Enhanced Expression of CD47 Is Associated With Off-Target Resistance to Tyrosine Kinase Inhibitor Gefitinib in NSCLC.
NSCLC 中 CD47 的表达增强与酪氨酸激酶抑制剂吉非替尼的脱靶耐药相关。
- 作者列表："Nigro A","Ricciardi L","Salvato I","Sabbatino F","Vitale M","Crescenzi MA","Montico B","Triggiani M","Pepe S","Stellato C","Casolaro V","Dal Col J
:Mutual interactions between cancer cells and the tumor microenvironment importantly contribute to the development of tyrosine kinase inhibitor (TKI) resistance in patients affected by EGFR-mutant NSCLC. In particular, immune recognition-associated proteins with impact on tumor cell clearance through phagocytosis, such as CD47 and calreticulin, could contribute to adaptive resistance and immune escape. Preclinical studies targeting the anti-phagocytic CD47 molecule showed promising results in different cancer types including lung cancer, but no data are available on its role in patients acquiring resistance to EGFR TKI treatment. We analyzed the functional contribution of CD47 and calreticulin to immune surveillance and evasion in a panel of NSCLC cell lines carrying sensitizing or resistant mutations in the EGFR gene, following treatment with the TKI gefitinib and after in vitro development of gefitinib resistance. While CD47 and calreticulin protein levels were markedly variable in both EGFR-mutant and wild-type cell lines, analysis of NSCLC transcriptomic dataset revealed selective overexpression of CD47 in patients carrying EGFR mutations. EGFR inhibition significantly reduced CD47 expression on the surface of pre-apoptotic cells, favoring more efficient engulfment of cancer cells by monocyte-derived dendritic cells. This was not necessarily associated with augmented surface exposure of calreticulin or other molecular markers of immunogenic cell death. Moreover, CD47 expression became up-regulated following in vitro drug resistance development, and blocking of this protein by a specific monoclonal antibody increased the clearance of EGFR-TKI resistant cells by phagocytes. Our study supports CD47 neutralization by specific monoclonal antibody as a promising immunotherapeutic option for naïve and resistant EGFR-mutant NSCLCs.
: 癌细胞和肿瘤微环境之间的相互作用重要地有助于 EGFR 突变 NSCLC 患者酪氨酸激酶抑制剂 (TKI) 耐药的发生。特别是通过吞噬作用影响肿瘤细胞清除的免疫识别相关蛋白，如 CD47 和钙网蛋白，可有助于适应性抵抗和免疫逃逸。针对抗吞噬 CD47 分子的临床前研究在包括肺癌在内的不同癌症类型中显示出有希望的结果，但没有关于其在获得 EGFR TKI 治疗耐药的患者中的作用的数据。我们在一组携带 EGFR 基因增敏或耐药突变的 NSCLC 细胞系中分析了 CD47 和钙网蛋白对免疫监视和逃避的功能贡献。 TKI 吉非替尼治疗后和吉非替尼体外耐药后。虽然 CD47 和钙网蛋白水平在 EGFR 突变型和野生型细胞系中均有显著差异，但对 NSCLC 转录组数据集的分析显示，携带 EGFR 突变的患者 CD47 选择性过表达。EGFR 抑制显著降低了凋亡前细胞表面的 CD47 表达，有利于单核细胞来源的树突状细胞更有效地吞噬癌细胞。这不一定与钙网蛋白或其他免疫原性细胞死亡的分子标志物的增强表面暴露相关。此外，CD47 表达随着体外耐药的发展而上调，特异性单克隆抗体阻断该蛋白可增加吞噬细胞对 EGFR-TKI 耐药细胞的清除。我们的研究支持特异性单克隆抗体中和 CD47 作为初治和耐药 EGFR 突变 NSCLCs 的一种有前途的免疫治疗选择。
METHODS::Macrophages (MΦ) play a critical role in tumor growth, immunosuppression and inhibition of adaptive immune responses in cancer. Hence, targeting signaling pathways in MΦs that promote tumor immunosuppression will provide therapeutic benefit. PI3Kγ has been recently established by our group and others as a novel immuno-oncology target. Herein, we report that a macrophage Syk-PI3K axis drives polarization of immunosuppressive MΦs which establish an immunosuppressive tumor microenvironment in in vivo syngeneic tumor models. Genetic or pharmacological inhibition of Syk and/or PI3Kγ in MΦs promotes a pro-inflammatory MΦphenotype, restores CD8+ T cell activity, destabilizes HIF under hypoxia, and stimulates an antitumor immune response. Assay for Transposase-accessible Chromatin using Sequencing (ATAC-seq) analyses on the bone marrow derived macrophages (BMDMs) show that inhibition of Syk kinase promotes activation and binding of NF-κB motif in SykMC-KO BMDMs, thus stimulating immunostimulatory transcriptional programming in MΦs to suppress tumor growth. Finally, we have developed in silico the "first in class" dual Syk/PI3K inhibitor, SRX3207, for the combinatorial inhibition of Syk and PI3K in one small molecule. This chemotype demonstrates efficacy in multiple tumor models and represents a novel combinatorial approach to activate antitumor immunity.
METHODS::Dysfunction of invariant natural killer T (iNKT) cells in tumor microenvironment hinders their anti-tumor efficacy, and the underlying mechanisms remain unclear. Here we report that iNKT cells increase lipid biosynthesis after activation, and that is promoted by PPARγ and PLZF synergically through enhancing transcription of Srebf1. Among those lipids, cholesterol is required for the optimal IFN-γ production from iNKT cells. Lactic acid in tumor microenvironment reduces expression of PPARγ in intratumoral iNKT cells and consequently diminishes their cholesterol synthesis and IFN-γ production. Importantly, PPARγ agonist pioglitazone, a thiazolidinedione drug for type 2 diabetes, successfully restores IFN-γ production in tumor-infiltrating iNKT cells from both human patients and mouse models. Combination of pioglitazone and alpha-galactosylceramide treatments significantly enhances iNKT cell-mediated anti-tumor immune responses and prolongs survival of tumor-bearing mice. Our studies provide a strategy to augment the anti-tumor efficacy of iNKT cell-based immunotherapies via promoting their lipid biosynthesis.
METHODS::Near-infrared photoimmunotherapy (NIR-PIT) is a newly developed and selective cancer treatment that induces necrotic and immunogenic cell death and utilizes a monoclonal antibody conjugated to a photo-absorber dye, IR700DX, activated by NIR light. Although CD44 is surface cancer marker associated with drug resistance, anti-CD44-IR700 NIR-PIT results in inhibited cell growth and prolonged survival in multiple tumor types. Meanwhile, anti-CD25-IR700-targeted NIR-PIT has been reported to achieve selective and local depletion of FOXP3+CD25+CD4+ regulatory T cells (Tregs), which are primary immunosuppressive cells in the tumor microenvironment (TME), resulting in activation of local antitumor immunity. Combined NIR-PIT with CD44- and CD25-targeted agents has the potential to directly eliminate tumor cells and also amplify the immune response by removing FOXP3+CD25+CD4+ Tregs from the TME. We investigated the difference in therapeutic effects of CD44-targeted NIR-PIT alone, CD25-targeted NIR-PIT alone, and the combination of CD44- and CD25-targeted NIR-PIT in several syngeneic tumor models, including MC38-luc, LL/2, and MOC1. The combined NIR-PIT showed significant tumor growth inhibition and prolonged survival compared with CD44-targeted NIR-PIT alone in all tumor models and showed prolonged survival compared with CD25-targeted NIR-PIT alone in MC38-luc and LL/2 tumors. Combined CD44/CD25 NIR-PIT also resulted in some complete remissions, whereas this was not achieved with either type of NIR-PIT alone. Therefore, combined NIR-PIT simultaneously targeting cancer antigens and immunosuppressive cells in the TME may be more effective than either type of NIR-PIT alone and may have potential to induce prolonged immune responses in treated tumors.