Targeting The Erβ/Her Oncogenic Network In Lung Cancer: Synergistic Antitumor Interaction And Potentiation Of Anti-Pd1 Efficacy
2020-04
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Targeting The Erβ/Her Oncogenic Network In Lung Cancer: Synergistic Antitumor Interaction And Potentiation Of Anti-Pd1 Efficacy
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2020-04
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Abstract
Lung cancer is the leading cause of cancer related mortality in the United States, accounting for more than 142,000 estimated deaths in 2019. The major subtype of lung cancer is non-small cell lung cancer (NSCLC), which represents 85% of all cases. Despite the advancement in understanding the molecular basis of NSCLC, the 5-year survival rate is less than 20%. The current treatment strategies for advanced stage patients rely on molecularly-targeted therapies, cytotoxic chemotherapy and immunotherapeutic agents. Because of the complexity and heterogeneity of lung tumors, intrinsic and acquired resistance mechanisms ultimately result in failure to respond to these therapies and early relapse. Combinatorial strategy that involves targeting multiple aspects of tumorigenesis may represent a new avenue for therapeutic intervention in lung cancer management. Estrogen signaling has been frequently shown to be an important mediator of lung cancer progression and metastasis. In a non-genomic fashion, ER mutually interacts with human epidermal growth factor receptors (HERs) to promote lung cancer proliferation and growth. Targeting ER signaling with the antiestrogen fulvestrant has shown moderate activities in preclinical models of lung cancer. The reciprocal interaction between ER and EGFR could limit the activity of using anti-ER agent alone. In a phase II clinical trial, combining fulvestrant with the selective EGFR tyrosine kinase inhibitor erlotinib showed an enhanced activity over erlotinib alone and improved the survival outcomes in NSCLC patients. However, the improvement in overall median survival was modest. Recent retrospective clinical analysis demonstrated that genes contained in the prediction analysis of microarray 50 (PAM50) provide prognostic information in high ERβ+ lung cancer patients. The 7-gene model includes c-Myc, MIA, CXXC5, FGFR4, Grb-7, FOXC1, and PgR. In high-risk patients, who tend to have a poor prognosis and short median survival, c-Myc, MIA, CXXC5, FGFR4, Grb-7, FOXC1 are overexpressed and PgR is downregulated. Importantly, the 7-gene model described one interacting network that includes ER and HER2/HER3 as the top upstream regulators for the 7-gene panel. In fact, a significant association between ERβ and HER2 expression was found, in which 70.2 % of ERβ-positive cases were positive for HER2 compared to 34.5% of ERβ-negative cases. HER3, when analyzed with HER2, showed also positive association with ERβ. These observations suggest that ERβ and HER2/HER3 pathways define lung tumors with very aggressive biology, indicating that blocking both pathways could be more efficacious than either one of them alone. These observations also could explain why the magnitude of response was modest when fulvestrant was combined with erlotinib, as erlotinib has weak activities against HER2 and HER3. Combining a pan-HER inhibitor such as dacomitinib (inhibits EGFR, HER2 and HER4) with fulvestrant could be more efficacious than either agent alone and could produce a gene signature that predicts better clinical outcomes in NSCLC. Immune checkpoint inhibitors have changed the treatment paradigm for several solid tumors, including lung cancer. Antibodies that target programmed death receptor 1(PD1) or its ligand (PD-L1) have proven great efficacy for certain patients with lung cancer. Patients who respond to these agents tend to have durable effects and longer survival outcomes; however, only 20-29% of patients are predicted to respond. Several combination strategies are being evaluated clinically to maximize the efficacy of these agents and improve the response rate. Preclinical evidence found that the ER blocker fulvestrant effectively sensitizes lung cancer cells to T cell and natural killer (NK) cell mediated cytotoxicity effects. In addition, estrogen signaling is a critical mediator for myeloid derived suppressor cells (MDSCs), which are largely known for their immunosuppressive effects. Evidence also demonstrated that EGFR inhibitors possess dual immunomodulatory effects that include upregulation of major histocompatibility complex I and II (MHC I and II), inducing the recruitment of immune cells, and inhibiting other tyrosine kinases essential for immune cells function. Synergy was observed when EGFR TKI and PD1 inhibitor was combined in an- EGFR-mutant model. Altogether, these previous observations encouragingly support the hypothesis that use of triple therapy (fulvestrant, pan-HER TKI and an immune checkpoint blocker) will be a promising treatment approach for lung cancer. Testing this hypothesis is the subject of this dissertation. To evaluate the therapeutic potential of combining ER blocker with pan-HER in NSCLC, we chose fulvestrant, as an antiestrogen, and dacomitinib, as a tyrosine kinase inhibitor that targets EGFR, HER2, and HER4. We first evaluated the efficacy of this combination in three different human NSCLC cell lines and assessed the ability of this treatment approach to produce a gene signature that predicts better clinical outcomes. We utilized three different cell lines that represent three different categories of NSCLC population; EGFR-mutant, KRAS-mutant and EGFR and KRAS wild-type. Next, we investigated the immunomodulatory effects of this combination treatment on macrophages and CD8+ T cells in vitro. We used a novel syngeneic lung cancer model (FVBW-17/FVB-N) to evaluate the therapeutic effectiveness of fulvestrant-dacomitinib in combination with a mouse anti-PD1. Major results The combination of fulvestrant and dacomitinib significantly suppressed NSCLC cell growth in vitro and produced a combination index < 0.5, indicating strong synergy. The combination also showed potent downregulation of HER activity and marked decrease in amphiregulin (AREG) and neuregulin (NRG1-β1) expression. Importantly, the combination, but not single agents, completely reversed the gene signature associated with poor prognosis. C-Myc, MIA, CXXC5, FGFR4, FOXC1 and Grb7 were downregulated and PgR was upregulated following the combination treatment. The combination significantly reduced c-Fos, JunB and pCREB DNA-binding activities. The c-Fos/Ap-1 inhibitor t-5224, but not CBP-CREB inhibitor, was able to partially mimic the effects of the combination in reversing the gene signature. In vivo, the combination treatment demonstrated a robust synergistic antitumor effect in NSCLC cell lines that were engrafted subcutaneously in immunodeficient mice. Tumor regression was observed in the majority of tumors following the combination treatment. A drastic decrease in HER activity and downstream signaling were observed with the combination, along with a significant decrease in AREG and NRG1-β1 expression. In situ proximity ligation assay revealed a significant decrease in the active dimerization of both p-HER2/p-HER3 and p-HER2/p-EGFR dimers following the combination treatment. Additionally, the gene signature was also completely reversed by the combination but not with single agents. In the EGFR mutant model, the survival of mice was improved after treatment discontinuation, tumors that recurred were less aggressive, and two mechanisms of resistance commonly associated with HER TKIs were blocked. To evaluate the immunomodulatory effects of the combination, bone marrow-derived macrophages and CD8+ T cells were treated with the combination. Surprisingly, macrophages lost their phagocytic function and behave more like M2-macrophages by expressing high IL-10, CD206 and PD1 following the combination treatment. Mechanistically, dacomitinib induced downregulation of phospho-Syk, and fulvestrant was not able to overcome this effect. In CD8+ T cells, the combination impaired the function of T cells, induced high PD1 expression, and severally reduced IFN-ϒ and TNF-α production. These debilitating effects were mostly attributed to the downregulation of Src Family kinases activities, as less phospho-SFK Y416 was detected following the combination treatment. In vivo, adding anti-PD1 antibody to the combination treatment enhanced the immune function and improved the antitumor effects. In a sequential approach, where anti-PD1 was administrated after the combination treatment, the average tumor volume was 4-fold less than placebo and this effect was synergistic. In comparison, the triple therapy given concomitantly showed a 2-fold decrease compared to placebo. Sequential triple therapy was also significantly better than concomitant triple therapy. None of the drugs alone show any sign of activities. Interestingly, after one week of administrating fulvestrant and dacomitinib, tumors showed high immune cell infiltration (inflamed tumor microenvironment), with relatively high PD1 expression on CD8+ T cells. In contrast, the concomitant triple therapy showed a significant increase in CD8+ T cells infiltration but with a decrease in PD1 expression, and fewer M2 macrophages. Conclusion and Significance Retrospective analysis of NSCLC patients revealed that the interaction between HER2/HER3 and ERβ contributes to poor outcomes in NSCLC patients with high ERβ expression. Here, we report that targeting ERβ with the antiestrogen fulvestrant, along with targeting multiple HER pathways with the pan-HER TKI dacomitinib produced synergistic antitumor effects in preclinical models of human ERβ+ NSCLC. The robust antitumor effect seen was accompanied by the ability of this combination treatment to produce a gene signature that predicts better clinical outcomes. The 7-gene model could serve as a predictive tool for identifying patients who will more likely respond to the treatment. These data strongly support its clinical use, giving the fact that both drugs are clinically used for cancer patients. Convincing evidence also suggest an immunomodulatory effect of ER signaling and HER pathways. The combination of fulvestrant and dacomitinib suppressed phagocytic macrophages and CD8+ T cell functions and upregulated PD1 expression. These effects were largely contributed by dacomitinib for its inhibitory effects on Syk and Src kinases on immune cells and might limit the clinical utility of the fulvestrant/dacomitinib combination. However, the ability of the combination to turn the TME into an inflamed microenvironment with upregulated PD1 could potentiate the tumor-mediated killing effects by immunotherapy. The synergistic antitumor effect observed with adding anti-PD1 in a sequential manner after the administration of fulvestrant and dacomitinib is highly encouraging. The significance of these observations is that the triple therapy has shown excellent antitumor effects in a highly aggressive tumor model that is unresponsive to immune checkpoint inhibitor alone or fulvestrant-dacomitinib combination. The clinical usefulness of this approach can have therapeutic applications for other solid tumor models, particularly in tumors that are less inflamed and are unlikely to respond to immune checkpoint inhibitors.
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University of Minnesota Ph.D. dissertation. April 2020. Major: Pharmacology. Advisor: Jill Siegfried. 1 computer file (PDF); xvi, 117 pages.
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Almotlak, Abdulaziz. (2020). Targeting The Erβ/Her Oncogenic Network In Lung Cancer: Synergistic Antitumor Interaction And Potentiation Of Anti-Pd1 Efficacy. Retrieved from the University Digital Conservancy, https://hdl.handle.net/11299/215171.
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