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Medulloblastoma (MB) is the most common malignant brain tumour in children and long-term sequelae in survivors caused by the harsh, non-targeted treatments is common. Current screening protocols for the detection of novel druggable vulnerabilities rely on 2D and 3D cell culture systems using established or primary tumour cells. However, these assays do neither provide the cellular, chemical and biophysical context of the tissue, nor reveal drug effects on the invasive behavior of the tumour cells. To overcome these limitations, we have established ex vivo Organotypic Cerebellum Slice Co-culture (OCSC) models for SHH and group 3 MB tumours, which provide spatio-temporal insights in the growth and dissemination behaviour of the tumour cells. OCSCs represent an excellent translational model for patient sample profiling, drug testing, and microenvironmental or explorative target identification studies in a physiologically relevant tissue context. We found that the AURKB inhibitor Barasertib (AZD-1152), which was ineffective to reduce ONS-76 cell invasion in vitro, effectively reduced tumour volume and proliferation of these SHH tumour cells in the tissue context. The AURKB inhibitor thus displays higher efficacy ex vivo, indicating an impact of the microenvironment on its functional activity. We further validated tumour suppressive activity of AURKB inhibition with Barasertib at low nanomolar concentration or with siRNA-mediated depletion of AURKB in Grp3 MB tumour cells and in primary tumour cells ex vivo, thereby confirming the therapeutic potential of AURKB inhibition against MB progression. We furthermore found that the combination of AURKB inhibition with irradiation or with the SRC inhibitor Dasatinib, lead to a near complete eradication of the tumour cells. Thus, OCSCs are a clinically relevant model where tumour material can be efficiently tested for drug sensitivities, which allowed us to identify AURKB inhibition as an effective treatment against tissue invasive MB.

FGF2, the ligand of FGF receptors (FGFRs), is expressed in the developing and adult brain. FGF2-FGFR1 signaling causes the induction and maintenance of cancer stem cells through ERK-dependent up-regulation of ZEB1 and Olig2 in glioblastoma. In SHH medulloblastoma, Olig2 triggers tumor initiation from GCPs, maintains quiescent stem-like cells during the disease and contributes to tumor outgrowth at recurrence. We found that FGF2-FGFR signaling causes increased growth and tissue invasion through the FGFR adaptor protein FRS2 in SHH and group-3 medulloblastoma 1. Thus, targeting of FGFR-FRS2 signaling could abrogate brain tumor growth and spread by repressing tumor-promoting functions that are induced by microenvironmental FGF2. Using virtual screening combined with functional validation, we identified protein-protein interaction inhibitors (F2i) that bind FRS2 and abrogate FGFR signaling to the MAP-ERK pathway. Consistent with the requirement of FRS2 for pro-invasive signaling downstream of FGFR1 in medulloblastoma, F2i also efficiently block FGF2-induced migration and invasion in medulloblastoma-derived cells. Selected F2i display excellent binding kinetics with a similar Kd as the natural ligand domain of FGFR and cause steric alterations in the targeted protein domain. On-target activity was confirmed by thermal proteome profiling. Neither in silico screening nor empirical testing revealed significant off-target activity of the compounds. No toxicity of F2i was observed in cell-based models with confirmed functional activity on invasion and MAPK activation. Thus, we identified novel, low molecular weight pharmacological protein-protein interaction inhibitors with an excellent potential to specifically block FGFR functions relevant for brain tumor progression. 1. Santhana Kumar et al., CellReports23, 3798–3812.e8 (2018).

The molecular mechanisms contributing to distant dissemination and local recurrence of medulloblastoma, the most common malignant brain tumor in childhood, are poorly understood and no targeted anti-invasion therapies exist till date. We explored regulators and effectors of MAP4K4, a pro-invasive kinase overexpressed in MB and associated with metastatic progression in different solid malignancies. MAP4K4 is upregulated both at mRNA and protein levels in primary pediatric brain tumors compared to normal cerebellum. MAP4K4 is required for growth factor- and irradiation-induced migration and invasion of medulloblastoma cells. It furthermore promotes turnover and activation of the receptor tyrosine kinase c-Met and of the ß1 integrin adhesion receptor 1. To characterize these clinically relevant consequences and to identify druggable targets of MAP4K4 function, we profiled the interactome of MAP4K4 in starved and growth factor stimulated medulloblastoma cells. To systematically address MAP4K4 impact on receptor expression and turnover, we determined the MAP4K4-dependent surface proteome in medulloblastoma cells. We found that MAP4K4 is part of the striatin-interacting phosphatase and kinase (STRIPAK) complex and that STRIPAK component striatin 4 is controlling cell motility and invasiveness in medulloblastoma cells. Invasiveness of medulloblastoma cells is abrogated by a truncation mutant of MAP4K4 lacking the striatin 4 interaction domain. We furthermore found that MAP4K4 mediates growth factor-induced surface expression of solute carriers and immunomodulatory proteins involved in chemoresistance and immune evasion. Thus, our study identified MAP4K4 as a missing link between pro-tumorigenic growth factor signaling and tumor cell functions relevant for disease progression. It may help identifying druggable vulnerabilities in medulloblastoma cells to restrict tumor growth and dissemination. 1. Tripolitsioti, D. et al., Oncotarget9, 23220–23236 (2018).

How pediatric medulloblastoma (MB), a metastatic tumor of the cerebellum, infiltrates the brain tissue prior to recurrence or distant spreading is poorly understood. We hypothesized that growth factors released from the tumor microenvironment trigger tumor cell migration and brain tissue infiltration. Using a medium-throughput in vitro 3D cell migration screen and ex vivo organotypic cerebellum slice culture, we identified and validated novel potential drug targets for preventing brain tissue infiltration and growth of MB. We found that the FGFR1 ligand bFGF, which we detected in the tumor microenvironment of clinical samples and in organotypic cultures, promoted tumor cell infiltration through the adaptor protein FRS2. Interestingly, FRS2 function was antagonized by TGFβ, which induced a contractile, non-motile cell phenotype through ROCK activation and direct FRS2 repression. In the absence of TGFβ, however, bFGF-FRS2 function attenuated ROCK and enabled motility and invasiveness through Rac-PAK and ERK1/2. In contrast, at high concentrations of bFGF as observed in certain areas of MB tumor tissue, TGFβ rescued tumor cell motility impeded by a FGFR1-dependent negative feed-back and triggered invasiveness. Using organotypic cerebellum slice culture, we demonstrated that pharmacological inhibition of FGFR or genetic ablation of FRS2 prevented infiltration and proliferation of MB cells. Using a computer-assisted molecular design approach, we are currently exploring low molecular weight compounds that might selectively block FRS2 function. Together, we describe an antagonistic crosstalk between bFGF and TGFβ signaling pathways in MB that tunes the motile and invasive properties of the tumor cells for brain infiltration under varying environmental conditions. Furthermore, we identified the FRS2 adaptor protein as a novel and promising drug target in this process.

Laboratory feeding experiments using transgenic Bacillus thuringiensis variety kurstaki (Berliner) corn plants have been carried out to study the effects of B. thuringiensis -fed herbivores (i.e., prey), on the predator Chrysoperla carnea Stephens. Host plants were a transgenic B. thuringiensis -expressing (CrylAb) corn hybrid and the corresponding untransformed, B. thuringiensis -free corn hybrid. Two different prey species were used in the experiments, the European corn borer, Ostrinia nubilalis (Hubner) (lepidopterous target pest), and Spodoptera littoralis (Boisduval) (lepidopterous nontarget pest for B. thuringiensis ). The objectives were to quantify the effects of B. thuringiensis -fed prey on chrysopid immature development and to determine whether observed effects were caused by sick, suboptimal prey (indirect effects) or associated with B. thuringiensis -related causes (direct effects). Mean total immature mortality for chrysopid larvae raised on B. thuringiensis -fed prey was 62% compared with 37% when raised on B. thuringiensis -free prey. There was no significant difference in mortality between chrysopid larvae reared on B. thuringiensis -fed O. nubilalis or B. thuringiensis -fed S. littoralis . Similarly, no significant difference in mortality was detected when chrysopid larvae were raised on B. thuringiensis -free O. nubilalis or B. thuringiensis -free S. littoralis . Development time of chrysopid larvae was prolonged when B. thuringiensis -fed O. nubilalis was given to the predators but not for B. thuringiensis -fed S. littoralis . Although some unnoticed adverse effects in S. littoralis may have occurred because of the B. thuringiensis corn, our results suggest that the reduced fitness of chrysopid larvae was associated with B. thuringiensis . The prolonged development time of chrysopid larvae raised on B. thuringiensis -fed O. nubilalis was probably because of a combined effect of B. thuringiensis exposure and nutritional deficiency caused by sick prey.