Targeting AXL kinase sensitizes leukemic stem and progenitor cells to venetoclax treatment in acute myeloid leukemia.

The abundance of genetic abnormalities and phenotypic heterogeneities in acute myeloid leukemia (AML) poses significant challenges to the development of improved treatments. Here, we demonstrated that a key growth arrest-specific gene 6/AXL axis is highly activated in cells from patients with AML, particularly in stem/progenitor cells. We developed a potent selective AXL inhibitor that has favorable pharmaceutical properties and efficacy against preclinical patient-derived xenotransplantation (PDX) models of AML. Importantly, inhibition of AXL sensitized AML stem/progenitor cells to venetoclax treatment, with strong synergistic effects in vitro and in PDX models. Mechanistically, single-cell RNA-sequencing and functional validation studies uncovered that AXL inhibition, alone or in combination with venetoclax, potentially targets intrinsic metabolic vulnerabilities of AML stem/progenitor cells and shows a distinct transcriptomic profile and inhibits mitochondrial oxidative phosphorylation. Inhibition of AXL or BCL-2 also differentially targets key signaling proteins to synergize in leukemic cell killing. These findings have a direct translational impact on the treatment of AML and other cancers with high AXL activity.

Evolutionary Ancestry of Eukaryotic Protein Kinases and Choline Kinases.

The reversible phosphorylation of proteins catalyzed by protein kinases in eukaryotes supports an important role for eukaryotic protein kinases (ePKs) in the emergence of nucleated cells in the third superkingdom of life. Choline kinases (ChKs) could also be critical in the early evolution of eukaryotes, because of their function in the biosynthesis of phosphatidylcholine, which is unique to eukaryotic membranes. However, the genomic origins of ePKs and ChKs are unclear. The high degeneracy of protein sequences and broad expansion of ePK families have made this fundamental question difficult to answer. In this study, we identified two class-I aminoacyl-tRNA synthetases with high similarities to consensus amino acid sequences of human protein-serine/threonine kinases. Comparisons of primary and tertiary structures supported that ePKs and ChKs evolved from a common ancestor related to glutaminyl aminoacyl-tRNA synthetases, which may have been one of the key factors in the successful of emergence of ancient eukaryotic cells from bacterial colonies.

A CRM1-dependent nuclear export signal controls nucleocytoplasmic translocation of HSCARG, which regulates NF-κB activity.

HSCARG is a newly identified nuclear factor-κB (NF-κB) inhibitor that plays important roles in cell growth. Our previous study found that HSCARG could shuttle between the nucleus and cytoplasm by sensing the change in cellular redox states. To further investigate the mechanism of HSCARG translocation and its effect on the regulation of NF-κB activity, we identified a previously uncharacterized nuclear export signal (NES) at residues 272-278 of HSCARG that is required for its cytoplasmic translocation. This leucine-rich NES was found to be mediated by chromosome region maintenance 1. More importantly, accumulation of HSCARG in the nucleus occurred following a mutation in the NES or oxidative stress, which attenuated the inhibition of NF-κB by HSCARG. These results indicate that nucleocytoplasmic translocation of HSCARG plays an important role in fine-tuning NF-κB signaling.

Regulatory roles of conserved phosphorylation sites in the activation T-loop of the MAP kinase ERK1.

The catalytic domains of most eukaryotic protein kinases are highly conserved in their primary structures. Their phosphorylation within the well-known activation T-loop, a variable region between protein kinase catalytic subdomains VII and VIII, is a common mechanism for stimulation of their phosphotransferase activities. Extracellular signal-regulated kinase 1 (ERK1), a member of the extensively studied mitogen-activated protein kinase (MAPK) family, serves as a paradigm for regulation of protein kinases in signaling modules. In addition to the well-documented T202 and Y204 stimulatory phosphorylation sites in the activation T-loop of ERK1 and its closest relative, ERK2, three additional flanking phosphosites have been confirmed (T198, T207, and Y210 from ERK1) by high-throughput mass spectrometry. In vitro kinase assays revealed the functional importance of T207 and Y210, but not T198, in negatively regulating ERK1 catalytic activity. The Y210 site could be important for proper conformational arrangement of the active site, and a Y210F mutant could not be recognized by MEK1 for phosphorylation of T202 and Y204 in vitro. Autophosphorylation of T207 reduces the catalytic activity and stability of activated ERK1. We propose that after the activation of ERK1 by MEK1, subsequent slower phosphorylation of the flanking sites results in inhibition of the kinase. Because the T207 and Y210 phosphosites of ERK1 are highly conserved within the eukaryotic protein kinase family, hyperphosphorylation within the kinase activation T-loop may serve as a general mechanism for protein kinase down-regulation after initial activation by their upstream kinases.

Determination of the Substrate Specificity of Protein Kinases with Peptide Micro- and Macroarrays.

Elucidation of the key determinants for the phosphorylation site specificities of protein kinases facilitates identification of their physiological substrates, and serves to better define their critical roles in the signaling networks that underlie a multitude of cellular activities. Albeit with some apparent limitations, such as the lack of contextual information for secondary substrate-binding sites, the synthetic peptide-based approach has been adopted widely for the kinase specificity profiling studies, especially when they are used in an array format, which permits the screening of large numbers of potential peptide substrates in parallel. In this chapter, we present detailed protocols for determining protein kinase substrate specificity using an approach that involves both peptide microarrays and macroarrays. In particular, SPOT synthesis on macroarrays can be used to follow up on in silico predictions of protein kinase substrate specificity with predictive algorithms.