Artificial Intelligence
Drone Research Group
We Made lots of Drone with Artificial Intelligence.
Carla Vasquez-Amos
Investigating the role of the T cell receptor (TCR) in ALCL pathogenesis towards the development of an innovative therapeutic approach
Masarykova Univerzita - Medical Faculty - CEITEC
Supervisor: Suzanne TURNER
Research objectives:
A defining feature of ALCL is the lack of expression of a TCR despite the cells of origin being T cells. The enigma as to why the TCR is not expressed has been studied in the host lab whereby the capacity to express a TCR is still an option for some ALCL, being internalised rather than absent, through a lack of transcription or genetic deletion of TCR-encoding genes. This data suggests that the TCR is actively internalised perhaps due to a tumour-derogatory role if it is expressed on the surface.
Indeed, our data shows that co-expression of a TCR and aberrant expression of ALK in primary T cells results in a signalling overload and cell death, particularly when the expressed TCR is engaged by cognate ligand. These findings will be investigated further as they might apply to:
- The origins of this largely paediatric malignancy and;
- The therapy of this cancer.
Employing a genetically engineered mouse model in which NPM-ALK is expressed in T cells through the CD4 promoter (CD4/NPM-ALK) together with forced expression of a TCR that specifically recognises ovalbumin antigen (OVA – OTI transgenic), mice will be exposed to murine herpes virus carrying OVA peptides of varying avidity for the TCR. The effects this has on tumour development in vivo will be monitored by MRI and ex vivo tumour phenotyping for tumour cell ontogeny and immune cell infiltration with a particular focus on comparing the ensuing tumours, should they develop, with the human malignancy.
In the second aim, these data will be interpreted towards a therapeutic outcome, e.g., if an antigen with high avidity drives apoptosis and prevents tumour growth, this might imply that established tumours (lacking a TCR) could be targeted therapeutically by forcing enhanced signalling through distal TCR signalling pathways rather than their inhibition. Towards this aim, patient derived xenografts (PDX) of ALCL developed in the lab of B#1 and 3 will be analysed for their response to select phosphatase inhibitors (in contrast to traditionally applied kinase inhibitors).
Expected results:
We predict that engaging the TCR with a high avidity peptide will prevent tumour growth in CD4/NPM-ALK/OTI transgenic mice whereas weakly binding peptides will be tolerated with tumour growth ensuing. This would suggest that an autoantigen or tonic stimulation of T cells might play a role in ALCL development. Alternatively, that high avidity stimulation results in TCR down-regulation as both the stimulated TCR and oncogenic ALK expression are ‘overload’ for the incipient tumour cells. In turn, this will allow us to speculate on therapeutic targets that, contrary to accepted dogma which tells us to inhibit proliferation-promoting signalling in tumour cells, might instead be over-stimulated by, for example, inhibiting phosphatase activity.
Planned secondments:
Year 2: CBMed: Analysis of murine tumour tissue sections applying technologies available at CBMed (3 months)
Year 3: University of Torino: Access to PDX models of ALCL that are uniquely available at the host institute (3 months)
Katarina Mišura
Epigenetics of ALCL Drug Resistance
Medizinische Universität Wien
Supervisor: Gerda EGGER
Research objectives:
Our lab has previously identified DNA methylation signatures of primary human ALCL that reflect biological tumour characteristics and provide some insight into the potential origin of ALCL. We could further demonstrate that inhibition of DNA methylation via genetic or pharmacological means resulted in loss of transformation of T cells and reduced tumour growth in mouse models. Thus, we hypothesize that epigenetic mechanisms might also be implicated in drug response/resistance in ALCL and that epigenetic therapies might be suitable for combination drug treatment of patients.
Within this project, we aim to develop more suitable models for drug testing of ALCL based on innovative 3D organoid models and to investigate the epigenetic mechanisms related to drug resistance:
- Generate lymphoid organoids from patient samples (primary/therapy refractory). Currently, only a few cell lines established from human ALCL patient samples are available, which only partially reflect in vivo tumours. Thus, we will generate organoid models from primary patient material(ascites, blood, tumour samples) together with clinical partners in the consortium and patient-derived xenograft models (PDX) that have been established by B#1 and 3. To establish the model and to test for its feasibility, we will also include tumours from the NPM-ALK transgenic mouse model;
- Perform phenotypic and molecular characterization (scRNA-seq, RNA-seq, WES, epigenomic profiling). Organoid models will be characterized in comparison to the original tumours for their morphology using histological analyses and for their (epi)genetic signatures using WES and DNA methylation profiling;
- Perform drug testing and induce therapy resistance by long-term treatment. By using chemotherapy and ALK-specific targeted therapy (e.g., Crizotinib) we will compare in vitro drug response to drug response in the patient. Further, we will induce therapy resistance in those models using long-term treatment with sub-lethal drug doses;
- Investigate the mechanisms of drug resistance based on (epi)genomic alterations. Organoid lines resistant to chemotherapy or ALK inhibitor treatment, will be analysed for their (epi)genetic adaptations and gene expression signatures using WES, DNA methylation profiling and RNA-Seq;
Resensitize resistant lines with epigenetic drugs. We will use epigenetic drugs including inhibitors of DNA methylation, chromatin modification (e.g., HDAC) or chromatin reader proteins (e.g., BRD4) on sensitive and resistant lines and test for their efficacy in combinatorial treatments with approved drugs.
Expected results:
Our work will provide novel models to study ALCL biology and will be made available for the whole consortium. Using these models, we expect to get a deeper insight into drug response/resistance mechanisms and to explore additional (epi)genetic vulnerabilities of ALCL.
Planned secondments:
Year 1: THT Biomaterials GmbH: Development of organoids together with the experts at the company who have significant experience of this technology (3 months)
Year 2: Masters Chancellors and Scholars of the university of Cambridge: Creation of organoids from PDX which are uniquely available in the host lab, also facilitating training in organoid development in the host lab to local students (2 months)
Nicola Mora
The role of STAT1 activation and immunity in ALCL oncogenesis
Medizinische Universität Wien
Supervisor: Olaf MERKEL
Research objectives:
The role of STAT1 has been understudied in ALCL because usually STAT1 is seen as an immunity-driven tumour suppressor.
In recent studies STAT1 has been shown to mediate the antitumor effect of aberrant ALK activity following intermittent ALK inhibitor dosing strategies, in keeping with another study arguing that STAT1 is deactivated by ALK. In contrast, we have recently shown that STAT1 mediates survival signals from the tyrosine kinase TYK2 to MCL1 suggesting an oncogenic role for this transcription factor. In FFPE tissue of primary patients we find high STAT1 and pYSTAT1 expression.
It is clear that the role of STAT1 requires further investigation. This will be achieved by:
- Knockout STAT1 in the well-established CD4/NPM-ALK mouse model of ALCL to study STAT1/STAT3 interactions and even more importantly the immunological effects of STAT1 knockout;
- To perform single cell RNAseq in wild-type and STAT1-/- murine tumours to assess anti-tumour immunity at a cellular level;
- Apply in vitro immune reporter systems to assess the effect of STAT1 knockout on the anti-tumour immune response;
- Investigate interactions of checkpoint proteins and their inhibitors that have recently been introduced into ALCL clinical trials, e.g., nivolumab with STAT1 will be assessed closely;
Clarify the role of STAT1 and the anti-tumour immune response in ALCL oncogenesis
Expected results:
Our data will provide insights into developing better strategies for ALCL immunotherapies as well as unique genetically engineered mouse models available to the consortium.
Planned secondments:
Year 1: Universitätsklinikum Freiburg: Analysis of immune cell profiles in the mouse model through learning the methodology that has been developed in the host lab (5 months)
Year 2: MLL Leukaemia lab: Conduct and analyse scRNAseq using equipment and expertise available at the MLL lab who will provide specific training (3 months)