MEK Inhibition in CAR-T Cells Prevents Exhaustion and Terminal Differentiation Via Downregulating C-Fos and JunB

Introduction: The effects of MEK inhibitors (MEKIs) in CAR-T cells are poorly understood and remain controversial. Some groups showed that MEKIs could impair CAR-T cells' function in vitro. However, another group reported that MEKIs could combine with GD2 CAR-T cells to provide additional efficacy. Yet, the mechanism for this combined effect is unclear. Considering that the CAR signaling pathway is similar to TCR, both involve the activation of MAPK signaling. We hypothesized that MEKIs might mitigate CAR-T cells' exhaustion and terminal differentiation by lessening redundant CAR signaling. The study aims to systematically evaluate the role and mechanism of MEK inhibitors on CAR-T cells.

Methods and Results: To explore whether MEKIs could mitigate the unbeneficial impact of antigen-independent CAR tonic signaling, we added 3 FDA-approved MEKIs: trametinib, cobimetinib, and binimetinib, respectively, to the culture medium of CD19.28z CAR-T cells for 9 days at the concentration approaching their clinically tolerable peak blood concentration. We found that all the 3 MEKIs could reduce CAR-T cells' terminal differentiation and restrain the expression of exhaustion and activation markers. Among them, trametinib was the most potent because it could achieve the comparable effect of cobimetinib and binimetinib at the lowest concentration. Thus, we chose trametinib for further research. We confirmed that compared to concentrations of 7.5nM and 30nM, 15nM was the optimal concentration of trametinib with mild inhibition on the proliferation of CAR-T cells and a potent effect on CAR-T cells' phenotype. Pre-treatment with trametinib didn't affect the in-vitro cytotoxicity of CD19.28z CAR-T cells. The above effects of trametinib were more significant as treatment time and the dose increased. Similar results could be obtained in analogous experiments with CD19.4-1BBz and GD2.28z CAR-T cells. Intriguingly, GD2.28z CAR-T cells cultured with trametinib had better proliferation and killing capacity because they were more exhausted than CD19.28z CAR-T cells. Using the Nalm-6-bearing leukemia xenograft model, we found that compared to DMSO pre-treated CD19.28z CAR-T cells, trametinib pre-treated CD19.28z CAR-T cells exerted more potent anti-leukemia activity, showed a less exhausted and differentiated state, proliferated better, and further extended mice survival (Fig.1). Similar results could be obtained in analogous in-vivo experiments with CD19.4-1BBz CAR-T cells. To evaluate whether trametinib could protect CAR-T cells from the exhaustion and terminal differentiation triggered by antigen stimulation, we cocultured CD19.28z CAR-T cells with Nalm-6 cells in a medium with or without trametinib. We demonstrated under antigen stimulation, trametinib could effectively inhibit CAR-T cells' activation, exhaustion, apoptosis, terminal differentiation, and phosphorylation of ERK and consequently promote the proliferation of total and CD8 CAR-T cells. In addition, after repetitive antigen stimulation, trametinib could rescue CAR-T cells' functional exhaustion and improve CAR-T cells' in-vitro cytotoxicity.

Mechanistically, Single-cell and bulk RNA-Seq revealed that the effect of MEK inhibition was associated with the downregulation of AP-1 and exhaustion-associated transcription factors (TFs) and the upregulation of memory-associated TFs (Fig.2). GSEA revealed the upregulation of naive/memory-associated genes and downregulation of genes involved in T cell activation/effector/exhaustion, AP-1 pathway and apoptosis in the trametinib-treated group. Additionally, single-cell transcriptional profiling demonstrated enrichment of memory and Ki67+cycling CAR-T clusters in the trametinib-treated group. Among the AP-1 TFs downregulated by trametinib, c-Fos and JunB are the direct downstream targets of the canonical MAPK signaling pathway, and both of them were reported to implicate T cell exhaustion. Thus we speculated both downregulation of c-Fos and JunB may contribute to the role of MEKIs. Consistent with our hypothesis, overexpression of c-Fos or JunB in CAR-T cells could partially, if not all, abrogated the effects of MEK inhibition.

Significance: Our research provides a strategy to optimize CAR-T antitumor efficacy by MEK inhibition and shed light on the role of c-Fos and JunB in driving CAR-T cell exhaustion and terminal differentiation.

No relevant conflicts of interest to declare.