ZBTB32 degradation increases T cell anti-tumor activity

Background
Zinc finger and BTB domain-containing protein 32 (ZBTB32) is a CXXCG motif-containing transcription factor with context-dependent roles in immune regulation. ZBTB32 represses CIITA during plasma cell differentiation, restrains CD8-positive T cell effector and memory responses, and supports natural killer cell proliferation following viral infection. In addition to these established functions, ZBTB32 has emerged as a potential regulator of tumor immunity and is enriched in terminally exhausted tumor-infiltrating CD8 T cells.

Methods
We analyzed clinical datasets to evaluate associations between ZBTB32 mRNA expression in T cells and clinical responses to anti-PD-1 and CAR-T therapies. To define the role of ZBTB32 in T cell antitumor activity, we generated genomic knockout mice and ZBTB32-deficient anti-CD19 human CAR-T cells. To enable pharmacologic interrogation, we developed selective CRBN-based molecular glue degraders of ZBTB32 with subnanomolar cellular degradation potency (DC50). Using these models and chemical tools, we performed CUT&RUN-seq and RASL-seq to define direct downstream targets of ZBTB32 and characterize its molecular activity in T cells.

Results
Elevated ZBTB32 expression in human T cells was associated with poorer responses to CAR-T and anti-PD-1 therapies. Consistent with these clinical associations, ZBTB32-deficient CAR-T cells mediated accelerated tumor clearance. In contrast, germline ZBTB32 knockout did not alter MC38 tumor growth, suggesting that prolonged systemic loss of ZBTB32 is not beneficial to enhance tumor control in this model. Mechanistically, ZBTB32 degraders showed that ZBTB32 directly represses genes associated with T cell stemness and effector function through chromatin binding. Acute pharmacologic degradation of ZBTB32 phenocopied genetic deletion in CAR-T cells, enhancing cytotoxicity in vitro and improving antitumor efficacy in vivo. A transcriptional signature of ZBTB32 deficiency or degradation in human T cells correlated with improved survival in patients treated with immune checkpoint inhibitors. Moreover, combining ZBTB32 degradation with anti-PD-1 therapy enhanced MC38 tumor control in humanized Zbtb32/Crbn mice.

Conclusions
Together, these findings identify ZBTB32 as a therapeutically actionable transcriptional regulator of T cell dysfunction and support transient ZBTB32 degradation as a promising strategy to enhance T cell-mediated cancer immunotherapy.