Qian Xie

Genetic alteration of the MET receptor tyrosine kinase frequently occurs in glioblastoma (GBM). Clinically, bevacizumab treatment results in MET signaling activation, leading to GBM recurrence with a more malignant phenotype. While MET has been a promising therapeutic target, MET inhibitors have not been successful in treating GBM patients. MET-directed chimeric antigen receptor (CAR) T cells hold the promise of targeting MET-positive GBM regardless of genetic alterations or kinase activity. GBM patient-derived xenografts (PDX) harboring MET amplification (METamp) or PTPRZ-MET fusion (ZM) were propagated in vivo followed by glioma stem cell (GSC) isolation. Cell-based assays were used for comparing GSC survival in response to MET inhibitors and CAR T cells. Multi-panel cytokine release was analyzed to profile MET-CAR T cell activation during co-culture with GBM. Orthotopic tumor growth and real-time imaging were performed to evaluate MET-CAR T cell therapeutic efficacy in vivo. Although GBM are heterogeneous tumors, neuro-sphere cells isolated from METamp or ZM fusion PDX tumors showed universal cognate genetic MET alteration along with GSC markers such as SOX2 and nestin. Both METamp and ZM fusion tumors showed MET overexpression but only the METamp cells presented activated MET signaling which was vulnerable to MET inhibitors. In contrast, MET-CAR T cells specifically inhibited all MET-positive tumor growth regardless of MET activation status. Whereas MET inhibitors are effective in MET-active tumors, MET-CAR T cells eradicate MET-positive GBM growth in an antigen-dependent manner, demonstrating a promising therapeutic approach for treating MET-positive GBM. MET overexpression, especially METamp and ZM fusion may be used to predefine the GBM patients for treating with MET-CAR T cell therapy.

Background: Recent progress in cancer immunotherapy encourages the expansion of chimeric antigen receptor (CAR) T cell therapy in solid tumors including hepatocellular carcinoma (HCC). Overexpression of MET receptor tyrosine kinase is common in HCC; however, MET inhibitors are effective only when MET is in an active form, making patient stratification difficult. Specific MET-targeting CAR-T cells hold the promise of targeting HCC with MET overexpression regardless of signaling pathway activity. Methods: MET-specific CARs with CD28ζ or 4-1BBζ as co-stimulation domains were constructed. MET-CAR-T cells derived from healthy subjects (HS) and HCC patients were evaluated for their killing activity and cytokine release against HCC cells with various MET activations in vitro, and for their tumor growth inhibition in orthotopic xenograft models in vivo. Results: MET-CAR.CD28ζ and MET-CAR.4-1BBζ T cells derived from both HS and HCC patients specifically killed MET-positive HCC cells. When stimulated with MET-positive HCC cells in vitro, MET-CAR.CD28ζ T cells demonstrated a higher level of cytokine release and expression of programmed cell death protein 1 (PD-1) than MET-CAR.4-1BBζ T cells. When analyzed in vivo, MET-CAR.CD28ζ T cells more effectively inhibited HCC orthotopic tumor growth in mice when compared to MET-CAR.4-1BBζ T cells. Conclusion: We generated and characterized MET-specific CAR-T cells for targeting HCC with MET overexpression regardless of MET activation. Compared with MET-CAR.4-1BBζ, MET-CAR.CD28ζ T cells showed a higher anti-HCC potency but also a higher level of T cell exhaustion. While MET-CAR.CD28ζ is preferred for further development, overcoming the exhaustion of MET-CAR-T cells is necessary to improve their therapeutic efficacy in vivo.

Glioblastoma (GBM) is the most malignant primary brain tumor without effective therapies. Since bevacizumab was FDA approved for targeting vascular endothelial growth factor receptor 2 (VEGFR2) in adult patients with recurrent GBM, targeted therapy against receptor tyrosine kinases (RTKs) has become a new avenue for GBM therapeutics. In addition to VEGFR, the epidermal growth factor receptor (EGFR), platelet-derived growth factor receptor (PDGFR), hepatocyte growth factor receptor (HGFR/MET), and fibroblast growth factor receptor (FGFR) are major RTK targets. However, results from clinical Phase II/III trials indicate that most RTK-targeting therapeutics including tyrosine kinase inhibitors (TKIs) and neutralizing antibodies lack clinical efficacy, either alone or in combination. The major challenge is to uncover the genetic RTK alterations driving GBM initiation and progression, as well as to elucidate the mechanisms toward therapeutic resistance. In this review, we will discuss the genetic alterations in these 5 commonly targeted RTKs, the clinical trial outcomes of the associated RTK-targeting therapeutics, and the potential mechanisms toward the resistance. We anticipate that future design of new clinical trials with combination strategies, based on the genetic alterations within an individual patient’s tumor and mechanisms contributing to therapeutic resistance after treatment, will achieve durable remissions and improve outcomes in GBM patients.

Helicobacter pylori infection is a leading cause of gastric cancer, which is the second-most common cancer-related death in the world. The chronic inflammatory environment in the gastric mucosal epithelia during H. pylori infection stimulates intracellular signaling pathways, namely inflammatory signals, which may lead to the promotion and progression of cancer cells. We herein report two important signal transduction pathways, the LPS-TLR4 and CagA-MET pathways. Upon H. pylori stimulation, lipopolysaccharide (LPS) binds to toll-like receptor 4 (TLR4) mainly on macrophages and gastric epithelial cells. This induces an inflammatory response in the gastric epithelia to upregulate transcription factors, such as NF-κB, AP-1, and IRFs, all of which contribute to the initiation and progression of gastric cancer cells. Compared with other bacterial LPSs, H. pylori LPS has a unique function of inhibiting the mononuclear cell (MNC)-based production of IL-12 and IFN-γ. While this mechanism reduces the degree of inflammatory reaction of immune cells, it also promotes the survival of gastric cancer cells. The HGF/SF-MET signaling plays a major role in promoting cellular proliferation, motility, migration, survival, and angiogenesis, all of which are essential factors for cancer progression. H. pylori infection may facilitate MET downstream signaling in gastric cancer cells through its CagA protein via phosphorylation-dependent and/or phosphorylation-independent pathways. Other signaling pathways involved in H. pylori infection include EGFR, FAK, and Wnt/β-Catenin. These pathways function in the inflammatory process of gastric epithelial mucosa, as well as the progression of gastric cancer cells. Thus, H. pylori infection-mediated chronic inflammation plays an important role in the development and progression of gastric cancer.

Glioblastoma multiforme (GBM) is the most common malignant brain cancer that invades normal brain tissue and impedes surgical eradication, resulting in early local recurrence and high mortality. In addition, most therapeutic agents lack permeability across the blood brain barrier (BBB), further reducing the efficacy of chemotherapy. Thus, effective treatment against GBM requires tumor specific targets and efficient intracranial drug delivery. With the most recent advances in immunotherapy, genetically engineered T cells with chimeric antigen receptors (CARs) are becoming a promising approach for treating cancer. By transducing T lymphocytes with CAR constructs containing a tumor-associated antigen (TAA) recognition domain linked to the constant regions of a signaling T cell receptor, CAR T cells may recognize a predefined TAA with high specificity in a non-MHC restricted manner, and is independent of antigen processing. Active T cells can travel across the BBB, providing additional advantage for drug delivery and tumor targeting. Here we review the CAR design and technical innovations, the major targets that are in pre-clinical and clinical development with a focus on GBM, and multiple strategies developed to improve CAR T cell efficacy.

Living organisms require complex signaling interactions and proper regulation of these interactions to influence biological processes. Of these complex networks, one of the most distinguished is the Notch pathway. Dysregulation of this pathway often results in defects during organismal development and can be a causative mechanism for initiation and progression of cancer. Despite previous research entailing the importance of this signaling pathway and the organismal processes that it is involved in, less is known concerning the major Notch downstream targets, especially the onset and sequence in which they are modulated during normal development. As timing of regulation may be linked to many biological processes, we investigated and established a model of temporal patterning of major Notch downstream targets including broad, cut, and hindsight during Drosophila melanogaster egg chamber development. We confirmed the sequential order of Broad upregulation, Hindsight upregulation, and Cut downregulation. In addition, we showed that Notch signaling could be activated at stage 4, one stage earlier than the stage 5, a previously long-held belief. However, our further mitotic marker analysis re-stated that mitotic cycle continues until stage 5. Through our study, we once again validated the effectiveness and reliability of our MATLAB toolbox designed to systematically identify egg chamber stages based on area size, ratio, and additional morphological characteristics.

Aberrant MET receptor tyrosine kinase (RTK) activation leads to invasive tumor growth in different types of cancer. Overexpression of MET and its ligand hepatocyte growth factor (HGF) occurs more frequently in glioblastoma (GBM) than in low grade gliomas. While we have shown previously that HGF-autocrine activation predicts sensitivity to MET tyrosine kinase inhibitors (TKIs) in GBM, whether it initiates tumorigenesis remains elusive. Using a well-established Sleeping Beauty (SB) transposon strategy, we injected human HGF and MET cDNA together with a short hairpin siRNA against Trp53 (SB-hHgf.Met.ShP53) into the lateral ventricle of neonatal mice to induce spontaneous glioma initiation and characterized the tumors with HE and immunohistochemistry (IHC) analysis. Glioma sphere cells also were isolated for measuring the sensitivity to specific MET TKIs. Mixed injection of SB-hHgf.Met.ShP53 plasmids induced de novo glioma formation with invasive tumor growth accompanied by HGF and MET overexpression. While glioma stem cells (GSCs) are considered as the tumor initiating cells in GBM, both SB-hHgf.Met.ShP53 tumor sections and glioma spheres harvested from these tumors expressed GSC markers nestin, GFAP, and Sox 2. Moreover, specific MET TKIs significantly inhibited tumor spheres’ proliferation and MET/MAPK/AKT signaling. Overexpression of the HGF/MET axis along with p53 attenuation may transform neural stem cells (NSCs) into GSCs, resulting in GBM formation in mice. These tumors are primarily driven by the MET RTK pathway activation and are sensitive to MET TKIs. The SB-hHgf.Met.ShP53 spontaneous mouse glioma model provides a useful tool for studying GBM tumor biology and MET-targeting therapeutics.

Aberrant MET tyrosine kinase signaling is known to cause cancer initiation and progression. While MET inhibitors are in clinical trials against several cancer types, the clinical efficacies are controversial and the molecular mechanisms toward sensitivity remain elusive. With the goal to investigate the molecular basis of MET amplification (METamp) and hepatocyte growth factor (HGF) autocrine-driven tumors in response to MET tyrosine kinase inhibitors (TKI) and neutralizing antibodies, we compared cancer cells harboring METamp (MKN45 and MHCCH97H) or HGF-autocrine (JHH5 and U87) for their sensitivity and downstream biological responses to a MET-TKI (INC280) and an anti-MET monoclonal antibody (MetMab) in vitro, and for tumor inhibition in vivo. We find that cancer cells driven by METamp are more sensitive to INC280 than are those driven by HGF-autocrine activation. In METamp cells, INC280 induced a DNA damage response with activation of repair through the p53BP1/ATM signaling pathway. Although MetMab failed to inhibit METamp cell proliferation and tumor growth, both INC280 and MetMab reduced HGF-autocrine tumor growth. In addition, we also show that HGF stimulation promoted human HUVEC cell tube formation via the Src pathway, which was inhibited by either INC280 or MetMab. These observations suggest that in HGF-autocrine tumors, the endothelial cells are the secondary targets MET inhibitors. Our results demonstrate that METamp and HGF-autocrine activation favor different molecular mechanisms. While combining MET TKIs and ATM inhibitors may enhance the efficacy for treating tumors harboring METamp, a combined inhibition of MET and angiogenesis pathways may improve the therapeutic efficacy against HGF-autocrine tumors.

Near-IR fluorescence (NIRF) imaging is becoming a promising approach in preclinical tumor detection and clinical image-guided oncological surgery. While heptamethine cyanine dye IR780 has excellent tumor targeting and imaging potential, its hydrophobic property limits its clinical use. In this study, we developed nanoparticle formulations to facilitate the use of IR780 for fluorescent imaging of malignant brain tumor. Self-assembled IR780-liposomes and IR780-phospholipid micelles were prepared and their NIRF properties were characterized. The intracellular accumulation of IR780-nanoparticles in glioma cells were determined using confocal microscopy. The in vivo brain tumor targeting and NIRF imaging capacity of IR780-nanoparticles were evaluated using U87MG glioma ectopic and orthotopic xenograft models and a spontaneous glioma mouse model driven by RAS/RTK activation. The loading of IR780 into liposomes or phospholipid micelles was efficient. The particle diameter of IR780-liposomes and IR780-phospholipid micelles were 95 and 26 nm, respectively. While stock solutions of each preparation were maintained at ready-to-use condition, the IR780-phospholipid micelles were more stable. In tissue culture cells, IR780-nanoparticles prepared by either method accumulated in mitochondria, however, in animals the IR780-phospholipid micelles showed enhanced intra-tumoral accumulation in U87MG ectopic tumors. Moreover, IR780-phospholipid micelles also showed preferred intracranial tumor accumulation and potent NIRF signal intensity in glioma orthotopic models at a real-time, non-invasive manner. The IR780-phospholipid micelles demonstrated tumor-specific NIRF imaging capacity in glioma preclinical mouse models, providing great potential for clinical imaging and image-guided surgery of brain tumors.