Targeting the Complement Pathway as a Therapeutic Strategy in Lung Cancer

Lung cancer is the leading cause of cancer death in men and women. Lung adenocarcinoma (LUAD), represents approximately 40% of all lung cancer cases. Advances in recent years, such as the identification of oncogenes and the use of immunotherapies, have changed the treatment of LUAD. Yet survival rates still remain low. Additionally, there is still a gap in understanding the molecular and cellular interactions between cancer cells and the immune tumor microenvironment (TME). Defining how cancer cells with distinct oncogenic drivers interact with the TME and new strategies for enhancing anti-tumor immunity are greatly needed. The complement cascade, a central part of the innate immune system, plays an important role in regulation of adaptive immunity. Initially it was proposed that complement activation on the surface of cancer cells would inhibit cancer progression via membrane attack complex (MAC)-dependent killing. However, data from several groups have shown that complement activation promotes cancer progression, probably through the actions of anaphylatoxins (C3a and C5a) on the TME and engagement of immunoevasive pathways. While originally shown to be produced in the liver, recent studies show localized complement production in numerous cell types including immune cells and tumor cells. These results suggest that complement inhibitory drugs may represent a powerful new approach for treatment of NSCLC, and numerous new anti-complement drugs are in clinical development. However, the mechanisms by which complement is activated and affects tumor progression are not well understood. Furthermore, the role of local complement production vs. systemic activation has not been carefully examined. This review will focus on our current understanding of complement action in LUAD, and describe gaps in our knowledge critical for advancing complement therapy into the clinic

Upregulation of complement proteins in lung cancer cells mediates tumor progression

IntroductionIn vivo, cancer cells respond to signals from the tumor microenvironment resulting in changes in expression of proteins that promote tumor progression and suppress anti-tumor immunity. This study employed an orthotopic immunocompetent model of lung cancer to define pathways that are altered in cancer cells recovered from tumors compared to cells grown in culture.MethodsStudies used four murine cell lines implanted into the lungs of syngeneic mice. Cancer cells were recovered using FACS, and transcriptional changes compared to cells grown in culture were determined by RNA-seq.ResultsChanges in interferon response, antigen presentation and cytokine signaling were observed in all tumors. In addition, we observed induction of the complement pathway. We previously demonstrated that activation of complement is critical for tumor progression in this model. Complement can play both a pro-tumorigenic role through production of anaphylatoxins, and an anti-tumorigenic role by promoting complement-mediated cell killing of cancer cells. While complement proteins are produced by the liver, expression of complement proteins by cancer cells has been described. Silencing cancer cell-specific C3 inhibited tumor growth In vivo. We hypothesized that induction of complement regulatory proteins was critical for blocking the anti-tumor effects of complement activation. Silencing complement regulatory proteins also inhibited tumor growth, with different regulatory proteins acting in a cell-specific manner.DiscussionBased on these data we propose that localized induction of complement in cancer cells is a common feature of lung tumors that promotes tumor progression, with induction of complement regulatory proteins protecting cells from complement mediated-cell killing

Mechanisms of rapid cancer cell reprogramming initiated by targeted receptor tyrosine kinase inhibitors and inherent therapeutic vulnerabilities

Abstract Receptor tyrosine kinase (RTK) pathways serve as frequent oncogene drivers in solid cancers and small molecule and antibody-based inhibitors have been developed as targeted therapeutics for many of these oncogenic RTKs. In general, these drugs, when delivered as single agents in a manner consistent with the principles of precision medicine, induce tumor shrinkage but rarely complete tumor elimination. Moreover, acquired resistance of treated tumors is nearly invariant such that monotherapy strategies with targeted RTK drugs fail to provide long-term control or cures. The mechanisms mediating acquired resistance in tumors at progression treated with RTK inhibitors are relatively well defined compared to the molecular and cellular understanding of the cancer cells that persist early on therapy. We and others propose that these persisting cancer cells, termed “residual disease”, provide the reservoir from which acquired resistance eventually emerges. Herein, we will review the literature that describes rapid reprogramming induced upon inhibition of oncogenic RTKs in cancer cells as a mechanism by which cancer cells persist to yield residual disease and consider strategies for disrupting these intrinsic responses for future therapeutic gain

Durable responses to alectinib in murine models of EML4-ALK lung cancer requires adaptive immunity

Abstract Lung cancers bearing oncogenic EML4-ALK fusions respond to targeted tyrosine kinase inhibitors (TKIs; e.g., alectinib), with variation in the degree of shrinkage and duration of treatment (DOT). However, factors that control this response are not well understood. While the contribution of the immune system in mediating the response to immunotherapy has been extensively investigated, less is known regarding the contribution of immunity to TKI therapeutic responses. We previously demonstrated a positive association of a TKI-induced interferon gamma (IFNγ) transcriptional response with DOT in EGFR-mutant lung cancers. Herein, we used three murine models of EML4-ALK lung cancer to test the role for host immunity in the alectinib therapeutic response. The cell lines (EA1, EA2, EA3) were propagated orthotopically in the lungs of immunocompetent and immunodeficient mice and treated with alectinib. Tumor volumes were serially measured by μCT and immune cell content was measured by flow cytometry and multispectral immunofluorescence. Transcriptional responses to alectinib were assessed by RNAseq and secreted chemokines were measured by ELISA. All cell lines were similarly sensitive to alectinib in vitro and as orthotopic tumors in immunocompetent mice, exhibited durable shrinkage. However, in immunodeficient mice, all tumor models rapidly progressed on TKI therapy. In immunocompetent mice, EA2 tumors exhibited a complete response, whereas EA1 and EA3 tumors retained residual disease that rapidly progressed upon termination of TKI treatment. Prior to treatment, EA2 tumors had greater numbers of CD8+ T cells and fewer neutrophils compared to EA1 tumors. Also, RNAseq of cancer cells recovered from untreated tumors revealed elevated levels of CXCL9 and 10 in EA2 tumors, and higher levels of CXCL1 and 2 in EA1 tumors. Analysis of pre-treatment patient biopsies from ALK+ tumors revealed an association of neutrophil content with shorter time to progression. Combined, these data support a role for adaptive immunity in durability of TKI responses and demonstrate that the immune cell composition of the tumor microenvironment is predictive of response to alectinib therapy

An Inducible TGF-β2-TGFβR Pathway Modulates the Sensitivity of HNSCC Cells to Tyrosine Kinase Inhibitors Targeting Dominant Receptor Tyrosine Kinases

<div><p>The epidermal growth factor receptor (EGFR) is overexpressed in approximately 90% of head and neck squamous cell carcinomas (HNSCC), and molecularly targeted therapy against the EGFR with the monoclonal antibody cetuximab modestly increases overall survival in head and neck cancer patients. We hypothesize that co-signaling through additional pathways limits the efficacy of cetuximab and EGFR-specific tyrosine kinase inhibitors (TKIs) in the clinical treatment of HNSCC. Analysis of gene expression changes in HNSCC cell lines treated 4 days with TKIs targeting EGFR and/or fibroblast growth factor receptors (FGFRs) identified transforming growth factor beta 2 (TGF-β2) induction in the three cell lines tested. Measurement of TGF-β2 mRNA validated this observation and extended it to additional cell lines. Moreover, TGF-β2 mRNA was increased in primary patient HNSCC xenografts treated for 4 weeks with cetuximab, demonstrating in vivo relevance of these findings. Functional genomics analyses with shRNA libraries identified TGF-β2 and TGF-β receptors (TGFβRs) as synthetic lethal genes in the context of TKI treatment. Further, direct RNAi-mediated silencing of TGF-β2 inhibited cell growth, both alone and in combination with TKIs. Also, a pharmacological TGFβRI inhibitor similarly inhibited basal growth and enhanced TKI efficacy. In summary, the studies support a TGF-β2-TGFβR pathway as a TKI-inducible growth pathway in HNSCC that limits efficacy of EGFR-specific inhibitors.</p></div

Comparison of genes upregulated after AZD8010 and Gefitinib treatment.

<p>RNA and protein was isolated from UMSCC25, 584-A2, and Ca9-22 HNSCC cell lines that were treated for 4 days with DMSO (control), 0.3M AZD8010, 0.1M gefitinib, or both AZD8010 and gefitinib. RNA was submitted to an Affymetrix GeneChip analysis and revealed 5 genes that were upregulated in common with AZD8010 and gefitinib. The genes identified had either a >2 (UMSCC25 and Ca9-22) or >1.5 (584-A2) fold change in expression levels compared to the DMSO control. <b>A.</b> A venn diagram showing the genes upregulated in all the cell lines. <b>B.</b> Tabulated values for the genes increased and decreased in the AZD8010 and gefitinib treated group. *Numbers are the fold change in the treated compared to the control. TKIs: AZD8010 is an FGF receptor inhibitor and gefitinib is an EGF receptor inhibitor.</p

RNAi-mediated silencing of TGF-2 inhibits clonogenic growth in UMSCC25 cells.

<p>2 independent shRNA sequences targeting TGF-β2 or a non-silencing control were transduced into UMSCC25 cells using a lentiviral construct to create 3 cell lines: shTGFB2.4, shTGFB2.5, and NSC. <b>A.</b> qRT-PCR was performed to demonstrate that TGF-β2 mRNA was decreased with the shRNA compared to the NSC. <b>B.</b> An ELISA performed on media from the knockdown cells demonstrates decreased TGF-β2 protein secretion into the media. <b>C.</b> Representative images of the clonogenic assay where cells were treated with DMSO, 0.3μM AZD8010, 0.1μM gefinitib, or both for 2 weeks. <b>D.</b> Graphical presentation of the quantified results from the clonogenic assay. ***denotes a p-value <0.0001.</p