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Neutrophils (Ns) influence cancer cell survival and metastasis through the release of chemokines, cytokines and interactions within the microenvironment and at distal sites from the primary tumour. Background: Patients with Breast Cancer (BC) often display elevated levels of circulating blood neutrophils and an elevated neutrophil: lymphocyte ratio (NLR) is associated with a shorter disease-free survival in patients with Early Breast Cancer (EBC). Evidence from mouse models of BC shows that Ns can either have a tumour-promoting or tumour-inhibitory role. CD62L (L-selectin) is a cell adhesion molecule found on the surface of leukocytes which is important for helping circulatory Ns bind to the endothelium and migrate into tissues and is rapidly shed upon N activation. Our laboratory has found a lower proportion of Ns expressing CD62L in pre-clinical models of BC, which implies that Ns from tumour-bearing mice have less endothelial- adhesive capacity and hence will remain in the circulation for a longer time. However, there is limited knowledge regarding whether different N subpopulations are present within the circulation of patients with BC, how early this occurs in the disease process and whether this is influenced by BC phenotype. In order to address these questions, we phenotyped Ns from BC patients. Objectives: Our primary objectives were to investigate changes in the surface marker CD62L expression via flow cytometry in neutrophils from patients and HVs to see if the changes seen in the pre-clinical mouse models were also seen in patients. Since kinases govern many aspects of intracellular signalling and functions, we also assessed the Serine Threonine Kinase (STK) and Phosphotyrosine Kinase (PK) activity within circulatory neutrophils in patients with Breast Cancer and paired HVs using a Pamgene kinase assay. This evaluated the activity of kinases (n = 340 kinases) in N lysates to compare N kinase activity in EBC patients and age-matched HVs. Methods: We recruited 28 patients with Early BC (EBC) (10 Node +, 18 node -; 10 T1 and 16 T2 and 2 T3) and paired healthy volunteers (HVs) who were age and sex-matched to compare phenotypical features of Ns isolated from the blood. 21 Patients with Oestrogen receptor positive (ER+) and 7 with Oestrogen receptor negative (ER -) EBC were recruited between June 2018 and June 2021. No patient had overt metastatic disease and all were screened for exclusion factors, such as infection, autoimmune conditions and certain medication known to influence neutrophils. Results: We found that ER+ patients had a lower level of the CD62Llow neutrophil subset compared to HVs (P< 0.0375). In contrast there was a significant increase in the CD62Llow neutrophil subset (2-fold higher) for ER - patients compared to HVs (P< 0.0316).The Pamgene kinase assay showed that there was a general upregulation in both STK and PK kinase activity, especially the tyrosine family of kinases for patients with ER positive breast cancer (Median kinase score which is the combined sensitivity score and specificity score > 1.2) and a general downregulation in kinase activity, especially the CMGC family of kinases for patients with ER negative disease (Median kinase score > 1.2) compared to paired HVs. The functional implications of these differences in kinase activity are being actively investigated. Conclusion: We have detected changes in CD62L expression and neutrophil kinase activity within the circulation of patients with EBC and the nature of these changes appear to be intrinsically linked to ER subtype. These findings may have important implications for use as part of an early diagnostic multi-omic platform. Citation Format: Anisha Ramessur, Bana Ambasager, Ilaria Malanchi, Raoul Charles Coombes. The multi switching activity of neutrophils in the circulation of patients with early breast cancer [abstract]. In: Proceedings of the 2021 San Antonio Breast Cancer Symposium; 2021 Dec 7-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2022;82(4 Suppl):Abstract nr P2-01-14.

CDK7 inhibition has emerged as a promising strategy in a range of cancer indications. CDK7 acts as a regulator of transcription through its role in phosphorylating and activating RNA Polymerase II (polII) as well as a master regulator of the cell cycle through phosphorylation of members of the CDK family. Inhibition of CDK7 in cancer cells has been shown to be effective in blocking transcriptional addiction to a defined cluster of genes (Wang et al 2015) and leads to decreased cell cycling, a decrease in the levels of proto-oncogenes such as c-Myc and induction of apoptosis. Here we describe the properties of a potent, selective and orally bioavailable CDK7 inhibitor CT7001 (formerly known as ICEC0942). Profiling across a pan-cancer cell line panel showed that CT7001 has anti-proliferative activity in a range of cancer types including acute myeloid leukaemia (AML), small cell lung (SCL) and hormone sensitive and triple negative breast cancers (TNBC).Mechanistic investigations revealed that CT7001s activity was associated with both a decrease in cell proliferation and an induction of apoptosis. Inhibition of cell proliferation was commensurate with a decrease in the levels of c-Myc, Mcl-1 and phospho-Pol II as determined by Western Blot analysis.CT7001 was then evaluated in a range of pre-clinical cancer models. CT7001 was shown to lead to cell death in a panel of patient derived models of small-cell lung cancer (SCLC) in 3D spheroid assays. In an in vivo orthotopic-patient derived xenograft model of TNBC in nu/nu mice, orally administered CT7001 monotherapy produced strong and sustained regression of the tumour that persisted during the dosing schedule, and strong suppression was still maintained upon cessation of treatment. At doses that produced regression CT7001 was well tolerated with little effect on body weight ( Citation Format: Edward K. Ainscow, Andrew Leishman, Elaine Sullivan, Bo Li, William Gallagher, Adam Peall, Kristopher Clark, Stuart Thomson, Simak Ali, Raoul C. Coombes, Ash Bahl. CT7001: An orally bioavailable CDK7 inhibitor is a potential therapy for breast, small-cell lung and haematological cancers [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 4834.

Background: [18F]ICMT-11 is an isatin analogue which has been developed by our group as a novel PET radiotracer for studies of apoptosis in vivo. Preclinical studies have demonstrated subnanomolar affinity to caspase 3, and validated the potential for imaging apoptosis in xenograft models. A first-in-man study showed that the agent was well tolerated with acceptable dosimetry. This is the first study of this agent to measure the effect of chemotherapy on radiotracer uptake in patients. As apoptosis is a dynamic process, one of the main objectives of the study was to determine the optimal time-point for imaging post-chemotherapy and compare the results with immunohistochemistry assessments at the same time-points. The study was approved by a regional ethics committee and ARSAC. Methods: 7 patients with breast tumour lesions measuring 15mm or more, due to undergo neoadjuvant chemotherapy with FEC (5FU, Epirubicin, Cyclophosphamide) had dynamic PET scans for 66mins 30seconds following intravenous injection of [18F]ICMT-11 with a mean activity of 340.82 Mbq±20.76 and Specific Activity range of 447.014-5128.34 Gbq/µmol prior to chemotherapy and 24h-2 weeks post-chemotherapy. A breast biopsy was also obtained within a few hours of the 2nd PET scan to correlate apoptosis in the breast tissue utilising TUNEL and Caspase 3 staining by immunohistochemistry. Volumes of interest were drawn manually and analyzed using Analyze software®. Results: The scans were well tolerated in all patients. Uptake of [18F]ICMT-11 was demonstrated in all tumor lesions. The tumours studied included ER positive and PR positive, HER2 positive and triple negative patients. The first cohort patients were imaged pre-chemotherapy and 24-48h post chemotherapy. Tumour to Breast ratio (TBR) showed an increase from 1.42±0.21(pre) to 1.71±0.33 (post). Tumour to muscle ratio (TMR) was not increased, 1.52±0.30(pre) and 1.22±0.09 (post). In addition, an increase the SUV was noted in the lymph nodes of patients, at both 24 and 48h. (SUVav 0.39±0.02 (pre), 0.45±0.03 (post), and SUVmax 0.87± 0.02(pre) ,1.22±0.12 (post). The lymph nodes were however not sampled for immunohistochemistry. A further cohort of patients had the follow-up scan 2 weeks post chemotherapy, TBR and TMR were both increased in this cohort 1.50±0.22 (pre), 2.52±0.48 (post),and 1.82±0.10 (pre), 2.08±0.0.04 (post), respectively. Caspase and TUNEL labelling with immunohistochemistry also showed increased in apoptosis in the breast biopsies at 2 weeks compared to baseline in keeping with the PET data. Conclusion: These preliminary data suggest that [18F]ICMT-11 is a promising marker for chemotherapy induced apoptosis in vivo, and correlates with findings in tumor biospsies using TUNEL and immunostaining for caspase 3. Further work is underway to study a larger cohort of patients, and identify the optimal PET pharmacokinetic parameter to describe [18F]ICMT-11 uptake and retention. Citation Format: Shairoz Merchant, Eric O Aboagye, Adrian Lim, Kasia Kozlowski, Naina Patel, Jennifer Steel, Susan Cleator, Sami Shousha, Vidhya Varghese, Raoul C Coombes, Laura Kenny. Evaluation of apoptosis in breast cancer using the novel PET probe [18F]ICMT-11 in patients treated with neoadjuvant FEC chemotherapy: Initial assessment of optimum imaging time and relation to caspase-3 immunostaining [abstract]. In: Proceedings of the Thirty-Seventh Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2014 Dec 9-13; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2015;75(9 Suppl):Abstract nr P5-01-02.

Background: Nicastrin (NCT) is the gate keeper of the gamma secretase (GS) enzyme complex. We have previously shown that high levels of NCT are observed in breast cancer patients. Moreover, we recently highlighted the importance of NCT expression for the expansion of breast cancer stem cells and their invasive features. The involvement of NCT in development of chemoresistance has been suggested by the evidence that 1) NCT is increased in response to oxaliplatin, irinotecan and 5-FU in colon cancer and 2) NCT regulates cell death within the p53 and AKT signalling pathway in human embryonic kidney (HEK293) cells. Here, we have explored the potential role of NCT in the chemoresistance of breast cancer cells. Methods: Stable NCT overexpressing MCF10A cells and control cells were produced by retroviral infections. siRNA strategy was used to silence NCT expression in a panel of p53wt and p53mut breast cancer cell lines. The sulphorhodamine B and Annexin V/PI staining were used to measure proliferation and apoptosis rate in breast cancer cells unstressed or treated with chemotherapeutic agents alone or in combination with DAPT, Wortmannin or Nutlin. p53 levels and activity were assessed by Western blotting, RT-qPCR and gene reporter assays. Results: Stable overexpression of NCT in MCF10A cells induced resistance to doxorubicin and cisplatin treatments as demonstrated by a decreased early and late apoptotis. Moreover, NCT lowers p53 expression, its phosphorylation at the phosphoSer15 site and its transcriptional activity in both unstressed cells and doxorubicin-treated cells. Using DAPT and Wortmannin as pharmacological inhibitors of Notch and AKT signalling pathways and Nutlin to inhibit Mdm2-mediated p53 degradation, we confirmed that NCT mediated chemoresistance in a p53 dependent manner partially through the PI3K/AKT pathways, while it is role is completely independent from GS activity. NCT knockdown was able to sensitize several breast cancer cell lines (MCF7, T47D, ZR75) with a wild type p53 status to doxorubicin. Moreover, NCT overexpression reduced the sensitivity to cisplatin in p53wt HTC116 colon cancer cells while it had no effect on p53mut cells. In order to translate these results to clinical settings, we have developed anti-NCT monoclonal antibodies which have been showed to inhibit proliferation and invasion of breast cancer cells. These antibodies used in combination with chemotherapy mirrored the positive effect of NCT siRNA in terms of response to doxorubicin in p53wt breast cancer cell lines. Conclusions: Our study indicates that in breast cancer cells NCT controls chemotherapy-induced cell death in a p53-dependent manner through the regulation of AKT pathway. This function is independent from the GS. We aim to investigate the association of NCT expression and p53 mutation status with clinical outcome in breast cancers in order to support potential combination of anti-NCT monoclonal antibodies with chemotherapy. Citation Format: Ylenia Lombardo, Aleksandra Filipovic, Monica Faronato, Raoul Charles Coombes. Role of nicastrin in the mediating chemoresistance of breast cancer cells. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 829. doi:10.1158/1538-7445.AM2013-829