An examination of the pharmacology of HER2 inhibitor responses in cellular models of breast cancer

So-called “targeted therapeutics”, agents with tumour-selective action, promise to revolutionise treatment of specific malignancies; however, realisation of the therapeutic promise of such drugs requires new methods of rapidly distinguishing patients who will derive treatment benefit. Tyrosine Kinase Inhibitors (TKIs), a group of small molecule therapeutics, inhibit specific aspects of the phosphorylation-mediated intracellular signalling pathways commonly altered in cancer. Overexpression of one such pathway, initiated by the HER2 growth factor receptor, occurs commonly in breast cancer. Lapatinib, a HER2 TKI, has been used in combinations with other cancer drugs for treating HER2 overexpressing breast cancer. The aim of this study was to evaluate gene expression changes in response to these targeted therapies to examine their ability to predict treatment response. In this thesis, microarray data from lapatinib-treated drug sensitive breast cancer cell lines was interrogated using an emerging bioinformatic technique, Co-inertia analysis (CIA). Using this technique, 512 genes were found to be altered in a specific response to lapatinib treatment in the cell lines. 27 gene targets were chosen for more detailed analysis using Taqman RT-PCR, of which five showed predictive response in a broader panel of breast cancer cell lines treated with lapatinib. Expression of the five genes was further examined in response to other HER2 targeted therapies and the analysis indicated that the gene expression changes remained consistent with these other treatments, demonstrating a more broadly representative anti-HER2 response pattern. An in vivo study sought to evaluate these gene expression responses in a more in vivo-relevant scenario and found that they were also conserved in this model. Our research indicates that there are commonalities among the gene expression response to HER2-targeting therapeutics in responsive cells which may extrapolate to HER2-amplified patient tumours and more broadly suggests that characterisation of gene changes shortly after treatment may provide a valuable rapid predictor of inhibitor response, potentially guiding a more specific use of such agents by identifying patients that will benefit from these therapies

Elucidating mechanism of action for clinical candidate therapeutic antibodies

Cancer is driven by numerous cellular dysregulations such as increased proliferation, decreased apoptosis, increased vascularization, and evasion of immune surveillance. As such, it is unlikely that inhibiting only one of these disease hallmarks will cause a lasting clinical response. The objective of this work is to modulate intracellular signaling, endothelial expansion, and immune cell activation in human and mouse models of cancer to better inform the development of new antibody therapeutics. The contribution of fourteen growth factors to receptor tyrosine kinase (RTK) driven chemotherapy resistance in nine pancreatic ductal adenocarcinoma and fifteen ovarian cancer cell lines was assessed using phosphorylation of Protein Kinase B (AKT) as a readout for pro-survival signaling via western blot and ELISA. Results revealed redundancy between Insulin-Like Growth Factor Receptor (IGF-1R) and Epidermal Growth Factor Receptor 3 (ErbB3) signaling. In pancreatic cancer cell lines, a tetravalent, bispecific antibody co-targeting IGF-1R and ErbB3 (istiratumab/MM-141) blocked growth factor induced pro-survival signaling and enhanced chemotherapy-induced apoptosis. Istiratumab also improved the in vivo efficacy of gemcitabine and nab-paclitaxel in two cell line-derived xenograft (CDX) models and one patient-derived xenograft (PDX) model of pancreatic cancer. In ovarian cancer cell lines, cell-surface IGF-1R expression correlated significantly with in vitro cisplatin and paclitaxel sensitivity, and istiratumab prevented chemotherapy induced AKT phosphorylation. Furthermore, istiratumab enhanced the in vivo efficacy of paclitaxel, pegylated-liposomal doxorubicin, and cisplatin in an ovarian cancer CDX model. The role of the cytokine Tumor Necrosis Factor (TNF) has been studied within the tumor microenvironment. Firstly, the in vitro human umbilical vein endothelial cell (HUVEC) model of angiogenesis revealed TNF-mediated TNF receptor 1 (TNFR1) activation to be a driver of endothelial tube maintenance. Secondly, an investigation into the driving mechanism of action for a TNFR2-targeting mouse IgG2a (Y9/MM-401) in eight in vivo mouse syngeneic models of cancer showed that cancer cell TNFR2 expression does not drive in vivo efficacy, rather it promotes Fc receptor mediated agonism of tumor infiltrating CD8 positive effector T cells. These data support TNFR2 as a possible therapeutic target for the treatment of cancer

‘ErbB activation and heterodimerisation is responsible for resistance upon PI3K-mTOR inhibition in metastatic prostate cancer’

My hypothesis is that resistance to PI3K-AKT-mTOR targeting in metastatic prostate cancer involves ErbB activation and heterodimerisation. Better description of the mechanisms implicated will allow the identification of appropriate predictive biomarkers. Current clinical trials are investigating the use of PI3K-AKT-mTOR inhibitors in metastatic castration-resistant prostate cancer (CRPC). 50-70% of metastatic CRPC patients have genomic aberrations of the PI3K pathway, mainly involving loss of PTEN, an important negative regulator of the PI3K-AKT pathway. Upregulation of HER3 was previously suggested to be an important resistance mechanism. Within the context of this project I have applied biophysical techniques to quantify protein-protein interactions i.e fluorescence lifetime imaging microscopy (FLIM) which is the gold-standard technique for measuring Forster resonance energy transfer (FRET). This is an established technology in our laboratory and was used to evaluate HER3 heterodimerisation in prostate cancer cells and mouse xenograft tissue, alongside biochemical methods to demonstrate changes in ErbB expression in response to PI3K-AKT-mTOR inhibition. In addition, I optimised this technology for use in cell line and patientderived exosomes. Different ErbB subtypes are upregulated in vitro as part of a potential resistance mechanism in response to PI3K-mTOR inhibition, depending on the cell line PTEN status. Concomitant upregulation of either AR or PSMA is also observed. In PTEN WT prostate cancer cells, the upregulation of PSMA is demonstrated to be HER2 dependent and can be inhibited by lapatinib. The clinical implications of my results propose the use of PI3K-AKT-mTOR inhibitors in the metastatic hormone-sensitive setting as well. In addition, tissue and/or exosomal ErbB heterodimerisation, together with the use of clinically available PSMA imaging probes, might prove an additional biomarker in resistance detection and subgroup classification. Some initial PSMA PET imaging analyses upon PI3K-mTOR inhibition in vivo will be presented. Finally, this might allow the design of prospective clinical trials using PSMA-targeted therapies

Resistance to HER2-targeted therapies in HER2-positive breast cancer

Breast cancer accounts for 522,000 deaths worldwide each year and is the most common cancer in women. It is classified according to the cell of origin and its expression of several receptors: oestrogen and progesterone receptors, and human epidermal growth factor receptor 2 (HER2). Historically, HER2-positive breast cancers had a worse survival prognosis than oestrogen or progesterone receptor-positive cancers, but the development of HER2-targeted therapies led to significant survival improvements. Despite this, patients often present with de novo resistance, or will develop acquired resistance to targeted therapies. Several resistance mechanisms have been identified but attempts to target them have failed. Thus, it is of paramount importance to identify the mechanisms used, to prevent development of resistance or resensitise tumours to HER2-targeted therapies. Objectives of this study were: to understand the link between epithelial to mesenchymal transition (EMT) and loss of HER2, seen in a model of acquired resistance to the HER2-targeted therapy, sapatinib, and to characterise and validate tumours from a sapatinib-treated spontaneous mouse model of HER2-positive breast cancer. The EMT-linked transcription factor ZEB1 was associated with acquired resistance to sapatinib in tumours that had undergone EMT and concurrently lost HER2. Generation of drug resistant cell lines failed to recapitulate the in vivo phenotype. Transient overexpression of ZEB1 in vitro did not induce clear EMT or loss of HER2, despite a trend towards lower HER2 expression. However, we found that treatment of cells with ERBB2 shRNA, the gene encoding HER2, increased levels of ZEB1 and enhanced migration, but did not induce overt EMT. This may be the result of differing PTEN status between in vivo and in vitro models. Treatment of a spontaneous mouse model of HER2-positive breast cancer with sapatinib revealed that progressing tumours had an increase in proteins associated with cellular iron homeostasis. Further investigation revealed increased heme oxygenase-1 (HO-1), iron exporter ferroportin and altered iron storage. To ascertain if modulation of dietary iron intake could affect the development of resistance to sapatinib, mice were given a control or iron-deficient diet and treated with vehicle or sapatinib. This showed that in sapatinib-treated mice fed an iron-deficient diet, HO-1 was not increased as in tumours from mice fed a iron-low control diet. We looked at the possibility of HER2-targeting therapies inducing ferroptosis, an iron-dependent form of cell death. Sapatinib-treated tumours from mice on a iron-low control diet had increased cyclooxygenase 2 (COX2), a marker of ferroptosis, which was not seen in sapatinib-treated tumours from mice on an iron-deficient diet. Additionally, in vitro drug treatments with HER2-targeting agents showed that SKBR3 cell death could be rescued by iron chelation. HO-1 overexpression in SKBR3 cells revealed increased autophagic flux and resistance to HER2-targeted therapies. Inhibition of autophagy reversed resistance, rendering them susceptible to sapatinib- and lapatinib-induced cell death. Further, increased autophagic flux was seen in all progressive tumours on sapatinib. The increased resistance to sapatinib in mice fed an iron-deficient diet was also associated with increased autophagic flux, although this was HO-1-independent. Taken together, the results presented here provide a novel mechanism of cell death induced by HER2-targeting agents in vitro and in vivo. We have shown that increased HO-1 and reducing dietary iron can affect the development of resistance to sapatinib, which is reliant on autophagy induction. Further, inhibiting autophagy can resensitise cells to sapatinib and lapatinib treatment

ANALOGUES OF (-)-PICROPODOPHYLLIN, SYNTHESIS AND USES THEREOF

Analogs of (-)-Picropodophyllin, synthesis thereof, and uses in pharmaceuticals as inhibitors of IGF1RK

Quantitative analysis of RET signaling dynamics and crosstalk

Most existing studies of receptor signaling are qualitative, which can lead scientists to misinterpret or overlook key information about the extent and timing of key events. To overcome these shortcomings, we have applied quantitative approaches to characterize receptor activation and signaling events. Most signaling studies focus on events occurring at a particular level in the system (e.g., on the membrane, at the level of phosphorylation of intracellular signaling molecules, or at the level of transcription). Instead, we are interested in taking a longitudinal view of signaling by achieving a quantitative understanding of a single signaling pathway from initial stimulation of the receptor by its growth factor (GF) ligand, through to gene expression, and functional cellular responses. As a model system for our studies, we used the growth factor receptor tyrosine kinase, REarranged during Transfection (RET), which requires a ligand and a glycosylphosphatidylinositol-anchored co-receptor for activation. RET mediates the response of cells to members of the glial cell-line derived neurotrophic factor (GDNF) family of neurotrophins, which are important in the development and maintenance of a subset of neuronal cells as well as in other cell types and tissues. We have characterized the molecular mechanisms of RET activation and signaling by pursuing the following four aims: 1) We developed a sensitive and robust luciferase reporter gene assay for RET signaling. 2) We characterized the dynamic relationship between receptor activation and downstream signaling events, including gene transcription and translation of three target genes. 3) We used the reporter gene assay, and other detection approaches, to test and quantify crosstalk between RET and other GF receptors. 4) We developed a FRET reporter system to enable monitoring of the assembly of the activated RET receptor complex on cells, as a means to distinguish between ligand-induced oligomerization and pre-associated oligomer mechanisms. Through these four aims, we have established new methods to quantitatively elucidate mechanisms of GF receptor activation, new insights into how signals are propagated from the receptor to the nucleus and into a functional response, and have established crosstalk between RET and other GF receptor pathways

Investigating mechanisms of acquired resistance to AT13148, an AGC kinase inhibitor

The PI3K/AKT/mTOR (PAM) pathway is a major driver of cell growth, proliferation and survival, and is frequently dysregulated in cancer. AKT, a member of the AGC family of serine/threonine kinases, is a key signalling node within the PAM pathway, and as such, an attractive therapeutic target for the treatment of cancer. AT13148 is an ATP-competitive inhibitor of AKT, currently in phase 1 clinical trial, which also potently inhibits several other clinically relevant AGC kinases, such as ROCK1/2 and p70S6K. Acquired resistance to kinase inhibitors has been a barrier to their success, but resistance to AT13148 is yet to be defined. Therefore, the aim of this thesis was to investigate mechanisms of acquired AT13148 resistance, using preclinical cell line models. Isogenic AT13148 resistant sub-clones were generated from the A2780 human ovarian carcinoma cell line, which harbours mutations in the PAM pathway. In these sub-clones, phosphorylation of S6RP (p70S6K substrate) and PRAS40 (AKT substrate) was refractory to AT13148 treatment, concurrent with increased ERK 1/2 phosphorylation, when compared to the parental A2780 cell line. Two of the resistant sub-clones were sensitised to AT13148 on exposure to the ERK inhibitor GDC-0994, with the combination of AT13148 and GDC-0994 shown to restore the inhibition of S6RP phosphorylation. This implicates ERK 1/2 as a driver of AT13148 resistance, potentially via the reactivation of the PAM pathway and suggests ERK inhibition as a strategy to overcome this resistance. A loss of DUSP6 expression, an ERK 1/2 phosphatase, was subsequently detected in AT13148 resistant sub-clones, but DUSP6 loss alone was not shown to cause a sustained increase in ERK 1/2 phosphorylation or confer AT13148 resistance, suggesting other factors are required for AT13148 resistance. In conclusion, the results presented in this thesis identify ERK 1/2 as a driver of AT13148 resistance. This discovery has the potential to aid the ongoing clinical development of AT13148 and provide a therapeutic strategy to overcome AT13148 resistance

Stromal Markers as Prognostic Factors in Primary Human Breast Cancer: Evaluation of Aldo-Keto Reductases AKR1C1 and AKR1C2

HINTERGRUND: Stromale Fibroblasten beeinflussen Tumorwachstum und -progression. Gegenstand der vorliegenden Arbeit war die Evaluation der zwei Aldo-Keto-Reduktasen AKR1C1 und AKR1C2 in Stromalfibroblasten und Karzinomzellen als prognostische Faktoren bei primärem menschlichen Brustkrebs. Sie sind am intratumoralen Progesteronstoffwechsel beteiligt. METHODEN: Die Immunhistochemie wurde an Gewebemikroarrays aus 504 Kernbiopsien von Brustkrebspatientinnen durchgeführt. Primäre Endpunkte waren das krankheitsfreie (DFS) und das Gesamtüberleben (OS). ERGEBNISSE: AKR1C1 und AKR1C2 Expression in Fibroblasten und Tumorzellen korreliert mit günstigen Tumoreigenschaften, wie z.B. kleine Tumorgröße und negativer Lymphknotenstatus. In der univariaten Analyse korrelierte die AKR1C1-Expression in Karzinomzellen positiv mit DFS und OS; die AKR1C2-Expression in Fibroblasten und Tumorzellen zeigte ebenfalls eine positive Korrelation mit DFS und OS. In der multivariaten Analyse war die AKR1C1-Expression in Karzinomzellen ein unabhängiger prognostischer Marker. FAZIT: Es ist davon auszugehen, dass unsere Beobachtungen auf die unabhängige regulatorische Funktion von AKR1C1/2 im Progesteronstoffwechsel zurückzuführen sind und damit die Grundlage für neue hormonbasierte Therapieoptionen für Brustkrebspatientinnen unabhängig vom klassischen Hormonrezeptorstatus bilden.BACKGROUND: Stromal fibroblasts influence tumor growth and progression. We evaluated two aldo-keto reductases, AKR1C1 and AKR1C2, in stromal fibroblasts and carcinoma cells as prognostic factors in primary human breast cancer. They are involved in intratumoral progesterone metabolism. METHODS: Immunohistochemistry was performed on tissue microarrays from 504 core biopsies from breast cancer patients. Primary endpoints were disease-free (DFS) and overall (OS) survival. RESULTS: AKR1C1 and AKR1C2 expression in fibroblasts and tumor cells correlated with favorable tumor characteristics, such as small tumor size and negative nodal status. In univariate analysis, AKR1C1 expression in carcinoma cells correlated positively with DFS und OS; AKR1C2 expression in both fibroblasts and tumor cells also showed a positive correlation with DFS and OS. In multivariate analysis, AKR1C1 expression in carcinoma cells was an independent prognostic marker. CONCLUSION: It can be assumed that our observations are due to the independent regulatory function of AKR1C1/2 in progesterone metabolism and therefore provide a basis for new hormone-based therapy options for breast cancer patients, independent of classic hormone receptor status