Cbl-b inhibitor NX-1607 activates MAPK/ERK signaling pathway and enhances T-cell activation.

in Journal for immunotherapy of cancer by Wenting Zhu, Shan Lu, Li Jia, Benjin Liu, Shanshan Song, Xubin Bao, Ting Yu, Yongliang Zhang, Zehong Miao, Jinxue He

TLDR

  • The study found that NX-1607 inhibits Cbl-b activity, boosting T cell activation and signaling pathways, and shows promise in treating cancer by modulating T cell function.

Abstract

The E3 ubiquitin ligase casitas B lymphoma-b (Cbl-b) is pivotal in modulating immune responses by attenuating T-cell activation and cytokine production. Inhibiting Cbl-b presents a potential therapeutic strategy in immuno-oncology by enhancing immune activity.A rapid Homogeneous Time-Resolved Fluorescence (HTRF) assay was employed to evaluate the inhibitory efficacy of NX-1607 on Cbl-b. The effects of NX-1607 on T cell activation, cytokine production, and proliferation were characterizedusing primary T cells and Jurkat T cellsA drug combination screening was performed utilizing the CD69 marker via flow cytometry to dentify signaling pathways involved in T cell activation by NX-1607. CRISPR/Cas9 technology was used to knock outandin Jurkat T cells, followed by the detection of p-PLCγ1 and p-ERK1/2 though Western blotting. The antitumor efficacy of NX-1607 was assessed in a murine model of A20 B-cell lymphoma using BALB/c mice, with subsequent flow cytometry analysis conducted to examine the phenotype of tumor-infiltrating lymphocytes (TILs).Our data show that NX-1607 effectively inhibits Cbl-b activity at low nanomolar levels, boosting PLCγ1 and HCSL1 phosphorylation, activating MAPK/ERK signaling, and elevating CD69 expression. Inhibiting PLCγ1 and ERK1/2 significantly reduces NX-1607's effect on T-cell activation. Oral administration of NX-1607 notably decreases tumor growth in the A20 B-cell lymphoma model, with immunophenotyping analyses of tumor-infiltrating lymphocytes revealing increased CD3, CD4, and CD8T cells in treated tumors. Furthermore, our results demonstrate that treatment with NX-1607 results in increased levels of phosphorylated PLCγ1 and ERK1/2 in circulating T cells.Taken together, these findings imply that the inhibition of Cbl-b by NX-1607 may enhance the activation of the MAPK/ERK signaling pathway, thereby sustaining T-cell activation. This provides compelling evidence for the molecular mechanism of NX-1607, underscoring the pivotal role of Cbl-b in controlling signal strength in T-cell activation after T-cell receptor (TCR) engagement.

Overview

  • The study investigated the efficacy of NX-1607 in inhibiting Cbl-b to enhance immune responses in immuno-oncology.
  • The study employed a rapid HTRF assay to measure Cbl-b inhibition, and characterized the effects of NX-1607 on T cell activation, cytokine production, and proliferation using primary T cells and Jurkat T cells.
  • The study aimed to identify the molecular mechanism of NX-1607 and its potential therapeutic applications in treating cancer by modulating T-cell activation and signaling pathways.

Comparative Analysis & Findings

  • NX-1607 effectively inhibited Cbl-b activity at low nanomolar levels and boosted PLCγ1 and HCSL1 phosphorylation.
  • Inhibiting PLCγ1 and ERK1/2 significantly reduced NX-1607's effect on T-cell activation.
  • Oral administration of NX-1607 decreased tumor growth in the A20 B-cell lymphoma model, and increased CD3, CD4, and CD8 T cells in treated tumors.

Implications and Future Directions

  • The inhibition of Cbl-b by NX-1607 may enhance the activation of the MAPK/ERK signaling pathway, thereby sustaining T-cell activation.
  • Future studies could explore the potential of NX-1607 in combination with other immunotherapies to enhance its efficacy in treating cancer.
  • The study highlights the importance of Cbl-b in controlling signal strength in T-cell activation, and suggests that targeting Cbl-b may be a promising therapeutic strategy in immuno-oncology.
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