Mechanistic basis of resistance to HDAC6 inhibitors reveals proteasome inhibition as a rational combination strategy in breast cancer

Introduction: Cancer treatment is increasingly driven by personalized medicine approaches, where targeted therapies are favored over traditional chemotherapy due to improved specificity and reduced toxicity. Although pan-histone deacetylase inhibitors show antitumor activity in several cancers, their clinical use is limited by toxicity. In contrast, HDAC6-specific inhibitors such as ricolinostat are well-tolerated and demonstrate clinical activity in subsets of breast cancer (BC) patients. However, intrinsic resistance restricts their broader therapeutic utility. Here, we present new evidence identifying proteasomal function as a critical determinant of resistance to HDAC6 inhibitors and show that combinatorial targeting of HDAC6 and the proteasome can overcome this resistance. Methods: We performed multi-omics analyses comparing the response to HDAC6 inhibitors of sensitive and resistant BC models. These studies led us to hypothesize that enhanced proteasomal capacity drives therapeutic resistance. To test this, we quantified steady-state proteasomal activity using fluorometric assays that measure chymotrypsin-like, trypsin-like, and caspase-like proteolytic functions in vitro. We also conducted therapeutic studies treating sensitive and resistant BC cells with HDAC6 inhibitors alone or in combination with proteasome inhibitors. Results: Omics profiling revealed that HDAC6 inhibition in sensitive cells induces the unfolded protein response (UPR), downregulates MYC, and upregulates ER-stress markers (BiP, CHOP) in both BC cell lines and MMTV-Neu tumors, indicating activation of a stress-adaptive transcriptional program. Given HDAC6's role in the aggresome-autophagy pathway and the proteasome's role in protein degradation, we assessed whether resistant cells compensate through elevated proteasomal activity. Our data revealed that resistant BC cells exhibited significantly higher basal activity across all three proteolytic subunits. Consistent with this, ricolinostat combined with the proteasome inhibitor bortezomib induced growth inhibition in resistant cells, reversing the resistance to HDAC6i/s. Conversely, chemically enhancing proteasomal function with oleuropein reduced sensitivity to ricolinostat in previously sensitive lines, supporting a mechanistic dependence on limited proteasomal activity for the responses to HDAC6 inhibition. Conclusions: These findings identify elevated proteasomal capacity as a key driver of resistance to HDAC6 inhibitors, supporting a therapeutic strategy that combines HDAC6 and proteasome inhibition to overcome resistance in breast cancer.