Background Resistant to immune checkpoint blockade (ICB) therapy, especially in a few solid tumor types including advanced prostate cancer, represents a formidable clinical challenge. The ketogenic diet can enhance ICB therapy in some cancer models (prostate cancer not included yet). However, the adverse effect associated with continuous KD was also observed, demanding better mechanistic understanding and optimized regimens of using KD as an immunotherapy sensitizer.

Materials and Methods We developed a series of ICB-resistant murine prostate cancer cell lines from an ICB-sensitive prostate cancer cell line previously developed from the PB-Cre⁺ Pten^L/L p53^L/L Smad4^L/L mouse model of metastatic prostate cancer. C57BL/6 mice inoculated with dual ICB-resistant subline PPS-ICBR were treated with epigenetic modulators or specialized diets in the presence or absence of anti-PD1 and anti-CTLA4 antibodies. The effect of the therapies were evaluated at both phenotypical levels and single cell profiling (including both CyTOF and scRNA-seq). Emerging mechanisms were tested with loss of function and gain of function experiments.

Results The major histocompatibility complex (MHC) class I protein levels were downregulated in the resistant sublines compared with the parental cell line. Using the most ICB-resistant subline model, we demonstrated that synergistic efficacy was achieved by combining anti-PD1 and anti-CTLA4 antibodies with histone deacetylase (HDAC) inhibitor vorinostat, a cyclic ketogenic diet (CKD), or supplementation of ketone body β-hydroxybutyrate (BHB, endogenous HDAC inhibitory metabolite) via 1,3-butanediol-admixed food. CKD and BHB supplementation delayed prostate cancer tumors as monotherapy, and both BHB and adaptive immunity are required for the anti-tumor activity of CKD. Single-cell transcriptomic and proteomic profiling revealed that the HDAC inhibitor and ketogenesis-enhanced ICB therapy was effectuated through both cancer-cell-intrinsic (upregulated MHC class I molecules) and extrinsic mechanisms (CD8⁺ T cell chemoattraction, M1/M2 macrophage rebalancing, monocyte differentiation toward antigen presenting cells, and diminished neutrophils).

Conclusions Epigenetic modulation with pharmacological HDAC inhibitor vorinostat or endogenous HDAC inhibitor BHB (via CKD or BD) sensitized ICB-refractory prostate cancer to PD1/CTLA4 dual ICB therapy. The two combination therapies share underlying mechanisms such as cancer cell MHC-I upregulation and concerted innate and adaptive immune landscape remodeling. Among the strategies, 1,3-butanediol-supplemented-diet may represent the most attractive option to enhance ICB therapy due to its high chance of patient compliance, low cost, and minimal toxicity.

X. Lu: None. S. Murphy: None. S. Rahmy: None. D. Gan: None. J. Li: None. X. Lu: None.