Selective HDAC6 inhibition perturbs autophagy and enhances integrated stress response-mediated immunogenic apoptosis in chronic myeloid leukemia.

Chronic myeloid leukemia (CML) is driven by the BCR-ABL1 fusion oncoprotein and is managed with tyrosine kinase inhibitors (TKIs). However, resistance and persistence of leukemic stem/progenitor cells remain major clinical challenges. Autophagy-mediated survival signaling contributes to therapeutic resistance in CML. We hypothesized that histone deacetylase 6 (HDAC6), a key regulator of protein homeostasis and autophagic flux, is a therapeutic target in this context. Transcriptomic analysis of CML bone marrow datasets revealed enrichment of HDAC6-associated autophagy and stress-response gene programs. The selective HDAC6 inhibitor 7b induced sustained α-tubulin acetylation at lower concentrations than ricolinostat or nexturastat A. 7b reduced primary CML PBMC viability while sparing healthy PBMCs and was active in vivo. Combining 7b with the allosteric BCR-ABL1 inhibitor asciminib synergistically suppressed cell viability and clonogenic growth. A tandem mCherry-GFP-LC3 reporter showed that 7b blocks autophagosome maturation, thereby decreasing autophagic degradation. Cotreatment engaged a maladaptive integrated stress response (ISR), characterized by eIF2α phosphorylation, ATF4 and CHOP induction, MCL-1 suppression, PUMA and NOXA upregulation, and BCL-xL-dependent mitochondrial apoptosis, accompanied by caspase-2 activation. ISR activation occurred downstream of the autophagy disruption: rapamycin attenuated ISR activation, whereas ATG7 silencing intensified ISR signaling and apoptosis. CHOP knockdown blunted the BCL-xL/PUMA/NOXA shift and caspase-3 cleavage, establishing CHOP as required. Caspase-10 acted upstream of caspases-9, -7, and -3. The combination elicited immunogenic cell death markers: calreticulin exposure, ATP and HMGB1 release, elevated TNF-α, and reduced IL-8. These findings identify HDAC6-driven autophagy as a therapeutically exploitable vulnerability in CML that, when combined with asciminib, triggers ISR-dependent immunogenic apoptosis.