Novel hydroxamic acid derivative induces apoptosis and constrains autophagy in leukemic cells

Marten A Fischer; Al-Hassan M Mustafa; Kristin Hausmann; Ramy Ashry; Anita G Kansy; Magdalena C Liebl; Christina Brachetti; Andrea Piée-Staffa; Matthes Zessin; Hany S Ibrahim; Thomas G Hofmann; Mike Schutkowski; Wolfgang Sippl; Oliver H Krämer

doi:10.1016/j.jare.2023.07.005

Novel hydroxamic acid derivative induces apoptosis and constrains autophagy in leukemic cells

J Adv Res. 2023 Jul 17:S2090-1232(23)00197-2. doi: 10.1016/j.jare.2023.07.005. Online ahead of print.

Authors

Affiliations

¹ Department of Toxicology, University Medical Center, 55131 Mainz, Germany. Electronic address: mfisch05@students.uni-mainz.de.
² Department of Toxicology, University Medical Center, 55131 Mainz, Germany; Department of Zoology, Faculty of Science, Aswan University, Aswan, Egypt. Electronic address: alabdeen@uni-mainz.de.
³ Department of Medicinal Chemistry, Institute of Pharmacy, Martin-Luther-University of Halle-Wittenberg, Halle (Saale), Germany. Electronic address: Kristin.hausmann@pharmazie.uni-halle.de.
⁴ Department of Toxicology, University Medical Center, 55131 Mainz, Germany; Department of Oral Pathology, Faculty of Dentistry, Mansoura University, Egypt. Electronic address: relashry@uni-mainz.de.
⁵ Department of Toxicology, University Medical Center, 55131 Mainz, Germany. Electronic address: akansy@students.uni-mainz.de.
⁶ Department of Toxicology, University Medical Center, 55131 Mainz, Germany. Electronic address: magliebl@uni-mainz.de.
⁷ Department of Toxicology, University Medical Center, 55131 Mainz, Germany. Electronic address: brachett@uni-mainz.de.
⁸ Department of Toxicology, University Medical Center, 55131 Mainz, Germany. Electronic address: pieean01@uni-mainz.de.
⁹ Department of Enzymology, Institute of Biochemistry, Martin-Luther-University of Halle-Wittenberg, Halle (Saale), Germany. Electronic address: matthes.zessin@googlemail.com.
¹⁰ Department of Medicinal Chemistry, Institute of Pharmacy, Martin-Luther-University of Halle-Wittenberg, Halle (Saale), Germany; Department of Pharmaceutical Chemistry, Faculty of Pharmacy, Egyptian Russian University, Badr City, Cairo, Egypt. Electronic address: hany.ibrahim@pharmazie.uni-halle.de.
¹¹ Department of Toxicology, University Medical Center, 55131 Mainz, Germany. Electronic address: thomas.hofmann@uni-mainz.de.
¹² Department of Enzymology, Institute of Biochemistry, Martin-Luther-University of Halle-Wittenberg, Halle (Saale), Germany. Electronic address: mike.schutkowski@biochemtech.uni-halle.de.
¹³ Department of Medicinal Chemistry, Institute of Pharmacy, Martin-Luther-University of Halle-Wittenberg, Halle (Saale), Germany. Electronic address: wolfgang.sippl@pharmazie.uni-halle.de.
¹⁴ Department of Toxicology, University Medical Center, 55131 Mainz, Germany. Electronic address: okraemer@uni-mainz.de.

PMID: 37467961
DOI: 10.1016/j.jare.2023.07.005

Abstract

Introduction: Posttranslational modification of proteins by reversible acetylation regulates key biological processes. Histone deacetylases (HDACs) catalyze protein deacetylation and are frequently dysregulated in tumors. This has spurred the development of HDAC inhibitors (HDACi). Such epigenetic drugs modulate protein acetylation, eliminate tumor cells, and are approved for the treatment of blood cancers.

Objectives: We aimed to identify novel, nanomolar HDACi with increased potency over existing agents and selectivity for the cancer-relevant class I HDACs (HDAC1,-2,-3,-8). Moreover, we wanted to define how such drugs control the apoptosis-autophagy interplay. As test systems, we used human leukemic cells and embryonic kidney-derived cells.

Methods: We synthesized novel pyrimidine-hydroxamic acid HDACi (KH9/KH16/KH29) and performed in vitro activity assays and molecular modeling of their direct binding to HDACs. We analyzed how these HDACi affect leukemic cell fate, acetylation, and protein expression with flow cytometry and immunoblot. The publicly available DepMap database of CRISPR-Cas9 screenings was used to determine sensitivity factors across human leukemic cells.

Results: Novel HDACi show nanomolar activity against class I HDACs. These agents are superior to the clinically used hydroxamic acid HDACi SAHA (vorinostat). Within the KH-series of compounds, KH16 (yanostat) is the most effective inhibitor of HDAC3 (IC₅₀ = 6 nM) and the most potent inducer of apoptosis (IC₅₀ = 110 nM; p < 0.0001) in leukemic cells. KH16 though spares embryonic kidney-derived cells. Global data analyses of knockout screenings verify that HDAC3 is a dependency factor in 115 human blood cancer cells of different lineages, independent of mutations in the tumor suppressor p53. KH16 alters pro- and anti-apoptotic protein expression, stalls cell cycle progression, and induces caspase-dependent processing of the autophagy proteins ULK1 and p62.

Conclusion: These data reveal that HDACs are required to stabilize autophagy proteins through suppression of apoptosis in leukemic cells. HDAC3 appears as a valid anti-cancer target for pharmacological intervention.

Keywords: Acetylation; Apoptosis; Autophagy; HDAC; HDACi; Isoenzyme specificity.