Hello, I am a research fellow in the Dr. Smith's lab at Boston Children's Hospital and it is a great pleasure to present my work on targeting neogenin as an anti-invasive therapy for diffuse intrinsic pontine gliomas. I have no disclosures. So diffuse intrinsic pontine gliomas, also called DIPG, is an orphan disease with an incidence of 2.3 per 1 million people, but this is a brain tumor with one of the highest mortality rates in neuro-oncology in children. Unfortunately, surgery is not an option due to the challenging location of these tumors as well as their diffuse behavior. Currently, radiotherapy is the only standard of care, so there is a real lack of efficient treatment despite the high number of clinical trials in progress in the country and beyond. One of the major challenges of these tumors is that they are very invasive and infiltrate the normal parent tumors. The origin of these tumors is not well understood, but some elegant studies show that they could arise from oligonucleotide lineage due to their genotype and, interestingly, a protein called NETREN1 is shown to promote oligodendrocyte differentiation and maintain specialized cells and junction during development. NETREN1 is an excellent guidance factor. It is a secreted protein very highly expressed during development to achieve its function in the brain, endothelial cells, and beyond. But after birth, the NETREN1 level is decreased and the expression is very low in adults. Many studies, including others, show that NETREN1 is elevated in tumors and promotes tumorigenesis, cell invasion, and tumor endogenesis, particularly in brain tumors such as medulloblastoma and glioblastoma. NETREN1 plays strong through multiple receptors, but interestingly, we have shown that it is preferentially through neogenin that NETREN1 acts in brain tumors. Neogenin is a cell surface receptor belonging to the immunoglobulin superfamily. It combines two sorts of ligands, the neutrinos, such as NETREN1, and the repulsive guidance molecules, RGM. It plays roles in the cell adhesion, cell guidance, and differentiation and endogenesis in development, but its involvement in tumors is not well understood. Due to the role of NETREN1 in promoting oligodendrocyte differentiation, we hypothesize that NETREN1 is lost during development of the pons and aborts differentiation of oligodendrocytes, leading to initiation of DIPG. We think also that its receptor, neogenin, is maintained in DIPG and promotes invasion of DIPG cells, and that the blockade of neogenin leads to a decrease of DIPG cell invasion. In order to validate this hypothesis, we used different cell lines, SAV628, as commercially available DIPG cells, and human primary DIPG cells from Mondilab at Stanford University. We used SVG and astrocyte as normal human astroviral cells and negative control, and DE556, a commercially available medulloblastoma cell line, as a positive control. We also obtained patient urinary sample and agents that match controls from multiple institutions to render ELISA. To validate our first hypothesis, which is the low level of NETREN1 in DIPG, we looked at the mRNA expression of NETREN1 in DIPG cells, SAV628, and we compared it to normal astroviral cells. The results showed that NETREN1 is indeed significantly lower in DIPG cells compared to SVG, and as low as in normal astrocytes. We then looked at the protein expression of NETREN1 and observed a lack of NETREN1 expression in DIPG cells, as well as in normal astroviral cells, compared to medulloblastoma cells used as a positive control here. These results confirm our previous finding with the pediatric brain tumor consumption earlier this year, where we showed that the urinary level of NETREN1 in DIPG patients were as low as in control, with a great accuracy, and could be used as a urinary biomarker. We then wanted to validate our second hypothesis about the high expression of NETREN1 receptor neogenin in DIPG. So we looked at neogenin in DIPG cells, and by RT-qPCR, we showed that neogenin mRNA levels are significantly higher in DIPG cells compared to controlled normal astroviral cells. We then confirmed these results, assessing the protein expression via western blot and immunoprocessants, and we also saw a higher neogenin cell surface expression in DIPG by flow cytometry. After validating the high level of neogenin in DIPG, we wanted to look at the functional effect of neogenin blockade. We first validated two techniques to shut down the expression of neogenin, first by silencing RNA. You can see in A and B that the down regulation is effective at 24, 48, and 72 hours. And secondly, the treatment with neutralizing antibody against neogenin, validated here in C by western blot. As mentioned earlier, one of the main challenges of DIPG is their invasive behavior. So the goal would be to stop the cell invasion. Also, we know that neogenin is involved in cell adhesion and invasion, so we assessed a cell invasion assay with DIPG cells in response to treatment with siRNA against neogenin first. And interestingly, we found that the down regulation of neogenin significantly decreased DIPG cells invasion. We ran the same experiment, but this time treating the cells with the blockade antibody and the results were even more striking. We decreased the DIPG cell invasion of 60% when we blocked neogenin, as you can see here in the graph and the pictures. After the promising effect on cell invasion with neogenin blockade in vitro, we wanted to verify the neogenin expression in patient samples in order to validate this target. So we first ran an ELISA looking at the expression of neogenin in urine of patients with DIPG compared to controls, and we found that neogenin is significantly higher in these DIPG patients compared to the controls. We then validate this higher expression in DIPG patient cells, here in purple, at mRNA levels and also the protein levels. In conclusion, we show that nutrinement is low in DIPG, as shown by in vitro data and patient samples. In cell surface receptor, neogenin is highly expressed in DIPG cells and in DIPG patients, and the blockade of neogenin leads to a significant decrease of DIPG cell invasion. Altogether, this data suggests that the loss of netrine 1 may lead to DIPG initiation via an aborted differentiation of oligodendrocyte progenitors and the loss of cell-cell junctions. The receptor neogenin remains high in DIPG cells and could participate to cancer cell infiltration and invasion of the normal parenchyma. And finally, that blocking this receptor via a neutralizing antibody is a promising therapeutic strategy and could stop the cell diffusion. To go further, the next step would be to investigate more the ligand and the downstream signaling pathway involved in this mechanism and to perform preclinical in vivo study to validate neogenin as a predictive target for DIPG treatments. To finish, I would like to thank my research team, the Smith's lab members, especially Jessica Driscoll, who helped a lot with this project, but also the former members of the lab and the Monge lab for the DIPG patient cells and the PBTC for the DIPG patient samples. And also, I thank you for your attention.

neogenin

anti-invasive therapy

diffuse intrinsic pontine gliomas

DIPG

NETREN1