Periodic Reporting for period 4 - RhabdoEvo (Deciphering the cellular origin and evolution of malignant rhabdoid tumors)
Reporting period: 2024-06-01 to 2025-05-31
Malignant rhabdoid tumours (MRT) are rare, but very aggressive childhood cancers. Although they may arise in any body part, MRT usually form in isolation or synchronously in the kidney and the brain (where they are referred to as atypical teratoid/rhabdoid tumours (AT/RT)). MRT, especially metastatic MRT, remain one of the most lethal childhood cancers, even following intense treatment regimens. Although current therapeutic regimens typically result in an initial reduction of tumour volume, resistance develops in nearly all cases. It remains unknown what is causing these tumours to develop and which biological processes are responsible for their aggressive behavior. Identifying such processes could help developing novel therapeutic opportunities. To do so, adequate experimental models are needed that faithfully capture disease in human patients. Furthermore, tools are required to analyze tumour development and behavior in great detail.
In this ERC-funded project, we aimed to study the processes that are involved in MRT initiation as well as key mechanisms driving disease progression and therapy resistance. For this, we make use of unique patient-derived tissues, pre-clinical cancer models (3D organoid technology) and state-of-the-art (single-cell) sequencing technologies. We combine these novel models and technologies to characterize the changes that normal cells have to acquire to become a tumour cell, as well as the changes tumour cells undergo when they progress and/or are exposed to different treatments (so-called tumour evolution). In doing so, we hope to identify therapeutic targets that in the future can be used to counteract the aggressive behavior of MRT and thereby provide a much-needed treatment option for patients suffering from this lethal disease. In parallel, the knowledge gained in these studies and the technological advances proposed will apply to other tumour types as well and will therefore be of great value for the tumour biology field.
In this ERC-funded project, we aimed to study the processes that are involved in MRT initiation as well as key mechanisms driving disease progression and therapy resistance. For this, we make use of unique patient-derived tissues, pre-clinical cancer models (3D organoid technology) and state-of-the-art (single-cell) sequencing technologies. We combine these novel models and technologies to characterize the changes that normal cells have to acquire to become a tumour cell, as well as the changes tumour cells undergo when they progress and/or are exposed to different treatments (so-called tumour evolution). In doing so, we hope to identify therapeutic targets that in the future can be used to counteract the aggressive behavior of MRT and thereby provide a much-needed treatment option for patients suffering from this lethal disease. In parallel, the knowledge gained in these studies and the technological advances proposed will apply to other tumour types as well and will therefore be of great value for the tumour biology field.
During the first half of our ERC-funded project, we devoted a lot of effort to 1) process MRT tissue in such a way that reliable data can be obtained from individual tumour cells and 2) develop and validate the tumour models to reliably and consistently monitor MRT progression. This has allowed us to obtain the following results:
For Objective I, we applied single-nucleus RNA-seq to 19 atypical teratoid rhabdoid tumor (ATRT) patient tumour tissues. For seven of these, we also generated single-nucleus ATAC-seq (i.e. single-nucleus Multiome (snMultiome)) data. Analysing these data revealed distinct subtype-specific differentiation trajectories, each resembling different brain progenitor lineages. Additionally, we discovered a proliferative, intermediate precursor cell (IPC)-like population that is consistently present across all subtypes, suggesting a shared transitional state within ATRT tumours. Importantly, we demonstrate that these differentiation pathways can be pharmacologically manipulated in ATRT-derived tumoroids. Our study, therefore, provides a framework for understanding ATRT heterogeneity and supports the feasibility of maturation-based therapeutic strategies tailored to the molecular subtype of the tumor (Blanco-Carmona et al., under review at Neuro-Oncology). Furthermore, we applied snMultiome to MRT (the extracranial counterpart of ATRT), revealing, for the first time, extensive patient-specific epigenetic reprogramming in MRT (the extracranial counterpart of ATRT). We show that his so-called intertumoral heterogeneity on the level of enhancer-mediated gene expression drives oncogene expression (Liu et al., Nat. Commun. 2023).
For Objective II, lentiviral lineage tracing in the first MRT organoid model revealed extensive clonal dynamics in vivo compared to matching in vitro-grown organoids. This could be validated in a second independent MRT model. Furthermore, we identified common transcriptional programs that could explain the highly aggressive nature of these tumours. For instance, we found genes specifically expressed in pro-metastatic clones and metastases that arose from it, which we validated in a patient-derived brain metastasis. Furthermore, we identified several transcription factors as putative drivers of in vivo tumorigenesis. Mechanistic investigations into the contribution of these genes and programs to tumour evolution are required to establish the role of these factors in MRT progression.
As part of Objective III, we performed phylogenetic lineage tracing and found the cellular origin of MRT and Wilms tumor. More specifically, having defined the origin of MRT allowed us to predict the normal counterpart of MRT to be neural crest-derived mesenchyme, and that repairing the oncogenic driver differentiates the tumour cells into harmless normal tissue in vitro (Custers et al., Nat. Commun. 2021). For Wilms tumor, we found an oncogenic gene fusion in infant cases that had not yet been found in Wilms tumor before, likely representing a distinct Wilms tumor subtype (Lee-Six et al., Nat. Commun. 2025). Finally, for yet another pediatric renal cancer, renal cell carcinoma, we identified the tubular epithelium as putative tissue of origin (Ganpat et al., iScience 2025).
The results of the project have been presented at international conferences as well as by giving seminars at international research institutes. Most of the results have already been published in peer-reviewed scientific journals. For objective 2, the barcode lineage tracing project, some final experiments are still ongoing. We hope to submit a manuscript describing our results by the end of 2025.
For Objective I, we applied single-nucleus RNA-seq to 19 atypical teratoid rhabdoid tumor (ATRT) patient tumour tissues. For seven of these, we also generated single-nucleus ATAC-seq (i.e. single-nucleus Multiome (snMultiome)) data. Analysing these data revealed distinct subtype-specific differentiation trajectories, each resembling different brain progenitor lineages. Additionally, we discovered a proliferative, intermediate precursor cell (IPC)-like population that is consistently present across all subtypes, suggesting a shared transitional state within ATRT tumours. Importantly, we demonstrate that these differentiation pathways can be pharmacologically manipulated in ATRT-derived tumoroids. Our study, therefore, provides a framework for understanding ATRT heterogeneity and supports the feasibility of maturation-based therapeutic strategies tailored to the molecular subtype of the tumor (Blanco-Carmona et al., under review at Neuro-Oncology). Furthermore, we applied snMultiome to MRT (the extracranial counterpart of ATRT), revealing, for the first time, extensive patient-specific epigenetic reprogramming in MRT (the extracranial counterpart of ATRT). We show that his so-called intertumoral heterogeneity on the level of enhancer-mediated gene expression drives oncogene expression (Liu et al., Nat. Commun. 2023).
For Objective II, lentiviral lineage tracing in the first MRT organoid model revealed extensive clonal dynamics in vivo compared to matching in vitro-grown organoids. This could be validated in a second independent MRT model. Furthermore, we identified common transcriptional programs that could explain the highly aggressive nature of these tumours. For instance, we found genes specifically expressed in pro-metastatic clones and metastases that arose from it, which we validated in a patient-derived brain metastasis. Furthermore, we identified several transcription factors as putative drivers of in vivo tumorigenesis. Mechanistic investigations into the contribution of these genes and programs to tumour evolution are required to establish the role of these factors in MRT progression.
As part of Objective III, we performed phylogenetic lineage tracing and found the cellular origin of MRT and Wilms tumor. More specifically, having defined the origin of MRT allowed us to predict the normal counterpart of MRT to be neural crest-derived mesenchyme, and that repairing the oncogenic driver differentiates the tumour cells into harmless normal tissue in vitro (Custers et al., Nat. Commun. 2021). For Wilms tumor, we found an oncogenic gene fusion in infant cases that had not yet been found in Wilms tumor before, likely representing a distinct Wilms tumor subtype (Lee-Six et al., Nat. Commun. 2025). Finally, for yet another pediatric renal cancer, renal cell carcinoma, we identified the tubular epithelium as putative tissue of origin (Ganpat et al., iScience 2025).
The results of the project have been presented at international conferences as well as by giving seminars at international research institutes. Most of the results have already been published in peer-reviewed scientific journals. For objective 2, the barcode lineage tracing project, some final experiments are still ongoing. We hope to submit a manuscript describing our results by the end of 2025.
We successfully developed a lentiviral barcode lineage tracing technology allowing for combined lineage tracing and transcriptomics on the single-cell level. In combination with our established orthotopic organoid transplantation methods, this presents a breakthrough achievement, as it for the first time allows for studying clonal evolution during tumour progression, metastasis, and therapy resistance on the single-cell level. Furthermore, we successfully implemented cutting-edge phylogenetic lineage tracing strategies, allowing for tracing back the cellular origin of cancer (Custer et al., Nat. Commun. 2021; Lee-Six et al., Nat. Commun. 2025). Our studies serve as blueprints for unraveling the origin of childhood cancers as well as defining the maturation blocks underpinning them.