Associate Professor Institute of Genetics and Animal Biotechnology, Polish Academy of Sciences Magdalenka, Mazowieckie, Poland
Abstract: Leukemia-initiating cells (LICs) are central to the pathogenesis and persistence of acute myeloid leukemia (AML) and are heavily influenced by epigenetic dysregulation. While ATRA has revolutionized treatment for APL-AML, its efficacy in non-APL AML remains limited due to epigenetic silencing of genes essential for ATRA signaling. This study aims to overcome these epigenetic barriers in AML by exploring ATRA-based combinatorial strategies with epigenetic modulators to restore pathway activity and target LIC populations. The RA-pathway activation was evaluated using HEK-293 cells transduced with the p-GreenFire-RARE-Tk-Luc construct, exposed to ATRA and a library of 650 epigenetic compounds. Selected compounds were assessed for their impact on cell viability, differentiation, and key signaling pathways (ERK1/2, AKT1, MAPK9, p38) in HL-60, NB4, KG1a, BMNC, and primary AML cells. RNAseq and CUT&Tag analyses were performed on ex vivo LIC-enriched AML samples and HL60 cells treated with the most effective combinations.
Eleven compounds demonstrated potential, with a dual PI3K-HDAC inhibitor and a pan-PKC inhibitor showing the most synergy with ATRA, enhancing RARE activity 4-5 fold. Both compounds were noncytotoxic to BMNC and primary MSC. Notably, the dual PI3K-HDAC inhibitor combined with ATRA achieved over 95% reduction in KG1α cell viability, significantly decreased the LIC population, and enhanced differentiation in AML samples (M1, M2, M4) by 2–4 fold compared to TCP (LSD1 inhibitor) or untreated controls. This combination also reduced the phosphorylation of LIC-associated survival pathways (p-MAPK and p-p38) while enhancing p53 activation. RNA sequencing revealed a distinct gene expression signature induced by the ATRA and drug combination treatment, highlighting its potential as a targeted therapeutic approach. Therefore, combining ATRA with dual PI3K-HDAC and pan-PKC inhibitors offers a promising strategy to target LICs and increase differentiation in AML by overcoming epigenetic resistance mechanisms. However, further validation using patient-derived xenograft models is crucial to confirm the potential of this approach in eradicating LIC populations and improving AML treatment outcomes.
Funding Source: The study was supported by the Polish National Science Centre grant no. 2019/33/B/NZ5/02399
