Periodic Reporting for period 1 - ORIGIN4MB (Uncovering the cell of origin of Group 4 Medulloblastoma)
Reporting period: 2024-04-01 to 2026-03-31
Medulloblastoma (MB) is the most common malignant paediatric brain tumour, affecting mostly infants and children, and to a lesser extent adults. It originates from neuronal stem or progenitor cell populations on the cerebellum or brainstem during development and/ or childhood, comprising around 60% of childhood intracranial embryonal tumours. The main risk factors associated with this condition are heritable factors, including germline mutations in genes that regulate developmental signaling pathways in the cerebellum. MB are classified in 4 subgroups (Wnt, Sonic Hedgeghog (SHH), Group 3 and Group 4) according to their gene expression, cell of origin, methylation patterns and clinical features. Group 4 MB is the most common subgroup of MB, affecting approximately 40% of the patients with this condition. The cellular origins of Group 4 MB are still under debate; however, recent papers using single cell RNA sequencing showed that Group 4 MBs are characterized by the expression of transcription factors such as Eomesodermin(EOMES) and LIM/Homeobox protein 2(LHX2) genes expressed by granule upper rhombic lip progenitors, more specifically Unipolar Brush Cells(UBCs) that are born during cerebellar morphogenesis. Therefore, one can hypothesize that Group 4 MB originates from UBCs progenitors. The main goals of the ORIGIN4MB project are to uncover the cell of origin of Group 4 MB and to develop the first preclinical model of Group 4 MB, revealing unprecendented insights in MB biology. To this end we will use lineage tracing techniques, lentiviral in utero infection, in vivo imaging and post-mortem analysis. We will also validate if the generated mouse model is able to recapitulate the human disease. This validation will determine if this model could be used to develop new treatments for Group 4 MB, opening the door to new therapeutic avenues for those pedeatric patients, ultimately improving their quality of life.
The ORIGIN4MB project aimed to define the cell of origin of Group 4 MB and generate the first preclinical model of this disease by targeting MYCN and CDK6 activation in unipolar brush cells (UBC) progenitors.
Specific scientific objectives were:
1. To infer which promoters could be used to target specifically UBCs.
2. To uncover whether activation of MYCN and/or CDK6 in the developing cerebellum leads to Group 4 MB formation.
3. To infer whether activation of MYCN and/or CDK6 specifically in UBC early or late progenitors leads to Group 4 MB formation.
4. To validate whether the generated mouse model for Group 4 MB mimics the human disease.
A brief summary of the progress made towards the achievement of each objective is given below.
Objective 1) Using immunofluorescence and lineage tracing in advanced genetic mouse models, we were able to define the spatial and temporal expression of key UBC progenitor markers, including Math1, Lmx1a, Lhx2, and EOMES, to distinguish early from late UBC progenitors. This was achieved by collecting embryos at all ages and performing immunofluorescence for the above-mentioned markers, showing that LHX2 starts being expressed at embryonic day 11 (E11), whereas Math1 starts being expressed at E12 in the rhombic lip, and Lmx1a and Eomes at E13.
In parallel, lineage tracing was performed by crossing Math1-CREER, Lhx2-CREER, Lmx1a-CREER or EOMES-CREER mice with Rosa-YFP reporter mice. Temporal control of recombination using tamoxifen allowed for an assessment of whether progeny from each promoter specifically gave rise to UBCs. It was possible to infer that Math1+ progenitors during embryonic days 13 – 14 (E13/E14) give rise to UBCs (but not after that, since Math1+ progenitors afterwards become restricted to granule cell progenitors), whereas Lmx1a+ progenitors give rise to UBCs from E13 onwards. In the case of LHX2+ progenitors these don’t seem to give rise to any UBCs. On the other hand, EOMES expression persists in UBC progenitors, suggesting EOMES as a UBC-specific marker at both embryonic and postnatal stages (being expressed in UBCs since their genesis at E13). This allowed for a selection of promoters that label UBCs to be used for the following experiments to drive oncogene activation selectively in UBC progenitors.
Objective 2) Lentiviral vectors encoding MYCN and/or CDK6, coupled to fluorescent reporters (mCherry and mCerulean, respectively), were injected in utero into the cerebellum of mouse embryos at different developmental stages (E15–E19) to identify critical temporal windows of tumor susceptibility. These were injected in the rhombic lip, the embryonic region where MBG4 arises from. The individual and combined contributions of MYCN and CDK6 to tumor initiation and progression were assessed by injecting each oncogene alone or together. Unfortunately, since in utero injections are very delicate and complex, many obstacles were encountered: pregnant females would open the surgery wound post-surgery (leading to their death), some embryos would die from the injection, or pregnant females would have spontaneous abortions post-surgery. Therefore, only a few injected embryos were later on able to be born and survive. However, these did not give rise to tumours – another type of cells (granule cells) were labelled with the reporter, indicating that the wrong cells were targeted by the lentivirus.
Objective 3) UBC progenitor cells were isolated from embryonic mouse cerebella using Math1-based reporter models (taking into consideration the embryonic timings inferred in objective 1). These cells were then sorted, and cultured to generate neurospheres. These neurospheres were characterized by immunocytochemistry, flow cytometry and qPCR to confirm UBC progenitor identity, reporter expression, and proliferative capacity. Lentiviral vectors expressing MYCN and/or CDK6, each coupled to fluorescent reporters and to a luciferase-encoding cassette, were used to infect dissociated neurosphere cells, with successful overexpression validated by fluorescence, qPCR, and proliferation assays.
Cells expressing the oncogenes were then grafted into the cerebellum of postnatal day 3 (P3) pups via stereotaxic surgery to mimic tumor initiation in the juvenile brain. Tumor formation was detected after cell grafting, and its development was then monitored using bioluminescence in vivo, followed by post-mortem analyses including immunostaining for Group 4 MB markers and single-cell RNA sequencing.
Specific scientific objectives were:
1. To infer which promoters could be used to target specifically UBCs.
2. To uncover whether activation of MYCN and/or CDK6 in the developing cerebellum leads to Group 4 MB formation.
3. To infer whether activation of MYCN and/or CDK6 specifically in UBC early or late progenitors leads to Group 4 MB formation.
4. To validate whether the generated mouse model for Group 4 MB mimics the human disease.
A brief summary of the progress made towards the achievement of each objective is given below.
Objective 1) Using immunofluorescence and lineage tracing in advanced genetic mouse models, we were able to define the spatial and temporal expression of key UBC progenitor markers, including Math1, Lmx1a, Lhx2, and EOMES, to distinguish early from late UBC progenitors. This was achieved by collecting embryos at all ages and performing immunofluorescence for the above-mentioned markers, showing that LHX2 starts being expressed at embryonic day 11 (E11), whereas Math1 starts being expressed at E12 in the rhombic lip, and Lmx1a and Eomes at E13.
In parallel, lineage tracing was performed by crossing Math1-CREER, Lhx2-CREER, Lmx1a-CREER or EOMES-CREER mice with Rosa-YFP reporter mice. Temporal control of recombination using tamoxifen allowed for an assessment of whether progeny from each promoter specifically gave rise to UBCs. It was possible to infer that Math1+ progenitors during embryonic days 13 – 14 (E13/E14) give rise to UBCs (but not after that, since Math1+ progenitors afterwards become restricted to granule cell progenitors), whereas Lmx1a+ progenitors give rise to UBCs from E13 onwards. In the case of LHX2+ progenitors these don’t seem to give rise to any UBCs. On the other hand, EOMES expression persists in UBC progenitors, suggesting EOMES as a UBC-specific marker at both embryonic and postnatal stages (being expressed in UBCs since their genesis at E13). This allowed for a selection of promoters that label UBCs to be used for the following experiments to drive oncogene activation selectively in UBC progenitors.
Objective 2) Lentiviral vectors encoding MYCN and/or CDK6, coupled to fluorescent reporters (mCherry and mCerulean, respectively), were injected in utero into the cerebellum of mouse embryos at different developmental stages (E15–E19) to identify critical temporal windows of tumor susceptibility. These were injected in the rhombic lip, the embryonic region where MBG4 arises from. The individual and combined contributions of MYCN and CDK6 to tumor initiation and progression were assessed by injecting each oncogene alone or together. Unfortunately, since in utero injections are very delicate and complex, many obstacles were encountered: pregnant females would open the surgery wound post-surgery (leading to their death), some embryos would die from the injection, or pregnant females would have spontaneous abortions post-surgery. Therefore, only a few injected embryos were later on able to be born and survive. However, these did not give rise to tumours – another type of cells (granule cells) were labelled with the reporter, indicating that the wrong cells were targeted by the lentivirus.
Objective 3) UBC progenitor cells were isolated from embryonic mouse cerebella using Math1-based reporter models (taking into consideration the embryonic timings inferred in objective 1). These cells were then sorted, and cultured to generate neurospheres. These neurospheres were characterized by immunocytochemistry, flow cytometry and qPCR to confirm UBC progenitor identity, reporter expression, and proliferative capacity. Lentiviral vectors expressing MYCN and/or CDK6, each coupled to fluorescent reporters and to a luciferase-encoding cassette, were used to infect dissociated neurosphere cells, with successful overexpression validated by fluorescence, qPCR, and proliferation assays.
Cells expressing the oncogenes were then grafted into the cerebellum of postnatal day 3 (P3) pups via stereotaxic surgery to mimic tumor initiation in the juvenile brain. Tumor formation was detected after cell grafting, and its development was then monitored using bioluminescence in vivo, followed by post-mortem analyses including immunostaining for Group 4 MB markers and single-cell RNA sequencing.
The ORIGIN4MB project has made significant conceptual and technical progress toward addressing key unresolved questions in Group 4 MB. Building on recent single-cell RNA sequencing studies from patient samples, the project has advanced the hypothesis that Group 4 MB originates from unipolar brush cell (UBC) progenitors derived from the upper rhombic lip.
A major scientific contribution of the project has been that both early and late UBC progenitors transcriptionally mirror distinct Group 4 MB tumor cell populations, narrowing the gap between human tumor profiling and cerebellar lineage specification. This represents a meaningful advance in the field, where the cellular origins of Group 4 MB have remained highly debated.
From a technological and innovative perspective, the project has designed a novel experimental strategy to overcome the lack of available genetic mouse models for Group 4 MB. Specifically, a grafting model was developed to enable constitutive activation of MYCN and CDK6 in UBC progenitor populations. This approach represents a significant methodological innovation, allowing spatially and temporally controlled oncogene activation in certain cell types.
Overall, the project has laid the groundwork for the development of the first faithful preclinical mouse model of Group 4 MB. When fully validated (with further research), this model will represent a major contribution to the state of the art, enabling mechanistic studies of Group 4 MB biology and providing an essential platform for testing targeted therapies, with the long-term potential to improve outcomes for paediatric patients. The results of the project (i.e. in vitro and in vivo models of Group 4 MB) could potentially be used by other groups in the field as a platform for testing targeted therapies.
A major scientific contribution of the project has been that both early and late UBC progenitors transcriptionally mirror distinct Group 4 MB tumor cell populations, narrowing the gap between human tumor profiling and cerebellar lineage specification. This represents a meaningful advance in the field, where the cellular origins of Group 4 MB have remained highly debated.
From a technological and innovative perspective, the project has designed a novel experimental strategy to overcome the lack of available genetic mouse models for Group 4 MB. Specifically, a grafting model was developed to enable constitutive activation of MYCN and CDK6 in UBC progenitor populations. This approach represents a significant methodological innovation, allowing spatially and temporally controlled oncogene activation in certain cell types.
Overall, the project has laid the groundwork for the development of the first faithful preclinical mouse model of Group 4 MB. When fully validated (with further research), this model will represent a major contribution to the state of the art, enabling mechanistic studies of Group 4 MB biology and providing an essential platform for testing targeted therapies, with the long-term potential to improve outcomes for paediatric patients. The results of the project (i.e. in vitro and in vivo models of Group 4 MB) could potentially be used by other groups in the field as a platform for testing targeted therapies.