Antitumor Activity of the Adamantyl Retinoid GEM144 in Acute Myeloid Leukemia: A Preclinical Study

Abstract

Introduction: Acute myeloid leukemia (AML) is classified as one of the most frequent hematological malignancies and represents the most common acute leukemia in adults. It is a complex and aggressive disease of undifferentiated hematopoietic progenitor cells. Several frontline treatments are available for AML, including induction therapy, targeted therapy, and stem cell transplantation. While many patients initially respond to these treatments, they are often limited by high relapse rates and resistance, underscoring the urgent need for novel therapeutic strategies. Retinoids play a crucial role in a wide range of biological processes, including cell growth and differentiation, which paved the way for evaluating their therapeutic potential in cancer. All-trans retinoic acid (ATRA) has been widely used as the standard treatment for acute promyelocytic leukemia, an AML subtype. However, ATRA treatment demonstrated limited efficacy in the majority of non-APL AML subtypes. Retinoid-related molecules, such as ST1926 –an adamantyl retinoid– have recently emerged as potent anticancer agents to overcome such limited efficacy. We have demonstrated that sub-micromolar (sub-µM) concentrations of ST1926 potently inhibited the growth of AML cells and blasts and reduced the tumor burden of xenograft mice. In addition, we have shown that ST1926 reduced the protein levels and inhibited the activity of DNA polymerase α (POLA1) –the first enzyme involved in DNA synthesis– in colorectal cancer. However, the clinical development of ST1926 was restricted due to its limited bioavailability and rapid excretion, resulting from its susceptibility to glucuronidation. To address these limitations, we developed GEM144, an ST1926 analogue with enhanced pharmacological properties and dual inhibitory activities against POLA1 and histone deacetylase 11 (HDAC11). Interestingly, POLA1 was shown to be upregulated in an AML mouse model, and HDAC11 in AML patients, whereby the upregulated expression of each was associated with poorer overall survival.

Methods: The effects of GEM144 on the viability of different human AML cell lines (OCI-AML3, THP-1, and MOLM-13) were assessed using the trypan blue exclusion dye assay, and confirmed by MTT assay (data not shown). Accordingly, the drug’s IC50 values were determined. The mechanism of action of GEM144 was studied using a propidium iodide (PI)-based flow cytometric analysis of DNA content to assess cell cycle regulation, western blotting and the TUNEL assay to assess apoptosis, and western blotting to evaluate DNA damage.

Results: GEM144 inhibited the viability of all tested AML cell lines in a time-dependent manner, at sub-µM concentrations. The IC50 value was determined to be 0.5 µM for all cell lines and thus was selected as the working concentration for all mechanistic studies. PI-based flow cytometric analysis revealed sub-G1 accumulation in all GEM144-treated cell lines, with G0/G1 cycle arrest observed only in GEM144 treated THP-1 and MOLM-13 cells. Western blotting and the TUNEL assay revealed the presence of PARP cleavage and DNA fragmentation, respectively, in all GEM144-treated cell lines. This validated that GEM144-induced cell death was apoptotic. Western blotting also revealed an increase in γH2AX expression in all cell lines post-GEM144 treatment.

Conclusion: These results highlight the promising antileukemic effects of GEM144 in AML. Future in vivo experiments, using an AML xenografted mouse model, are needed to validate these findings and assess GEM144’s therapeutic potential.