Recent data have led to a re-classification of pHGGs based on molecular subgrouping. In adult gliomas, IDH1 and TERTp mutations represent key molecular alterations, whereas in pHGGs are rare. A significant difference between pediatric and adult gliomas is the frequency of histone H3F3A mutations, frequently reported in pHGGs, while are nearly absent in adults. The most common variants affect amino acid 34 and 27 (G34R/V and K27M), representing different clinico-pathological and biological subgroups. Elongation of telomeres, necessary for tumor cell immortalization, can be performed via reactivation of telomerase or ALT. TERTp mutations and hyper-methylation result in up-regulation of telomerase activity; neoplastic cells, which do not reactivate telomerase, trigger ALT. Previous studies have identified a connection of ATRX and H3F3A mutations with ALT phenotype in gliomas. However, an extensive evaluation of the association of telomeres elongation with specific molecular subgroups of pHGG is still lacking, the comparison between pHGG K27M and G34R/V mutated, in terms of ALT activation, was not elucidated.
The aim of the project is to investigate which genetic alterations trigger ALT in different molecular subgroups of pHGG, in order to evidence the involvement of each genetic alteration in tumor cell senescence escape by telomeres elongation, defining the differences of each molecular subgroup and extending the knowledge on the biology of the H3.3-mutated (G34, K27) pHGGs. We will screen a cohort of histological revised pHGGs using Telo-FISH to evaluate telomeres length; loss of ATRX expression will be analyzed by IHC, H3F3A and TERTp mutations by sequencing, TERTp methylation by MS-PCR. Finally, data will be correlated to patients survival using Kaplan-Meier method. With this project we hope to translate our results into patient benefit, contributing to the risk stratification definition, and to application of new targeted treatments for a subset of these tumors.
Given the clinical and biological heterogeneity of pHGGs, improvements in therapy will require patient stratification based upon their molecular hallmarks [22; 24; 26]. The aim of this project is to investigate which genetic alterations trigger the mechanisms of telomeres elongation in pHGG, in order to define the differences of the histone-defined subgroups G34R/V and K27M in terms of ALT activation and telomere lengthening control [4; 5].
The present study could help in translating the emerging molecular insights into patient benefit, showing the activation of telomeres maintenance mechanism via ALT or telomerase in different molecular subgroups of pediatric gliomas with H3G34-mut, H3K27-mut, ATRX inactivation and TERTp status [8; 9; 10; 13; 17; 18].
We will investigate which molecular alteration is associated with ALT pathway in a cohort of 50 IDH-wild type paediatric high-grade gliomas, highlighting the differences between two histone-mutated subgroups, G34R and K27M, and H3.3-wild type cases. To this end, we will evaluate telomeres length in association with ATRX nuclear loss, H3F3A mutations and TERTp mutations and methylation status [4; 8; 14; 15; 17; 18; 19]. We expect to extend the knowledge on the biology of the H3.3-mutated pHGGs, aiming to figure out the involvement of each genetic alteration in tumor cell senescence escape by telomere lengthening.
Currently, new therapies targeting molecules involved in telomere maintenance mechanism are under development, and initial trials are producing encouraging results in glioma subtypes that do not contain histone H3.3 mutations, as telomerase inhibitors for TERTp mutations and ATR inhibitors for ALT positive tumours with ATRX loss [22; 27] Interestingly, recent publications report potential ALT-targeted drugs to hit heterochromatin formation that drives the development of ALT, ATRX/DAXX/H3.3 complex, inhibitors of recombination factors (ATR, RAD52, SETDB1, FANCM), histone deacetylase inhibitors, G-quadruplex stabilizer, inhibitors of APBs formation, and other chromatin remodelling strategies [13; 21; 22; 23; 24; 25; 26; 27].
If such ALT-targeted treatments will enter novel therapeutic protocols for molecular subgroups of paediatric gliomas, the study of telomeres maintenance mechanisms in pHGG may contribute to the patient¿s risk stratification and application of new targeted treatments for a subset of these tumors, with the hope of improving survival rates [12; 24; 26]. Initial trials have produced encouraging results in subtypes of pediatric gliomas that do not contain histone mutations. Therefore, in the hope to improve survival of pediatric patients with H3F3A mutations, extend the knowledge of the biology that underlies histone mutations will be necessary. Interestingly, since histone mutations seem to be associated with the activation of ALT, but the differences between the two histone-defined subgroups H3G34-mut and K27-mut in terms of telomeric profiles and activation of ALT are not well defined yet, our findings might, therefore, contribute to the improvement of therapeutic strategies using specific targeted treatments on a subset of ALT positive gliomas H3.3G34-mut and K27-mut, since the personalized therapeutic approach based on individual tumor characteristics is becoming the gold standard treatment strategy [4; 5; 7; 13; 20; 24]. This will increase the scientific level of the Italian pediatric neuro-oncology community and will be useful to implement the participation in international research consortium on childhood CNS tumors and particularly on pHGG.