Background
CAR-T cell efficacy in solid tumors is dampened by T-cell dysfunction, especially exhaustion, which is driven by chronic exposure to tumor antigens and the immunosuppressive, nutrient-limited tumor microenvironment. Transcription factor (TF) overexpression or knockout are promising approaches to enhance CAR-T cell durability. However, given that a single genetic modulation is unlikely to sufficiently counter the multiple triggers of CAR-T cell dysfunction, efforts are needed to manipulate T-cell function by engineering pairs of TFs that synergize to enhance CAR-T cell performance. Here, we employ interactome-based networks derived by integrating phenotypic data with protein-protein interaction networks and differential gene coexpression from TF perturbation to prioritize TF pairs that resist exhaustion. Interrogating the interactome-based networks for biological insights has the potential to move beyond correlation to causation, and identify factors that complement the overexpression of a beneficial TF to induce an additive or synergistic effect on T-cell proliferation.
Methods
We apply systems-level approaches to uncover basic leucine zipper (bZIP) TFs modulating T-cell exhaustion. To identify regulators of CD8+ and CD4+ CAR-T cell proliferation, a barcoded, pooled library of bZIP variants that includes ~300 full-length bZIPs, bZIP subdomains, and synthetic bZIP inhibitors was screened in GD2 CAR-T cells subjected to a chronic stimulation assay with a neuroblastoma tumor cell line. Top candidates were subsequently validated in an IncuCyte assay under chronic stimulation conditions and characterized by RNA-seq. Differential gene expression caused by TF overexpression was integrated into a protein-protein interaction (PPI)-regulatory network resulting in unique exhaustion stage-specific CAR-T cell interactomes (early vs intermediate).
Results
In the library screen, bZIP TFs including BACH2 and BATF2 were enriched as top regulators of CAR-T cell proliferation that promote T-cell persistence under chronic tumor stimulation. The early and intermediate exhaustion interactome networks of validated hits including Batf2, Bach2 and Jun are compared to gain mechanistic insights into their pro-proliferative effects. Network centrality metrics are used to predict candidates that synergize with bZIP TFs to enhance CAR-T cell proliferation. Furthermore, as a case study, interactomes of Jun and its protein-based inhibitor AEL-FOS which was de-enriched in the screen are compared to gain a deeper understanding of the Jun anti-exhaustion phenotype.
Conclusions
This systems-level analysis has yielded high-confidence candidates for TF multiplexing to counter CAR-T cell dysfunction.