Human aneuploid cells depend on the RAF/MEK/ERK pathway for overcoming increased DNA damage.

Aneuploidy is a hallmark of human cancer, yet the molecular mechanisms to cope with aneuploidy-induced cellular stresses remain largely unknown. Here, we induce chromosome mis-segregation in non-transformed RPE1-hTERT cells and derive multiple stable clones with various degrees of aneuploidy. We perform a systematic genomic, transcriptomic and proteomic profiling of 6 isogenic clones, using whole-exome DNA, mRNA and miRNA sequencing, as well as proteomics. Concomitantly, we functionally interrogate their cellular vulnerabilities, using genome-wide CRISPR/Cas9 and large-scale drug screens. Aneuploid clones activate the DNA damage response and are more resistant to further DNA damage induction. Aneuploid cells also exhibit elevated RAF/MEK/ERK pathway activity and are more sensitive to clinically-relevant drugs targeting this pathway, and in particular to CRAF inhibition. Importantly, CRAF and MEK inhibition sensitize aneuploid cells to DNA damage-inducing chemotherapies and to PARP inhibitors. We validate these results in human cancer cell lines. Moreover, resistance of cancer patients to olaparib is associated with high levels of RAF/MEK/ERK signaling, specifically in highly-aneuploid tumors. Overall, our study provides a comprehensive resource for genetically-matched karyotypically-stable cells of various aneuploidy states, and reveals a therapeutically-relevant cellular dependency of aneuploid cells.

Promotion of Raf-1/ASK1 complex formation by corylin inhibits cell apoptosis in myocardial ischemia/reperfusion injury.

Effective treatment of myocardial ischemia-reperfusion (MIR) injury remains an unmet clinical need. Cardiomyocyte apoptosis is common at this stage and poses a significant risk. Corylin, a flavonoid compound extracted from Psoralea corylifolia L., has been shown to have anti-inflammatory, anticancer, and antiatherosclerotic properties. However, whether and how corylin affects MIR injury remain unclear. In this study, we explored the mechanism of corylin as a potent therapeutic agent for MI/R injury, using a left anterior descending (LAD) coronary artery ligation and oxygen-glucose deprivation and reperfusion (OGD/R) model in vivo and in vitro. TUNEL, Annexin-V/PI double staining,Ki67 immunohistochemistry, western blot analysis, and immunofluorescence were used to validate cell apoptosis level and Raf-1/ASK1 complex activity. The interaction between corylin and Raf-1/ASK1 complex was detected using molecular docking, corylin-Raf-1 binding assays, and coimmunoprecipitation (Co-IP). Moreover, TTC staining, echocardiography, HE staining, Masson trichrome staining and serological testing were performed to assess the cardioprotective effects of corylin in vivo. These findings showed that corylin reduces MIR injury-induced cardiomyocyte apoptosis and improves cardiac function. Mechanistically, corylin can interact with Raf-1 and promote the formation of the Raf-1/ASK1 complex, thus inhibiting cardiomyocyte apoptosis. In conclusion, our results demonstrate that corylin ameliorated cardiac dysfunction after MIR injury by reducing myocardial apoptosis.

Insight into molecular basis and dynamics of full-length CRaf kinase in cellular signaling mechanisms.

Raf kinases play key roles in signal transduction in cells for regulating proliferation, differentiation, and survival. Despite decades of research into functions and dynamics of Raf kinases with respect to other cytosolic proteins, understanding Raf kinases is limited by the lack of their full-length structures at the atomic resolution. Here, we present the first model of the full-length CRaf kinase obtained from artificial intelligence/machine learning algorithms with a converging ensemble of structures simulated by large-scale temperature replica exchange simulations. Our model is validated by comparing simulated structures with the latest cryo-EM structure detailing close contacts among three key domains and regions of the CRaf. Our simulations identify potentially new epitopes of intramolecule interactions within the CRaf and reveal a dynamical nature of CRaf kinases, in which the three domains can move back and forth relative to each other for regulatory dynamics. The dynamic conformations are then used in a docking algorithm to shed insight into the paradoxical effect caused by vemurafenib in comparison with a paradox breaker PLX7904. We propose a model of Raf-heterodimer/KRas-dimer as a signalosome based on the dynamics of the full-length CRaf.

A specific super-enhancer actuated by berberine regulates EGFR-mediated RAS-RAF1-MEK1/2-ERK1/2 pathway to induce nasopharyngeal carcinoma autophagy.

Nasopharyngeal carcinoma (NPC), primarily found in the southern region of China, is a malignant tumor known for its highly metastatic characteristics. The high mortality rates caused by the distant metastasis and disease recurrence remain unsolved clinical problems. In clinic, the berberine (BBR) compound has widely been in NPC therapy to decrease metastasis and disease recurrence, and BBR was documented as a main component with multiple anti-NPC effects. However, the mechanism by which BBR inhibits the growth and metastasis of nasopharyngeal carcinoma remains elusive. Herein, we show that BBR effectively inhibits the growth, metastasis, and invasion of NPC via inducing a specific super enhancer (SE). From a mechanistic perspective, the RNA sequencing (RNA-seq) results suggest that the RAS-RAF1-MEK1/2-ERK1/2 signaling pathway, activated by the epidermal growth factor receptor (EGFR), plays a significant role in BBR-induced autophagy in NPC. Blockading of autophagy markedly attenuated the effect of BBR-mediated NPC cell growth and metastasis inhibition. Notably, BBR increased the expression of EGFR by transcription, and knockout of EGFR significantly inhibited BBR-induced microtubule associated protein 1 light chain 3 (LC3)-II increase and p62 inhibition, proposing that EGFR plays a pivotal role in BBR-induced autophagy in NPC. Chromatin immunoprecipitation sequencing (ChIP-seq) results found that a specific SE existed only in NPC cells treated with BBR. This SE knockdown markedly repressed the expression of EGFR and phosphorylated EGFR (EGFR-p) and reversed the inhibition of BBR on NPC proliferation, metastasis, and invasion. Furthermore, BBR-specific SE may trigger autophagy by enhancing EGFR gene transcription, thereby upregulating the RAS-RAF1-MEK1/2-ERK1/2 signaling pathway. In addition, in vivo BBR effectively inhibited NPC cells growth and metastasis, following an increase LC3 and EGFR and a decrease p62. Collectively, this study identifies a novel BBR-special SE and established a new epigenetic paradigm, by which BBR regulates autophagy, inhibits proliferation, metastasis, and invasion. It provides a rationale for BBR application as the treatment regime in NPC therapy in future.

Defining the variant-phenotype correlation in patients affected by Noonan syndrome with the RAF1:c.770C>T p.(Ser257Leu) variant.

Hypertrophic cardiomyopathy (HCM) is the major contributor to morbidity and mortality in Noonan syndrome (NS). Gain-of-function variants in RAF1 are associated with high prevalence of HCM. Among these, NM_002880.4:c.770C > T, NP_002871.1:p.(Ser257Leu) accounts for approximately half of cases and has been reported as associated with a particularly severe outcome. Nevertheless, comprehensive studies on cases harboring this variant are missing. To precisely define the phenotype associated to the RAF1:c.770C > T, variant, an observational retrospective analysis on patients carrying the c.770C > T variant was conducted merging 17 unpublished patients and literature-derived ones. Data regarding prenatal findings, clinical features and cardiac phenotypes were collected to provide an exhaustive description of the associated phenotype. Clinical information was collected in 107 patients. Among them, 92% had HCM, mostly diagnosed within the first year of life. Thirty percent of patients were preterm and 47% of the newborns was admitted in a neonatal intensive care unit, mainly due to respiratory complications of HCM and/or pulmonary arterial hypertension. Mortality rate was 13%, mainly secondary to HCM-related complications (62%) at the average age of 7.5 months. Short stature had a prevalence of 91%, while seizures and ID of 6% and 12%, respectively. Two cases out of 75 (3%) developed neoplasms. In conclusion, patients with the RAF1:c.770C > T pathogenic variant show a particularly severe phenotype characterized by rapidly progressive neonatal HCM and high mortality rate suggesting the necessity of careful monitoring and early intervention to prevent or slow down the progression of HCM.

Vitamin D Relieves Epilepsy Symptoms and Neuroinflammation in Juvenile Mice by Activating the mTOR Signaling Pathway via RAF1: Insights from Network Pharmacology and Molecular Docking Studies.

Epilepsy is a common neurological disorder, and the exploration of potential therapeutic drugs for its treatment is still ongoing. Vitamin D has emerged as a promising treatment due to its potential neuroprotective effects and anti-epileptic properties. This study aimed to investigate the effects of vitamin D on epilepsy and neuroinflammation in juvenile mice using network pharmacology and molecular docking, with a focus on the mammalian target of rapamycin (mTOR) signaling pathway. Experimental mouse models of epilepsy were established through intraperitoneal injection of pilocarpine, and in vitro injury models of hippocampal neurons were induced by glutamate (Glu) stimulation. The anti-epileptic effects of vitamin D were evaluated both in vivo and in vitro. Network pharmacology and molecular docking analysis were used to identify potential targets and regulatory pathways of vitamin D in epilepsy. The involvement of the mTOR signaling pathway in the regulation of mouse epilepsy by vitamin D was validated using rapamycin (RAPA). The levels of inflammatory cytokines (TNF-α, IL-1ß, and IL-6) were assessed by enzyme-linked immunosorbent assay (ELISA). Gene and protein expressions were detected by quantitative real-time polymerase chain reaction (qRT-PCR) and Western blot, respectively. The terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick end-labeling (TUNEL) staining was used to analyze the apoptosis of hippocampal neurons. In in vivo experiments, vitamin D reduced the Racine scores of epileptic mice, prolonged the latency of epilepsy, and inhibited the production of TNF-α, IL-1ß, and IL-6 in the hippocampus. Furthermore, network pharmacology analysis identified RAF1 as a potential target of vitamin D in epilepsy, which was further confirmed by molecular docking analysis. Additionally, the mTOR signaling pathway was found to be involved in the regulation of mouse epilepsy by vitamin D. In in vitro experiments, Glu stimulation upregulated the expressions of RAF1 and LC3II/LC3I, inhibited mTOR phosphorylation, and induced neuronal apoptosis. Mechanistically, vitamin D activated the mTOR signaling pathway and alleviated mouse epilepsy via RAF1, while the use of the pathway inhibitor RAPA reversed this effect. Vitamin D alleviated epilepsy symptoms and neuroinflammation in juvenile mice by activating the mTOR signaling pathway via RAF1. These findings provided new insights into the molecular mechanisms underlying the anti-epileptic effects of vitamin D and further supported its use as an adjunctive therapy for existing anti-epileptic drugs.

The USP10/13 inhibitor, spautin-1, attenuates the progression of glioblastoma by independently regulating RAF-ERK mediated glycolysis and SKP2.

Glioblastoma is a malignant brain tumor with poor prognosis. Though several dysregulated pathways were found to mediate the tumor progression, hyperactivation of RAS-RAF-ERK pathway, enhanced glycolysis and SKP2 are associated with several glioblastomas. Recent findings on the role of USP10 in the transition from pro-neural to mesenchymal subtype of glioblastoma and, USP13 in the stabilization of RAF1 in mouse embryonic stem cells prompted us to examine their role in the mechanisms mediating the progression of glioblastoma. In the present study, we have examined the role of spautin-1, a pharmacological inhibitor of USP10 and USP13 in the mechanisms mediating glioblastoma. Our results indicate that spautin-1 as well as knockdown of its downstream targets, USP10 and USP13, reduced the proliferation and migration of glioblastoma cells. Also, spautin-1 mediated inhibition of RAF-ERK pathway or inhibition of RAF1 and MEK1 per se reduced the glycolytic function via PKM2/Glut-1 and inhibited the progression of glioblastoma. Further, the protooncogene, SKP2, which was shown to be a direct target of USP10 /USP13 was also reduced by spautin-1. While inhibition of SKP2 enhanced its downstream target p21, no apparent changes in the RAF-ERK levels or glycolytic function were evident. Also, inhibition of MEK1 did not affect SKP2 levels, indicating that these two pathways act independent of each other. Overall, our findings indicate that spautin-1 by virtue of its inhibitory effects on USP10/13 counteracts RAS-RAF-ERK mediated glycolysis and SKP2 that are critical in the progression of glioblastoma. Hence, further preclinical validation is warranted for taking the present observations forward.

Regulation of RAF family kinases: new insights from recent structural and biochemical studies.

The RAF kinases are required for signal transduction through the RAS-RAF-MEK-ERK pathway, and their activity is frequently up-regulated in human cancer and the RASopathy developmental syndromes. Due to their complex activation process, developing drugs that effectively target RAF function has been a challenging endeavor, highlighting the need for a more detailed understanding of RAF regulation. This review will focus on recent structural and biochemical studies that have provided 'snapshots' into the RAF regulatory cycle, revealing structures of the autoinhibited BRAF monomer, active BRAF and CRAF homodimers, as well as HSP90/CDC37 chaperone complexes containing CRAF or BRAFV600E. In addition, we will describe the insights obtained regarding how BRAF transitions between its regulatory states and examine the roles that various BRAF domains and 14-3-3 dimers play in both maintaining BRAF as an autoinhibited monomer and in facilitating its transition to an active dimer. We will also address the function of the HSP90/CDC37 chaperone complex in stabilizing the protein levels of CRAF and certain oncogenic BRAF mutants, and in serving as a platform for RAF dephosphorylation mediated by the PP5 protein phosphatase. Finally, we will discuss the regulatory differences observed between BRAF and CRAF and how these differences impact the function of BRAF and CRAF as drivers of human disease.

RAF1 facilitates KIT signaling and serves as a potential treatment target for gastrointestinal stromal tumor.

The aberrant activation of RAS/RAF/MEK/ERK signaling is important for KIT mutation-mediated tumorigenesis of gastrointestinal stromal tumor (GIST). In this study, we found that inhibition of RAF1 suppresses the activation of both wild-type KIT and primary KIT mutations in GIST, with primary KIT mutations showing greater sensitivity. This suggests a positive feedback loop between KIT and RAF1, wherein RAF1 facilitates KIT signaling. We further demonstrated that RAF1 associates with KIT and the kinase activity of RAF1 is necessary for its contribution to KIT activation. Accordingly, inhibition of RAF1 suppressed cell survival, proliferation, and cell cycle progression in vitro mediated by both wild-type KIT and primary KIT mutations. Inhibition of RAF1 in vivo suppressed GIST growth in a transgenic mouse model carrying germline KIT/V558A mutation, showing a similar treatment efficiency as imatinib, the first-line targeted therapeutic drug of GIST, while the combination use of imatinib and RAF1 inhibitor further suppressed tumor growth. Acquisition of drug-resistant secondary mutation of KIT is a major cause of treatment failure of GIST following targeted therapy. Like wild-type KIT and primary KIT mutations, inhibition of RAF1 suppressed the activation of secondary KIT mutation, and the cell survival, proliferation, cell cycle progression in vitro, and tumor growth in vivo mediated by secondary KIT mutation. However, the activation of secondary KIT mutation is less dependent on RAF1 compared with that of primary KIT mutations. Taken together, our results revealed that RAF1 facilitates KIT signaling and KIT mutation-mediated tumorigenesis of GIST, providing a rationale for further investigation into the use of RAF1 inhibitors alone or in combination with KIT inhibitor in the treatment of GIST, particularly in cases resistant to KIT inhibitors.

A Method to Conditionally Measure Target Engagement at Intracellular RAS and RAF Complexes.

Dysfunction of the RAS/mitogen-activated protein kinase (MAPK) pathway is a common driver of human cancers. As such, both the master regulator of the pathway, RAS, and its proximal kinase effectors, RAFs, have been of interest as drug targets for decades. Importantly, signaling within the RAS/MAPK pathway is highly coordinated due to the formation of a higher-order complex called the RAS/RAF signalosome, which may minimally contain dimers of both RAS and RAF protomers. In the disease state, RAS and RAF assemble in homo- and/or heterodimeric forms. Traditionally, drug development campaigns for both RAS and RAF have utilized biochemical assays of purified recombinant protein. As these assays do not query the RAS or RAF proteins in their full-length and complexed forms in cells, potency results collected using these assays have often failed to correlate with inhibition of the MAPK pathway. To more accurately quantify engagement at this signaling components, we present a bioluminescence resonance energy transfer (BRET)-based method to conditionally measure target engagement at individual protomers within the RAS/RAF signalosome in live cells.

RAF1 gene fusions are recurrent driver events in infantile fibrosarcoma-like mesenchymal tumors.

Infantile fibrosarcomas (IFS) and congenital mesoblastic nephroma (CMN) are rare myofibroblastic tumors of infancy and early childhood commonly harboring the ETV6::NTRK3 gene fusion. IFS/CMN are considered as tumors with an 'intermediate prognosis' as they are locally aggressive, but rarely metastasize, and generally have a favorable outcome. A fraction of IFS/CMN-related neoplasms are negative for the ETV6::NTRK3 gene rearrangement and are characterized by other chimeric proteins promoting MAPK signaling upregulation. In a large proportion of these tumors, which are classified as IFS-like mesenchymal neoplasms, the contributing molecular events remain to be identified. Here, we report three distinct rearrangements involving RAF1 among eight ETV6::NTRK3 gene fusion-negative tumors with an original histological diagnosis of IFS/CMN. The three fusion proteins retain the entire catalytic domain of the kinase. Two chimeric products, GOLGA4::RAF1 and LRRFIP2::RAF1, had previously been reported as driver events in different cancers, whereas the third, CLIP1::RAF1, represents a novel fusion protein. We demonstrate that CLIP1::RAF1 acts as a bona fide oncoprotein promoting cell proliferation and migration through constitutive upregulation of MAPK signaling. We show that the CLIP1::RAF1 hyperactive behavior does not require RAS activation and is mediated by constitutive 14-3-3 protein-independent dimerization of the chimeric protein. As previously reported for the ETV6::NTRK3 fusion protein, CLIP1::RAF1 similarly upregulates PI3K-AKT signaling. Our findings document that RAF1 gene rearrangements represent a recurrent event in ETV6::NTRK3-negative IFS/CMN and provide a rationale for the use of inhibitors directed to suppress MAPK and PI3K-AKT signaling in these cancers. © 2024 The Pathological Society of Great Britain and Ireland.

Exploring Molecular Genetic Alterations and RAF Fusions in Melanoma: A Belvarafenib Expanded Access Program in Patients with RAS/RAF-Mutant Melanoma.

BACKGROUND: Melanoma incidence is on the rise in East Asia, yet studies of the molecular landscape are lacking in this population. We examined patients with melanoma who underwent next-generation sequencing (NGS) at a single tertiary center in South Korea, focusing on patients harboring NRAS or RAF alterations who received belvarafenib, a pan-RAF dimer inhibitor, through the Expanded Access Program (EAP). PATIENTS AND METHODS: Data were collected from 192 patients with melanoma who underwent NGS between November 2017 and May 2023. Variant call format data were obtained and annotated. Patients in the EAP received 450 mg twice daily doses of belvarafenib. RESULTS: Alterations in the RAS/RTK pathway were the most prevalent, with BRAF and NRAS alteration rates of 22.4% and 17.7%, respectively. NGS enabled additional detection of fusion mutations, including 6 BRAF and 1 RAF1 fusion. Sixteen patients with NRAS or RAF alterations received belvarafenib through the EAP, and disease control was observed in 50%, with 2 patients demonstrating remarkable responses. CONCLUSIONS: Our study highlights the value of NGS in detecting BRAF, NRAS mutations and RAF fusions, expanding possibilities for targeted therapies in malignant melanoma. Belvarafenib showed clinical benefit in patients harboring these alterations. Ongoing trials will provide further insights into the safety and efficacy of belvarafenib.

Cryo-EM Structures of CRAF2/14-3-32 and CRAF2/14-3-32/MEK12 Complexes.

RAF protein kinases are essential effectors in the MAPK pathway and are important cancer drug targets. Structural understanding of RAF activation is so far based on cryo-electron microscopy (cryo-EM) and X-ray structures of BRAF in different conformational states as inactive or active complexes with KRAS, 14-3-3 and MEK1. In this study, we have solved the first cryo-EM structures of CRAF2/14-3-32 at 3.4 Å resolution and CRAF2/14-3-32/MEK12 at 4.2 Å resolution using CRAF kinase domain expressed as constitutively active Y340D/Y341D mutant in insect cells. The overall architecture of our CRAF2/14-3-32 and CRAF2/14-3-32/MEK12 cryo-EM structures is highly similar to corresponding BRAF structures in complex with 14-3-3 or 14-3-3/MEK1 and represent the activated dimeric RAF conformation. Our CRAF cryo-EM structures provide additional insights into structural understanding of the activated CRAF2/14-3-32/MEK12 complex.

Inhibition of the RAF/MEK/ERK Signaling Cascade in Pancreatic Cancer: Recent Advances and Future Perspectives.

Pancreatic cancer represents a formidable challenge in oncology, primarily due to its aggressive nature and limited therapeutic options. The prognosis of patients with pancreatic ductal adenocarcinoma (PDAC), the main form of pancreatic cancer, remains disappointingly poor with a 5-year overall survival of only 5%. Almost 95% of PDAC patients harbor Kirsten rat sarcoma virus (KRAS) oncogenic mutations. KRAS activates downstream intracellular pathways, most notably the rapidly accelerated fibrosarcoma (RAF)/mitogen-activated protein kinase kinase (MEK)/extracellular signal-regulated kinase (ERK) signaling axis. Dysregulation of the RAF/MEK/ERK pathway is a crucial feature of pancreatic cancer and therefore its main components, RAF, MEK and ERK kinases, have been targeted pharmacologically, largely by small-molecule inhibitors. The recent advances in the development of inhibitors not only directly targeting the RAF/MEK/ERK pathway but also indirectly through inhibition of its regulators, such as Src homology-containing protein tyrosine phosphatase 2 (SHP2) and Son of sevenless homolog 1 (SOS1), provide new therapeutic opportunities. Moreover, the discovery of allele-specific small-molecule inhibitors against mutant KRAS variants has brought excitement for successful innovations in the battle against pancreatic cancer. Herein, we review the recent advances in targeted therapy and combinatorial strategies with focus on the current preclinical and clinical approaches, providing critical insight, underscoring the potential of these efforts and supporting their promise to improve the lives of patients with PDAC.

RAF1 mutation leading to hypertrophic cardiomyopathy in a Chinese family with a history of sudden cardiac death: A diagnostic insight into Noonan syndrome.

BACKGROUND: Hypertrophic cardiomyopathy (HCM) is predominantly caused by mutations in sarcomeric genes. However, a subset of cases is attributed to genetic disorders unrelated to sarcomeric genes, such as Noonan syndrome (NS) and other RASopathies. In this study, we present a family with a history of sudden cardiac death (SCD) and focus on two adults with syndromic left ventricular hypertrophy (LVH). METHODS: Clinical evaluations, including echocardiography, were conducted to assess cardiac manifestations. Whole-exome sequencing was performed to identify potential genetic variants underlying syndromic LVH in the study participants. RESULTS: Whole-exome sequencing revealed a missense variant in the RAF1 gene, c.782C>T (p.Pro261Leu). This variant confirmed the diagnosis of NS in the affected individuals. CONCLUSION: The findings of this study underscore the importance of family history investigation and genetic testing in diagnosing syndromic LVH. By identifying the underlying genetic cause, clinicians can better understand the etiology of RAS-HCM and its association with SCD in young adults.

Targeting CRAF kinase in anti-cancer therapy: progress and opportunities.

The RAS/mitogen-activated protein kinase (MAPK) signaling cascade is commonly dysregulated in human malignancies by processes driven by RAS or RAF oncogenes. Among the members of the RAF kinase family, CRAF plays an important role in the RAS-MAPK signaling pathway, as well as in the progression of cancer. Recent research has provided evidence implicating the role of CRAF in the physiological regulation and the resistance to BRAF inhibitors through MAPK-dependent and MAPK-independent mechanisms. Nevertheless, the effectiveness of solely targeting CRAF kinase activity remains controversial. Moreover, the kinase-independent function of CRAF may be essential for lung cancers with KRAS mutations. It is imperative to develop strategies to enhance efficacy and minimize toxicity in tumors driven by RAS or RAF oncogenes. The review investigates CRAF alterations observed in cancers and unravels the distinct roles of CRAF in cancers propelled by diverse oncogenes. This review also seeks to summarize CRAF-interacting proteins and delineate CRAF's regulation across various cancer hallmarks. Additionally, we discuss recent advances in pan-RAF inhibitors and their combination with other therapeutic approaches to improve treatment outcomes and minimize adverse effects in patients with RAF/RAS-mutant tumors. By providing a comprehensive understanding of the multifaceted role of CRAF in cancers and highlighting the latest developments in RAF inhibitor therapies, we endeavor to identify synergistic targets and elucidate resistance pathways, setting the stage for more robust and safer combination strategies for cancer treatment.

ITPR1-AS1 promotes small cell lung cancer metastasis by facilitating P21HRAS splicing and stabilizing DDX3X to activate the cRaf-MEK-ERK cascade.

The mechanisms underlying the involvement of long non-coding RNAs (lncRNAs) in the metastasis of small cell lung cancer (SCLC) remain largely unknown. Here, we identified that the lncRNA ITPR1-AS1 was upregulated in SCLC and lymph node metastasis tissues and positively correlated with SCLC malignant features. The overexpression of ITPR1-AS1 in SCLC was an independent risk factor for the overall survival of patients with SCLC. Our data confirmed that ITPR1-AS1 induces SCLC cell metastasis both in vitro and in vivo. Mechanistically, ITPR1-AS1 acts as a scaffold to enhance the interaction between SRC-associated in mitosis 68 kDa and heterogeneous nuclear ribonucleoprotein A1, which facilitates the alternative splicing of the H-Ras proto-oncogene (HRAS) pre-mRNA (P21HRAS). Moreover, we observed that ITPR1-AS1 could associate in a complex with and maintain the stability of DEAD-box polypeptide 3 (DDX3X), which inhibited the latter's ubiquitination and degradation. Our data provide evidence that ITPR1-AS1 activates the cRaf-MEK-ERK cascade by upregulating P21HRAS production and stabilizing DDX3X, to promote SCLC metastasis.

Sex disparities in non-small cell lung cancer: mechanistic insights from a cRaf transgenic disease model.

BACKGROUND: Women are at greater risk of developing non-small cell lung cancer (NSCLC), yet the underlying causes remain unclear. METHODS: We performed whole genome scans in lung tumours of cRaf transgenic mice and identified miRNA, transcription factor and hormone receptor dependent gene regulations. We confirmed hormone receptors by immunohistochemistry and constructed regulatory gene networks by considering experimentally validated miRNA-gene and transcription factor-miRNA/gene targets. Bioinformatics, genomic foot-printing and gene enrichment analysis established sex-specific circuits of lung tumour growth. Translational research involved a large cohort of NSCLC patients. We evaluated commonalities in sex-specific NSCLC gene regulations between mice and humans and determined their prognostic value in Kaplan-Meier survival statistics and COX proportional hazard regression analysis. FINDINGS: Overexpression of the cRaf kinase elicited an extraordinary 8-fold increase in tumour growth among females, and nearly 70% of the 112 differentially expressed genes (DEGs) were female specific. We identified oncogenes, oncomirs, tumour suppressors, cell cycle regulators and MAPK/EGFR signalling molecules, which prompted sex-based differences in NSCLC, and we deciphered a regulatory gene-network, which protected males from accelerated tumour growth. Strikingly, 41% of DEGs are targets of hormone receptors, and the majority (85%) are oestrogen receptor (ER) dependent. We confirmed the role of ER in a large cohort of NSCLC patients and validated 40% of DEGs induced by cRaf in clinical tumour samples. INTERPRETATION: We report the molecular wiring that prompted sex disparities in tumour growth. This allowed us to propose the development of molecular targeted therapies by jointly blocking ER, CDK1 and arginase 2 in NSCLC. FUNDING: We gratefully acknowledge the financial support of the Lower Saxony Ministry of Culture and Sciences and Volkswagen Foundation, Germany to JB (25A.5-7251-99-3/00) and of the Chinese Scholarship Council to SZ (202008080022). This publication is funded by the Deutsche Forschungsgemeinschaft (DFG) as part of the "Open Access Publikationskosten" program.

RAS-independent ERK activation by constitutively active KSR3 in non-chordate metazoa.

During early development of the sea urchin embryo, activation of ERK signalling in mesodermal precursors is not triggered by extracellular RTK ligands but by a cell-autonomous, RAS-independent mechanism that was not understood. We discovered that in these cells, ERK signalling is activated through the transcriptional activation of a gene encoding a protein related to Kinase Suppressor of Ras, that we named KSR3. KSR3 belongs to a family of catalytically inactive allosteric activators of RAF. Phylogenetic analysis revealed that genes encoding kinase defective KSR3 proteins are present in most non-chordate metazoa but have been lost in flies and nematodes. We show that the structure of KSR3 factors resembles that of several oncogenic human RAF mutants and that KSR3 from echinoderms, cnidarians and hemichordates activate ERK signalling independently of RAS when overexpressed in cultured cells. Finally, we used the sequence of KSR3 factors to identify activating mutations of human B-RAF. These findings reveal key functions for this family of factors as activators of RAF in RAS-independent ERK signalling in invertebrates. They have implications on the evolution of the ERK signalling pathway and suggest a mechanism for its co-option in the course of evolution.