Concurrent inhibition of oncogenic and wild-type RAS-GTP for cancer therapy.

RAS oncogenes (collectively NRAS, HRAS and especially KRAS) are among the most frequently mutated genes in cancer, with common driver mutations occurring at codons 12, 13 and 611. Small molecule inhibitors of the KRAS(G12C) oncoprotein have demonstrated clinical efficacy in patients with multiple cancer types and have led to regulatory approvals for the treatment of non-small cell lung cancer2,3. Nevertheless, KRASG12C mutations account for only around 15% of KRAS-mutated cancers4,5, and there are no approved KRAS inhibitors for the majority of patients with tumours containing other common KRAS mutations. Here we describe RMC-7977, a reversible, tri-complex RAS inhibitor with broad-spectrum activity for the active state of both mutant and wild-type KRAS, NRAS and HRAS variants (a RAS(ON) multi-selective inhibitor). Preclinically, RMC-7977 demonstrated potent activity against RAS-addicted tumours carrying various RAS genotypes, particularly against cancer models with KRAS codon 12 mutations (KRASG12X). Treatment with RMC-7977 led to tumour regression and was well tolerated in diverse RAS-addicted preclinical cancer models. Additionally, RMC-7977 inhibited the growth of KRASG12C cancer models that are resistant to KRAS(G12C) inhibitors owing to restoration of RAS pathway signalling. Thus, RAS(ON) multi-selective inhibitors can target multiple oncogenic and wild-type RAS isoforms and have the potential to treat a wide range of RAS-addicted cancers with high unmet clinical need. A related RAS(ON) multi-selective inhibitor, RMC-6236, is currently under clinical evaluation in patients with KRAS-mutant solid tumours (ClinicalTrials.gov identifier: NCT05379985).

CPEB2 inhibit cell proliferation through upregulating p21 mRNA stability in glioma.

Glioma is the most common primary malignant brain tumor in adults and remains an incurable disease at present. Thus, there is an urgent need for progress in finding novel molecular mechanisms that control the progression of glioma which could be used as therapeutic targets for glioma patients. The RNA binding protein cytoplasmic polyadenylate element-binding protein 2 (CPEB2) is involved in the pathogenesis of several tumors. However, the role of CPEB2 in glioma progression is unknown. In this study, the functional characterization of the role and molecular mechanism of CPEB2 in glioma were examined using a series of biological and cellular approaches in vitro and in vivo. Our work shows CPEB2 is significantly downregulated in various glioma patient cohorts. Functional characterization of CPEB2 by overexpression and knockdown revealed that it inhibits glioma cell proliferation and promotes apoptosis. CPEB2 exerts an anti-tumor effect by increasing p21 mRNA stability and inducing G1 cell cycle arrest in glioma. Overall, this work stands as the first report of CPEB2 downregulation and involvement in glioma pathogenesis, and identifies CPEB2 as an important tumor suppressor gene through targeting p21 in glioma, which revealed that CPEB2 may become a promising predictive biomarker for prognosis in glioma patients.

The possibility of mutations of RAS signaling genes and/or TP53 in combination as a negative prognostic impact on pathological stage I non-small cell lung cancer.

BACKGROUND: The recurrence rate of non-small cell lung cancer (NSCLC) is as high as 30%, even in the cancer with pathological stage I disease. Therefore, identifying factors predictive of high-risk pathological recurrence is important. However, few studies have examined the genetic status of these tumors and its relationship to prognosis. MATERIALS AND METHODS: A cohort of 328 cases of primary lung cancer that underwent complete resection at Tokyo Medical and Dental University (TMDU) was screened for 440 cancer-associated genes using panel testing. Further analyses included 92 cases of pathological stage I NSCLC who did not receive adjuvant chemotherapy. Ridge regression was performed to identify association studies mutational status and postoperative recurrence. These data were then validated using clinical and genetic data from 56 patients in The Cancer Genome Atlas (TCGA). RESULTS: Mutations in TP53, RAS signaling genes KRAS and HRAS, and EGFR were recurrently detected. Ridge regression analysis relevant to recurrence, as well as survival analysis, performed using data from the TMDU cohort revealed significantly shorter relapse-free survival (RFS) for patients with RAS signaling or TP53 gene mutations than for those without (log-rank test, p = 0.00090). This statistical trend was also suggested in the TCGA cohort (log-rank test, p = 0.10). CONCLUSION: Mutations in RAS signaling genes and/or TP53 could be useful for the prediction of shorter RFS of patients with stage I NSCLC.

Recent Developments in Targeting RAS Downstream Effectors for RAS-Driven Cancer Therapy.

Aberrant activity of oncogenic rat sarcoma virus (RAS) protein promotes tumor growth and progression. RAS-driven cancers comprise more than 30% of all human cancers and are refractory to frontline treatment strategies. Since direct targeting of RAS has proven challenging, efforts have been centered on the exploration of inhibitors for RAS downstream effector kinases. Two major RAS downstream signaling pathways, including the Raf/MEK/Erk cascade and the phosphatidylinositol-3-kinase (PI3K) pathway, have become compelling targets for RAS-driven cancer therapy. However, the main drawback in the blockade of a single RAS effector is the multiple levels of crosstalk and compensatory mechanisms between these two pathways that contribute to drug resistance against monotherapies. A growing body of evidence reveals that the sequential or synergistic inhibition of multiple RAS effectors is a more convenient route for the efficacy of cancer therapy. Herein, we revisit the recent developments and discuss the most promising modalities targeting canonical RAS downstream effectors for the treatment of RAS-driven cancers.

EPHA2-dependent outcompetition of KRASG12D mutant cells by wild-type neighbors in the adult pancreas.

As we age, our tissues are repeatedly challenged by mutational insult, yet cancer occurrence is a relatively rare event. Cells carrying cancer-causing genetic mutations compete with normal neighbors for space and survival in tissues. However, the mechanisms underlying mutant-normal competition in adult tissues and the relevance of this process to cancer remain incompletely understood. Here, we investigate how the adult pancreas maintains tissue health in vivo following sporadic expression of oncogenic Kras (KrasG12D), the key driver mutation in human pancreatic cancer. We find that when present in tissues in low numbers, KrasG12D mutant cells are outcompeted and cleared from exocrine and endocrine compartments in vivo. Using quantitative 3D tissue imaging, we show that before being cleared, KrasG12D cells lose cell volume, pack into round clusters, and E-cadherin-based cell-cell adhesions decrease at boundaries with normal neighbors. We identify EphA2 receptor as an essential signal in the clearance of KrasG12D cells from exocrine and endocrine tissues in vivo. In the absence of functional EphA2, KrasG12D cells do not alter cell volume or shape, E-cadherin-based cell-cell adhesions increase and KrasG12D cells are retained in tissues. The retention of KRasG12D cells leads to the early appearance of premalignant pancreatic intraepithelial neoplasia (PanINs) in tissues. Our data show that adult pancreas tissues remodel to clear KrasG12D cells and maintain tissue health. This study provides evidence to support a conserved functional role of EphA2 in Ras-driven cell competition in epithelial tissues and suggests that EphA2 is a novel tumor suppressor in pancreatic cancer.

Non-cell-autonomous migration of RasV12-transformed cells towards the basal side of surrounding normal cells.

Oncogenic transformation enables cells to behave differently from their neighboring normal cells. Both cancer and normal cells recognize each other, often promoting the extrusion of the former from the epithelial cell layer. Here, we show that RasV12-transformed normal rat kidney 52E (NRK-52E) cells are extruded towards the basal side of the surrounding normal cells, which is concomitant with enhanced motility. The active migration of the basally extruded RasV12 cells is observed when surrounded by normal cells, indicating a non-cell-autonomous mechanism. Furthermore, specific inhibitor treatment and knockdown experiments elucidate the roles of PI3K and myosin IIA in the basal extrusion of Ras cells. Our findings reveal a new aspect of cancer cell invasion mediated by functional interactions with surrounding non-transformed cells.

A phospho-switch controls RNF43-mediated degradation of Wnt receptors to suppress tumorigenesis.

Frequent mutation of the tumour suppressor RNF43 is observed in many cancers, particularly colon malignancies. RNF43, an E3 ubiquitin ligase, negatively regulates Wnt signalling by inducing degradation of the Wnt receptor Frizzled. In this study, we discover that RNF43 activity requires phosphorylation at a triplet of conserved serines. This phospho-regulation of RNF43 is required for zebrafish development and growth of mouse intestinal organoids. Cancer-associated mutations that abrogate RNF43 phosphorylation cooperate with active Ras to promote tumorigenesis by abolishing the inhibitory function of RNF43 in Wnt signalling while maintaining its inhibitory function in p53 signalling. Our data suggest that RNF43 mutations cooperate with KRAS mutations to promote multi-step tumorigenesis via the Wnt-Ras-p53 axis in human colon cancers. Lastly, phosphomimetic substitutions of the serine trio restored the tumour suppressive activity of extracellular oncogenic mutants. Therefore, harnessing phospho-regulation of RNF43 might be a potential therapeutic strategy for tumours with RNF43 mutations.

ERas regulates cell proliferation and epithelial-mesenchymal transition by affecting Erk/Akt signaling pathway in pancreatic cancer.

Pancreatic cancer is the fourth most common lethal malignancy with an overall 5-year survival rate of less than 5%. ERas, a novel Ras family member, was first identified in murine embryonic stem cells and is upregulated in various cancers. However, the expression and potential role of ERas in pancreatic cancer have not been investigated. In this study, we found that ERas mRNA and protein were upregulated in pancreatic cancer tissues and cells compared with controls. Knockdown of ERas in pancreatic cancer cells by siRNA significantly decreased cell proliferation, colony formation, migration, and invasion and promoted cell apoptosis in vitro. Epithelial-mesenchymal transition (EMT) is closely related to tumor progression. We observed a significant decrease in N-cadherin expression in pancreatic cancer cells in response to ERas gene silencing by immunofluorescence assay and western blot. Furthermore, tumor growth and EMT were inhibited in xenografts derived from pancreatic cancer cells with ERas downregulation. We further investigated the regulatory mechanisms of ERas in pancreatic cancer and found that ERas may activate the Erk/Akt signaling pathway. Moreover, Erk inhibitor decreased pancreatic cancer cells proliferation and colony formation activities. Our data suggest that targeting ERas and its relevant signaling pathways might represent a novel therapeutic approach for the treatment of pancreatic cancer.

Single-cell analysis based dissection of clonality in myelofibrosis.

Cancer development is an evolutionary genomic process with parallels to Darwinian selection. It requires acquisition of multiple somatic mutations that collectively cause a malignant phenotype and continuous clonal evolution is often linked to tumor progression. Here, we show the clonal evolution structure in 15 myelofibrosis (MF) patients while receiving treatment with JAK inhibitors (mean follow-up 3.9 years). Whole-exome sequencing at multiple time points reveal acquisition of somatic mutations and copy number aberrations over time. While JAK inhibition therapy does not seem to create a clear evolutionary bottleneck, we observe a more complex clonal architecture over time, and appearance of unrelated clones. Disease progression associates with increased genetic heterogeneity and gain of RAS/RTK pathway mutations. Clonal diversity results in clone-specific expansion within different myeloid cell lineages. Single-cell genotyping of circulating CD34 + progenitor cells allows the reconstruction of MF phylogeny demonstrating loss of heterozygosity and parallel evolution as recurrent events.

Co-occurring Alterations in the RAS-MAPK Pathway Limit Response to MET Inhibitor Treatment in MET Exon 14 Skipping Mutation-Positive Lung Cancer.

PURPOSE: Although patients with advanced-stage non-small cell lung cancers (NSCLC) harboring MET exon 14 skipping mutations (METex14) often benefit from MET tyrosine kinase inhibitor (TKI) treatment, clinical benefit is limited by primary and acquired drug resistance. The molecular basis for this resistance remains incompletely understood. EXPERIMENTAL DESIGN: Targeted sequencing analysis was performed on cell-free circulating tumor DNA obtained from 289 patients with advanced-stage METex14-mutated NSCLC. RESULTS: Prominent co-occurring RAS-MAPK pathway gene alterations (e.g., in KRAS, NF1) were detected in NSCLCs with METex14 skipping alterations as compared with EGFR-mutated NSCLCs. There was an association between decreased MET TKI treatment response and RAS-MAPK pathway co-occurring alterations. In a preclinical model expressing a canonical METex14 mutation, KRAS overexpression or NF1 downregulation hyperactivated MAPK signaling to promote MET TKI resistance. This resistance was overcome by cotreatment with crizotinib and the MEK inhibitor trametinib. CONCLUSIONS: Our study provides a genomic landscape of co-occurring alterations in advanced-stage METex14-mutated NSCLC and suggests a potential combination therapy strategy targeting MAPK pathway signaling to enhance clinical outcomes.

KRAS: A Promising Therapeutic Target for Cancer Treatment.

Kirsten rat sarcoma 2 viral oncogene homolog (KRAS) is the most commonly mutated oncogene in human cancer. The developments of many cancers depend on sustained expression and signaling of KRAS, which makes KRAS a high-priority therapeutic target. Scientists have not successfully developed drugs that target KRAS, although efforts have been made last three decades. In this review, we highlight the emerging experimental strategies of impairing KRAS membrane localization and the direct targeting of KRAS. We also conclude the combinatorial therapies and RNA interference technology for the treatment of KRAS mutant cancers. Moreover, the virtual screening approach to discover novel KRAS inhibitors and synthetic lethality interactors of KRAS are discussed in detail.

Overview of Current Immunotherapies Targeting Mutated KRAS Cancers.

The occurrence of somatic substitution mutations of the KRAS proto-oncogene is highly prevalent in certain cancer types, which often leads to constant activation of proliferative pathways and subsequent neoplastic transformation. It is often seen as a gateway mutation in carcinogenesis and has been commonly deemed as a predictive biomarker for poor prognosis and relapse when conventional chemotherapeutics are employed. Additionally, its mutational status also renders EGFR targeted therapies ineffective owing to its downstream location. Efforts to discover new approaches targeting this menacing culprit have been ongoing for years without much success, and with incidences of KRAS positive cancer patients being on the rise, researchers are now turning towards immunotherapies as the way forward. In this scoping review, recent immunotherapeutic developments and advances in both preclinical and clinical studies targeting K-ras directly or indirectly via its downstream signal transduction machinery will be discussed. Additionally, some of the challenges and limitations of various K-ras targeting immunotherapeutic approaches such as vaccines, adoptive T cell therapies, and checkpoint inhibitors against KRAS positive cancers will be deliberated.

Targeting Mutant KRAS for Anticancer Therapy.

Over the past decades, designing therapeutic strategies to target KRAS-mutant cancers, which is one of the most frequent mutant oncogenes among all cancer types, have proven unsuccessful regardless of many concerted attempts. There are key challenges for KRAS-mutant anticancer therapy, as the complex cellular processes involved in KRAS signaling has present. Herein, we highlight the emerging therapeutic approaches for inhibiting KRAS signaling and blocking KRAS functions, in hope to serve as a more effective guideline for future development of therapeutics.

Recent Advances in Developing K-Ras Plasma Membrane Localization Inhibitors.

The Ras proteins play an important role in cell growth, differentiation, proliferation and survival by regulating diverse signaling pathways. Oncogenic mutant K-Ras is the most frequently mutated class of Ras superfamily that is highly prevalent in many human cancers. Despite intensive efforts to combat various K-Ras-mutant-driven cancers, no effective K-Ras-specific inhibitors have yet been approved for clinical use to date. Since K-Ras proteins must be associated to the plasma membrane for their function, targeting K-Ras plasma membrane localization represents a logical and potentially tractable therapeutic approach. Here, we summarize the recent advances in the development of K-Ras plasma membrane localization inhibitors including natural product-based inhibitors achieved from high throughput screening, fragment-based drug design, virtual screening, and drug repurposing as well as hit-to-lead optimizations.

Drug Combinatorial Therapies for the Treatment of KRAS Mutated Lung Cancers.

KRAS is the most common oncogene to be mutated in lung cancer, and therapeutics directly targeting KRAS have proven to be challenging. The mutations of KRAS are associated with poor prognosis, and resistance to both adjuvant therapy and targeted EGFR TKI. EGFR TKIs provide significant clinical benefit for patients whose tumors bear EGFR mutations. However, tumors with KRAS mutations rarely respond to the EGFR TKI therapy. Thus, combination therapy is essential for the treatment of lung cancers with KRAS mutations. EGFR TKI combined with inhibitors of MAPKs, PI3K/mTOR, HDAC, Wee1, PARP, CDK and Hsp90, even miRNAs and immunotherapy, were reviewed. Although the effects of the combination vary, the combined therapeutics are one of the best options at present to treat KRAS mutant lung cancer.

Mitophagy mediated by BNIP3 and BNIP3L/NIX in urothelial cells of the urinary bladder of cattle harbouring bovine papillomavirus infection.

Autophagy is a powerful tool that host cells use to defend against viral infection. Mitophagy, the selective autophagic removal of dysfunctional mitochondria was upregulated in urothelial cancer cells harbouring bovine papillomavirus (BPV) infection, as detected by the expression of BPV E5 protein, the major oncoprotein of bovine Deltapapillomavirus genus. HIF-1α-induced mitophagy receptors, BNIP3 and BNIP3L/Nix, were found to be overexpressed in these cells. The BNIP3 and BNIP3L/Nix receptors were amplified, and amplicon sequencing showed homology between bovine BNPI3 and BNIP3L/Nix sequences deposited in GenBank (accession number: NM_001076366.1 and NM_001034614.2, respectively). The transcripts and protein levels of BNIP3 and BNIP3L/Nix were significantly overexpressed in hypoxic neoplastic cells relative to healthy, non-neoplastic cells. BNIP3 and BNIP3L/Nix interacted with the LC3 protein, a marker of autophagosome (mitophagosome) membrane, ERAS, a small GTPase, and p62, known to be a specific autophagy receptor protein, that plays a role in mitochondrial priming for mitophagy and subsequent elimination. ERAS also interacted with the BPV E5 oncoprotein at mitochondrial level. Furthermore, in anti-Bag3 mitochondrial immunoprecipitates, a complex composed of the Hsc70/Hsp70 chaperone, CHIP co-chaperone, Synpo2, ERAS, LC3, p62, BNPI3, and BNIP3L/Nix was also detected. Bag3 may play a role in mitophagosome formation together with the Synpo2 protein and may be involved in the degradation of Hsc70/Hsp70-bound CHIP-ubiquitinated cargo, in association with its chaperone. ERAS may be involved in mitophagosome maturation via the PI3K signalling pathway. Ultrastructural findings revealed the presence of mitochondria exhibiting severe fragmentation and loss of cristae, as well as numerous mitochondria-containing autophagosomes.

The Application of the RNA Interference Technologies for KRAS: Current Status, Future Perspective and Associated Challenges.

KRAS is a member of the murine sarcoma virus oncogene-RAS gene family. It plays an important role in the prevention, diagnosis and treatment of tumors during tumor cell growth and angiogenesis. KRAS is the most commonly mutated oncogene in human cancers, such as pancreatic cancers, colon cancers, and lung cancers. Detection of KRAS gene mutation is an important indicator for tracking the status of oncogenes, highlighting the developmental prognosis of various cancers, and the efficacy of radiotherapy and chemotherapy. However, the efficacy of different patients in clinical treatment is not the same. Since RNA interference (RNAi) technologies can specifically eliminate the expression of specific genes, these technologies have been widely used in the field of gene therapy for exploring gene function, infectious diseases and malignant tumors. RNAi refers to the phenomenon of highly specific degradation of homologous mRNA induced by double-stranded RNA (dsRNA), which is highly conserved during evolution. There are three classical RNAi technologies, including siRNA, shRNA and CRISPR-Cas9 system, and a novel synthetic lethal interaction that selectively targets KRAS mutant cancers. Therefore, the implementation of individualized targeted drug therapy has become the best choice for doctors and patients. Thus, this review focuses on the current status, future perspective and associated challenges in silencing of KRAS with RNAi technology.

Impact of circulating tumor DNA mutant allele fraction on prognosis in RAS-mutant metastatic colorectal cancer.

Despite major advances in the treatment of metastatic colorectal cancer (mCRC), the survival rate remains very poor. This study aims at exploring the prognostic value of RAS-mutant allele fraction (MAF) in plasma in mCRC. Forty-seven plasma samples from 37 RAS-mutated patients with nonresectable metastases were tested for RAS in circulating tumor DNA using BEAMing before first- and/or second-line treatment. RAS MAF was correlated with several clinical parameters (number of metastatic sites, hepatic volume, carcinoembryonic antigen, CA19-9 levels, primary site location, and treatment line) and clinical outcome [progression-free survival (PFS) and overall survival (OS)]. An independent cohort of 32 patients from the CAPRI-GOIM trial was assessed for clinical outcome based on plasma baseline MAF. RAS MAF analysis at baseline revealed a significant correlation with longer OS [Hazard ratios (HR) = 3.514; P = 0.00066]. Patients with lower MAF also showed a tendency to longer PFS, although not statistically significant. Multivariate analysis showed RAS MAFs as an independent prognostic factor in both OS (HR = 2.73; P = 0.006) and first-line PFS (HR = 3.74; P = 0.049). Tumor response to treatment in patients with higher MAF was progression disease (P = 0.007). Patients with low MAFs at baseline in the CAPRI-GOIM group also showed better OS [HR = 3.84; 95% confidence intervals (CI) 1.5-9.6; P = 0.004] and better PFS (HR = 2.5; 95% CI: 1.07-5.62; P = 0.033). This minimally invasive test may help in adding an independent factor to better estimate outcomes before initiating treatment. Further prospective studies using MAF as a stratification factor could further validate its utility in clinical practice.

The Extract of Cordyceps Militaris Inhibited the Proliferation of Cisplatin-Resistant A549 Lung Cancer Cells by Downregulation of H-Ras.

We investigated the antitumor effect of Cordyceps militaris extract (CME) on A549 cisplatin-resistant (CR) lung cancer cells. The proliferation of A549/CR cells was suppressed by CME. Apoptosis of the cells was induced by CME. The cell cycle arrest was observed in the sub-G1 phase in the cells treated with CME. Proteomic profile analysis showed that H-Ras was downregulated in CME-treated cells and it was confirmed by western blot analysis. Collectively, these data demonstrated that CME is an alternative treatment for the anticancer effect.