Targeting Epigenetic–Metabolic crosstalk in CML via butyrate-induced HDAC inhibition

Abstract

Abstract Introduction Tyrosine kinase inhibitors (TKIs) have improved Chronic Myeloid Leukemia (CML) patient outcomes; however, a subset of patients develops resistance, partly due to adaptive changes in metabolism and epigenetic regulation within the hypoxic bone marrow niche. In this context butyrate, a short-chain fatty acid produced in the human colon through bacterial fermentation, has gained significant attention for its epigenetic regulatory and anticancer properties. As a histone deacetylase (HDAC) inhibitor, butyrate has been shown to induce cancer cell death at concentrations >1.5 mM. Moreover, its ability to reduce inflammation, reverse epigenetic aberrations, and suppress cancer stem cell proliferation, makes it a promising adjunct in chemotherapy. Methods To evaluate the impact of butyrate on differentiation and proliferation, K562 cell lines were used. Cells were grown with different concentrations of butyrate and imatinib (0,0048 uM to 20 uM for imatinib and 0,048 mM to 100 mM for butyrate, respectively). Viability was evaluated using assays based on Resazurin after 72 hours. The differentiation status was validated by FACS analysis, and genes linked to metabolism and TKi resistance were examined by RT-PCR. Protein levels of several targets of interest were evaluated using Wester blot. Results To explore the effects of butyrate in CML, we first established the IC50 values of butyrate and imatinib in K562 cells after 72 hours of treatment. Based on these data, we selected sub-lethal concentrations of 0.25 µM for imatinib and 1 mM for butyrate concentrations that preserved viability while eliciting biological activity, and exposed cells to these conditions for 48 hours. Flow cytometry analysis showed that butyrate-treated cells maintained high viability, but exhibited a marked increase in CD235a expression, indicating erythroid differentiation. Notably, butyrate also reduced the proliferation rate of K562 cells, consistent with both differentiation induction and epigenetic reprogramming. At the transcriptional and protein levels, butyrate induced a substantial downregulation of HDAC1 and HDAC3, as assessed by qRT-PCR and Western blot. This was accompanied by a strong increase in acetylated histone H4, confirming effective HDAC inhibition and global chromatin remodelling. BCR :ABL mRNA expression was remarkably reduced by butyrate treatment. Western blot analysis further demonstrated that butyrate reduced BCR::ABL protein levels to an extent comparable to imatinib, highlighting its potential to interfere with the oncogenic driver of CML at both transcriptional and post-transcriptional levels. Finally, we investigated the metabolic effects of butyrate and observed a decrease in HIF-1α mRNA and downregulation of glycolytic gene expression, suggesting inhibition of hypoxia-driven metabolic reprogramming in leukemic cells. Conclusion In summary, our data demonstrate that butyrate exerts diverse anti-leukemic effects in CML cells: it slows proliferation, promotes erythroid differentiation, downregulates HDAC1/3 and BCR::ABL expression, increases histone H4 acetylation, and suppresses both HIF-1α signaling and glycolysis. Notably, butyrate is a short-chain fatty acid naturally produced by gut microbiota, this suggests that a microbiome high in bacteria that produce butyrate may be a good predictive factor for CML. Given its endogenous origin, butyrate could be a promising drug to explore further in terms of efficacy, safety, and teratogenicity, particularly in clinical contexts where TKI therapy is contraindicated, such as during pregnancy. Acknowledge ESH and the John Goldman Award on all publications/ communication related to the Research Project