Abstract Purpose: Enzymatic pockets, such as those found in histone deacetylases (HDACs), have long served as attractive targets for drug discovery. However, conventional HDAC inhibitors often lack selectivity and cause systemic toxicity due to paralog redundancy and their incorporation into multi-subunit transcriptional regulatory complexes. To identify more selective modulators, we performed an unbiased yeast genetic screen of ∼52,000 compounds by interrogating the activity of the conserved HDAC/Rpd3L complex. Follow-up mechanistic studies uncovered hits that do not directly inhibit HDAC catalytic activity but instead modulate repression through alternative mechanisms. We subsequently evaluated the lead compound, E6R, in human neuroblastoma cells and mouse xenografts, benchmarking against the enzymatic inhibitor TSA in vitro and Vorinostat (SAHA) in vivo. These studies demonstrate that E6R, a first-in-class non-enzymatic SIN3-HDAC modulator, achieves comparable anti-tumor efficacy with far greater selectivity and minimal global transcriptional disruption. Methods: E6R was evaluated in yeast and SK-N-BE(2)-C neuroblastoma cells using bulk and single-cell RNA-seq (Seq-Well S3), SIN3A ChIP-seq, viability and invasion assays, and mouse xenografts. Results: In yeast, E6R disrupts Sin3/Rpd3L-dependent transcriptional repression without inhibiting HDAC catalytic activity. In human neuroblastoma cells, E6R produced anti-tumor activity comparable to TSA. Transcriptomically, E6R modulated ∼14-fold fewer genes than TSA and caused minimal global perturbation. Interestingly, E6R selectively activated stress- and senescence-associated programs governed by the ATF4-driven integrated stress response (ISR), including GDF15, DDIT3, ATF3, and FGF21, while inducing minimal off-target effects. SIN3A ChIP-seq revealed promoter-proximal loss of SIN3A binding at several ISR loci, most notably GDF15 (∼55 bp upstream of the TSS), consistent with direct de-repression through dissociation of the SIN3-HDAC complex. Although both compounds shared repression of E2F, MYC, and glycolytic targets and activation of p53, TNFα/NF-κB, and apoptotic signaling, E6R induced a distinct stress-adaptive state through an HDAC-independent mechanism. Functionally, E6R significantly reduced neuroblastoma cell invasion and tumor growth with limited cytotoxicity. In vivo, E6R inhibited neuroblastoma xenograft growth comparably to Vorinostat, supporting non-enzymatic HDAC modulation as a therapeutic alternative. Conclusions: Together, these data strongly suggest that E6R is a selective, non-enzymatic SIN3-HDAC modulator that reprograms chromatin from a repressive to a stress-adaptive, anti-proliferative state, offering a mechanistically distinct and potentially safer framework for HDAC-targeted cancer therapy. Citation Format: Olivia Debnath, Julien Olivet, Soon Gang Choi, Yasmine Bramerloo, Jeremy Blavier, TINA O'GRADY, Florent Laval, Vladimir V. Botchkarev, Bin Hu, Anthony C. Varca, Jonathan Bruyr, Samira Ibrahim, Tasneem Jivanjee, Joshua D. Bromley, Sarah K. Nyquist, Natalia Calonghi, Alessandra Stefan, Alejandro Hochkoeppler, Maria Francesca Baietti, Eleonora Leucci, Michael A. Calderwood, Tong Hao, Alex K. Shalek, David E. Hill, Sara J. Buhrlage, Sirano Dhe-Paganon, Franck Dequiedt, Jean Claude Twizere, Marc Vidal. Targeting non-enzymatic HDAC-mediated repression reveals a selective stress-adaptive mechanism for cancer therapy abstract. In: Proceedings of the American Association for Cancer Research Annual Meeting 2026; Part 1 (Regular Abstracts); 2026 Apr 17-22; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2026;86(7 Suppl):Abstract nr 6776.
Debnath et al. (Fri,) studied this question.