Impaired ligand-dependent MET activation caused by an extracellular SEMA domain missense mutation in lung cancer.

Aberrant activation of the MET/hepatocyte growth factor (HGF) receptor participates in the malignant behavior of cancer cells, such as invasion-metastasis and resistance to molecular targeted drugs. Many mutations in the MET extracellular region have been reported, but their significance is largely unknown. Here, we report the dysregulation of mutant MET originally found in a lung cancer patient with Val370 to Asp370 (V370D) replacement located in the extracellular SEMA domain. MET-knockout cells were prepared and reconstituted with WT-MET or V370D-MET. HGF stimulation induced MET dimerization and biological responses in cells reconstituted with WT-MET, but HGF did not induce MET dimerization and failed to induce biological responses in V370D-MET cells. The V370D mutation abrogated HGF-dependent drug resistance of lung cancer cells to epidermal growth factor receptor-tyrosine kinase inhibitors (EGFR-TKI). Compared with WT-MET cells, V370D-MET cells showed different activation patterns in receptor tyrosine kinases upon exposure to survival/growth-stressed conditions. Surface plasmon resonance analysis indicated that affinity between the extracellular region of V370D-MET and HGF was reduced compared with that for WT-MET. Further analysis of the association between V370D-MET and the separate domains of HGF indicated that the SP domain of HGF was unchanged, but its association with the NK4 domain of HGF was mostly lost in V370D-MET. These results indicate that the V370D mutation in the MET receptor impairs the functional association with HGF and is therefore a loss-of-function mutation. This mutation may change the dependence of cancer cell growth/survival on signaling molecules, which may promote cancer cell characteristics under certain conditions.

Distinct Localization of Mature HGF from its Precursor Form in Developing and Repairing the Stomach.

Hepatocyte growth factor (HGF) is secreted as an inactive single-chain HGF (scHGF); however, only proteolytically processed two-chain HGF (tcHGF) can activate the MET receptor. We investigated the localization of tcHGF and activated/phosphorylated MET (pMET) using a tcHGF-specific antibody. In day 16.5 mouse embryos, total HGF (scHGF + tcHGF) was mainly localized in smooth muscle cells close to, but separate from, MET-positive epithelial cells in endodermal organs, including the stomach. In the adult stomach, total HGF was localized in smooth muscle cells, and tcHGF was mainly localized in the glandular base region. Immunostaining for pMET and Lgr5-driven green fluorescent protein (GFP) indicated that pMET localization overlapped with Lgr5+ gastric stem cells. HGF promoted organoid formation similar to EGF, indicating the potential for HGF to promote the survival and growth of gastric stem cells. pMET and tcHGF localizations changed during regeneration following gastric injury. These results indicate that MET is constantly activated in gastric stem cells and that the localization of pMET differs from the primary localization of precursor HGF but has a close relationship to tcHGF. Our results suggest the importance of the microenvironmental generation of tcHGF in the regulation of development, regeneration, and stem cell behavior.

Co-stimulation of CD28/CD40 signaling molecule potentiates CAR-T cell efficacy and stemness.

CD19 chimeric antigen receptor T (CD19CAR-T) cells have achieved promising outcomes in relapsed/refractory B cell malignancies. However, recurrences occur due to the loss of CAR-T cell persistence. We developed dual T/B cell co-stimulatory molecules (CD28 and CD40) in CAR-T cells to enhance intense tumoricidal activity and persistence. CD19.28.40z CAR-T cells promoted pNF-κB and pRelB downstream signaling while diminishing NFAT signaling upon antigen exposure. CD19.28.40z CAR-T cells demonstrated greater proliferation, which translated into effective anti-tumor cytotoxicity in long-term co-culture assay. Repetitive weekly antigen stimulation unveiled continuous CAR-T cell expansion while preserving central memory T cell subset and lower expression of exhaustion phenotypes. The intrinsic genes underlying CD19.28.40z CAR-T cell responses were compared with conventional CARs and demonstrated the up-regulated genes associated with T cell proliferation and memory as well as down-regulated genes related to apoptosis, exhaustion, and glycolysis pathway. Enrichment of genes toward T cell stemness, particularly SELL, IL-7r, TCF7, and KLF2, was observed. Effective and continuing anti-tumor cytotoxicity in vivo was exhibited in both B cell lymphoblastic leukemia and B cell non-Hodgkin lymphoma xenograft models while demonstrating persistent T cell memory signatures. The functional enhancement of CD37.28.40z CAR-T cell activities against CD37+ tumor cells was further validated. The modification of dual T/B cell signaling molecules remarkably maximized the efficacy of CAR-T cell therapy.

Aberrant Upregulation of RUNX3 Activates Developmental Genes to Drive Metastasis in Gastric Cancer.

Gastric cancer metastasis is a major cause of mortality worldwide. Inhibition of RUNX3 in gastric cancer cell lines reduced migration, invasion, and anchorage-independent growth in vitro. Following splenic inoculation, CRISPR-mediated RUNX3-knockout HGC-27 cells show suppression of xenograft growth and liver metastasis. We interrogated the potential of RUNX3 as a metastasis driver in gastric cancer by profiling its target genes. Transcriptomic analysis revealed strong involvement of RUNX3 in the regulation of multiple developmental pathways, consistent with the notion that Runt domain transcription factor (RUNX) family genes are master regulators of development. RUNX3 promoted "cell migration" and "extracellular matrix" programs, which are necessary for metastasis. Of note, we found pro-metastatic genes WNT5A, CD44, and VIM among the top differentially expressed genes in RUNX3 knockout versus control cells. Chromatin immunoprecipitation sequencing and HiChIP analyses revealed that RUNX3 bound to the enhancers and promoters of these genes, suggesting that they are under direct transcriptional control by RUNX3. We show that RUNX3 promoted metastasis in part through its upregulation of WNT5A to promote migration, invasion, and anchorage-independent growth in various malignancies. Our study therefore reveals the RUNX3-WNT5A axis as a key targetable mechanism for gastric cancer metastasis. SIGNIFICANCE: Subversion of RUNX3 developmental gene targets to metastasis program indicates the oncogenic nature of inappropriate RUNX3 regulation in gastric cancer.

Computational discovery of binding mode of anti-TRBC1 antibody and predicted key amino acids of TRBC1.

Peripheral T-cell lymphoma (PTCL) is a type of non-Hodgkin lymphoma that progresses aggressively with poor survival rate. CAR T cell targeting T-cell receptor ß-chain constant domains 1 (TRBC1) of malignant T cells has been developed recently by using JOVI.1 monoclonal antibody as a template. However, the mode of JOVI.1 binding is still unknown. This study aimed to investigate the molecular interaction between JOVI.1 antibody and TRBC1 by using computational methods and molecular docking. Therefore, the TRBC protein crystal structures (TRBC1 and TRBC2) as well as the sequences of JOVI.1 CDR were chosen as the starting materials. TRBC1 and TRBC2 epitopes were predicted, and molecular dynamic (MD) simulation was used to visualize the protein dynamic behavior. The structure of JOVI.1 antibody was also generated before the binding mode was predicted using molecular docking with an antibody mode. Epitope prediction suggested that the N3K4 region of TRBC1 may be a key to distinguish TRBC1 from TCBC2. MD simulation showed the major different surface conformation in this area between two TRBCs. The JOVI.1-TRBC1 structures with three binding modes demonstrated JOVI.1 interacted TRBC1 at N3K4 residues, with the predicted dissociation constant (Kd) ranging from 1.5 × 108 to 1.1 × 1010 M. The analysis demonstrated JOVI.1 needed D1 residues of TRBC1 for the interaction formation to N3K4 in all binding modes. In conclusion, we proposed the three binding modes of the JOVI.1 antibody to TRBC1 with the new key residue (D1) necessary for N3K4 interaction. This data was useful for JOVI.1 redesign to improve the PTCL-targeting CAR T cell.