Use of genetically engineered mouse models in preclinical drug development

The paucity of well validated preclinical models is frequently cited as a contributing factor to the high attrition rates seen in clinical oncological trials. There remains a critical need to develop models which are accurately able to recapitulate the features of human disease. The aims of this study were to use genetically engineered mouse models (GEMMs) to explore the efficacy of novel treatment strategies in HER2 positive breast cancer and to further develop the model to facilitate the study of mechanisms underpinning drug resistance. Using the BLG--HER2KI-PTEN+/- model, we demonstrated that Src plays an important role in the early stages of tumour development. Chemopreventative treatment with dasatinib delayed tumour inititation (p= 0.046, Wilcoxon signed rank test) and prolonged overall survival (OS) (p=0.06, Wilcoxon signed rank test). Dasatinib treatment also induced squamous metaplasia in 66% of drug treated tumours. We used 2 cell lines derived from this model to further explore dasatinib’s mechanism of action and demonstrated reduced proliferation, migration and invasion following in vitro treatment. Due to the prolonged tumour latency and the low metastatic rate seen in this model, further studies were undertaken with the MMTV-NIC model. This model also allowed us to study the impact of PTEN loss on therapeutic response. We validated this model by treating a cohort of MMTV-NIC PTEN+/- mice with paclitaxel and demonstrated prolonged OS (p=0.035, Gehan Breslow Wilcoxon test). AZD8931 is an equipotent signalling inhibitor of HER2, HER3 and EGFR. We observed heterogeneity in tumour response but overall AZD8931 treatment prolonged OS in both MMTV-NIC PTEN FL/+ and MMTV-NIC PTEN+/- models. PTEN loss was associated with reduced sensitivity to AZD8931 and failure to suppress Src activity, suggesting these may be suitable predictive biomarkers of AZD8931 response. To facilitate further studies exploring resistance, we transplanted MMTV-NIC PTEN+/- fragments into syngeneic mice and generated 3 tumours with acquired resistance to AZD8931. These tumours displayed differing resistance strategies; 1 tumour continued to express HER2 whilst the remaining 2 underwent EMT and lost HER2 expression reflecting to a very limited degree some of the heterogeneity of resistance strategies seen in human disease. To further explore resistance to HER2 targeting tyrosine kinase inhibitors, we generated a panel of human cell lines with acquired resistance to AZD8931 and lapatinib. Western blotting demonstrated loss of HER2, HER3 and PTEN in all resistant lines. Acquisition of resistance was associated with a marked change in phenotype and western blotting confirmed all lines had undergone EMT. We used a combination of RPPA and mass spectrometry to further characterise the AZD8931 resistant lines and identified multiple potential novel proteins involved in the resistant phenotype, including several implicated in EMT. In conclusion, when coupled with appropriate in vitro techniques, the MMTV-NIC model is a valuable tool for selection of emerging drugs to carry forward into clinical trials of HER2 positive breast cancer

Kindlin-1 promotes pulmonary breast cancer metastasis

Abstract In breast cancer, increased expression of the cytoskeletal adaptor protein Kindlin-1 has been linked to increased risks of lung metastasis, but the functional basis is unknown. Here, we show that in a mouse model of polyomavirus middle T antigen–induced mammary tumorigenesis, loss of Kindlin-1 reduced early pulmonary arrest and later development of lung metastasis. This phenotype relied on the ability of Kindlin-1 to bind and activate β integrin heterodimers. Kindlin-1 loss reduced α4 integrin–mediated adhesion of mammary tumor cells to the adhesion molecule VCAM-1 on endothelial cells. Treating mice with an anti–VCAM-1 blocking antibody prevented early pulmonary arrest. Kindlin-1 loss also resulted in reduced secretion of several factors linked to metastatic spread, including the lung metastasis regulator tenascin-C, showing that Kindlin-1 regulated metastatic dissemination by an additional mechanism in the tumor microenvironment. Overall, our results show that Kindlin-1 contributes functionally to early pulmonary metastasis of breast cancer. Significance: These findings provide a mechanistic proof in mice that Kindin-1, an integrin-binding adaptor protein, is a critical mediator of early lung metastasis of breast cancer. Cancer Res; 78(6); 1484–96. ©2018 AACR.</jats:p

Exploring mechanisms of acquired resistance to HER2 (human epidermal growth factor receptor 2)-targeted therapies in breast cancer

HER2 (human epidermal growth factor receptor 2)-targeted therapy in breast cancer is one of the earliest and arguably most successful examples of the modern class of targeted drugs. Initially identified in the 1980s, the observation that HER2 acts as an independent predictor of poor prognosis in the 20% of breast cancer cases carrying a gene amplification or protein overexpression cemented its place at the forefront of research in this field. The outlook for patients with HER2-positive breast cancer has been revolutionized by the introduction of HER2-targeted agents, such as trastuzumab and lapatinib, yet resistance is frequently encountered and multiple different resistance mechanisms have been identified. We have explored resistance to a novel pan-HER inhibitor, AZD8931, and we examine mechanisms of resistance common to trastuzumab, lapatinib and AZD8931, and discuss the current problems associated with translating the wealth of pre-clinical data into clinical benefit.</jats:p

Identification of novel pathways linking epithelial-to-mesenchymal transition with resistance to HER2-targeted therapy

Resistance to human epidermal growth factor receptor 2 (HER2)-targeted therapies in the treatment of HER2-positive breast cancer is a major clinical problem. To identify pathways linked to resistance, we generated HER2-positive breast cancer cell lines which are resistant to either lapatinib or AZD8931, two pan-HER family kinase inhibitors. Resistance was HER2 independent and was associated with epithelial-to-mesenchymal transition (EMT), resulting in increased proliferation and migration of the resistant cells. Using a global proteomics approach, we identified a novel set of EMT-associated proteins linked to HER2-independent resistance. We demonstrate that a subset of these EMT-associated genes is predictive of prognosis within the ERBB2 subtype of human breast cancers. Furthermore, targeting the EMT-associated kinases Src and Axl potently inhibited proliferation of the resistant cells, and inhibitors to these kinases may provide additional options for the treatment of HER2-independent resistance in tumors

Distinct histopathological features are associated with molecular subtypes and outcome in low grade serous ovarian carcinoma

Abstract Low grade serous ovarian carcinoma (LGSOC) demonstrates unique clinical and molecular features compared to other ovarian cancer types. The relationship between common histological features of LGSOC and molecular events, such as hormone receptor expression patterns and MAPK gene mutation status, remains poorly understood. Recent data suggest some of these molecular features may be biomarkers of response to recently introduced biologically-targeted therapies, namely endocrine therapy and MEK inhibitors. We utilize a cohort of 63 pathologically-confirmed LGSOC cases with whole exome sequencing and hormone receptor expression data to investigate these relationships. LGSOC cases demonstrated uniformly high oestrogen receptor (ER) expression, but variable progesterone receptor (PR) expression intensity. 60% and 37% of cases demonstrated micropapillary and macropapillary patterns of stromal invasion, respectively. 63% of cases demonstrated desmoplasia, which was significantly associated with advanced disease stage and visible residual disease after cytoreductive surgery. MAPK-mutant cases (KRAS, BRAF, NRAS) more frequently demonstrated macropapillary stromal invasion, while Chr1p loss was associated with desmoplasia and low PR expression. Presence of micropapillary stromal invasion and low PR expression were associated with significantly poorer survival after accounting for stage and residual disease status. Together, these data identify novel relationships between histopathological features and molecularly-defined subgroups in LGSOC

Whole exome sequencing of low grade serous ovarian carcinoma identifies genomic events associated with clinical outcome

Objectives: Low-grade serous ovarian carcinoma (LGSOC) is a distinct, rare, ovarian cancer type characterised by younger patient age and intrinsic chemoresistance. Understanding the molecular landscape is crucial for optimising targeted therapy. Methods: Genomic data from whole exome sequencing of tumour tissue was analysed in a LGSOC cohort with detailed clinical annotation. Results: 63 cases were analysed and three subgroups identified based on single nucleotide variants: canonical MAPK mutant (cMAPKm: 52%, KRAS/BRAF/NRAS), MAPK-associated gene mutation (MAPK-assoc: 27%) and MAPK wild-type (MAPKwt: 21%). NOTCH pathway disruption occurred across all subgroups. Tumour mutational burden (TMB), mutational signatures and recurrent copy number (CN) changes varied across the cohort with co-occurrence of chromosome 1p loss and 1q gain (CN Chr1pq) a recurrent feature. Low TMB and CN Chr1pq were associated with inferior disease-specific survival (HR 6.43; p < 0.001 and HR 3.29, p = 0.011 respectively). Stepwise genomic classification in relation to outcome resulted in four groups (TMB low; CN Chr1pq; MAPKwt/MAPKassoc; cMAPKm). 5 year disease-specific survival was 46%, 55%, 79% and 100% respectively for these groups. The two most favourable genomic subgroups were enriched for the SBS10b mutational signature, particularly the cMAPKm subgroup. Conclusions: LGSOC comprises multiple genomic subgroups with distinct clinical and molecular features. Chr1pq CN arm disruption and TMB represent promising methods to identify individuals with poorer prognosis. Further investigation of the molecular basis for these observations is required. MAPKwt cases represent around a fifth of patients. NOTCH inhibitors represent a candidate therapeutic strategy worthy of exploration across these cases