Periodic Reporting for period 1 - CoMAnD (Contribution of the Epstein-Barr Virus and the Tumour Microenvironment to Anti-Apoptotic Mechanisms in DLBCL)
Reporting period: 2023-01-03 to 2025-01-02
This MSCA-Global Fellowship, “Contribution of the Epstein-Barr Virus and the Tumour Microenvironment to Anti-Apoptotic Mechanisms in DLBCL” (CoMAnD), aims to explore the synergistic role of the proto-oncogene MYC and the oncogenic Epstein-Barr virus (EBV) in the development and treatment of Diffuse large B cell Lymphoma (DLBCL). The post-doctoral fellow (PF) is exploring this by first generating novel models of DLBCL from primary human tonsillar germinal centre B cells through immortalisation with different proto-oncogenes (MYC, BCL-2, BCL-6) and/or EBV. These novel DLBCL models would then be characterised, with a specific focus on 1) the impact of MYC and EBV on the expression of members of the BCL-2 family of proteins (members of the intrinsic apoptotic pathway) and 2) the sensitivity of the novel DLBCL models of BH3-mimetics, a family of small molecule inhibitors that act on pro-survival members of the BCL-2 family of proteins (WP1). Next, the impact of MYC and EBV on the evolution of the tumour microenvironment (TME) and pro-/anti-apoptotic signals would be explored by transplanting the novel DLBCL models into humanised mouse models. These mice would then be treated with BH3-mimetics to target the intrinsic apoptotic pathway (WP2). Finally, the TME of the novel humanised mouse models of MYC and/or EBV+ DLBCL would be compared to patient samples using ultrahigh multiplex immunohistochemistry to uncover the relationship between MYC and EBV, and the evolution of the TME and anti-/pro-apoptotic signalling in DLBCL (WP3).
This research project has significant societal impact as currently, EBV+ DLBCL is associated with worse survival outcomes than the EBV- counterpart. Despite this, no treatment stratification exists for patients with EBV+ DLBCL. Without tractable models of EBV+ DLBCL, developing novel therapeutic strategies is severely impeded. To address this barrier, this project uses novel isogenic models of EBV+ and EBV- DLBCL, genetically engineered to express different cellular and viral oncogenes, recapitulating human disease. Beyond the scope of this project, which aims to explore intrinsic apoptosis and the tumour microenvironment in EBV+ DLBCL, these models have the potential to be employed to explore a diverse range of mechanisms and pathways involved in tumour progression and treatment resistance in EBV+ DLBCL.
This project aims to elucidate the relationship between EBV, the proto-oncogene MYC and their effects on the intrinsic apoptosis pathway and the tumour microenvironment. The PF will explore the potential to treat EBV+ DLBCL by targeting the intrinsic apoptosis pathway using BH3-mimetic drugs and evaluate how EBV and MYC modulate the tumour microenvironment to promote therapy resistance. Towards this aim, this fellowship had 6 objectives, 3 scientific and 3 training and development:
O1: Explore how EBV infection and MYC synergise to transform B cells in vitro.
O2: Using humanised mouse models, determine how different viral and cellular oncogenic drivers impact the TME.
O3: Validate TME changes in primary tumours and explore how selected TME features regulate apoptosis.
O4: Project management.
O5: Training and knowledge transfer.
O6: Dissemination, exploitation and communication.
This research project has significant societal impact as currently, EBV+ DLBCL is associated with worse survival outcomes than the EBV- counterpart. Despite this, no treatment stratification exists for patients with EBV+ DLBCL. Without tractable models of EBV+ DLBCL, developing novel therapeutic strategies is severely impeded. To address this barrier, this project uses novel isogenic models of EBV+ and EBV- DLBCL, genetically engineered to express different cellular and viral oncogenes, recapitulating human disease. Beyond the scope of this project, which aims to explore intrinsic apoptosis and the tumour microenvironment in EBV+ DLBCL, these models have the potential to be employed to explore a diverse range of mechanisms and pathways involved in tumour progression and treatment resistance in EBV+ DLBCL.
This project aims to elucidate the relationship between EBV, the proto-oncogene MYC and their effects on the intrinsic apoptosis pathway and the tumour microenvironment. The PF will explore the potential to treat EBV+ DLBCL by targeting the intrinsic apoptosis pathway using BH3-mimetic drugs and evaluate how EBV and MYC modulate the tumour microenvironment to promote therapy resistance. Towards this aim, this fellowship had 6 objectives, 3 scientific and 3 training and development:
O1: Explore how EBV infection and MYC synergise to transform B cells in vitro.
O2: Using humanised mouse models, determine how different viral and cellular oncogenic drivers impact the TME.
O3: Validate TME changes in primary tumours and explore how selected TME features regulate apoptosis.
O4: Project management.
O5: Training and knowledge transfer.
O6: Dissemination, exploitation and communication.
To date, we have generated a range of isogenic models of EBV+ and EBV- DLBCL, using primary human tonsillar germinal centre B cell. These models can be used to explore the mechanisms underpinning EBV+ B cell lymphomagenesis and treatment resistance. Specifically, this project has investigated the effect of EBV on the intrinsic apoptosis pathway. Main findings suggest that EBV+ models have an increased expression of pro-survival proteins and a reduction in the expression of anti-apoptotic proteins compared to isogenic EBV- models. The EBV+ models also demonstrate a reduction in sensitivity to BH3-mimetic, small molecule inhibitors of the pro-survival proteins which are currently being investigated for the treatment of a range of haematological malignancies.
Furthermore, these EBV+ DLBCL models are readily transplantable into the lymph nodes of NSG mice that have been reconstituted with a human immune system (so called "humanised mice"). In these humanised mouse models of EBV+ DLBCL, we see immune infiltration into the tumours, in particular CD3+ T cells. These humanised mouse models of EBV+ DLBCL also displayed increased tumour latency compared to transplantation in mice lacking an immune system, thereby indicating immune mediated control of tumour outgrowth in the humanised mice.
Next steps for this project include ultrahigh multiplex immunohistochemistry to investigate the microenvironment of these EBV+ DLBCL models. These will be compared to patient samples to 1) validate the utility of these models to investigate to tumour microenvironment of EBV+ DLBCL and 2) explore the synergistic impact of EBV with the proto-oncogene MYC on the evolution of the tumour microenvironment.
Furthermore, these EBV+ DLBCL models are readily transplantable into the lymph nodes of NSG mice that have been reconstituted with a human immune system (so called "humanised mice"). In these humanised mouse models of EBV+ DLBCL, we see immune infiltration into the tumours, in particular CD3+ T cells. These humanised mouse models of EBV+ DLBCL also displayed increased tumour latency compared to transplantation in mice lacking an immune system, thereby indicating immune mediated control of tumour outgrowth in the humanised mice.
Next steps for this project include ultrahigh multiplex immunohistochemistry to investigate the microenvironment of these EBV+ DLBCL models. These will be compared to patient samples to 1) validate the utility of these models to investigate to tumour microenvironment of EBV+ DLBCL and 2) explore the synergistic impact of EBV with the proto-oncogene MYC on the evolution of the tumour microenvironment.
Patients with EBV+ DLBCL have worse overall prognosis compared to patients with EBV- DLBCL. Despite this, there is no treatment stratification of EBV+ DLBCL. Identifying novel therapeutics for EBV+ DLBCL is impeded, in part, due to a lack of availability of reliable pre-clinical models for drug discovery. To address this, this project has successfully developed isogenic models of of EBV+ and EBV- DLBCL which can be used to determine the role EBV plays in treatment resistance and tumourigenesis in vitro. We have also validated that these models are readily transplantable directly into the lymph nodes of NSG mice reconstituted with a human immune system. These models could therefore be exploited for both in vitro and in vivo pre-clinical assessment of novel therapeutic agents for the treatment of EBV+ DLBCL.