Ras Proteins Research Articles

NMR 1H, 13C, 15N backbone resonance assignments of 14-3-3ζ binding region of human FOXO3a (residues 1-284)

In tumors, mutation in Ras proteins stimulates a signaling cascade through phosphorylation. Downstream of the cascade, many transcription and translation factors are up- or down-regulated by phosphorylation, leading to cancer progression. This phosphorylation cascade is sustained by 14-3-3ζ protein. 14-3-3ζ binds to its client proteins that are Ser/Thr-phosphorylated and prevents their dephosphorylation. One of those transcription factors is FOXO3a, whose transcriptional activity is suppressed in the phosphorylation cascade. FOXO3a binds to specific DNA sequences and activates the transcription of apoptosis-related proteins. In cancer cells, however, FOXO3a is phosphorylated, bound to 14-3-3ζ, and dissociated from the DNA, resulting in FOXO3a inactivation. To elucidate the mechanism of FOXO3a inactivation by the 14-3-3ζ binding, we aim to perform NMR analysis of the interaction between 14-3-3ζ and di-phosphorylated FOXO3a residues 1-284 (dpFOXO3a). Here, we report the backbone resonance assignments of dpFOXO3a, which are transferred from those of the N-terminal domain (NTD) and the DNA-binding domain (DBD) of dpFOXO3a.

Locally advanced rectal cancer in a young adult affected with dyskeratosis congenita (Zinsser–Cole–Engman syndrome): a case report

BackgroundDyskeratosis congenita (DKC), also known as Zinsser–Cole–Engman syndrome, is a progressive genetic disease with a triad of reticulate skin pigmentation, nail dystrophy, and leukoplakia. Approximately 8–10% of patients with DKC develop malignancies, and cases of colorectal cancer with DKC in young people have been reported previously.Case presentationA 25-year-old man with DKC since approximately 10 years of age developed fever and lower abdominal discomfort. Diagnostic imaging revealed locally advanced rectal cancer with lymph node metastasis, direct invasion of the prostate, and pelvic abscess due to tumor microperforation (cT4bN2M0 cStage IIIC). Biopsy showed well to moderately differentiated ductal adenocarcinoma. Genetic testing was negative for RAS and BRAF gene mutations, and microsatellite instability (MSI) testing was also negative. After sigmoid colostomy, the patient was treated with total neoadjuvant therapy (TNT) with systemic chemotherapy (six courses of FOLFOX + panitumumab) followed by chemoradiation therapy (50.4 Gy with capecitabine). After TNT, the primary tumor and metastatic lymph nodes shrank. According to the findings of colonoscopy and magnetic resonance image (MRI), we diagnosed near complete response (near-CR) and decided to follow the patient without surgery by every 3 months re-evaluation. However, 5 months after TNT, tumor regrowth was detected on colonoscopy and imaging, and the patient underwent total pelvic exenteration. He developed paralytic ileus as a postoperative complication, and was discharged on the 38th postoperative day. Pathological examination revealed a residual tumor with invasion of the periprostatic tissue. There was no metastasis in the pararectal and lateral pelvic lymph nodes, but one extramural non-contiguous cancerous extension (tumor deposit) was observed (ypT4bN1cM0 ypStage IIIC). The patient has been free of recurrence for one year after surgery.ConclusionsDKC often develops into various tumors in the digestive system at an early age; therefore, appropriate surveillance may be required. In addition, considering that cancers in patients with DKC occur at a young age, fertility preservation and survivorship are also important, and adequate explanations and care should be provided to patients before and after treatment.

PPM1D activity promotes cellular transformation by preventing senescence and cell death

Cell cycle checkpoints, oncogene-induced senescence and programmed cell death represent intrinsic barriers to tumorigenesis. Protein phosphatase magnesium-dependent 1 (PPM1D) is a negative regulator of the tumour suppressor p53 and has been implicated in termination of the DNA damage response. Here, we addressed the consequences of increased PPM1D activity resulting from the gain-of-function truncating mutations in exon 6 of the PPM1D. We show that while control cells permanently exit the cell cycle and reside in senescence in the presence of DNA damage caused by ionising radiation or replication stress induced by the active RAS oncogene, RPE1-hTERT and BJ-hTERT cells carrying the truncated PPM1D continue proliferation in the presence of DNA damage, form micronuclei and accumulate genomic rearrangements revealed by karyotyping. Further, we show that increased PPM1D activity promotes cell growth in the soft agar and formation of tumours in xenograft models. Finally, expression profiling of the transformed clones revealed dysregulation of several oncogenic and tumour suppressor pathways. Our data support the oncogenic potential of PPM1D in the context of exposure to ionising radiation and oncogene-induced replication stress.

Survival Analysis, Clinical Characteristics, and Predictors of Cerebral Metastases in Patients with Colorectal Cancer.

Colorectal cancer (CRC) is the third most common cancer globally and a leading cause of cancer-related deaths. While liver metastasis is common, brain metastasis (BM) is rare, occurring in 0.1% to 14% of cases. Risk factors for BM include lung metastasis at diagnosis, rectal cancer, and mutations in RAS and KRAS genes. Due to its rarity, guidelines for BM screening and treatment are limited. The aim of this study is to identify the clinical characteristics and predictors of BM at the time of the initial diagnosis of CRC. We evaluated patients ≥18 years old with metastatic colorectal cancer and brain metastases at diagnosis from the SEER database (2010-2021). A retrospective cohort study was conducted to analyze overall survival and predictive factors for brain metastasis, utilizing multivariate logistic regression, Kaplan-Meier survival analysis, and the Cox proportional hazards models, with p-values < 0.05 considered significant. Out of 24,703 patients with metastatic colorectal cancer (mCRC), 228 (0.92%) had brain metastasis (BM) at diagnosis. BM was more prevalent in average-onset mCRC (≥50 years) compared to early-onset (<50 years) (1% vs. 0.55%, p = 0.004). Certain factors, such as older age and adenocarcinoma subtype, were associated with BM. Additionally, Asians/Pacific-Islanders (HR 1.83 CI: 1.01-3-33, p = 0.045) and American Indians/Alaska Natives (HR 4.79 CI 1.15-19.97, p = 0.032) had higher mortality rates, while surgical treatment and chemotherapy were linked to decreased mortality. Patients with BM had significantly worse overall survival (6 months vs. 21 months, p < 0.001). BM in mCRC is uncommon, but it is associated with significantly worse outcomes, including markedly reduced overall survival. Our study highlights several critical factors associated with the presence of BM, such as older age and specific racial/ethnic groups, which may inform risk stratification and early-detection strategies. Our findings emphasize the need for heightened awareness and screening for BM in high-risk mCRC patients, as well as the inclusion of these patients in clinical trials to explore tailored therapeutic approaches aimed at improving survival and quality of life.

Abstract MP05: A Preliminary 10X Genomics Xenium Profiler Spatial Transcriptomics Map of Renin-Angiotensin System Genes in the Mouse Subfornical Organ

The subfornical organ (SFO) is a brain region rich in angiotensin-II (ANG) AT1 receptors (AT1R) which regulates water and salt intake and communicates with other hypothalamic nuclei to regulate blood pressure (BP). Activation of the classical G protein-mediated AT1R signaling in the brain induces pressor and natri-dipsogenic responses. TRV027, a synthetic AT1R β-arrestin-biased agonist, decreases BP and increases saline avoidance. We examined the spatial transcriptomic makeup of the SFO to balanced or biased AT1R activation using 10X Genomics Xenium. We treated C57BL/6J mice with either aCSF, ANG (0.64 µg/h), or TRV027 (0.56 µg/h) through continuous ICV infusion for 11-days. Water intake was monitored on days 6-11. ANG caused an increase in water intake compared to aCSF or TRV027 groups (7.0±2.0, 3.0±0.2, and 1.9±0.6 mL/day, respectively, n=4, p&lt;0.05). On day 11, brains were perfused, collected, and fixed. Subsequently, 5 µm paraffin-embedded coronal sections were mounted on Xenium slides and tested with the 10X mouse brain set of 247 genes plus 100 custom add-on genes including all RAS genes. A third of the cells expressed AT1R, and the pattern of AT1R-expressing cells Xenium-detected was qualitatively similar to NZ44 mice, which express GFP under the control of the AT1R locus. Expression of the prorenin receptor ( Atp6ap2 ) was abundant throughout the SFO. A few cells in the SFO and more prominently in the adjacent blood vessels were angiotensinogen positive. Single cells were identified that expressed the ANG type 2 receptors (AT2R). Expression of Mas receptor was undetectable. Most of the AT1R-expressing cells were excitatory glutamatergic neurons (expressing Slc17a6 , VGLUT2) but a few were inhibitory GABAergic neurons co-expressing Slc32a1 (VGAT). Each section had cells classified as astrocytes, neurons, ependymal cells, and immune cells (e.g., microglia or macrophages). Increased numbers of immune cells were detected in the SFO of ANG-treated mice. Interestingly, although there was no evidence for expression of renin in the SFO, renin-expressing cells were identified in the choroid plexus which is consistent with highly sensitive in situ RNA hybridization data. Additional qualitative and quantitative analyses are ongoing to compare gene expression patterns in AT1R-positive cell types. Future studies will identify gene expression patterns in SFO neurons with connectivity to other brain regions controlling water/salt intake and BP.

Identification and Characterization of Novel Small-Molecule Enhancers of the CUL3LZTR1 E3 Ligase KRAS Complex.

The RAS family of GTPases is among the most frequently mutated proteins in human cancer, creating a high clinical demand for therapies that counteract their signaling activity. An important layer of regulation that could be therapeutically exploited is the proteostatic regulation of the main RAS GTPases KRAS, NRAS, and HRAS, as well as the closely related members, MRAS and RIT1, by the leucine zipper-like transcriptional regulator 1 cullin 3 RING E3 ubiquitin ligase complex (CUL3LZTR1). Genetic inactivation of LZTR1, as observed in different cancer entities and Noonan syndrome leads to enhanced RAS GTPase abundance and altered MAPK pathway activation state. Novel therapeutic approaches to interfere with hyperactive RAS signaling, thereby complementing existing treatments, are highly sought after. Motivated by the growing arsenal of molecular glue degraders, we report the identification of novel chemical fragments that enhance the protein-protein interaction (PPI) of the KRAS-LZTR1 complex. We established a split-luciferase-based reporter assay that monitors the RAS GTPase-LZTR1 interaction in a scalable format, capable of capturing chemical, as well as mutational perturbations. Using this screening system, in combination with a small fragment library, we identified two fragments, C53 and Z86, that enhance the interaction of the KRAS-LZTR1 complex in a dose-dependent manner. Further orthogonal validation experiments using proximity biotinylation (BioID), thermal shift assays, and NMR spectroscopy demonstrated fragment-dependent enhanced recruitment of endogenous LZTR1 and physical engagement of KRAS. The two fragments, which potentiate the KRAS-LZTR1 interaction, serve as starting points for fragment-based drug discovery. Additionally, the assay we introduced is amenable to high-throughput screening to further explore the pharmacological modulation of the CUL3LZTR1-RAS GTPase complex.

The pathological significance of HRAS gene mutation in colorectal cancer patients in Erbil province

Background and objective: RAS gene mutations, including Harvey RAS, are a biomarker in studying many types of cancer, including colorectal cancer. Studying the molecular and mutations of the HRAS gene help in understanding the nature of the tumor and helps to formulate a suitable plan for prevention and treatment. The aim of the present study is to identify HRAS mutations among a sample of colorectal cancer. Methods: The sample for the present study involved 20 Formalin fixed embedded tissue of colorectal carcinoma and 10 adjacent normal tissues were obtained from Rizgary Teaching Hospital in Erbil. Results: The sample of the present study involved 20 patients (60% males and 40% females) with CRC. The mean age was 54.20 years, with an SD ±19.80. The study found a single HRAS mutation in the sample study. The size of the tumor mutated by HRAS is significantly different than other tumors (P = 0.009). Conclusion: there was an HRAS mutation in a sample of colorectal cancers and the size of the tumor was different than other tumors in the study sample. Further studies needed about the mutation of the HRAS gene.

Molecular profiling of low-risk papillary thyroid carcinoma (mPTC) on active surveillance.

The active surveillance (AS) program for papillary thyroid carcinoma (≤ 1 cm) at low-risk (mPTC) showed a low percentage of progression. The aim of this study was to find a molecular signature of cases that showed disease progression during AS, which would allow their early identification. We performed next generation sequencing of 95 fine needle aspiration cytology specimens from cases prospectively enrolled in the AS program to analyze key somatic driver alterations or gene fusions implicated in PTC tumorigenesis. TERT promoter analysis was performed using Sanger sequencing or droplet digital PCR. BRAF p.V600E was found in 66.3% (63/95) of mPTC and was the most common somatic alteration, followed by RAS oncogene mutations detected in 3.2% of mPTC (3/95: 2 NRAS and 1 KRAS) and gene fusions detected in 3.2% of mPTC (3/95: 1 RET-PTC1, 1 TFG-NTRK1, 1 ALK imbalance). No TERT promoter mutations (C228T and C250T) were found in the analyzed mPTC (84/95). The comparison between the molecular profile and the clinical outcome of the mPTC (stable versus progressive disease) showed no correlation (p-value=0.6) and did not identify a molecular signature able to identify progressive mPTC. The molecular profile of mPTC is like that of bigger PTC with the exception that none of them showed a TERT promoter mutation. The identification of the most common driver mutations, such as BRAF, RAS, or gene fusions, is not helpful for the early identification of mPTC that will show disease progression during follow-up in the AS program.

Capturing RAS oligomerization on a membrane

RAS GTPases associate with the biological membrane where they function as molecular switches to regulate cell growth. Recent studies indicate that RAS proteins oligomerize on membranes, and disrupting these assemblies represents an alternative therapeutic strategy. However, conflicting reports on RAS assemblies, ranging in size from dimers to nanoclusters, have brought to the fore key questions regarding the stoichiometry and parameters that influence oligomerization. Here, we probe three isoforms of RAS [Kirsten Rat Sarcoma viral oncogene (KRAS), Harvey Rat Sarcoma viral oncogene (HRAS), and Neuroblastoma oncogene (NRAS)] directly from membranes using mass spectrometry. We show that KRAS on membranes in the inactive state (GDP-bound) is monomeric but forms dimers in the active state (GTP-bound). We demonstrate that the small molecule BI2852 can induce dimerization of KRAS, whereas the binding of effector proteins disrupts dimerization. We also show that RAS dimerization is dependent on lipid composition and reveal that oligomerization of NRAS is regulated by palmitoylation. By monitoring the intrinsic GTPase activity of RAS, we capture the emergence of a dimer containing either mixed nucleotides or GDP on membranes. We find that the interaction of RAS with the catalytic domain of Son of Sevenless (SOScat) is influenced by membrane composition. We also capture the activation and monomer to dimer conversion of KRAS by SOScat. These results not only reveal the stoichiometry of RAS assemblies on membranes but also uncover the impact of critical factors on oligomerization, encompassing regulation by nucleotides, lipids, and palmitoylation.

Effector Binding Sequentially Alters KRAS Dimerization on the Membrane: New Insights Into RAS-Mediated RAF Activation.

RAS proteins are peripheral membrane GTPases that activate multiple downstream effectors for cell proliferation and differentiation. The formation of a signaling RAS-RAF complex at the plasma membrane is implicated in a quarter of all human cancers; however, the underlying mechanism remains unclear. In this work, nanodisc platforms and paramagnetic relaxation enhancement (PRE) analyses to determine the structure of a hetero-tetrameric complex comprising KRAS and the RAS-binding domain (RBD) and cysteine-rich domain (CRD) of activated RAF1 are employed. The binding of the RBD or RBD-CRD differentially alters the dimerization modes of KRAS on both anionic and neutral membranes, validated by interface-specific mutagenesis. Notably, the RBD binding allosterically generated two distinct KRAS dimer interfaces in equilibrium, favored by KRAS free and in complex with the RBD-CRD, respectively. Additional interactions of the CRD with both KRAS protomers are mutually cooperative to stabilize a new dimer configuration of KRAS bound to the RBD-CRD. The RAF binding sequentially alters KRAS dimerization, providing new insights into RAF activation, including a configurational transition of the KRAS dimer to provide an interaction site for the CRD and release the autoinhibited RAF complex. These methods are applicable to many other signaling protein complexes on the membrane.

Hypermethylated RASAL1’s promotive role in chemoresistance and tumorigenesis of choriocarcinoma was regulated by TET2 but not DNMTs

BackgroundPatients with choriocarcinoma (CC) accompanying chemoresistance conventionally present a poor prognosis. Whether ras protein activator like-1 (RASAL1) functions as a tumor promoter or suppressor depends on tumor types. However, the role of RASAL1 in process of chemoresistance of CC and underlying molecular mechanism remain elusive.MethodsThe expression pattern of RASAL1 in CC cells and tissues was measured using Western blotting, immunohistochemistry and qRT-PCR. Cell viability and proliferative ability were assessed by MTT assay, Tunnel assay and flow cytometric analysis. Additionally, the stemness was evaluated by the colony formation and tumor sphere formation. Methotrexate (MTX) was applied to exam the chemosensitivity of CC cells.ResultsThe expression of RASAL1 was reduced both at the protein and mRNA levels in CC tissues and cells compared to hydatidiform mole (HM) and invasive mole (IM). Loss of RASAL1 was attributed to its promoter hypermethylation and could be restored by 5-Aza. Knock-down of RASAL1 promoted the viability, proliferative potential, stemness and EMT phenotype of JEG-3 cells. However, induced overexpression of RASAL1 by 5-Aza significantly prohibited cell proliferation and stemness potential of the JAR cell. Additionally, the xenograft model indicated that knockdown of RASAL1 led to a remarkable increase of tumor volume and weight in comparison with its counterpart. Moreover, the stimulatory activity brought by decrease of RASAL1 could be deprived by β-catenin inhibitor XAV 939, yet the suppressive activity resulted from its promoter demethylation could be rescued by β-catenin activator BML-284, indicating that function of RASAL1 depends on β-catenin. Besides, the co-immunoprecipitation assay confirmed the physical binding between RASAL1 and β-catenin. Further investigations showed hypermethylated RASAL1 was regulated by TET2 but not DNMTs.ConclusionTaken together, the present data elucidated that reduced RASAL1 through its promoter hypermethylation regulated by TET2 promoted the tumorigenicity and chemoresistance of CC via modulating β-catenin both in vitro and in vivo.

Titration of RAS alters senescent state and influences tumour initiation

Oncogenic RAS-induced senescence (OIS) is an autonomous tumour suppressor mechanism associated with premalignancy1,2. Achieving this phenotype typically requires a high level of oncogenic stress, yet the phenotype provoked by lower oncogenic dosage remains unclear. Here we develop oncogenic RAS dose-escalation models in vitro and in vivo, revealing a RAS dose-driven non-linear continuum of downstream phenotypes. In a hepatocyte OIS model in vivo, ectopic expression of NRAS(G12V) does not induce tumours, in part owing to OIS-driven immune clearance3. Single-cell RNA sequencing analyses reveal distinct hepatocyte clusters with typical OIS or progenitor-like features, corresponding to high and intermediate levels of NRAS(G12V), respectively. When titred down, NRAS(G12V)-expressing hepatocytes become immune resistant and develop tumours. Time-series monitoring at single-cell resolution identifies two distinct tumour types: early-onset aggressive undifferentiated and late-onset differentiated hepatocellular carcinoma. The molecular signature of each mouse tumour type is associated with different progenitor features and enriched in distinct human hepatocellular carcinoma subclasses. Our results define the oncogenic dosage-driven OIS spectrum, reconciling the senescence and tumour initiation phenotypes in early tumorigenesis.

KCTD17-mediated Ras stabilization promotes hepatocellular carcinoma progression.

Potassium channel tetramerization domain containing 17 (KCTD17) protein, an adaptor for the cullin3 (Cul3) ubiquitin ligase complex, has been implicated in various human diseases; however, its role in hepatocellular carcinoma (HCC) remains elusive. Here, we aimed to elucidate the clinical features of KCTD17, and investigate the mechanisms by which KCTD17 affects HCC progression. We analyzed transcriptomic data from patients with HCC. Hepatocyte-specific KCTD17 deficient mice were treated with diethylnitrosamine (DEN) to assess its effect on HCC progression. Additionally, we tested KCTD17-directed antisense oligonucleotides for their therapeutic potential in vivo. Our investigation revealed the upregulation of KCTD17 expression in both tumors from patients with HCC and mouse models of HCC, in comparison to non-tumor controls. We identified the leucine zipper-like transcriptional regulator 1 (Lztr1) protein, a previously identified Ras destabilizer, as a substrate for KCTD17-Cul3 complex. KCTD17-mediated Lztr1 degradation led to Ras stabilization, resulting in increased proliferation, migration, and wound healing in liver cancer cells. Hepatocyte-specific KCTD17 deficient mice or liver cancer xenograft models were less susceptible to carcinogenesis or tumor growth. Similarly, treatment with KCTD17-directed antisense oligonucleotides (ASO) in a mouse model of HCC markedly lowered tumor volume as well as Ras protein levels, compared to those in control ASO-treated mice. KCTD17 induces the stabilization of Ras and downstream signaling pathways and HCC progression and may represent a novel therapeutic target for HCC.

Functional distinction in oncogenic Ras variant activity in Caenorhabditis elegans.

Ras genes are important oncogenes that are frequently mutated in cancer. Human oncogenic variants exhibit functional distinctions in terms of their representation in different cancer types, impact on cellular targets and sensitivity to pharmacological treatments. However, how these distinct variants influence and respond to the cellular networks in which they are embedded is poorly understood. To identify novel participants in the complex interplay between Ras genotype and cell interaction networks in vivo, we have developed and tested an experimental framework using a simple vulva-development assay in the nematode C. elegans. Using this system, we evaluated a set of Ras oncogenic substitution changes at G12, G13 and Q61. We found that these variants fall into distinct groups based on phenotypic differences, sensitivity to gene dosage and inhibition of the downstream kinase MEK and their response to genetic modulators that influence Ras activity in a non-autonomous manner. Together, our results demonstrated that oncogenic C. elegans Ras variants exhibit clear distinctions in how they interface with the vulva-development network and showed that extracellular modulators yield variant-restricted effects in vivo.

The genetic duet of concurrent RASAL1 and PTEN alterations promotes cancer aggressiveness by cooperatively activating the PI3K-AKT pathway.

The significance of the prominent tumor suppressor gene for RAS protein activator-like 1 (RASAL1) could be better understood by combined genetic, clinical, and functional studies. Here, we investigated the oncogenic and clinical impacts of genetic alterations of RASAL1, particularly when coexisting with genetic alterations of the gene for phosphatase and tensin homolog (PTEN), in 9924 cancers of 33 types in the TCGA database. We found common concurrent genetic alterations of the two genes, which were cooperatively associated with activation of the phosphatidylinositol 3-kinase (PI3K)-AKT pathway, with cancer progression and mortality rates being 46.36% and 31.72% with concurrent gene alterations, versus 29.80% and 16.93% with neither gene alteration (HR 1.64, 95% CI 1.46-1.84 and 1.77, 95% CI 1.53-2.05), respectively. This was enhanced by additional tumor protein p53 (TP53) gene alterations, with cancer progression and mortality rates being 47.65% and 34.46% with coexisting RASAL1, PTEN, and TP53 alterations versus 25.30% and 13.11% with no alteration (HR 2.21, 95% CI 1.92-2.56 and 2.76, 95% CI 2.31-3.30), respectively. In the case of breast cancer, this genetic trio was associated with a triple-negative risk of 68.75% versus 3.83% with no genetic alteration (RR 17.94, 95% CI 9.60-33.51), consistent with the aggressive nature of triple-negative breast cancer. Mice with double knockouts of Rasal1 and Pten displayed robust Pi3k pathway activation, with the development of metastasizing malignancies, while single gene knockout resulted in only benign neoplasma. These results suggest that RASAL1, like PTEN, is a critical player in negatively regulating the PI3K-AKT pathway; defect in RASAL1 causes RAS activation, thus initiating the PI3K-AKT pathway signaling, which cannot terminate with concurrent PTEN defects. Thus, the unique concurrent RASAL1 and PTEN defects drive oncogenesis and cancer aggressiveness by cooperatively activating the PI3K-AKT pathway. This represents a robust genetic mechanism to promote human cancer.

Design, synthesis, and antitumor activity evaluation of BF3-o, m, p-phenylenediamine bridged with pyrimidine-indole BF3 adduction derivatives.

Fluorescent drugs and pyrimidine-indole scaffolds have been shown to have advantages in cancer treatment. Fluorescent antitumor drugs BF3-o, m, p-phenylenediamine pyrimidine-indole derivatives (PYB1, PYB2, and PYB3) were synthesized by linking pyrimidine and indole groups with aniline through a simple step and introducing BF3. The drugs exhibit promising antitumor activity and their fluorescent properties make them useful for imaging purposes. The optical properties of the three compounds have been investigated. All of them have fluorescent properties and compound PYB2 has good fluorescent properties. Additionally, the in vitro cytotoxicity of these compounds was evaluated against the human cancer cell line HeLa and the human normal cell line L02. The inhibition rates of HeLa cells treated with PYB1, PYB2, and PYB3 at a concentration of 19.2μg/mL were 80.91%, 77.72%, and 65.94%, respectively. These results indicate a strong inhibitory effect on cancer cells. Further through the cell imaging experiment, we can see that PYB2 can enter the cell through the cell membrane through the fluorescence scattering diagram, which has good biocompatibility. In addition, the possible interactions between the compounds and Ras protein active sites were analyzed by molecular docking. The three compounds can bind well to Ras protein through hydrogen bonding. This study provides a basis for the development and modification of pyrimidine-indole fluorescent anticancer drugs. Compound PYB2 shows potential as a fluorescent anticancer drug.

Complex interplay between RAS GTPases and RASSF effectors regulates subcellular localization of YAP

RAS GTPases bind effectors to convert upstream cues to changes in cellular function. Effectors of classical H/K/NRAS are defined by RBD/RA domains which recognize the GTP-bound conformation of these GTPases, yet the specificity of RBD/RAs for over 160 RAS superfamily proteins remains poorly explored. We have systematically mapped interactions between BRAF and four RASSF effectors, the largest family of RA-containing proteins, with all RAS, RHO and ARF small GTPases. 39 validated complexes reveal plasticity in RASSF binding, while BRAF demonstrates tight specificity for classical H/K/NRAS. Complex between RASSF5 and diverse RAS GTPases at the plasma membrane can activate Hippo signalling and sequester YAP in the cytosol. RASSF8 undergoes liquid-liquid phase separation and resides in YAP-associated membraneless condensates, which also engage several RAS and RHO GTPases. The poorly studied RASSF3 has been identified as a first potential effector of mitochondrial MIRO proteins, and its co-expression with these GTPases impacts mitochondria and peroxisome distribution. These data reveal the complex nature of GTPase-effector interactions and show their systematic elucidation can reveal completely novel and biologically relevant cellular processes.

Targeting Hyperactive Ras Signaling in Pediatric Cancer.

Somatic RAS mutations are among the most frequent drivers in pediatric and adult cancers. Somatic KRAS, NRAS, and HRAS mutations exhibit distinct tissue-specific predilections. Germline NF1 and RAS mutations in children with neurofibromatosis type 1 and other RASopathy developmental disorders have provided new insights into Ras biology. In many cases, these germline mutations are associated with increased cancer risk. Promising targeted therapeutic strategies for pediatric cancers and neoplasms with NF1 or RAS mutations include inhibition of downstream Ras effector pathways, directly inhibiting the signal output of oncogenic Ras proteins and associated pathway members, and therapeutically targeting Ras posttranslational modifications and intracellular trafficking. Acquired drug resistance to targeted drugs remains a significant challenge but, increasingly, rational drug combination approaches have shown promise in overcoming resistance. Developing predictive preclinical models of childhood cancers for drug testing is a high priority for the field of pediatric oncology.

Intrinsically Disordered Membrane Anchors of Rheb, RhoA, and DiRas3 Small GTPases: Molecular Dynamics, Membrane Organization, and Interactions.

Protein structure has been well established to play a key role in determining function; however, intrinsically disordered proteins and regions (IDPs and IDRs) defy this paradigm. IDPs and IDRs exist as an ensemble of structures rather than a stable 3D structure yet play essential roles in many cell-signaling processes. Nearly all Ras superfamily GTPases are tethered to membranes by a lipid tail at the end of a flexible IDR. The sequence of the IDR is a key determinant of membrane localization, and interaction between the IDR and the membrane has been shown to affect signaling in RAS proteins through the modulation of dynamic membrane organization. Here, we utilized atomistic molecular dynamics simulations to study the membrane interaction, conformational dynamics, and lipid sorting of three IDRs from small GTPases Rheb, RhoA, and DiRas3 in model membranes representing their physiological target membranes. We found that complementarity between the lipidated IDR sequence and target membrane lipid composition is a determinant of conformational plasticity. We also show that electrostatic interactions between anionic lipids and basic residues on IDRs are correlated with sampling of semistable conformational substates, and lack of these interactions is associated with greater conformational diversity. Finally, we show that small GTPase IDRs with a polybasic domain alter local lipid composition by segregating anionic lipids and, in some cases, excluding other lipids from their immediate vicinity in favor of anionic lipids.

Efficacy and safety of trifluridine/tipiracil plus bevacizumab across different subgroups of patients with refractory colorectal cancer: a meta-analysis.

Metastatic colorectal cancer (mCRC) patients who are refractory to initial treatment lines exhibit a challenging clinical scenario characterised by a poor prognosis and constrained therapeutic options. This systematic review and meta-analysis assess the integration of bevacizumab into trifluridine-tipiracil (TFD/TPI) therapy for mCRC, examining its benefits across patient subgroups and evaluating safety relative to TFD/TPI monotherapy. Following preferred reporting items for systematic reviews and meta-analysis statements, we conducted a thorough literature search from 15 October to 11 November 2023, covering MEDLINE, Embase and the Cochrane database. Data extraction and quality assessment followed Cochrane guidelines, and hazard or odds ratios with 95% confidence intervals (CI) were pooled (p < 0.05 significance threshold). The study protocol is registered in PROSPERO (CRD42023484695). Analysing 770 database results, we included two randomised controlled trials and five observational studies covering over 4,000 patients. Combined therapy exhibited significant improvements in overall survival (OS) hazard ratios (HR 0.60; 95% CI 0.49-0.72; p < 0.01) and progression-free survival (HR 0.48; 95% CI 0.40-0.59; p < 0.01). Subgroups, including prior bevacizumab exposure (HR 0.70; 95% CI 0.64-0.77; p < 0.01) and mutated RAS gene (HR 0.64; 95% CI 0.53-0.77; p < 0.01), demonstrated improvements in OSwith bevacizumab. This meta-analysis underscores the heightened efficacy of TFD/TPI combined with bevacizumab for refractory mCRC compared to TFD/TPI monotherapy across diverse subgroups. Combined therapy has increased grade ≥3 neutropenia and hypertension, while monotherapy is associated with fatigue and anemia.