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COMMON PART

Project Number18-15-00061

Project titleIdentification of factors that affect efficacy of HER2 targeted therapy for breast cancer patients and development of antitumor drug that block HER ligands.

Project LeadBuzdin Anton

AffiliationShemyakin - Ovchinnikov Institute of Bioorganic Chemistry of the Russian Academy of Sciences,

PROJECT EXTENSION CARD

Research area 05 - FUNDAMENTAL RESEARCH IN MEDICINE, 05-310 - Personalized medicine

KeywordsBreast cancer, oncology, epidermal growth factor receptor, HER2, EGFR, tyrosin kinase receptors, prognostic test, biomarkers, chemotherapy, target anticancer drugs, growth factors, targeted therapy, transcriptome analysis, blood serum, recombinant proteins

PROJECT CONTENT

Annotation
The HER/HER receptor family is made up of four structurally related receptor tyrosine kinases.In humans, these include: EGFR (HER1), HER2 (Neu), HER3, and HER4.Several growth factors are known to be able to bind receptors and to activate them, named HER ligands. Approximately 20% of all breast cancers (BC) have gene amplification or overexpression (or both) of human epidermal growth factor receptor 2 (HER2). A therapeutic approach has been developed to block receptor activity, HER2-targeted drugs. Several humanized monoclonal antibodies were developed to target extracellular receptor domains. For therapy of BC with HER2 overexpression (HER2-positive or HER2+BC) antibodies trastuzumab (Herceptin), ado-trastuzumab emtansine (Kadcyla) and pertuzumab (Perjeta), were approved as well as tyrosin-kinase inhibitor lapatinib (Tykerb). Hundreds of thousands of BC patients take these drugs; their annual sales are billions of dollars. At the same time, not all women with tumors expressing high levels of HER2 respond to trastuzumab. If trastuzumab is used as monotherapy, an objective response rate is estimated at 20 to 35%; if trastuzumab is combined with chemotherapy – at 50 to 65%. We are intent to study intra- and extracellular factors that affect tumour response on the HER2-targeted drugs treatment; we are intent to elucidate molecular mechanisms of the tumour resistance to HER2-targeted drugs. Our objective is to develop tests aimed to predict the patient response on HER2-targeted drugs used for BC HER2+. We expect, patient pre-screening based on the proposed test will lead to the increased response rate of HER2-targeted cure and will greatly decrease failure of the targeted therapy. 1) HER ligands and HER2-targeted drugs have the same target, while exert an opposite impact on the receptor. While patient is treated by HER2-targeted therapy, the drugs are being able to withstand endogenous HER ligands which are present in human body. Earlier we have shown that HER ligands affect an anti-proliferative effect of HER2-targeted drugs in vitro. Moreover, we have demonstrated association between high HER ligands concentration and results of the targeted therapy using samples of 25 lung cancer patients. Others demonstrated this possibility in clinic for Erbitux treatment. We are intent to measure concentration of six known HER ligands in patients’ blood by ELISA technique. It is known that concentration of each ligand can differ between patients as much as 1000-fold. We are intent to study the correlation between HER ligands concentration in patients’ peripheral blood with the patients’ response on the targeted therapy with trastuzumab. The basic idea of the proposed approach is using the decreased endogenous HER ligands level of patients relative to the threshold level as criteria for the HER2-targeted cure. Thresholds for the ligand concentrations will be determined in this study. 2) Our study will demonstrate that HER ligands can be used as targets for the novel approach to the BC therapy. This approach consists of targeting soluble HER ligands by drugs that temporary decrease the whole-body ligands concentration. Such decrease in HER ligands concentration will render HER-targeted drugs more effective in its anti-tumour activity. We are intent to produce a recombinant protein that bind HER ligands specifically. We will measure how human serum influence an inhibitory effect of HER-targeted drugs erlotinib, gefitinib and lapatinib on the cell growth for lines originated from BC and lung cancer in the presence and absence of the recombinant protein. We propose to use of the recombinant protein as antitumor therapy for the cure of BC and lung cancer in combination with tyrosine kinase inhibitors. Of note, now HER-targeted therapy is not employed for the lung cancer treatment in 85% of cases in consequence of its low efficacy. 3) Tumour response on the targeted therapy can be affected by variety of factors (proteins or metabolites) present in patient body. As these factors affect anti-proliferative action of the drugs during the patient treatment, it seems to be possible that they will be able to affect anti-proliferative action of the drug on the cultivated cells in vitro. Thus, we believe that the negative effect of the patient’s blood on the targeted drug efficacy can be rapidly and cost-effectively estimated before administration of the time-consuming and expensive therapy course. We are intent to measure the inhibitor impact of anticancer drug trastuzumab on the growth of the BC-originated cell lines in the presence of the patient’s serum. If serum does not essentially affect inhibitory effect of trastuzumab on the clonogenity, one can predict that tumour progression will be effectively inhibited by this drug. Our preliminary experiments have shown that human serum decreases the anti-proliferative impact of targeted drugs. The basic idea of the proposed approach is using the decreased influence of patient sera on the drug inhibitory effect relative to average population level as criteria for the HER2-targeted cure. We are intent to measure anti-proliferative action of trastuzumab in the presence of the blood samples of HER2-positive BC patients with known tumour response on the trastuzumab treatment. Our aim is to check if inhibitory effect of patient’s sera on the anti-proliferative action of trastuzumab in vitro is correlated with trastuzumab therapy results. 4) More and more new targeted drugs are proposed by pharmaceutical companies and novel molecular diagnostics methods are highly demanded. We are intent to perform transcriptome profiling of BC tumour samples. Recently we developed a new bioinformatic method for the processing of gene expression data leading to quantitative characterization of cellular signaling and metabolic pathways activities (www.oncofinder.com). We will employ this method to find out how pathways activity profiles correlates with clinical results of HER2-targeted therapy. Analysis of molecular mechanisms of cancer cell response to the therapy must help doctors to choose the right medicine for effective BC treatment and, hopefully helps researchers to find ways to overcome tumour resistance to targeted drugs.

Expected results
The objective of this proposal is to study intra- and extracellular factors that affect tumour response on the HER2-targeted drugs treatment. BC is the most frequently diagnosed cancer and the leading cause of cancer death in females worldwide. In 2013, there were approx. 67 000 (85 per 100,000 women per year) new cases of breast cancer diagnosed and 23 000 cancer deaths in Russia [Злокачественные новообразования в России в 2015 году, под ред. А.Д. Карпина, В.В. Старинского, Г.В. Петровой, Москва, 2017.]. Approximately 20% of all breast cancers have gene amplification or overexpression (or both) of HER2, HER2+ BC. A therapeutic approach has been developed to block receptor activation, HER2-targeted drugs. For therapy of HER2+ BC antibodies trastuzumab are commonly used; usually it is applied in combination with chemotherapy. Objective response rate of HER2-targeted therapy combination with chemotherapy is evaluated at 30-65% [Baselga J, Manikhas A, Cortes J, Llombart A, Roman L, Semiglazov VF, et al. Phase III trial of nonpegylated liposomal doxorubicin in combination with trastuzumab and paclitaxel in HER2-positive metastatic breast cancer. Annals of oncology: official journal of the European Society for Medical Oncology / ESMO. 2014;25(3):592–598]. That is to say, these high-priced drugs bring no benefit to a half of patients. In the framework of this project, we will study mechanisms of tumour resistance and develop test-systems for selection of BC patients for the HER2-targeted drugs treatment. Far not all the HER2+ BC patients are treated with targeted drugs as subventions for purchasing of these expansive drugs are limited. Our objective is to establish an objective criterion for the trastuzumab chargeless prescription. Many efforts world-wide are directed towards understanding how activation of intracellular signaling pathways links with the machinery that controls fundamental cellular processes and cancer development. Modern experimental technologies make it possible to determine complete transcriptome profiles of biosamples, which increases productivity of such efforts. Our studies will be a part of this mainstream research. Originality of our approach is a method of the data proceeding. We developed a new bioinformatic method termed OncoFinder that for the first time enables quantitative measuring of the activity of intracellular signaling pathways basing on transcriptomic data. Clinically annotated biological materials to be collected include blood samples and biopsies. Samples will be obtained from HER2+ BC patients with known trastuzumab treatment outcomes. For the collected tissue samples (corresponding to minimum 40 individual patients) the transcriptome profiling will be performed using hybridization with high-density customized microarrays (CustomArray platform, USA). The data on the gene expression will be processed using bioinformatic method OncoFinder. As a result we obtain a list of intracellular signaling and metabolic pathways whose activity is characteristic of the individual patient. Associative analysis could show how pathway activity profile is correlated with BC treatment outcome of the trastuzumab therapy. We hope to reveal intracellular signaling and metabolic pathways whose activity can serve as predictor for the tumor response on the trastuzumab therapy. This knowledge is also important for understanding of molecular mechanisms of tumour resistance to trastuzumab treatment and is suitable for possible clinical applications. Tumour transcriptome profiling permit to acquire knowledge of molecular mechanisms of signal transduction launched by the cellular receptors and has an extreme scientific significance. We study the mechanisms of cellular resistance to the HER2-targeted drugs. Studies on the relation between receptors’ inhibition/activation, signaling pathways activities and cancer progression is a rapidly developing area of medicine and biology. We are intent to study extracellular factors in the patient blood that could influence cancer progression and affect targeted therapy efficacy. Search for reliable prognostic markers for tumour progression and for tumour response to the targeted drugs therapy is strong objective worldwide. HER ligands specifically bind receptors; binding induces receptor activation and autophosphorylation that leads to the launch of the intracellular signaling cascades [Izrailita J, Bermana H, Dattid A. High throughput kinase inhibitor screens reveal TRB3 and MAPK/ERK/ TGFβ pathways as fundamental Notch regulators in breast cancer.(2013) Proc Natl Acad Sci U S A 110:1714–9].. HER2-targeted drugs block ligand binding (antibodies) to receptor or inhibit receptor autophosphorylation (tyrosine kinase inhibitors) [Baker, A. et al., Notch-EGFR/HER2 bidirectional crosstalk in breast cancer Front. Oncol.,(2014) 12:234-52]. Thus, HER2-targeted drugs action is opposite to the action of the HER ligands. We suggest that high endogenous ligand concentration decreases anti-tumour impact of HER2-targeted drugs. For some HER ligands we have already demonstrated a correlation between ligand concentration and targeted therapy results of the lung cancer. Our preliminary results show that each ligand, EGF, TGF-alpha and HB-EGF, blocks cell growth inhibition effect of receptor-targeted drugs, erlotinib, lapatinib and trastuzumab. We are intent to analyze blood sera samples of a hundred HER2+ BC patients with known objective response on the trastuzumab therapy. For HER ligand we will find how the objective response rate depends on the serum ligand concentration. To reach this goal we will measure by ELISA technique sample concentrations of the following six ligands (shown to be most important HER ligands for BC progression): EGF, TGF-alpha, amphiregulin, HB-EGF, betacellulin, and epiregulin. Based on the results of measurements we will find ligands which concentrations in sera significantly affect the therapy outcome and we will determine the thresholds for the concentrations of each ligand. We will develop a test-system that includes measurement of HER ligands concentration by ELISA technique in the peripheral blood serum of patient and estimation criteria for tumour response rate to the HER2-targeted therapy. Estimation criteria include thresholds for ligand concentration (or a threshold for a combination of several ligand concentrations). If patient serum contains concentrations of HER ligands that exceed the threshold, then the patient must NOT be treated with HER-targeted drugs because the probability this drug will help is very low. Prediction of HER2-targeted therapy efficacy may be important for tens of thousands of HER2-positive BC patients only in Russia. We estimate a number of the patients with revealed HER2-positive BC as 80 thousand in Russia. Patent application based on our study results will be prepared as the test-system utilization has a commercial value for efficacy prediction of HER2-targeted drugs that are already on the marked as well as for development of novel drugs. We expect that our study could be a basis for development of novel methods to help doctors to choose the right medicine for BC treatment. Personalized therapy in BC will improve quality of life, disease-free and overall survival and to decrease treatment cost. Tumour response on the targeted therapy can be affected by variety of factors (proteins or metabolites) present in patient body. Our study takes into account numerous factors in patient’s serum that can affect the therapy results, not only HER ligands. Again, we are intent to analyze blood sera samples of a hundred HER2+ BC patients with known objective response on the trastuzumab therapy. We will measure cell clonogenity for cell lines, originated from BC and expressing HER2 (examples are MDA-MB-453, SK-BR-3, BT-474, ZR-75 versus MCF7 or T-47D) in the presence of trastuzumab and sera. In this way we will determine how the inhibitory impact of HER2-targeted drug is affected by the blood sera in vitro. As a result we will find a correlation between the sera influence on cell clonogenity in the presence of trastuzumab and the objective response rate to the trastuzumab therapy. Thus, as a result of the proposed studies we will find how clinical outcome of the HER2-targeted therapy of HER2-positive breast cancer depends on 1) signaling and metabolic pathway activity in the tumour cells, 2) concentration of HER ligands in the patient serum, 3) trastuzumab anti-proliferative effect inhibition by the patient serum in vitro (clonogenity of BC-originated cell lines). Potentially, all three studies can be employed to develop test-systems for HER2-targeted therapy outcome prediction before the therapy administration. Test-system based on ELISA technique can be immediately used in clinic as ELISA is a routine technique in the majority of clinics. For clonogenity analysis, samples must be delivered in specialized labs. Although this test is easy-to-perform and cost-effective (cost of this test is about 1500 rubbles, comparable with ELISA). . Factors associated with clinical outcome (biomarkers) in patient’s blood is very important for any research that concerns anti-tumour therapy. Predictive biomarkers can be used for patient selection to increase tumour response rate. Tumour sequencing revealed changes in gene-drivers of somatic cells that cause cell transformation and tumour progression. For lung cancer it was demonstrated that targeted therapy is successful only for tumors with mutant targeted gene, EGFR (HER1) that render EGFR active even in the absence of HER ligands. This finding led to the test-system development and resulted in the increase of tumour response to targeted drugs treatment from 12 to 70%, while patient population to be treated with targeted drugs decreased from practically 100 % to 10 – 15%. As a result, the targeted therapy for the lung cancer treatment is prescribed now only for the tumors bearing activating EGFR mutations. Thus, this promising therapeutic method is not available for 85% of lung cancer patients. Here, we propose a therapeutic approach based on the temporary decrease of the HER ligands’ concentration. We believe that this approach essentially increase HER-targeted therapy efficacy for this major cohort of patients and renders targeted therapy available for these patients. We are intent to produce recombinant protein that specifically binds HER ligands. We will investigate how human blood serum influence an inhibitory impact of HER targented drugs erlotinib, gefitinib, and lapatinib on the cell proliferation for the cell lines originated from BC and lung cancer in the presence or absence of aforementioned recombinant protein. The produced recombinant protein can be used as anti-tumor drug for BC and lung cancer cure in combination with tyrosine kinase inhibitors. Patent application based on our study results and developed test-systems will be prepared as the test-system utilization has a commercial value. We expect that patient pre-screening based on the proposed tests will lead to the increased response rate of HER2-targeted cure and will greatly decrease failure of the targeted therapy. Thus, it will be possible to reduce the burdens of illness and reduce the healthcare cost saving hundreds million of the dollars only in Russia. Expensive targeted drugs will be prescribed to the patients that will benefit from therapy. The clinical trials of the new targeted drugs could use the proposed tests for patients’ selection to obtain better results; drugs will be accepted faster by physicians and society to save human lives. We hope that recombinant protein that we are intent to produce for decrease of HER ligand concentration will be applicable for the lung cancer cure as well as for BC treatment. Results of this study can be applied in medicine (diagnostics), pharmacology (novel anticancer drugs development), and in biotechnology (test-system production).

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