In Vivo Expansion and Antitumor Activity of Coinfused CD28- and 4-1BB-Engineered CAR-T Cells in Patients with B Cell Leukemia.

Several recent clinical trials have successfully incorporated a costimulatory domain derived from either CD28 or 4-1BB with the original CD3ζ T cell activating domain to form second-generation chimeric antigen receptors (CARs) that can increase the responsiveness and survival of CAR-engineered T (CAR-T) cells. However, a rigorous assessment of the individual benefits of these costimulatory components relative to the in vivo performance of infused T cells in patients is still lacking. Therefore, we have designed a study that allows us to investigate and compare the impact of different costimulatory signal domains on CAR-T cells in vivo. Patients with B cell leukemia were infused with a mixture of two types of CD19-specific CAR-T cells, individually bearing CD28 (28ζ) and 4-1BB (BBζ) costimulatory signaling domains. We found that such a clinical procedure was feasible and safe. Complete remission (CR) was observed in five of seven enrolled patients, with two patients exhibiting durable CR lasting more than 15 months. The in vivo expansion pattern of 28ζ and BBζ CAR-T cells varied significantly among individual patients. These results confirm a feasible method of comparing different CAR designs within individual patients, potentially offering objective insights that may facilitate the development of optimal CAR-T cell-based immunotherapies.

Targeting endogenous DLK1 exerts antitumor effect on hepatocellular carcinoma through initiating cell differentiation.

Cancer stem cells (CSCs) are responsible for tumor initiation and progression. We previously showed that Delta-like homolog 1 (DLK1) may be a therapeutic target against the CSCs of human hepatocellular carcinoma (HCC). However, the therapeutic efficacy and underlying mechanism remain unclear. Here we demonstrated that knockdown of DLK1 using a tet-inducible short hairpin RNA (shRNA) system significantly inhibited proliferation, spheroid formation and in vivo xenograft tumor growth of human HCC cells. Furthermore, in an orthotopic xenograft mouse model, adenovirus-mediated DLK1 knockdown could significantly reduce tumor size, as shown by in vivo imaging approach. Subsequently, an adenoviral vector harboring mouse Dlk1 shRNA was applied. The results showed that Dlk1 knockdown also could inhibit tumor progression in a diethylnitrosamine (DEN) induced mouse HCC model. At cellular mechanism, DLK1 knockdown delayed the cell cycle G1-S transition, along with the decreased expression of cyclin E1 and D1. Significantly, DLK1 knockdown resulted in the decrease of molecular markers such as AFP and EpCAM for hepatic progenitor cells, but the increase of KRT18 and KRT19 for the differentiated hepatocytes. The collective data indicated that targeting endogenous DLK1 may exert antitumor effect on HCCs possibly through initiating cell differentiation.

Insulin receptor tyrosine kinase substrate activates EGFR/ERK signalling pathway and promotes cell proliferation of hepatocellular carcinoma.

Insulin receptor tyrosine kinase substrate (IRTKS) is closely associated with actin remodelling and membrane protrusion, but its role in the pathogenesis of malignant tumours, including hepatocellular carcinoma (HCC), is still unknown. In this study, we showed that IRTKS was frequently upregulated in HCC samples, and its expression level was significantly associated with tumour size. Enforced expression of IRTKS in human HCC cell lines significantly promoted their proliferation and colony formation in vitro, and their capacity to develop tumour xenografts in vivo, whereas knockdown of IRTKS resulted in the opposite effects. Furthermore, the bromodeoxyuridine (BrdU) incorporation analyses and propidium iodide staining indicated that IRTKS can promote the entry into S phase of cell cycle progression. Significantly, IRTKS can interact with epidermal growth factor receptor (EGFR), results in the phosphorylation of extracellular signal-regulated kinase (ERK). By contrast, inhibition of ERK activation can attenuate the effects of IRTKS overexpression on cellular proliferation. Taken together, these data demonstrate that IRTKS promotes the proliferation of HCC cells by enhancing EGFR-ERK signalling pathway.

Deficiency of IRTKS as an adaptor of insulin receptor leads to insulin resistance.

IRTKS encodes a member of the IRSp53/MIM homology domain family, which has been shown to play an important role in the formation of plasma membrane protrusions. Although the phosphorylation of IRTKS occurs in response to insulin stimulation, the role of this protein in insulin signaling remains unknown. Here we show that IRTKS-deficient mice exhibit insulin resistance, including hyperglycemia, hyperinsulinemia, glucose intolerance, decreased insulin sensitivity, and increased hepatic glucose production. The administration of ectopic IRTKS can ameliorate the insulin resistance of IRTKS-deficient and diabetic mice. In parallel, the expression level of IRTKS was significantly decreased in diabetic mouse model. Furthermore, DNA hypermethylation of the IRTKS promoter was also observed in these subjects. We also show that IRTKS, as an adaptor of the insulin receptor (IR), modulates IR-IRS1-PI3K-AKT signaling via regulating the phosphorylation of IR. These findings add new insights into our understanding of insulin signaling and resistance.