Bone is a dynamic tissue that is continuously remodeled by tightly-coordinated actions of bone-resorbing osteoclasts and bone-forming osteoblasts. Uncontrolled osteoclastogenesis has a severe impact on skeletal health and thus represents an important therapeutic target in osteolytic diseases. VprBP is a recently identified kinase and plays an important role in stimulating osteoclast differentiation and bone resorption through histone H2AT120 phosphorylation (H2AT120p). We previously developed B32B3 as the first VprBP inhibitor, but its high IC50 value has been a major challenge in using it for osteoclast-triggered bone diseases. In this study, we designed and characterized a series of small molecule compounds derived from B32B3 to identify a novel second-generation inhibitor of VprBP. Our initial screening of B32B3 derivatives identified three compounds, designated as B1486, B3703, and B6756, that are effective in interfering with VprBP kinase activity toward H2AT120p and RANKL-induced differentiation of osteoclast precursor cells (OCPs). However, when their cellular efficacy was systematically accessed over different concentration ranges, B1486 was more potent than B3703 and B6756 at hindering VprBP function in the process of OCP cell differentiation. Consistent with these in vitro findings, B1486 showed significantly improved inhibitory activity against VprBP-driven bone resorption and low bone mass phenotypes in our preclinical studies using zebrafish and mouse models. Taken together, our results indicate that B1486 is a highly potent inhibitor for blocking VprBP-mediated H2AT120p and osteoclastogenic gene silencing, as well as for overcoming osteoporosis generated by excessive osteoclast differentiation.
Natural killer (NK) cells are innate lymphocytes that directly eliminate tumor and virus-infected cells by integrating signals from activating and inhibitory receptors, and their effector functions are tightly coupled to cellular metabolism. Given that the inhibitory receptor PD-1 reprograms T cell metabolism to shape functional fate, the bioenergetic consequences of inhibitory receptor engagement on human NK cells remain largely unexplored, particularly for sialic acid-binding immunoglobulin-like lectin (Siglec-7), a glyco-immune checkpoint receptor. Here, we investigated metabolic programs and effector functions associated with Siglec-7 expression and antibody-mediated Siglec-7 ligation in primary NK cells and NK-92MI cells. Siglec-7POS NK cells exhibited selectively impaired CD107a degranulation under glycolytic and oxidative phosphorylation inhibition, whereas Siglec-7NEG cells remained relatively resistant, indicating distinct energetic wiring between these subsets. Engagement of Siglec-7 by an agonistic antibody induced mitochondrial fission with altered Drp1 phosphorylation, transient mitochondrial depolarization, and broadly suppressed mitochondrial respiration, while concurrently enhancing glycolytic capacity, consistent with a dual metabolic shift upon Siglec-7 ligation. In contrast, sustained Siglec-7 expression in NK-92MI-S cells was associated with globally enhanced mitochondrial respiratory capacity, indicating that sustained Siglec-7 expression and short-term treatment with an agonistic anti-Siglec-7 antibody were associated with distinct metabolic profiles in NK cells. Furthermore, Siglec-7POS NK cells showed increased accumulation of autophagic vacuole, reduced proliferation, and heightened apoptotic susceptibility compared with Siglec-7NEG counterparts. Collectively, these findings support an association between Siglec-7 status, mitochondrial homeostasis, and metabolic fitness in NK cells, with Siglec-7NEG cells retaining a metabolically robust, cytotoxic phenotype.
Chronic inflammation drives many diseases, including cancer, where inflammation is associated with metastasis, the cause of death in 90% of cancer fatalities. Tumor inflammation drives inducible nitric oxide synthase (NOS2) and cyclooxygenase 2 (COX2) expression, each of which is associated with poor outcomes in cancer. Here, we knocked out the NOS2 gene in the murine triple-negative breast cancer allograft tumor model 4T1 and examined metastatic spread from primary mammary tumors to lung in BALB/c mice with intact immune systems. Remarkably, while the parental 4T1 tumors were highly metastatic, metastasis from 4T1 NOS2-/- tumors was nearly eliminated. In cell culture, we find that nitric oxide (NO) from NOS2 induces a glycolytic phenotype, epitrascriptomic dysregulation, and prostaglandin E2 synthesis by stimulating COX2. Cytokine addition led to DNA damage, presumably from NO. Human colorectal cancer cell line DLD1 yielded similar results. RNA sequencing of 4T1 cells revealed a NOS2-dependent reduction in mRNAs associated with regulation of chromosome and DNA replication, including cell cycle checkpoints, as well as mRNAs associated with RNA demethylation, including tRNA demethylation. Immunoprofiling of the tumor microenvironment revealed immunosuppressed primary tumors that were low in cytotoxic T cells and high in myeloid derived suppressor cells expressing interferon gamma, which induces NOS2 expression. Taken together, these data suggest high NOS2 in tumor cells may drive metastasis through a broad accumulation of cancer-associated factors - including metabolic dysregulation, genetic dysregulation, and an increase in inflammatory prostaglandins - all of which contribute to aggressive cancers.
The integral membrane protease Rce1 cleaves prenylated CaaX proteins, including all RAS GTPases, as part of their post-translational maturation. This cleavage, which removes the -aaX residues, is necessary for the proper cellular location and signaling properties of RAS and its oncogenic forms. Poor biochemical stabilities of yeast and mammalian Rce1 have hindered the purification of Rce1 in active form and limited studies addressing its catalytic properties. Here we identify that Drosophila melanogaster Rce1 has good biochemical stability and is catalytically active when purified. The purified protein recapitulates known properties of Rce1, including specificity for prenylated CaaX substrates. Like other integral membrane proteases, the enzyme has a low turnover number (kcat), of approximately 20 hr-1. Pre-steady-state kinetic analysis reveals that product release is the rate limiting step. Our data indicate that Rce1 is inhibited by nanomolar levels of Zn2+, but that metals are not involved in catalysis. The data suggest that Zn2+ is a competitive inhibitor with respect to the substrate. The signaling lipid PA also inhibits the enzyme in vitro, raising the possibility that the cellular activity of Rce1 may be modulated by lipids and/or Zn2+. These studies combined with structural predictions and prior investigation lead to the hypothesis that the active site contains a catalytic Glu-His-His triad of amino acids that coordinate the substrate and form the inhibitory Zn2+ site. This work enhances the understanding of the enzyme and gives insight into means for Rce1 inhibition.
Lysophosphatidylcholine (LPC) is an essential mammalian phospholipid that serves multiple physiological functions. We have previously shown that plasma LPC levels are rapidly reduced after cardiac arrest (CA) in humans and in rodent models, where exogenous supplementation improves survival and neurologic outcomes. However, the specific mechanisms driving this depletion remain undefined, precluding rational design of targeted therapies to preserve LPC levels and limiting clinical translation. Here, we provide the first pharmacokinetic analysis of acute LPC reduction through a novel stable-isotope tracer approach. Our findings demonstrate that saturation status of the fatty acyl chain in LPC species determines the degree of reduction after CA, with polyunsaturated species showing the greatest declines. Further, we found that in situ metabolism, not decreased hepatic production nor increased tissue distribution, underlies the observed depletion in plasma LPC after cardiac arrest. Polyunsaturated LPC species showed correlated changes with lysophosphatidic acid and choline-containing metabolites. These correlations likely reflect shared vulnerability to post-CA pathological stress rather than direct metabolic conversion, as conventional metabolic pathways could not account for the temporal patterns and magnitude of LPC reduction, suggesting a broader, systemic dysregulation of plasma lipid metabolism. These findings indicate that therapeutic strategies must focus on either identifying and targeting the novel metabolic mechanisms responsible for species-selective elimination or implementing LPC supplementation protocols designed to overcome accelerated plasma elimination.
