Blocking LAIR1 signaling in immune cells inhibits tumor development.

The current immune checkpoint blockade therapy has been successful in treating some cancers but not others. New molecular targets and therapeutic approaches of cancer immunology need to be identified. Leukocyte associated immunoglobulin like receptor 1 (LAIR1) is an immune inhibitory receptor expressing on most immune cell types. However, it remains a question whether we can specifically and actively block LAIR1 signaling to activate immune responses for cancer treatment. Here we report the development of specific antagonistic anti-LAIR1 monoclonal antibodies and studied the effects of LAIR1 blockade on the anti-tumor immune functions. The anti-LAIR1 antagonistic antibody stimulated the activities of T cells, natural killer cells, macrophages, and dendritic cells in vitro. The single-cell RNA sequencing analysis of intratumoral immune cells in syngeneic human LAIR1 transgenic mice treated with control or anti-LAIR1 antagonist antibodies indicates that LAIR1 signaling blockade increased the numbers of CD4 memory T cells and inflammatory macrophages, but decreased those of pro-tumor macrophages, regulatory T cells, and plasmacytoid dendritic cells. Importantly, the LAIR1 blockade by the antagonistic antibody inhibited the activity of immunosuppressive myeloid cells and reactivated T cells from cancer patients in vitro and impeded tumor metastasis in a humanized mouse model. Blocking LAIR1 signaling in immune cells represents a promising strategy for development of anti-cancer immunotherapy.

USP25 inhibits DNA damage by stabilizing BARD1 protein in a house dust mite-induced asthmatic model in vitro and in vivo.

House dust mites (HDM) can cause DNA double-strand breaks in the lungs of asthmatic patients. However, the molecular mechanisms driving DNA damage and repair in HDM-induced asthma are yet to be elucidated. Thus, in this study, HDM treatment was applied to BEAS-2B cells and mice to mimic the pathological process of asthma in vitro and in vivo, respectively. γ-H2AX foci and expression were measured by immunofluorescence staining and western blot, respectively. The levels of interleukin (IL)-4, IL-6, IL-13, and tumour necrosis factor α (TNFα) were measured using enzyme-linked immunoassay. The expression of USP25 and BARD1 was measured by reverse transcription quantitative PCR and western blot. Co-immunoprecipitation and ubiquitination assays were employed to detect the relationship between USP25 and BARD1. As per the results, it was found that the deubiquitylating enzyme USP25 repressed HDM-induced DNA damage and the production of proinflammatory cytokines, including TNF-α, IL-4, IL-8, and IL-13, in BEAS-2B cells; in contrast, the depletion of USP25 led to the opposite effects. USP25-mediated inhibition of DNA damage and inflammation was facilitated by the stabilizing protein BARD1, which is a tumor suppressor that principally functions by promoting DNA repair and replication in BEAS-2B cells. Furthermore, USP25 was found to robustly augment BARD1 protein abundance and prevent HDM-induced DNA damage and inflammation in vivo. Taken together, these results suggest a novel mechanism contributing to DNA damage and repair in HDM-induced asthma and that selectively modulating this pathway could lead to a novel therapeutic approach for controlling and managing asthma due to HDM exposure.

Acylpeptide hydrolase is a novel regulator of KRAS plasma membrane localization and function.

The primary site for KRAS signaling is the inner leaflet of the plasma membrane (PM). We previously reported that oxanthroquinone G01 (G01) inhibited KRAS PM localization and blocked KRAS signaling. In this study, we identified acylpeptide hydrolase (APEH) as a molecular target of G01. APEH formed a stable complex with biotinylated G01, and the enzymatic activity of APEH was inhibited by G01. APEH knockdown caused profound mislocalization of KRAS and reduced clustering of KRAS that remained PM localized. APEH knockdown also disrupted the PM localization of phosphatidylserine (PtdSer), a lipid critical for KRAS PM binding and clustering. The mislocalization of KRAS was fully rescued by ectopic expression of APEH in knockdown cells. APEH knockdown disrupted the endocytic recycling of epidermal growth factor receptor and transferrin receptor, suggesting that abrogation of recycling endosome function was mechanistically linked to the loss of KRAS and PtdSer from the PM. APEH knockdown abrogated RAS-RAF-MAPK signaling in cells expressing the constitutively active (oncogenic) mutant of KRAS (KRASG12V), and selectively inhibited the proliferation of KRAS-transformed pancreatic cancer cells. Taken together, these results identify APEH as a novel drug target for a potential anti-KRAS therapeutic.

HRAS-driven cancer cells are vulnerable to TRPML1 inhibition.

By serving as intermediaries between cellular metabolism and the bioenergetic demands of proliferation, endolysosomes allow cancer cells to thrive under normally detrimental conditions. Here, we show that an endolysosomal TRP channel, TRPML1, is necessary for the proliferation of cancer cells that bear activating mutations in HRAS Expression of MCOLN1, which encodes TRPML1, is significantly elevated in HRAS-positive tumors and inversely correlated with patient prognosis. Concordantly, MCOLN1 knockdown or TRPML1 inhibition selectively reduces the proliferation of cancer cells that express oncogenic, but not wild-type, HRAS Mechanistically, TRPML1 maintains oncogenic HRAS in signaling-competent nanoclusters at the plasma membrane by mediating cholesterol de-esterification and transport. TRPML1 inhibition disrupts the distribution and levels of cholesterol and thereby attenuates HRAS nanoclustering and plasma membrane abundance, ERK phosphorylation, and cell proliferation. These findings reveal a selective vulnerability of HRAS-driven cancers to TRPML1 inhibition, which may be leveraged as an actionable therapeutic strategy.

An oxanthroquinone derivative that disrupts RAS plasma membrane localization inhibits cancer cell growth.

Oncogenic RAS proteins are commonly expressed in human cancer. To be functional, RAS proteins must undergo post-translational modification and localize to the plasma membrane (PM). Therefore, compounds that prevent RAS PM targeting have potential as putative RAS inhibitors. Here we examine the mechanism of action of oxanthroquinone G01 (G01), a recently described inhibitor of KRAS PM localization. We show that G01 mislocalizes HRAS and KRAS from the PM with similar potency and disrupts the spatial organization of RAS proteins remaining on the PM. G01 also inhibited recycling of epidermal growth factor receptor and transferrin receptor, but did not impair internalization of cholera toxin, indicating suppression of recycling endosome function. In searching for the mechanism of impaired endosomal recycling we observed that G01 also enhanced cellular sphingomyelin (SM) and ceramide levels and disrupted the localization of several lipid and cholesterol reporters, suggesting that the G01 molecular target may involve SM metabolism. Indeed, G01 exhibited potent synergy with other compounds that target SM metabolism in KRAS localization assays. Furthermore, G01 significantly abrogated RAS-RAF-MAPK signaling in Madin-Darby canine kidney (MDCK) cells expressing constitutively activated, oncogenic mutant RASG12V. G01 also inhibited the proliferation of RAS-less mouse embryo fibroblasts expressing oncogenic mutant KRASG12V or KRASG12D but not RAS-less mouse embryo fibroblasts expressing oncogenic mutant BRAFV600E. Consistent with these effects, G01 selectively inhibited the proliferation of KRAS-transformed pancreatic, colon, and endometrial cancer cells. Taken together, these results suggest that G01 should undergo further evaluation as a potential anti-RAS therapeutic.

AMPK and Endothelial Nitric Oxide Synthase Signaling Regulates K-Ras Plasma Membrane Interactions via Cyclic GMP-Dependent Protein Kinase 2.

K-Ras must localize to the plasma membrane and be arrayed in nanoclusters for biological activity. We show here that K-Ras is a substrate for cyclic GMP-dependent protein kinases (PKGs). In intact cells, activated PKG2 selectively colocalizes with K-Ras on the plasma membrane and phosphorylates K-Ras at Ser181 in the C-terminal polybasic domain. K-Ras phosphorylation by PKG2 is triggered by activation of AMP-activated protein kinase (AMPK) and requires endothelial nitric oxide synthase and soluble guanylyl cyclase. Phosphorylated K-Ras reorganizes into distinct nanoclusters that retune the signal output. Phosphorylation acutely enhances K-Ras plasma membrane affinity, but phosphorylated K-Ras is progressively lost from the plasma membrane via endocytic recycling. Concordantly, chronic pharmacological activation of AMPK → PKG2 signaling with mitochondrial inhibitors, nitric oxide, or sildenafil inhibits proliferation of K-Ras-positive non-small cell lung cancer cells. The study shows that K-Ras is a target of a metabolic stress-signaling pathway that can be leveraged to inhibit oncogenic K-Ras function.

Rare Streptomyces N-formyl amino-salicylamides inhibit oncogenic K-Ras.

During a search for inhibitors of oncogenic K-Ras, we detected two known and two new examples of the rare neoantimycin structure class from a liquid cultivation of Streptomyces orinoci, and reassigned/assigned structures to all based on detailed spectroscopic analysis and microscale C3 Marfey's and C3 Mosher chemical degradation/derivatization/analysis. SAR investigations inclusive of the biosynthetically related antimycins and respirantin, and synthetic benzoxazolone, documented a unique N-formyl amino-salicylamide pharmacophore as a potent inhibitor of oncogenic K-Ras.