAF*, *KRAS*, and *FGFR1* at lower frequencies [88, 99, 100]. G35R/V mutations coexpress with mutations in *TP53*, *ATRX/DAXX* and *PDGFRA* [83].

#### *8.3.1.4. Mutations of chromatin modifiers*

Chromatin writers or erasers are the enzymes which catalyze the post-translational modifications of histone tails like methylation, acetylation and ubiquitylation of lysine residues, phosphorylation of serine or threonine residues and methylation of arginine residues. The effector proteins called readers are recruited to the chromatin by the resultant histone code which helps in localization of functional complexes that affect transcriptional regulation [101]. Numerous recurrent mutations are observed in chromatin writers, erasers, readers and remodelers in DIPG and other tumors [102].

lower mutation burden [84]. At the other end of the spectrum are HGG from patients with inherited mutations in mismatch repair genes. Germline mutations in tumor suppressor genes like *TP53* and neurofibromin 1 (*NF1*) predispose to the development of HGG [110]. The hypermutated tumors arising in the context of these germline mutations show a very high number of somatic SNVs; these may be more than 100-fold higher than in 95% of pediatric HGG [84]. DIPG show similar mutation burden as other pediatric HGG and their genomic complexity is indicative of multiple genetic mechanisms generating numerous mutations which provide the tumor with diverse potential pathways to therapeutic resistance [77].

Diffuse Intrinsic Pontine Glioma

47

http://dx.doi.org/10.5772/intechopen.78578

*ACVR1*, also known as *ALK2* encodes the serine kinase, Activin receptor type 1 A (ACVR1) [111]. It is a type 1 BMP receptor which belongs to the mammalian TGF-β signaling family [112]. ACVR1 binds to a diverse set of ligands, including TGF-β, activins and multiple BMP [113]. ACVR1 is essential for signaling and after ligand it is phosphorylated by ACVR2 with formation a stable ACVR1/2 complex [114]. This results in the phosphorylation and activation of growth promoting genes through SMAD transcription factors [77]. ACVR1 mutations are constitutionally activating, leading to increased expression of activin signal-

Germline *ACVR1* mutations cause the congenital malformation syndrome fibrodysplasia ossificans progressive (FOP) [116]. Seven somatic mutations of this gene have been identified in 13–32% of DIPG samples leading to either ligand-independent kinase activation or gain-of-function effects [84, 111, 112, 115]. However, there is no increased cancer risk in FOP despite having similar germline mutations as those seen in DIPG which suggests that *ACVR1* mutations on their own are not tumor initiating and lead to DIPG only in the presence of other

*ACVR1* mutant tumors commonly co-segregate with *HIST1H3B* mutations [115]. *ACVR1- HIST1H3B* co-segregating tumors do not show *TP53* loss or *PDGFRA* amplifications but

*ACVR1* mutations signify a distinct subset of DIPG patients. They occurred more frequently in females (F:M ratio of 1.75:1) and are associated with younger age and longer survival (median OS of 14.9 months) [84, 111]. There was a significant pharmacologic inhibition of ACVR1 by a selective ALK2 inhibitor, LDN-193189, leading to dose-dependent cytotoxicity across all the tested DIPG cell lines [111]. Due to its role in DIPG pathogenesis and targetable potential,

around 60% have mutations in the PI3K signaling pathway [114].

ACVR1 inhibition represents a novel therapeutic option.

*8.4.2. Abnormalities of cellular proliferation pathways*

ing targets *ID1* and *ID2* [115].

mutations [77, 116].

*8.4.2.1.2. ACVR1 co-mutations*

*8.4.2.1.3. Clinical implications*

*8.4.2.1.1. ACVR1 mutations and DIPG*

*8.4.2.1. Bone morphogenetic protein (BMP) signaling and ACVR1 mutations*

The discovery of histone mutations which are present in up to 80% of DIPG is one of the most remarkable breakthroughs in terms of understanding DIPG biology and identification of actionable targets [90, 92, 99].

#### *8.3.2. Polycomb repressive complex (PRC) abnormalities*

Polycomb group proteins remodel chromatin enabling epigenetic silencing of genes. There are two main Polycomb group complexes found in mammals-PRC1 and PRC2. PRC1 catalyzes the monoubiquitylation of histone H2A and PRC2 catalyzes the methylation of H3K27 [103]. Some PRC1 complexes also act independent of enzymatic activity to regulate gene expression by compacting chromatin [104]. PRC1 functions downstream of PRC2 by binding specifically to H3K27me3 [103]. By inducing such sequential histone modifications, PRC1 and PRC2 achieve stable silencing of gene expression [105]. Dysregulation of PRC and its downstream targets has been implicated in many cancers [83]. B cell-specific Moloney murine leukemia virus integration site 1 (BMI-1) is a component of PRC1 complex. It was found to be highly expressed in DIPG tumor cells and its downregulation inhibited various cellular processes like cell proliferation, cell cycle signaling, telomerase expression and activity, and cell migration [105].

#### **8.4. Gene abnormalities**

Molecular profiling of DIPG samples has provided new insights [61]. In the past, candidate gene approaches were utilized to identify gene abnormalities associated with adult highgrade gliomas (HGG) [106]. Although, these studies were limited in defining the biology of DIPG due to their small numbers, they still highlighted some differences between adult HGG and DIPG. Recently, studies performed with next-generation sequencing approaches have confirmed that DIPG are molecularly distinct from adult HGG and non-DIPG pediatric HGG [18]. The current technologies utilize whole-genome sequencing (WGS), whole-exome sequencing (WES), and RNA-sequencing in addition to copy number, gene expression, and methylation profiles and histopathology.

#### *8.4.1. Mutational burden of DIPG compared to other tumors*

The genomic signatures of the most pediatric HGG are complex and involve significant copy number alterations (CNAs), single nucleotide variants (SNVs) and structural variants [107– 109]. HGG have a higher mutation burden than many other pediatric cancers but it is still lower than common adult cancers [77]. HGG commonly show structural variants like simple rearrangements and abnormalities caused by chromothripsis [84]. But there is a wide range of genomic complexity in pediatric HGG. At one end of the spectrum is infant non-brainstem HGG (NBS-HGG) arising in children less than 3 years old. These tumors have significantly lower mutation burden [84]. At the other end of the spectrum are HGG from patients with inherited mutations in mismatch repair genes. Germline mutations in tumor suppressor genes like *TP53* and neurofibromin 1 (*NF1*) predispose to the development of HGG [110]. The hypermutated tumors arising in the context of these germline mutations show a very high number of somatic SNVs; these may be more than 100-fold higher than in 95% of pediatric HGG [84]. DIPG show similar mutation burden as other pediatric HGG and their genomic complexity is indicative of multiple genetic mechanisms generating numerous mutations which provide the tumor with diverse potential pathways to therapeutic resistance [77].

#### *8.4.2. Abnormalities of cellular proliferation pathways*

phosphorylation of serine or threonine residues and methylation of arginine residues. The effector proteins called readers are recruited to the chromatin by the resultant histone code which helps in localization of functional complexes that affect transcriptional regulation [101]. Numerous recurrent mutations are observed in chromatin writers, erasers, readers and

The discovery of histone mutations which are present in up to 80% of DIPG is one of the most remarkable breakthroughs in terms of understanding DIPG biology and identification

Polycomb group proteins remodel chromatin enabling epigenetic silencing of genes. There are two main Polycomb group complexes found in mammals-PRC1 and PRC2. PRC1 catalyzes the monoubiquitylation of histone H2A and PRC2 catalyzes the methylation of H3K27 [103]. Some PRC1 complexes also act independent of enzymatic activity to regulate gene expression by compacting chromatin [104]. PRC1 functions downstream of PRC2 by binding specifically to H3K27me3 [103]. By inducing such sequential histone modifications, PRC1 and PRC2 achieve stable silencing of gene expression [105]. Dysregulation of PRC and its downstream targets has been implicated in many cancers [83]. B cell-specific Moloney murine leukemia virus integration site 1 (BMI-1) is a component of PRC1 complex. It was found to be highly expressed in DIPG tumor cells and its downregulation inhibited various cellular processes like cell proliferation, cell cycle signaling, telomerase expression and activity, and

Molecular profiling of DIPG samples has provided new insights [61]. In the past, candidate gene approaches were utilized to identify gene abnormalities associated with adult highgrade gliomas (HGG) [106]. Although, these studies were limited in defining the biology of DIPG due to their small numbers, they still highlighted some differences between adult HGG and DIPG. Recently, studies performed with next-generation sequencing approaches have confirmed that DIPG are molecularly distinct from adult HGG and non-DIPG pediatric HGG [18]. The current technologies utilize whole-genome sequencing (WGS), whole-exome sequencing (WES), and RNA-sequencing in addition to copy number, gene expression, and

The genomic signatures of the most pediatric HGG are complex and involve significant copy number alterations (CNAs), single nucleotide variants (SNVs) and structural variants [107– 109]. HGG have a higher mutation burden than many other pediatric cancers but it is still lower than common adult cancers [77]. HGG commonly show structural variants like simple rearrangements and abnormalities caused by chromothripsis [84]. But there is a wide range of genomic complexity in pediatric HGG. At one end of the spectrum is infant non-brainstem HGG (NBS-HGG) arising in children less than 3 years old. These tumors have significantly

remodelers in DIPG and other tumors [102].

*8.3.2. Polycomb repressive complex (PRC) abnormalities*

of actionable targets [90, 92, 99].

46 Brain Tumors - An Update

cell migration [105].

**8.4. Gene abnormalities**

methylation profiles and histopathology.

*8.4.1. Mutational burden of DIPG compared to other tumors*

#### *8.4.2.1. Bone morphogenetic protein (BMP) signaling and ACVR1 mutations*

*ACVR1*, also known as *ALK2* encodes the serine kinase, Activin receptor type 1 A (ACVR1) [111]. It is a type 1 BMP receptor which belongs to the mammalian TGF-β signaling family [112]. ACVR1 binds to a diverse set of ligands, including TGF-β, activins and multiple BMP [113]. ACVR1 is essential for signaling and after ligand it is phosphorylated by ACVR2 with formation a stable ACVR1/2 complex [114]. This results in the phosphorylation and activation of growth promoting genes through SMAD transcription factors [77]. ACVR1 mutations are constitutionally activating, leading to increased expression of activin signaling targets *ID1* and *ID2* [115].

#### *8.4.2.1.1. ACVR1 mutations and DIPG*

Germline *ACVR1* mutations cause the congenital malformation syndrome fibrodysplasia ossificans progressive (FOP) [116]. Seven somatic mutations of this gene have been identified in 13–32% of DIPG samples leading to either ligand-independent kinase activation or gain-of-function effects [84, 111, 112, 115]. However, there is no increased cancer risk in FOP despite having similar germline mutations as those seen in DIPG which suggests that *ACVR1* mutations on their own are not tumor initiating and lead to DIPG only in the presence of other mutations [77, 116].

#### *8.4.2.1.2. ACVR1 co-mutations*

*ACVR1* mutant tumors commonly co-segregate with *HIST1H3B* mutations [115]. *ACVR1- HIST1H3B* co-segregating tumors do not show *TP53* loss or *PDGFRA* amplifications but around 60% have mutations in the PI3K signaling pathway [114].

#### *8.4.2.1.3. Clinical implications*

*ACVR1* mutations signify a distinct subset of DIPG patients. They occurred more frequently in females (F:M ratio of 1.75:1) and are associated with younger age and longer survival (median OS of 14.9 months) [84, 111]. There was a significant pharmacologic inhibition of ACVR1 by a selective ALK2 inhibitor, LDN-193189, leading to dose-dependent cytotoxicity across all the tested DIPG cell lines [111]. Due to its role in DIPG pathogenesis and targetable potential, ACVR1 inhibition represents a novel therapeutic option.

#### *8.4.2.2. Receptor tyrosine kinase (RTK) pathway*

RTKs are transmembrane protein receptors containing intrinsic enzymatic activity. Their ligands include growth factors, hormones and cytokines [117] and they play an critical role in mediating key signaling pathways involving cell proliferation, differentiation, survival and migration [118]. The human RTK family has 20 subfamilies and 58 known members including platelet-derived growth factor receptors (PDGFR), epidermal growth factor receptors (EGFR) and fibroblast growth factor receptors (FGFR) [118–120]. Upon ligand binding, the RTKs are activated leading to signal transduction to the nucleus and subsequent protein transcription. This is achieved by downstream activation of various RTK substrates like mitogen-activated protein kinase (MAPK) and phosphatidylinositol-3-kinase (PI3K) [121].

*8.4.2.2.2. RTK pathway co-mutations in DIPG*

may not be effective on their own [124].

*8.4.2.3. MYC and MYCN aberrations*

*8.4.2.4. Hedgehog (Hh) signaling*

tial DIPG cancer stem cells (CSC).

tions [124].

*8.4.2.2.3. Clinical significance*

*PDGFRA* gains and amplifications co-segregate with H3.3 mutations [88, 99] and *TP53* muta-

Diffuse Intrinsic Pontine Glioma

49

http://dx.doi.org/10.5772/intechopen.78578

RTK signaling dysregulation, particularly PDGFR pathway either overexpression or mutation, may have an important role in the pathophysiology of DIPG and provide therapeutic targets [47]. Identification of PDGFRA mutations may be beneficial in developing targeted therapies. But some particular mutations like PDGFR D842V in the gastrointestinal stromal tumors confers resistance to imatinib [127]. Others may have only cytostatic effects so they

*MYCN* proto-oncogene is a member of the MYC family encoding the protein MYCN. MYCN plays a critical role during embryogenesis and is involved in cellular proliferation and differentiation [128]. *MYCN* amplification is seen in DIPG [84, 92, 99] and is associated with hypermethylation, high-grade histology and chromothripsis on chromosome 2p [115]. *MYCN* amplifications are transcriptional regulators that affect the epigenetic landscape by enhancing gene expression across the whole genome [84]. MYCN pathway maybe induced by H3.3 K27 M [99] or H3.3G34V [83, 102] but may act independent of H3 mutations [92].

Hh signaling pathway plays a major role in regulation of developmental processes like cell proliferation, cell differentiation, tissue polarity and stem cell maintenance. Aberrant activation of Hh pathway has been implicated in the pathogenesis of cancers like medulloblastoma. No structural mutation involving this pathway resulting in the development of DIPG has been identified so far. However, in pre-clinical murine models [35] upregulation of Hh pathway activity resulted in ventral pontine hyperplasia. Hh pathway is essential for the normal development of PPC in humans. Also, Hh pathway activation stimulates and blockade reduces the self-renewal capacity of DIPG neurosphere cells. These findings indicate that Hh signaling, which drives the development of neural precursors in the ventral pons, may play a role in tumor formation in a subset of DIPG. Patients with Gorlin syndrome, a genetic entity occurring secondary to unregulated Hh activity, usually do not develop DIPG [35]. So, in addition to unregulated Hh pathway activity, a second "hit" may be necessary for DIPG transformation. Hh signaling role in the pathogenesis of DIPG was further investigated in a study [92] which identified upregulation of Hh signaling. DIPG samples showed upregulation of Patched (PTCH) and nuclear translocation of Glioma Associated Oncogene 1 (GLI1); both PTCH and GLI1 are key Hh pathway molecules. In summary, Hh pathway may play a significant role in DIPG tumorigenesis by stimulating PPC and transforming them into poten-

#### *8.4.2.2.1. RTK pathway aberrations and DIPG*

Amplifications and mutations in components of RTK-RAS-PI3K pathway are seen in up to 60% of DIPG [18]. The most common affected component is *PDGFRA*. Other abnormalities involve *AKT1, AKT3*, *c-MET*, epidermal growth factor receptor (*EGFR*), erythroblastic leukemia viral oncogene homolog-4 (*ERBB4*), hepatocyte growth factor, Kirsten rat sarcoma viral oncogene homolog (*KRAS*), *PIK3CA, PIKC2G, PIK3R1, PTEN*, insulin-like growth factor 2, and insulin-like growth factor receptor [18, 45, 53, 111].

#### *8.4.2.2.1.1. PDGFRA amplifications*

Whole-genome profiling of DIPG tumors have identified recurrent amplifications of *PDGFA* and *PDGFRA* with overexpression of PDGFR-α in 28–50% tumors [47, 48, 122, 123].

#### *8.4.2.2.1.2. PDGFRA mutations*

Somatic activating mutations including missense mutations and in-frame deletions and insertions were identified in 4.7% DIPG tumors and were found to be oncogenic *in vivo* [124]. Concurrent amplification was seen in 40% of tumors with mutations and 60% had heterozygous mutations [124]. Similar mutations were identified in other studies in 8.8–25% samples [54, 123]. Downstream activation of the PDGFR pathway has been shown by phospho-mammalian target of rapamycin (m-TOR) immunopositivity [47] as well as activation of MAPK and PI3K pathways [124]. PDGFR-α is expressed by the oligodendrocyte precursor cell derived from the candidate cell of origin PCC [35] and hence the precursor cell may be responsive to PDGF. Also, human DIPG cell culture yield was better after the addition of PDGF [35] and upregulation of PDGF pathway was associated with dorsal pontine glioblastoma in mouse models [125, 126].

#### *8.4.2.2.1.3. EGFR aberrations*

EGFR immunopositivity and gene amplification were seen in about 27% [47] and 7–9% [48] of cases respectively.

### *8.4.2.2.2. RTK pathway co-mutations in DIPG*

*PDGFRA* gains and amplifications co-segregate with H3.3 mutations [88, 99] and *TP53* mutations [124].

#### *8.4.2.2.3. Clinical significance*

*8.4.2.2. Receptor tyrosine kinase (RTK) pathway*

48 Brain Tumors - An Update

*8.4.2.2.1. RTK pathway aberrations and DIPG*

*8.4.2.2.1.1. PDGFRA amplifications*

*8.4.2.2.1.2. PDGFRA mutations*

models [125, 126].

of cases respectively.

*8.4.2.2.1.3. EGFR aberrations*

and insulin-like growth factor receptor [18, 45, 53, 111].

RTKs are transmembrane protein receptors containing intrinsic enzymatic activity. Their ligands include growth factors, hormones and cytokines [117] and they play an critical role in mediating key signaling pathways involving cell proliferation, differentiation, survival and migration [118]. The human RTK family has 20 subfamilies and 58 known members including platelet-derived growth factor receptors (PDGFR), epidermal growth factor receptors (EGFR) and fibroblast growth factor receptors (FGFR) [118–120]. Upon ligand binding, the RTKs are activated leading to signal transduction to the nucleus and subsequent protein transcription. This is achieved by downstream activation of various RTK substrates like mitogen-activated

Amplifications and mutations in components of RTK-RAS-PI3K pathway are seen in up to 60% of DIPG [18]. The most common affected component is *PDGFRA*. Other abnormalities involve *AKT1, AKT3*, *c-MET*, epidermal growth factor receptor (*EGFR*), erythroblastic leukemia viral oncogene homolog-4 (*ERBB4*), hepatocyte growth factor, Kirsten rat sarcoma viral oncogene homolog (*KRAS*), *PIK3CA, PIKC2G, PIK3R1, PTEN*, insulin-like growth factor 2,

Whole-genome profiling of DIPG tumors have identified recurrent amplifications of *PDGFA*

Somatic activating mutations including missense mutations and in-frame deletions and insertions were identified in 4.7% DIPG tumors and were found to be oncogenic *in vivo* [124]. Concurrent amplification was seen in 40% of tumors with mutations and 60% had heterozygous mutations [124]. Similar mutations were identified in other studies in 8.8–25% samples [54, 123]. Downstream activation of the PDGFR pathway has been shown by phospho-mammalian target of rapamycin (m-TOR) immunopositivity [47] as well as activation of MAPK and PI3K pathways [124]. PDGFR-α is expressed by the oligodendrocyte precursor cell derived from the candidate cell of origin PCC [35] and hence the precursor cell may be responsive to PDGF. Also, human DIPG cell culture yield was better after the addition of PDGF [35] and upregulation of PDGF pathway was associated with dorsal pontine glioblastoma in mouse

EGFR immunopositivity and gene amplification were seen in about 27% [47] and 7–9% [48]

and *PDGFRA* with overexpression of PDGFR-α in 28–50% tumors [47, 48, 122, 123].

protein kinase (MAPK) and phosphatidylinositol-3-kinase (PI3K) [121].

RTK signaling dysregulation, particularly PDGFR pathway either overexpression or mutation, may have an important role in the pathophysiology of DIPG and provide therapeutic targets [47]. Identification of PDGFRA mutations may be beneficial in developing targeted therapies. But some particular mutations like PDGFR D842V in the gastrointestinal stromal tumors confers resistance to imatinib [127]. Others may have only cytostatic effects so they may not be effective on their own [124].

#### *8.4.2.3. MYC and MYCN aberrations*

*MYCN* proto-oncogene is a member of the MYC family encoding the protein MYCN. MYCN plays a critical role during embryogenesis and is involved in cellular proliferation and differentiation [128]. *MYCN* amplification is seen in DIPG [84, 92, 99] and is associated with hypermethylation, high-grade histology and chromothripsis on chromosome 2p [115]. *MYCN* amplifications are transcriptional regulators that affect the epigenetic landscape by enhancing gene expression across the whole genome [84]. MYCN pathway maybe induced by H3.3 K27 M [99] or H3.3G34V [83, 102] but may act independent of H3 mutations [92].

#### *8.4.2.4. Hedgehog (Hh) signaling*

Hh signaling pathway plays a major role in regulation of developmental processes like cell proliferation, cell differentiation, tissue polarity and stem cell maintenance. Aberrant activation of Hh pathway has been implicated in the pathogenesis of cancers like medulloblastoma. No structural mutation involving this pathway resulting in the development of DIPG has been identified so far. However, in pre-clinical murine models [35] upregulation of Hh pathway activity resulted in ventral pontine hyperplasia. Hh pathway is essential for the normal development of PPC in humans. Also, Hh pathway activation stimulates and blockade reduces the self-renewal capacity of DIPG neurosphere cells. These findings indicate that Hh signaling, which drives the development of neural precursors in the ventral pons, may play a role in tumor formation in a subset of DIPG. Patients with Gorlin syndrome, a genetic entity occurring secondary to unregulated Hh activity, usually do not develop DIPG [35]. So, in addition to unregulated Hh pathway activity, a second "hit" may be necessary for DIPG transformation. Hh signaling role in the pathogenesis of DIPG was further investigated in a study [92] which identified upregulation of Hh signaling. DIPG samples showed upregulation of Patched (PTCH) and nuclear translocation of Glioma Associated Oncogene 1 (GLI1); both PTCH and GLI1 are key Hh pathway molecules. In summary, Hh pathway may play a significant role in DIPG tumorigenesis by stimulating PPC and transforming them into potential DIPG cancer stem cells (CSC).

#### *8.4.3. Abnormalities of cell cycle regulation pathways*

#### *8.4.3.1. TP53 pathway*

The TP53 pathway is a complex network of genes which respond to diverse internal and external stress signals and have an impact on the normal cellular homeostasis [129]. The p53 protein is activated by stress signals transmitted as post-translational modifications leading to apoptosis [130]. In addition, the TP53 pathway produces proteins which aid directly in DNA repair processes and alter cellular environment enabling inter-cellular communication [131]. In the critical role of safeguarding the genomic integrity, it functions as a tumor suppression pathway [132]. *TP53* is the most commonly mutated gene found in a broad variety of human cancers [129, 133].

[122, 133, 143] causing a dysfunctional G1

**8.5. Chromosomal number abnormalities**

**8.6. Immune checkpoint abnormalities**

neuroblastoma using intrathecal 131I-8H9 [150].

**8.7. Tumor microenvironment abnormalities**

*8.7.1. Neuroligin-3 (NLGN3) role*

into investigating microenvironment alteration for better results.

in cell death.

the treatment effect.

*8.6.1. B7-H3 abnormalities*

post-mortem DIPG samples [73]. Abrogation of the G2

*8.4.3.5. Poly (ADP-ribose) polymerase (PARP)-1 abnormalities*

arrest. So, these cells rely heavily on G2 checkpoint

checkpoint achieved by WEE1 kinase

http://dx.doi.org/10.5772/intechopen.78578

Diffuse Intrinsic Pontine Glioma

51

to repair DNA damage caused by irradiation. WEE1 protein is significantly overexpressed in

inhibition pushes DIPG cells with unrepaired DAN damage into mitotic catastrophe resulting

PARP-1 is a nuclear protein involved in the DNA damage repair processes [144]. PARP-1 activity provides an escape mechanism for cancer cells to avoid apoptosis and its overexpression may be associated with temozolomide and radiation resistance [47]. Gain of *PARP-1* is

Copy number abnormalities (CNAs) reported in DIPG include gain in chromosomes 1q, 2q, 8q, 9q, 7p/7q and loss in chromosomes 16q, 17p, 20p, 21q, 10q and 4q [47, 48, 122, 123, 139, 145]. The CNAs may represent the initial mutations responsible for DIPG tumorigenesis as well as

B7-H3 or CD276, a member of the B7-CD28 family, is a type I transmembrane glycoprotein [146]. Many malignant neuroectodermal tumors including adult HGG over-express B7-H3. B7-H3 was found to be overexpressed in a small panel of DIPG samples obtained at autopsy [147]. A monoclonal antibody 8H9 recognizes it and binds specifically to the tumor cells [148, 149] enabling therapeutic cell selectivity. B7H3 was targeted safely in the salvage therapy of stage IV

Therapies targeted at intrinsic cellular pathways have yielded poor results in DIPG. Tumor microenvironment plays a vital role in tumorigenesis and progression, so studies have looked

Neuroligin-3 (NLGN3) is a synaptic adhesion molecule which is cleaved from neurons and oligodendrocyte precursor cells via the ADAM10 sheddase and released into the tumor microenvironment. This important neuronal activity promotes many types of brain cancers including DIPG, pediatric and adult HGG and anaplastic oligodendroglioma. NLGN3 release activates oncogenic pathways like focal adhesion kinase activation resulting in the downstream PI3K-mTOR pathway induction. This in turn causes upregulation of several synapse-related genes resulting in the proliferation of glioma cells [151]. HGG glioma growth in xenograft models was blocked by ADAM10 inhibitors by preventing NLGN3 release into

seen in DIPG tumors and provides a potentially targetable therapeutic option [47].

### *8.4.3.1.1. TP53 mutations and DIPG*

*TP53* mutations are commonly found in DIPG with the reported incidence between 9 and 77% [45, 99]. They are more common in higher grade histology tumors (grades III and IV) [53]. About 50% of *TP53* wild-type grade II DIPG show presence of *PPM1D* mutations [134]. *PPM1D* is an oncogene associated with cancers like neuroblastoma [135] and lung cancer [136] which codes for wild-type p53-induced phosphatase 1D (WIP1). WIP1 is a negative regulator of *TP53* as it inactivates p53 and promotes termination of stress-induced responses. So *PPM1D* mutations have the same functional significance as *TP53* mutations [137]. *PPM1D* and *TP53* mutations are mutually exclusive and may ultimately lead to dysregulated homeostasis and tumorigenesis [134].

#### *8.4.3.1.2. TP53 co-mutations in DIPG*

*TP53* mutations more commonly co-segregate with H3.3 K27 M than H3.1 K27 M [112] and frequently occur in the setting of *PDGFRA* aberrations [124].

#### *8.4.3.2. The RB pathway*

Cyclins and cyclin-dependent kinases (CDKs) control the G1 /S transition of the cell cycle [138]. The abnormalities involving these regulators observed in DIPG include cyclin-dependent kinase inhibitor 2A or 2B (*CDKN2A* or *CDKN2B*) deletions [48, 122] and *CDK4*, *CDK6* or cyclin D1 (*CCND1*), *CCND2,* and *CCND3* amplifications [48, 122, 139].

#### *8.4.3.3. Aurora kinase pathway*

Aurora kinase family include three highly homologous serine/threonine kinases required during mitosis and which are linked to many cancers [140]. AURKB forms the catalytic component of the chromosomal passenger complex (CPC) which plays a critical role during mitosis [141]. Almost 70% of DIPG have demonstrated overexpression of *AURKB* [142].

#### *8.4.3.4. WEE1 kinase pathway*

WEE1 kinase is an important part of G2 checkpoint. DIPG cells, unlike normal cells, have aberrations in genes regulating the G1 checkpoint, including *TP53*, *MDM2*, *CDKN2A*, and *ATM* [122, 133, 143] causing a dysfunctional G1 arrest. So, these cells rely heavily on G2 checkpoint to repair DNA damage caused by irradiation. WEE1 protein is significantly overexpressed in post-mortem DIPG samples [73]. Abrogation of the G2 checkpoint achieved by WEE1 kinase inhibition pushes DIPG cells with unrepaired DAN damage into mitotic catastrophe resulting in cell death.
