Near infrared fluorescent choline kinase alpha inhibitors for cancer imaging and therapy

Choline kinase alpha (ChoKα) deregulation is associated with a more aggressive phenotype and greater malignancy in many human cancers. Inhibitors of ChoKα induce apoptosis in tumorigenic cells, but validation of their specificity is difficult in vivo. Alterations in the profile of choline metabolites are detectable by magnetic resonance spectroscopy (MRS), but because of competing catabolic contributions from the phospholipases, the relative role of ChoKα is not absolutely discernable in a clinical setting. The goal of this work was to develop ChoKα-specific imaging probes to assist in the development of ChoKα as a diagnostic biomarker and therapeutic target. A series of compounds were synthesized for this purpose, and JAS239 was identified as the most promising choline-mimetic with inherent near infrared fluorescence. Attenuation of choline phosphorylation by JAS239 in human breast cancer cells was observed using 14C-choline radiotracing and high-resolution 1H MRS. Microscopy was used to explore the interaction of JAS239 with the ChoKα protein. These in vitro studies, using the established MN58b as a positive control, indicated that JAS239 functions as a competitive inhibitor of ChoKα. Athymic nude mice inoculated with human breast cancer xenografts were injected i.v. with trace doses of JAS239 for imaging studies. In vivo optical imaging of JAS239 accumulation delineated breast tumor margins, and the signal intensity was capable of distinguishing both genetic overexpression and pharmacologic inhibition of ChoKα in breast xenografts. At therapeutic doses, JAS239 and MN58b reduced murine xenograft growth rates, and JAS239 was more effective than MN58b at reducing tumor total choline levels. Histological assessment found both JAS239 and MN58b reduced tumor cell density, decreased proliferation, and elicited an apoptotic response. In a parallel study, ChoKα inhibition was shown for the first time to be an effective therapeutic strategy in glioma tumors, however, JAS239 was not found to cross the blood-brain barrier. A library of derivatives were synthesized and these are being investigated to improve the potency, biodistribution, and tumor specificity. These results represent a new paradigm of multifunctional small molecules that can be used for ChoKα measurement, as companion diagnostics to validate new ChoKα inhibitors, and as therapeutically-effective inhibitors of choline metabolism

IRF3 and type I interferons fuel a fatal response to myocardial infarction

Interferon regulatory factor 3 (IRF3) and type I interferons (IFNs) protect against infections and cancer, but excessive IRF3 activation and type I IFN production cause autoinflammatory conditions such as Aicardi–Goutières syndrome and STING-associated vasculopathy of infancy (SAVI)3. Myocardial infarction (MI) elicits inflammation5, but the dominant molecular drivers of MI-associated inflammation remain unclear. Here we show that ischemic cell death and uptake of cell debris by macrophages in the heart fuel a fatal response to MI by activating IRF3 and type I IFN production. In mice, single-cell RNA-seq analysis of 4,215 leukocytes isolated from infarcted and non-infarcted hearts showed that MI provokes activation of an IRF3–interferon axis in a distinct population of interferon-inducible cells (IFNICs) that were classified as cardiac macrophages. Mice genetically deficient in cyclic GMP-AMP synthase (cGAS), its adaptor STING, IRF3, or the type I IFN receptor IFNAR exhibited impaired interferon-stimulated gene (ISG) expression and, in the case of mice deficient in IRF3 or IFNAR, improved survival after MI as compared to controls. Interruption of IRF3-dependent signaling resulted in decreased cardiac expression of inflammatory cytokines and chemokines and decreased inflammatory cell infiltration of the heart, as well as in attenuated ventricular dilation and improved cardiac function. Similarly, treatment of mice with an IFNAR-neutralizing antibody after MI ablated the interferon response and improved left ventricular dysfunction and survival. These results identify IRF3 and the type I IFN response as a potential therapeutic target for post-MI cardioprotection