Targeting MYC in high risk medulloblastoma

Medulloblastoma (MB), arising in the cerebellum, is one of the most common malignant brain tumors in children. MB comprises four distinct molecular subgroups. One of them, Group 3, is enriched in oncogene MYC amplifications resulting in dismal prognosis and reduced survival rates. The treatment of MB includes surgical tumor-resection, cranio-spinal irradiation, and chemotherapy. However, even if the treatment is effective, a large population of surviving patients suffer from various sequelae. Therefore, there is a necessity for the development of therapeutics and their combinations targeted against the drivers of MB. In this thesis, a combination of two compounds indirectly targeting MYC is discussed. We and others have previously demonstrated that MYC-amplified MB cells are highly susceptible towards treatment with class I histone deacetylase (HDAC) inhibitors, potentially due to MYC and HDAC2 co-localization in a protein complex in MB cells. We used a class I HDACi entinostat as the first compound in the combination. In order to determine the second candidate, we applied two target discovery techniques, elucidating the protein complex members by mass spectrometry and examining the potential targets in the transcriptional landscape changes induced by entinostat treatment. The elucidated candidate was tested for the selectivity for MYC-amplified MB cells assessing cell metabolic activity, cell cycle, cell death and viability. The interaction between entinostat and a second target inhibitor was examined and validated. Finally, the mechanism of interaction was investigated. We determined that cell cycle regulator polo-like kinase 1 (PLK1) is a target for the combination therapy in MYC-amplified MB. PLK1 was found overexpressed in MYC-amplification-associated molecular subgroups and subtypes of MB underscoring the translational potential of PLK1 targeting. Moreover, MYC-amplified cells were more susceptible to the PLK1 inhibition as confirmed by cell metabolic activity, viability, and apoptosis induction. Entinostat and PLK1 inhibitors volasertib and GSK461364 were interacting synergistically in clinically relevant concentrations only in MYC-amplified MB cells. In addition, entinostat and volasertib synergistically induced apoptosis and reduced the cell viability in MYC-amplified cells only. Finally, we show that volasertib and its’ combination with entinostat exert their activity via MYC axis. However, the details of interaction mechanism remain to be examined. The validation experiments involving on-target confirmation by PLK1 knockdown as well as in vivo assessment of the combination therapy are planned. In summary, in this thesis, the synergistic interaction of entinostat and PLK1 inhibitors was demonstrated. This combination offers a potential for clinical development with MYC amplification serving as a predictive biomarker in MB and possibly other entities with MYC overexpression

Rebound growth of BRAF mutant pediatric glioma cells after MAPKi withdrawal is associated with MAPK reactivation and secretion of microglia-recruiting cytokines

International audienceAbstract Introduction Patients with pediatric low-grade gliomas (pLGGs), the most common primary brain tumors in children, can often benefit from MAPK inhibitor (MAPKi) treatment. However, rapid tumor regrowth, also referred to as rebound growth, may occur once treatment is stopped, constituting a significant clinical challenge. Methods Four patient-derived pediatric glioma models were investigated to model rebound growth in vitro based on viable cell counts in response to MAPKi treatment and withdrawal. A multi-omics dataset (RNA sequencing and LC-MS/MS based phospho-/proteomics) was generated to investigate possible rebound-driving mechanisms. Following in vitro validation, putative rebound-driving mechanisms were validated in vivo using the BT-40 orthotopic xenograft model. Results Of the tested models, only a BRAF V600E -driven model (BT-40, with additional CDKN2A/B del) showed rebound growth upon MAPKi withdrawal. Using this model, we identified a rapid reactivation of the MAPK pathway upon MAPKi withdrawal in vitro, also confirmed in vivo. Furthermore, transient overactivation of key MAPK molecules at transcriptional (e.g. FOS ) and phosphorylation (e.g. pMEK) levels, was observed in vitro. Additionally, we detected increased expression and secretion of cytokines (CCL2, CX3CL1, CXCL10 and CCL7) upon MAPKi treatment, maintained during early withdrawal. While increased cytokine expression did not have tumor cell intrinsic effects, presence of these cytokines in conditioned media led to increased attraction of microglia cells in vitro. Conclusion Taken together, these data indicate rapid MAPK reactivation upon MAPKi withdrawal as a tumor cell intrinsic rebound-driving mechanism. Furthermore, increased secretion of microglia-recruiting cytokines may play a role in treatment response and rebound growth upon withdrawal, warranting further evaluation

BH3 mimetics targeting BCL-XL impact the senescent compartment of pilocytic astrocytoma

BACKGROUND: Pilocytic astrocytoma (PA) is the most common pediatric brain tumor and a mitogen-activated protein kinase (MAPK)-driven disease. Oncogenic MAPK-signaling drives the majority of cells into oncogene-induced senescence (OIS). While OIS induces resistance to anti-proliferative therapies, it represents a potential vulnerability exploitable by senolytic agents. METHODS: We established new patient-derived PA cell lines that preserve molecular features of the primary tumors and can be studied in OIS and proliferation depending on expression ore repression of the SV40 large T antigen. We determined expression of anti-apoptotic BCL-2 members in these models and primary PA. Dependence of senescent PA cells on anti-apoptotic BCL-2 members was investigated using a comprehensive set of BH3-mimetics. RESULTS: Senescent PA cells upregulate BCL-XL upon senescence induction and show dependency on BCL-XL for survival. BH3 mimetics with high affinity for BCL-XL (BCL-XLi) reduce metabolic activity and induce mitochondrial apoptosis in senescent PA cells at nano-molar concentrations. In contrast, BH3 mimetics without BCL-XLi activity, conventional chemotherapy and MEK inhibitors show no effect. CONCLUSIONS: Our data demonstrates that BCL-XL is critical for survival of senescent PA tumor cells and provides proof-of-principle for the use of clinically available BCL-XL-dependent senolytics