Apelin Alleviates Meniscus Endothelial Cell Apoptosis in Osteoarthritis

Osteoarthritis (OA) is a degenerative disease characterized by articular cartilage and/or chondrocyte destruction, and although it has long been considered as a primary disease, the importance of meniscus endothelial cell modulation in the subchondral microenvironment has recently drawn attention. Previous studies have shown that apelin could potentially inhibit cellular apoptosis; however, it remains unclear whether apelin could play a protective role in protecting the endothelium in the OA meniscus. In this study, with the advantages of single-cell RNA sequencing (scRNA-seq) data, in combination with flow cytometry, we identified two endothelial subclusters in the meniscus, featured by high expression of Homeobox A13 (HOXA13) and Ras Protein-Specific Guanine Nucleotide Releasing Factor 2 (RASGRF2), respectively. Compared with control patients, both subclusters decreased in absolute cell numbers and exhibited downregulated APJ endogenous ligand (APLN, coding for apelin) and upregulated apelin receptor (APLNR, coding apelin receptor). Furthermore, we confirmed that in OA, decreased endothelial cell numbers, including both subclusters, were related to intrinsic apoptosis factors: one more relevant to caspase 3 (CASP3) and the other to BH3-Interacting Domain Death agonist (BID). In vitro culturing of meniscal endothelial cells purified from patients proved that apelin could significantly inhibit apoptosis by downregulating these two factors in endothelial cell subclusters, suggesting that apelin could potentially serve as a therapeutic target for patients with OA.

Equilibria between conformational states of the Ras oncogene protein revealed by high pressure crystallography

In this work, we experimentally investigate the allosteric transitions between conformational states on the Ras oncogene protein using high pressure crystallography. Ras protein is a small GTPase involved in central...

Anlotinib Suppresses Oral Squamous Cell Carcinoma Growth and Metastasis by Targeting the RAS Protein to Inhibit the PI3K/Akt Signalling Pathway

Oral squamous cell carcinoma (OSCC) is a malignant tumour originating from the mucosal lining of the oral cavity. Its characteristics include hidden onset, high recurrence, and distant metastasis after operation. At present, clinical treatment usually includes surgery, chemotherapy, radiotherapy, or the joint use of these modalities. Unfortunately, multidrug resistant is one of the important obstacles that causes cancer chemotherapy failure. Anlotinib, which has recently been proven to have good antitumour effects, is a novel multitargeted tyrosine kinase inhibitor. However, there are few studies of the anlotinib-associated mechanism in OSCC and its underlying molecular mechanism. In our study, in vitro models of human oral squamous cell carcinoma HSC-3 cells were used to determine the efficacy of anlotinib. On the one hand, we showed that anlotinib treatment significantly reduced the viability and proliferation of HSC-3 cells and decreased cell migration by inhibiting the activation of the Akt phosphorylation pathway. On the other side, anlotinib inhibited PI3K/Akt/Bad phosphorylation and promoted apoptosis of HSC-3 cells by activating RAS protein expression. In brief, these results indicated that anlotinib had prominent antitumour activity in OSCC, mainly by inhibiting the PI3K/Akt phosphorylation pathway. This work provides evidences and a basic principle for using anlotinib to treat patients with OSCC for clinical research.

A chemorepellent inhibits local Ras activation to inhibit pseudopod formation to bias cell movement away from the chemorepellent

The ability of cells to sense chemical gradients is essential during development, morphogenesis, and immune responses. Although much is known about chemoattraction, chemorepulsion remains poorly understood. Proliferating Dictyostelium cells secrete a chemorepellent protein called AprA. AprA prevents pseudopod formation at the region of the cell closest to the source of AprA, causing the random movement of cells to be biased away from the AprA. Activation of Ras proteins in a localized sector of a cell cortex helps to induce pseudopod formation, and Ras proteins are needed for AprA chemorepulsion. Here we show that AprA locally inhibits Ras cortical activation through the G protein-coupled receptor GrlH, the G protein subunits Gβ and Gα8, Ras protein RasG, protein kinase B, the p-21 activated kinase PakD, and the extracellular signal-regulated kinase Erk1. Diffusion calculations and experiments indicate that in a colony of cells, high extracellular concentrations of AprA in the center can globally inhibit Ras activation, while a gradient of AprA that naturally forms at the edge of the colony allow cells to activate Ras at sectors of the cell other than the sector of the cell closest to the center of the colony, effectively inducing both repulsion from the colony and cell differentiation. Together, these results suggest that a pathway that inhibits local Ras activation can mediate chemorepulsion. [Media: see text] [Media: see text] [Media: see text] [Media: see text] [Media: see text] [Media: see text]

Oncogenic KRAS: Signaling and Drug Resistance

RAS proteins play a role in many physiological signals transduction processes, including cell growth, division, and survival. The Ras protein has amino acids 188-189 and functions as GTPase. These proteins are switch molecules that cycle between inactive GDP-bound and active GTP-bound by guanine nucleotide exchange factors (GEFs). KRAS is one of the Ras superfamily isoforms (N-RAS, H-RAS, and K-RAS) that frequently mutate in cancer. The mutation of KRAS is essentially performing the transformation in humans. Since most RAS proteins belong to GTPase, mutated and GTP-bound active RAS is found in many cancers. Despite KRAS being an important molecule in mostly human cancer, including pancreatic and breast, numerous efforts in years past have persisted in cancer therapy targeting KRAS mutant. This review summarizes the biological characteristics of these proteins and the recent progress in the exploration of KRAS-targeted anticancer, leading to new insight.

Small-Molecule Inhibitors and Degraders Targeting KRAS-Driven Cancers

Drug resistance continues to be a major problem associated with cancer treatment. One of the primary causes of anticancer drug resistance is the frequently mutated RAS gene. In particular, considerable efforts have been made to treat KRAS-induced cancers by directly and indirectly controlling the activity of KRAS. However, the RAS protein is still one of the most prominent targets for drugs in cancer treatment. Recently, novel targeted protein degradation (TPD) strategies, such as proteolysis-targeting chimeras, have been developed to render “undruggable” targets druggable and overcome drug resistance and mutation problems. In this study, we discuss small-molecule inhibitors, TPD-based small-molecule chemicals for targeting RAS pathway proteins, and their potential applications for treating KRAS-mutant cancers. Novel TPD strategies are expected to serve as promising therapeutic methods for treating tumor patients with KRAS mutations.

Impaired RAS Proteolysis Drives Clonal Hematopoietic Transformation

Recently, the protein LZTR1 (leucine zipper-like transcriptional regulator 1) was discovered as an adaptor for a cullin 3 complex responsible for ubiquitin-mediated degradation of RAS proteins. While these data provided a novel mechanism for RAS protein regulation, there is considerable controversy as to which RAS paralogs are physiologic substrates of LZTR1. In parallel, dysregulated LZTR1 expression via aberrant splicing and mutations in both LZTR1 as well as the RAS GTPase and LZTR1 substrate RIT1 were identified in patients with clonal hematopoietic disorders. However, the effects of these alterations on normal and maliganant hematopoiesis have not been evaluated. Here we utilized a series of genetically engineered murine models for germline and conditional deletion of LZTR1, RIT1, and expression of oncogenic RIT1 mutant which revealed a key role for LZTR1 in the regulation of hematopoietic stem cell (HSC) self-renewal and delineated a series of LZTR1-regulated substrates in hematopoietic cells. Consistent with a role for LZTR1 alterations in the Noonan Syndrome, germline homozygous deletion of Lztr1 was associated with lethality between embryonic day 17.5 and birth. Lztr1-/- fetuses had massive dyserythropoiesis and apoptosis of fetal liver hematopoietic cells. Competitive transplantation of E14.5 Lztr1 null fetal liver or bone marrow from 6-week-old Mx1-cre Lztr1 conditional knockout (cKO) mice resulted in striking increased self-renewal in primary and secondary competitive transplantation assays in vivo (Fig.A-B). Interestingly, recipient animals reconstituted with Lztr1-/- cells developed fatal myeloid and lymphoid leukemias characterized by anemia, thrombocytopenia, and increased myeloid and B-lymphoid cells (Fig.C-D). In order to identify the LZTR1 substrates responsible for effects on HSCs, we evaluated levels of all RAS GTPases in Lztr1 null HSCs. This revealed elevated KRas, NRas, MRas, and Rit1 protein in LZTR1 KO cells (Fig.E), with RIT1 being most prominently elevated. Evaluation of a cohort of 4,113 patients with hematologic malignancies identified 41 patients with somatic RIT1 mutations, the majority of which cluster in the switch II region and escape LZTR1-mediated ubiquitination, resulting in RIT1 protein accumulation (Fig.F-H). Given that the impact of RIT1 mutations on hematopoiesis is unknown, we next compared Lztr1 cKO with conditional expression of one of the most common leukemia-associated RIT1 mutants that escapes LZTR1-mediated ubiquitin (Rit1 M90I). Both Lztr1 cKO and Rit1 M90I conditional expression conferred GM-CSF hypersensitivity to HSCs in vitro, cytokine independent growth to human AML cell lines in vitro, and strong competitive self-renewal in vivo (Fig. I-J). Consistent with RIT1 mutations being found primarily in myeloid neoplasm patients, aged Mx1-cre Rit1M90I/WT mice developed fatal MPN, MDS, and mixed MDS/MPN disorders (Fig.K), which were serially transplantable into sublethally irradiated recipients. Despite convergent effects of LZTR1 and RIT1 on clonal HSC advantage, LZTR1 null cell lines did not solely require RIT1 for HSC advantage as revealed by Lztr1/Rit1 double KO mice. We therefore next carried out a series of experiments in RAS-less cells and whole genome CRISPR screens to delineate factors required for LZTR1 mediated hematopoietic transformation. This revealed that KRAS as well as MRAS and its RAF phosphatase partner SHOC2 were selective dependencies for LZTR1-mediated transformation. These data indicate that multiple RAS GTPases as well as RAF activation are required for LZTR1-mediated transformation (Fig.L). While considerable prior research has evaluated oncogenic alleles of RAS which alter RAS-GTP hydrolysis on hematopoiesis, the role of modulating RAS protein abundance on hematopoiesis is unknown. Here we identify RAS proteolysis as a novel regulator of HSC function, define the spectrum of RIT1 mutations in leukemia, and identify LZTR1 and RIT1 mutations as drivers of leukemogenesis. The discovery of RAS proteolysis as a novel driver of leukemogenesis has important therapeutic implications given efforts to therapeutically degrade RAS family members. Finally, the clinical importance of K/NRAS mutations on resistance to therapies in AML motivates future studies on the potential clinical impact of LZTR1 and RIT1 alterations in myeloid neoplasm patients. Figure 1 Figure 1. Disclosures Abdel-Wahab: H3B Biomedicine: Consultancy, Research Funding; Merck: Consultancy; Foundation Medicine Inc: Consultancy; Prelude Therapeutics: Consultancy; LOXO Oncology: Consultancy, Research Funding; Lilly: Consultancy; AIChemy: Current holder of stock options in a privately-held company, Membership on an entity's Board of Directors or advisory committees; Envisagenics Inc.: Current holder of stock options in a privately-held company, Membership on an entity's Board of Directors or advisory committees.

Ras protein abundance correlates with Ras isoform mutation patterns in cancer.

Activating mutations of Ras genes are often observed in cancer. The protein products of the three Ras genes are almost identical. However, for reasons that remain unclear, KRAS is far more frequently mutated than the other Ras isoforms in cancer and RASopathies. We have quantified HRAS, NRAS, KRAS4A and KRAS4B protein abundance across a large panel of cell lines and healthy tissues. We observe consistent patterns of KRAS>NRAS>>HRAS protein expression in cells that correlate with the rank order of Ras mutation frequencies in cancer. Our data provide support for the model of a sweet-spot of Ras dosage mediating isoform-specific contributions to cancer and development. However, they challenge the notion that rare codons mechanistically underpin the predominance of KRAS mutant cancers. Finally, direct measurement of mutant versus wildtype KRAS protein abundance revealed a frequent imbalance that may suggest additional non-gene duplication mechanisms for optimizing oncogenic Ras dosage.