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PO-221 Mouse models of lung squamous cell carcinoma for preclinical intervention studies

Introduction Lung squamous cell carcinoma (LSCC) is a lethal disease accounting for 30% of all lung cancer cases. Currently, there are no effective treatments: patients are treated with a combination of surgery, chemotherapy and radiotherapy. So far numerous high-throughput screenings have been carried out in order to identify the representative mutations of this tumour. Their contribution is precious but remains descriptive. Material and methods Mice were engineered by using Flp-Recombinase mediated cassette exchange technology in ES cells. Mutations were activated by Intratracheal injection of adenoviruses carrying Cre recombinase under different promoters. Genome profiling and histological analysis were performed on lung isolated from mice that showed signs of distress due to tumour development. Results and discussions We published that mice carrying SOX2 over-expression combined with PTEN and CDKN2AB loss (hereafter SOX2PC) developed central and peripheral LSCC according to the targeted cell-of-origin, fully mimicking the human counterpart. To examine gene contribution, we uncoupled the inactivation of tumour suppressor genes and found that mice defective either of PTEN or CDKN2AB in combination with SOX2 over-expression, only showed epithelial hyperplasia, indicating that the combination is necessary for the tumour to progress. In order to use SOX2PC autochthonous model for intervention studies, we attempted at reducing tumour latency by combining other oncogenes with SOX2 over-expression. Our data pointed at FGFR1 and NFE2L2 as candidate drivers of human LSCC. Surprisingly, FGFR1-SOX2PC mice were resistant to tumour formation, indicating that FGFR1 downstream pathways may be detrimental to squamous differentiation. However, NFE2L2, a transcription factor involved in the anti-oxidant pathway and found altered in 12% of human LSCC cases, strongly enhanced tumour progression without affecting the overall tumour morphology. Most importantly, in both NFE2L2-SOX2PC and SOX2PC mice, we observed a highly immunogenic microenvironment, as reported for human patients, with high level of PD-L1 and extensive immune infiltrations of leukocytes mostly of myeloid lineage. Conclusion Our data show that neoplastic squamous differentiation is strongly associated to the oncogenic activation of SOX2. Concomitant activation of the anti-oxidant pathway accelerates tumour progression, making NFE2L2-SOX2PC autochthonous model, with its characteristic immunophenotype (massive myeloid immune infiltrations) suitable for a diversity of intervention studies.

Introduction Heparanase (HPSE) is a beta-d-endoglucuronidase and the only mammalian enzyme that cleaves heparan sulphate (HS), a major structural and regulatory component of the extracellular matrix (ECM) and the vascular basement membrane (BM). The expression of HPSE is tightly controlled and under physiological conditions is limited to immune cells, endothelial cells, placental trophoblasts and keratinocytes. However, during pathological conditions such as cancer, HPSE expression is dysregulated and high expression often correlates with poor patient survival. The cleavage of HS by HPSE expressing tumours degrades the ECM/BM and releases HS bound growth factors and cytokines, promoting cellular signalling in a positive feed-back mechanism. This in turn leads to enhanced primary tumour growth, metastasis, angiogenesis and inflammation. HPSE is therefore a critical promoter of the hallmarks of cancer and has generated significant interest as an anti-cancer drug target. However, despite decades of research, the precise mechanistic role of HPSE in the tumour microenvironment remains poorly defined. Breast cancer is the most prevalent malignancy in women worldwide. Clinical data reveals that the expression of HPSE in mammary tumours results in poor patient survival. Material and methods We recently generated a HPSE-deficient C57Bl/6 mouse strain (C57Bl/6xHPSE -/-) that were crossed with spontaneous mammary tumour developing PyMT-MMTV mice to generate the PyMT-MMTVxHPSE -/strain, providing us with a valuable in vivo model to characterise the role of HPSE in early mammary tumour development, tumour progression and metastasis.
Results and discussions Our data indicate that although HPSE promoted tumour angiogenesis, overall tumour development and metastasis between PyMT-MMTV and PyMT-MMTVxHPSE -/mice remained comparable. By examination of the tumour microenvironment, we also demonstrate that the tumour stroma positively contributes to mammary tumour HPSE activity, promoting tumour angiogenesis. In contrast, HPSE expressed in the tumour-bearing host appeared to play no significant role in tumour growth and metastasis. These data suggest that in the PyMT-MMTV model and indeed, in certain cancer settings such as breast cancer, HPSE may not play as significant a role to what has been proposed over the last two decades. Introduction Lung squamous cell carcinoma (LSCC) is a lethal disease accounting for 30% of all lung cancer cases. Currently, there are no effective treatments: patients are treated with a combination of surgery, chemotherapy and radiotherapy. So far numerous high-throughput screenings have been carried out in order to identify the representative mutations of this tumour. Their contribution is precious but remains descriptive. Material and methods Mice were engineered by using Flp-Recombinase mediated cassette exchange technology in ES cells. Mutations were activated by Intratracheal injection of adenoviruses carrying Cre recombinase under different promoters.
Genome profiling and histological analysis were performed on lung isolated from mice that showed signs of distress due to tumour development. Results and discussions We published that mice carrying SOX2 over-expression combined with PTEN and CDKN2AB loss (hereafter SOX2PC) developed central and peripheral LSCC according to the targeted cell-of-origin, fully mimicking the human counterpart.
To examine gene contribution, we uncoupled the inactivation of tumour suppressor genes and found that mice defective either of PTEN or CDKN2AB in combination with SOX2 over-expression, only showed epithelial hyperplasia, indicating that the combination is necessary for the tumour to progress.
In order to use SOX2PC autochthonous model for intervention studies, we attempted at reducing tumour latency by combining other oncogenes with SOX2 over-expression. Our data pointed at FGFR1 and NFE2L2 as candidate drivers of human LSCC. Surprisingly, FGFR1-SOX2PC mice were resistant to tumour formation, indicating that FGFR1 downstream pathways may be detrimental to squamous differentiation. However, NFE2L2, a transcription factor involved in the antioxidant pathway and found altered in 12% of human LSCC cases, strongly enhanced tumour progression without affecting the overall tumour morphology. Most importantly, in both NFE2L2-SOX2PC and SOX2PC mice, we observed a highly immunogenic microenvironment, as reported for human patients, with high level of PD-L1 and extensive immune infiltrations of leukocytes mostly of myeloid lineage. Conclusion Our data show that neoplastic squamous differentiation is strongly associated to the oncogenic activation of SOX2. Concomitant activation of the anti-oxidant pathway accelerates tumour progression, making NFE2L2-SOX2PC autochthonous model, with its characteristic immunophenotype (massive myeloid immune infiltrations) suitable for a diversity of intervention studies. Introduction Lymphoid cancers are a biologically diverse group of neoplasms. Disease aetiology is multifactorial, implicating viral and infectious agents, immune dysfunction, and genetic factors. Lymphoid cancers affect domestic and laboratory animals; among members of Mustelidae, lymphoma has been reported in domestic ferrets, yet rarely in sea otters, with only 3 reported cases since 2002. However, since 2006, 3 sea otters at the Vancouver Aquarium (British Columbia, Canada) developed lymphoma. All 3 sea otters were rescued as young animals and have lived at the aquarium for the majority of their lives. A 21 year old female was diagnosed with chronic lymphocytic leukaemia, while 2 males, aged 12 and 16, were diagnosed with lymphoma. Both males received chemotherapy before being humanely euthanized due to declining quality of life. A high rate of blood cancers in this population motivated the molecular characterisation of the most recent lymphoma. Material and methods Whole genome sequencing was performed on peripheral blood and a lymphoma needle biopsy. The tumour biopsy was sequenced on the Illumina HiSeq X and Oxford Nanopore MinION, generating 264.5 and 4.63 passed filtered Gbp, respectively. De novo assembly was performed on the Illumina DNA and RNA data using ABySS 1.3.4. Structural variants were called with trans-abyss 1.4.10. Somatic variants were called by Strelka (v1.0.15) using matched tumour-normal samples aligned against the Enhydra lutris kenyoni reference genome. Variants were annotated by snpEFF (v4.3) using an in-house custom gene annotation database. The mutations in the sea otter tumour were subsequently compared to human and canine lymphomas. Results and discussions The sea otter genome contains 24 129 genes, of which 17 421 were mapped to the human genome. We identified several mutated genes with a known involvement in human lymphomas. We observed a stop-gain mutation in EP400, a paralog of human EP300. EP300 is a tumour suppressor which has been previously implicated in human Diffuse Large B-Cell and Follicular lymphomas. We also observed an indel in SPEN, a NOTCH pathway regulator, the deregulation of which is associated with human hematopoietic neoplasms. Conclusion These observations suggest gene and pathway deregulation are a shared genetic commonality between sea otter and human lymphomas. Comparing the mutational spectrum in human, canine, and sea otter lymphoid cancers may elucidate aetiology and treatment options, and facilitate a One Health approach to neoplasia.

PO-222
Introduction Malignant mesothelioma(MM) is a highly aggressive tumour of serosal surfaces. The vast majority of cases of mesothelioma are linked to asbestos exposure. Loss of BAP1, NF2, and CDKN2AB tumour suppressor gene function is frequently observed in malignant mesothelioma. There is limited treatment option available with cisplatin/pemetrexed as the main frontline therapy with modest survival advantage. There is an urgent need of preclinical models that are fast, reproducible and that recapitulates human disease to test new treatment modalities. Here, we describe a mouse model of MM based upon the co-deletion of Bap1, Nf2, and Cdkn2ab. Material and methods Mesotheliomas were induced by deleting Bap1, Nf2, and Cdkn2ab in the thoracic cavity of mice. The tumour and its microenvironment were characterised histologically. Furthermore, we performed gene expression, chromatin profiling and drug response in tumor-derived primary cells. In addition, we employed this autochthonous model for testing current standard of care as a proof of concept treatment. Results and discussions The combined inactivation of Bap1, Nf2, and Cdkn2ab in thoracic cavity led to a highly aggressive primarily epithelioid mesothelioma with histopathological features and gene expression profile similar to human MM. This includes the distinct inflammatory phenotype. Bap1 deletion accelerated mesothelioma dramatically when combined with Nf2, Cdkn2a, Cdkn2b and p19 Arf disruption (hereafter refer as BNC loss). The accelerated tumour development is invariably connected with EZH2 mediated redistribution of H2K27me3 towards promoter sites, and activation of the PI3K, MAPK pathway. The primary cells derived from mouse mesothelioma showed drug response profile similar to those observed in human MM. Treatment of BNC tumours with cisplatin and pemetrexed, the current frontline treatment modestly prolonged survival of the mice. Conclusion We have developed a mouse model that is fast and recapitulates human mesothelioma. The mouse model is an ideal model system to study the biology and pathogenesis of mesothelioma. Furthermore, the immune-competence of this