Background: Brain tumors are the deadliest solid tumors in children and adolescents. Most of these tumors are glial in origin and exhibit strong heterogeneity, hampering the development of effective therapeutic strategies. In the past decades, patient-derived tumor organoids (PDT-O) have emerged as powerful tools for modeling tumoral cell diversity and dynamics, and they could then help define new therapeutic options for pediatric brain tumors. Methods: Through an integrative approach based on our expertise and a careful review of the literature about glioblastoma 3D primary cultures, we set up a standardized methodological pipeline for the establishment, characterization, and biobanking of PDT-O through direct 3D in vitro culture of the deadliest pediatric glial brain tumors. To assess PDT-O fidelity and validate their preclinical relevance, we performed comprehensive histological, molecular, and drug-response analyses. Results: Our methodological pipeline allowed the rapid and efficient generation of PDT-O recapitulating their parental tumor features, including intratumoral heterogeneity, even after several passages and cryopreservation/revival as 3D cultures. Moreover, we successfully performed preclinical test responses on these PDT-O to standard-of-care therapies and new therapeutic options. Finally, we identified ONC201 as a selective drug for pediatric glial tumor types not restricted to H3K27-altered glial tumors, as well as combination strategies to increase the therapeutic response to ONC201. Conclusions: Hence, we describe a fast and robust process to biobank PDT-O for pediatric glial brain tumors. These PDT-O models have the potential for patient-specific modeling even after long-term expansion in vitro, and we established the proof-of-concept of their usefulness to support powerful preclinical studies.

Pediatric diffuse midline gliomas (pDMG) are an aggressive type of childhood cancer with a fatal outcome. Their major epigenetic determinism has become clear, notably with the identification of K27M mutations in histone H3. However, the synergistic oncogenic mechanisms that induce and maintain tumor cell phenotype have yet to be deciphered. In 20 to 30% of cases, these tumors have an altered BMP signaling pathway with an oncogenic mutation on the BMP type I receptor ALK2, encoded by ACVR1. However, the potential impact of the BMP pathway in tumors non-mutated for ACVR1 is less clear. By integrating bulk, single-cell, and spatial transcriptomic data, we show here that the BMP signaling pathway is activated at similar levels between ACVR1 wild-type and mutant tumors and identify BMP2 and BMP7 as putative activators of the pathway in a specific subpopulation of cells. By using both pediatric isogenic glioma lines genetically modified to overexpress H3.3K27M and patients-derived DIPG cell lines, we demonstrate that BMP2/7 synergizes with H3.3K27M to induce a transcriptomic rewiring associated with a quiescent but invasive cell state. These data suggest a generic oncogenic role for the BMP pathway in gliomagenesis of pDMG and pave the way for specific targeting of downstream effectors mediating the K27M/BMP crosstalk.

The BMP pathway is one of the major signaling pathways in embryonic development, ontogeny and homeostasis, identified many years ago by pioneers in developmental biology. Evidence of the deregulation of its activity has also emerged in many cancers, with complex and sometimes opposing effects. Recently, its role has been suspected in Diffuse Midline Gliomas (DMG), among which Diffuse Intrinsic Pontine Gliomas (DIPG) are one of the most complex challenges in pediatric oncology. Genomic sequencing has led to understanding part of their molecular etiology, with the identification of histone H3 mutations in a large proportion of patients. The epigenetic remodeling associated with these genetic alterations has also been precisely described, creating a permissive context for oncogenic transcriptional program activation. This review aims to describe the new findings about the involvement of BMP pathway activation in these tumors, placing their appearance in a developmental context. Targeting the oncogenic synergy resulting from this pathway activation in an H3K27M context could offer new therapeutic perspectives based on targeting treatment-resistant cell states.

Ma thèse a été consacrée à l’étude des états cellulaires impliqués dans l’agressivité des gliomes et des rhabdomyosarcomes pédiatriques, deux cancers caractérisés par une forte hétérogénéité cellulaire et une plasticité tumorale importante. L’objectif de ce travail a été de mieux comprendre comment certaines altérations génétiques et le microenvironnement tumoral contribuent à la mise en place de programmes cellulaires favorisant la progression tumorale. Dans un premier axe, je me suis intéressé aux gliomes diffus de la ligne médiane, caractérisés par la mutation H3.3K27M, et à leur interaction avec la voie de signalisation des Bone Morphogenetic Proteins (BMP). Les résultats obtenus mettent en évidence une coopération fonctionnelle entre la mutation H3.3K27M et la signalisation BMP, conduisant à l’induction d’un état de quiescence dans une sous-population de cellules tumorales. Cet état quiescent est associé à une augmentation des capacités invasives, suggérant un lien entre quiescence et dissémination tumorale. Ces travaux soulignent ainsi le rôle clé des états quiescents dans l’agressivité des gliomes pédiatriques et leur implication potentielle dans la résistance aux traitements ciblant les cellules prolifératives. Dans un second axe, ma thèse s’est focalisée sur l’étude d’une protéine de la matrice extracellulaire impliquée dans l’homéostasie et les pathologies musculaires. Les résultats montrent que cette protéine modifie la régénération musculaire et réduit l’agressivité tumorale des rhabdomyosarcomes en modifiant l'hétérogénéité intra-tumorale, et en limitant la transition vers des états invasifs. Dans leur ensemble, ces travaux mettent en lumière le rôle central de l'hétérogénéité intra-tumorale et de sa dynamique dans l’agressivité de cancers pédiatriques, et ouvrent des perspectives thérapeutiques visant à cibler non seulement les altérations génétiques, mais également les mécanismes non-génétiques associés à l'émergence d'états agressifs.