Abstract
Targeted signaling inhibitors for hematologic malignancies may lead to limited clinical efficacy due to the outgrowth of subpopulations with alternative pathways independent of the drug target. Relapse/refractory disease that results from treatment with targeted signaling inhibitors is a major hurdle in obtaining curative responses. Interestingly, work over the past decade or more has shown that chronic myelogenous leukemia (CML) stem cells (CD34+CD38-) are resistant to targeted signaling inhibitors, such as the BCR-ABL kinase class of inhibitors, often a problematic source of resistance leading to residual disease that may precipitate later progression (Hamilton et al., 2012).
Recent studies have shown that some forms of lymphoma and leukemia cell have an energy metabolism highly dependent on mitochondrial oxidative phosphorylation (Ashton et al., 2018). Tigecycline, a US FDA approved antibiotic, has been shown to inhibit synthesis of mitochondrion-encoded proteins due to the similarity of bacterial and mitochondrial ribosomes, leading to selective lethality in hematologic malignancies reliant on enhanced oxidative phosphorylation (Norberg et al., 2017). Indeed, it was established that CML stem cells are reliant on upregulated oxidative phosphorylation, and combination treatment with the tyrosine-kinase inhibitor (TKI) imatinib and tigecycline eradicated therapy-resistant CML, both in vitro and in animal models (Kuntz et al., 2017).
We have previously reported that elatol, the major compound from the red alga Laurencia microcladia, is effective against several non-Hodgkin lymphomas and primary chronic myelogenous leukemia cells (Peters et al., 2018). In vitro studies showed that elatol inhibits eIF4A1 helicase activity, suppressing cytoplasmic cap-dependent translation initiation. Further assessments using 35-S-methionine incorporation in HEK293T cells with or without single-digit micromolar concentrations of elatol for short time periods revealed strong downregulation of mitochondrion-encoded proteins as in Figure 1, (with no effect on mitochondrial transcription). This was confirmed in CML and acute lymphoblastic leukemia (ALL) cell lines whose 24-hour elatol LD50 ranged from high nanomolar to low micromolar concentrations. This potency was 10-40x higher than for tigecycline in side-by-side comparisons across several leukemia cell lines when compared at 72h. Additionally, we established that elatol does not affect integrity of small and large mitochondrial ribosomal units through sedimentation property analysis using sucrose gradients. Although the specific target on the mitochondrial translation apparatus remains elusive, we have uncovered that its mechanism of action differs from that of chloramphenicol, which inhibits translation elongation.
In summary, we have performed proof-of-concept studies using HEK293T and HeLa cell lines, isolated mitochondria from HEK293T, and CML and ALL cell lines to reveal that elatol is a potent inhibitor of mitochondrial protein synthesis at concentrations that do not affect cytoplasmic protein synthesis and that this mechanism differs from chloramphenicol. Tigecycline's compelling preclinical data in combination with TKI informed design of a pending clinical trial (NCT02883036). Elatol's greatly improved potency provide a potential starting point for further optimization of this paradigm.
Figure 1 Figure 1.
Disclosures
No relevant conflicts of interest to declare.
Inhibition of Human Peptide Deformylase Disrupts Mitochondrial Function