Dual HDAC3/8 PROTAC degraders exert anti-tumor and immunomodulating effects in diffuse large B-cell lymphoma

Critical challenges remain in the clinical management of diffuse large B-cell lymphoma (DLBCL), as up to 40% of patients experience disease relapse or refractoriness. Despite the use of novel therapies, relapsed/refractory DLBCL results in a high risk of mortality, especially in the activated B-cell-like (ABC) subtype. Developing new therapeutic options for those patients becomes a pressing and clinically unmet need. Here, we explore the combined targeting of histone deacetylase (HDAC) 3 and 8 by targeted protein degradation to combat epigenetic deregulation in DLBCL. Through a comprehensive screen using single or dual HDAC3/8-targeting compounds in 12 DLBCL cell lines, we discovered that a highly selective, potent, first-in-class dual HDAC3/8 proteolysis-targeting chimera (PROTAC) degrader YX968 potently suppressed cell survival and proliferation. This effect was particularly more prominent and significantly stronger than single HDAC3 or 8 PROTACs in the ABC-DLBCL cell lines. Further functional evaluations revealed that YX968 specifically induced caspase-3-mediated apoptosis and caused cell cycle arrest. Mechanistically, YX968 profoundly upregulated H3K27 acetylation (H3K27ac), a key epigenetic mark for enhancer regulation, and generated a unique transcriptomic profile with downregulated genes associated with cell cycle regulation and upregulated genes implicated in immune activation. Moreover, we validated the gene expression results using flow cytometry for upregulated surface protein expression of the antigen presentation markers MHC-I and II, and detected enhanced CD8 + T-cell-induced cytotoxicity by YX968 in an in vitro T-cell co-culture assay, indicating its immunomodulating activities to potentiate anti-tumor immunity. Although YX968 showed in vitro efficacy, we found that it had poor metabolic stability, which precluded its potential in vivo application. To enhance the translation value of our approach, we developed a second-generation dual HDAC3/8 PROTAC (YX226) with improved metabolic stability while maintaining potent degradation, cell-killing, and immunomodulating activities. Using global proteomics, we confirmed the on-target activity of YX226 to selectively degrade HDAC3 and 8 but not the other HDAC isoforms. Importantly, we observed significant in vivo HDAC3/8 protein degradation and tumor growth inhibition by YX226 in a xenograft lymphoma model without adverse reduction of body weight, providing proof-of-principle evidence for in vivo efficacy and safety of a dual HDAC3/8 PROTAC. In conclusion, our findings demonstrate that dual HDAC3/8 PROTAC degraders induce significant anti-tumor and immunomodulating effects in our preclinical DLBCL models, supporting further development and evaluation of our second-generation bioavailable compound YX226 as a novel treatment option for DLBCL.