Phosphorylation Of Raf-1 Research Articles

Phenylephrine Enhances the Mitogenic Effect of S-Allyl-L-cysteine on Primary Cultured Hepatocytes through Protein Kinase C-Induced B-Raf Phosphorylation.

The co-mitogenic effects of the α1-adrenoceptor agonist phenylephrine on S-allyl-L-cysteine (SAC)-induced hepatocyte proliferation were examined in primary cultures of adult rat hepatocytes. The combination of phenylephrine (10-10-10-6 M) and SAC (10-6 M) exhibited a significant dose-dependent increase in the number of hepatocyte nuclei and viable cells compared to SAC alone. This combination also increased the progression of hepatocyte nuclei into the S-phase. The potentiating effect of phenylephrine on SAC-induced cell proliferation was counteracted by prazosin (an α1-adrenergic receptor antagonist) and GF109203X (selective protein kinase C (PKC) inhibitor). In addition, PMA (direct PKC activator) potentiated the proliferative effects of SAC similarly to phenylephrine. In essence, these findings suggest that PKC activity plays a crucial role in enhancing SAC-induced cell proliferation. Moreover, the effects of phenylephrine on SAC-induced Ras activity, Raf phosphorylation, and extracellular signal-regulated kinase 2 (ERK2) phosphorylation were investigated. Phenylephrine (or PMA) in combination with SAC did not augment Ras activity, but further increased ERK2 phosphorylation and its upstream B-Raf phosphorylation. These results indicate that PKC activation, triggered by stimulating adrenergic α1 receptors, further amplifies SAC-induced cell proliferation through enhanced ERK2 phosphorylation via increased B-Raf-specific phosphorylation in primary cultured hepatocytes.

Raddeanin A augments the cytotoxicity of natural killer cells against chronic myeloid leukaemia cells by modulating MAPK and Ras/Raf signalling pathways.

Natural killer (NK) cell therapy, a developing approach in cancer immunotherapy, involves isolating NK cells from peripheral blood. However, due to their limited number and activity, it is essential to significantly expand these primary NK cells and enhance their cytotoxicity. In this study, we investigated how Raddeanin A potentiate NK activity using KHYG-1 cells. The results indicated that Raddeanin A increased the expression levels of cytolytic molecules such as perforin, granzymes A and granzymes B, granulysin and FasL in KHYG-1 cells. Raddeanin A treatment increased CREB phosphorylation, p65 phosphorylation, NFAT1 and acetyl-histone H3 expression. Raddeanin A elevated caspase 3 and PARP cleavage, increased t-Bid expression, promoting apoptosis in K562 cells. Furthermore, it reduced the expression of HMGB2, SET and Ape1, impairing the DNA repair process and causing K562 cells to die caspase-independently. Additionally, Raddeanin A increased ERK, p38 and JNK phosphorylation at the molecular level, which increased granzyme B production in KHYG-1 cells. Raddeanin A treatment increased Ras, Raf phosphorylation, MEK phosphorylation, NKG2D, NKp44 and NKp30 expression in KHYG-1 cells. Collectively, our data indicate that Raddeanin A enhances the cytotoxic activity of NK cells against different cancer cells.

The expression of congenital Shoc2 variants induces AKT-dependent crosstalk activation of the ERK1/2 pathway.

The Shoc2 scaffold protein is crucial in transmitting signals within the Epidermal Growth Factor Receptor (EGFR)-mediated Extracellular signal-Regulated Kinase (ERK1/2) pathway. While the significance of Shoc2 in this pathway is well-established, the precise mechanisms through which Shoc2 governs signal transmission remain to be fully elucidated. Hereditary variants in Shoc2 are responsible for Noonan Syndrome with Loose anagen Hair (NSLH). However, due to the absence of known enzymatic activity in Shoc2, directly assessing how these variants affect its function is challenging. ERK1/2 phosphorylation is used as a primary parameter of Shoc2 function, but the impact of Shoc2 mutants on the pathway activation is unclear. This study investigates how the NSLH-associated Shoc2 variants influence EGFR signals in the context of the ERK1/2 and AKT downstream signaling pathways. We show that when the ERK1/2 pathway is a primary signaling pathway activated downstream of EGFR, Shoc2 variants cannot upregulate ERK1/2 phosphorylation to the level of the WT Shoc2. Yet, when the AKT and ERK1/2 pathways were activated, in cells expressing Shoc2 variants, ERK1/2 phosphorylation was higher than in cells expressing WT Shoc2. In cells expressing the Shoc2 NSLH mutants, we found that the AKT signaling pathway triggers the PAK activation, followed by phosphorylation of Raf-1/MEK1/2 and activation of the ERK1/2 signaling axis. Hence, our studies reveal a previously unrecognized feedback regulation downstream of the EGFR and provide additional evidence for the role of Shoc2 as a "gatekeeper" in controlling the selection of downstream effectors within the EGFR signaling network.

TCF19 promotes cell proliferation and tumor formation in lung cancer by activating the Raf/MEK/ERK signaling pathway

ObjectiveThis study aimed to investigate TCF19′s role in lung cancer development, specifically its involvement in the RAF/MEK/ERK signaling pathway. MethodsLung cancer tissue analysis revealed significant TCF19 overexpression. In vitro experiments using A549 and Hop62 cells with TCF19 overexpression demonstrated enhanced cell growth. Transgenic mouse models confirmed TCF19′s role in primary tumor development. Transcriptome sequencing identified altered gene expression profiles, linking TCF19 to RAF/MEK/ERK pathway activation. Functional assays elucidated underlying mechanisms, revealing increased phosphorylation of Raf1, MEK1/2, and ERK1/2. Inhibiting RAF1 or ERK through shRaf1 or ERK inhibitor reduced cell cycle-related proteins and inhibited TCF19-overexpressing cell growth. ResultsTCF19 was identified as an oncogene in lung carcinoma, specifically impacting the RAF/MEK/ERK pathway. Elevated TCF19 levels in lung cancer suggest targeting TCF19 or its associated pathways as a promising strategy for disease management. ConclusionThis study unveils TCF19′s oncogenic role in lung cancer, emphasizing its modulation of the RAF/MEK/ERK pathway and presenting a potential therapeutic target for TCF19-overexpressing lung cancers.

The antipsychotic drug pimozide promotes apoptosis through the RAF/ERK pathway and enhances autophagy in breast cancer cells

ABSTRACT The antipsychotic drug pimozide has been demonstrated to inhibit cancer. However, the precise anti-cancer mechanism of pimozide remains unclear. The purpose of this study was to investigate the effects of pimozide on human MCF-7 and MDA-MB-231 breast cancer cell lines, and the potential involvement in the RAF/ERK signaling. The effects of pimozide on cells were examined by 4,5-dimethylthiazol-2-yl-3,5-diphenylformazan, wound healing, colony formation, transwell assays, and caspase activity assay. Flow cytometry and acridine orange and ethidium bromide staining were performed to assess changes in cells. Transmission electron microscopy and monodansylcadaverine staining were used to observe autophagosomes. The cyclic adenosine monophosphate was evaluated using the FRET system. Immunohistochemistry, immunofluorescence, RNA interference, and western blot investigated the expression of proteins. Mechanistically, we focus on the RAF1/ERK signaling. We detected pimozide was docked to RAF1 by Schrodinger software. Pimozide down-regulated the phosphorylation of RAF1, ERK 1/2, Bcl-2, and Bcl-xl, up-regulated Bax, and cleaved caspase-9 to induce apoptosis. Pimozide might promote autophagy by up-regulating cAMP. The enhancement of autophagy increased the conversion of LC3-I to LC3-II and down-regulated p62 expression. But mTOR signaling was not involved in promoting autophagy. The knockdown of RAF1 expression induced autophagy and apoptosis in breast cancer cells, consistent with the results of pimozide or sorafenib alone. Blocked autophagy by chloroquine resulted in the impairment of pimozide-induced apoptosis. These data showed that pimozide inhibits breast cancer by regulating the RAF/ERK signaling pathway and might activate cAMP-induced autophagy to promote apoptosis and it may be a potential drug for breast cancer treatment.

Potential Plant Secondary Metabolites for the Prevention of Skin Cancers and their Mechanism: A Review

Abstract: Skin carcinoma is the most common malignancy, including malignant melanoma and non-malignant melanoma throughout the world, and in the last three decades, its incidences have been increasing drastically. High cost, severe side effects and development of resistance from conventional chemotherapy have decreased its acceptability and increased the urge for the search for alternate options. Therefore, plant derived phytochemicals have come up with cost effectiveness, ease of availability and promising potential for skin related cancers. In spite of the lack of clinical data involving humans to support the positive effects of phytochemicals on different types of skin cancers, topical application of these plant derived anticancer phytochemicals is extremely promising. Many mechanisms and pathways underlie the antitumor activity of plant-derived phytochemicals , such as loss of mitochondrial membrane potential, the release of cytochrome-c, suppression of antiapoptotic proteins and induction of proapoptotic proteins, activation of caspases, Fas, FADD and p53 signaling pathway, inhibition of Akt signaling pathway, phosphorylation of ERK, P13K, Raf, survivin gene, STAT 3 and NF-kB. The use of cell line models in the in vitro study of skin cancer offers the opportunity to identify the mechanisms of action of plant derived phytochemicals against various types of skin cancer. As such, this article aims at providing a comprehensive review of plant extracts and phytochemicals, which have been reported to exhibit promising anti-carcinogenic properties in animal models and against various skin cancer cell lines.

RAF1 deficiency causes a lethal syndrome that underscores RTK signaling during embryogenesis

Somatic and germline gain‐of‐function point mutations in RAF, one of the first oncogenes to be discovered in humans, delineate a group of tumor‐prone syndromes known as the RASopathies. In this study, we document the first human phenotype resulting from the germline loss‐of‐function of the proto‐oncogene RAF1 (a.k.a. CRAF). In a consanguineous family, we uncovered a homozygous p.Thr543Met variant segregating with a neonatal lethal syndrome with cutaneous, craniofacial, cardiac, and limb anomalies. Structure‐based prediction and functional tests using human knock‐in cells showed that threonine 543 is essential to: (i) ensure RAF1's stability and phosphorylation, (ii) maintain its kinase activity toward substrates of the MAPK pathway, and (iii) protect from stress‐induced apoptosis mediated by ASK1. In Xenopus embryos, mutant RAF1T543M failed to phenocopy the effects of normal and overactive FGF/MAPK signaling, confirming its hypomorphic activity. Collectively, our data disclose the genetic and molecular etiology of a novel lethal syndrome with progeroid features, highlighting the importance of RTK signaling for human development and homeostasis.

Abstract 570: Dual acting bone defender agents inhibit prostate cancer-mediated bone destruction

Abstract Introduction: Bone metastasis is clinically observed in approximately 90% of patients with advanced prostate cancer (PCa). Metastatic bone destruction is driven by a self-reinforcing process coupling osteoclast (OC)-mediated bone degradation to PCa growth, and where cell movement acts to keep these two cooperating cell types in close proximity and to move them across the bone surface. We sought to create a new class of therapeutics that would be highly effective at inhibiting PCa-mediated bone destruction by targeting both OC function and cell motility. Methods: We synthesized new chemical entities, termed Dual Acting Bone Defender agents (DABDs), that couple the small molecule anti-motility agent, KBU2046, with bisphosphonate (bis), through a chemical linker. Assays for in vitro efficacy included: cell migration and invasion, binding to bone mineral, inhibiting osteoclast (OC)-mediated bone destruction, inhibition of Raf1 phosphorylation and inhibition of protein prenylation. In vivo assays included intra-cardiac (IC) injection of human PCa cells into mice, followed by real time monitoring of tumor growth and bone destruction by IVIS and CT imaging, respectively. Findings: We successfully created a novel route of chemical synthesis allowing us to access and probe unique chemical space, providing for single small molecules with bis functional groups linked to a KBU2046 core through a linker moiety. DABDs retain the cellular and molecular characteristics of KBU2046 and bis. Like KBU2046, they inhibit PCa cell motility and phosphorylation of Raf1 on its activation motif. They also inhibit the motility of breast and lung cancer cells. Like bis, they inhibit OC-mediated bone mineral destruction, prenylation of Ras small GTPase proteins, and chemically bind hydroxyapatite. Administered prior to IC injection, DABDs are so effective that they prolong animal survival and inhibit bone destruction in a dose-dependent manner. In mice with established bone metastasis to the femur, after IC injection, DABDs inhibit the growth of tumors. Doses 3,000 fold higher than that required for efficacy had no clinical or critical organ toxicity. Conclusions: DABDs represent a new multifunctional class of therapeutic agents that inhibit PCa-mediated bone destruction and have a high potential for advancing clinical efficacy in PCa patients with bone metastasis. Citation Format: Fangfang Qiao, Ryan Gordon, Abhinandan Pattanayak, Katelyn O’Neill, Nina Chaika, Weining Chen, Rachel Rhatigan, Raymond Bergan. Dual acting bone defender agents inhibit prostate cancer-mediated bone destruction [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 570.

Bag-1 Protects Nucleus Pulposus Cells from Oxidative Stress by Interacting with HSP70

Bcl-2-associated athanogene 1 (Bag-1) is a multifunctional prosurvival protein that binds to several intracellular targets and promotes cell survival. HSP70 and Raf-1 are important targets of Bag-1; however, the protective function of Bag-1 in nucleus pulposus (NP) cells remains unclear. In this study, we determined the effects of Bag-1 on NP cells under oxidative stress induced by treatment with hydrogen peroxide (H2O2). We found that Bag-1 was bound to HSP70, but Bag-1-Raf1 binding did not occur in NP cells. Bag-1 overexpression in NP cells enhanced cell viability and mitochondrial function and significantly suppressed p38/MAPKs phosphorylation during oxidative stress, although NP cells treated with a Bag-1 C-terminal inhibitor, which is the binding site of HSP70 and Raf-1, decreased cell viability and mitochondrial function during oxidative stress. Furthermore, the phosphorylation of the ERK/MAPKs was significantly increased in Bag-1 C-terminal inhibitor-treated NP cells without H2O2 treatment but did not change with H2O2 exposure. The phosphorylation of Raf-1 was not influenced by Bag-1 overexpression or Bag-1 C-terminal binding site inhibition. Overall, the results suggest that Bag-1 preferentially interacts with HSP70, rather than Raf-1, to protect NP cells against oxidative stress.

Licoricidin combats gastric cancer by targeting the ICMT/Ras pathway in vitro and in vivo.

Licoricidin, a type of isoflavonoid, is extracted from the root of Glycyrrhiza glabra. It has been widely proven that licoricidin possesses multiple biological activities, including anti-cancer effects and a powerful antimicrobial effect against Helicobacter pylori (H. pylori). However, the exact mechanism of licoricidin against gastric cancer remains unclear. In this study, we comprehensively explored the effects of licoricidin on MGC-803 gastric cancer cells in vitro and in vivo and further elucidated its mechanism of action. Our results revealed that licoricidin exhibited multiple anti-gastric cancer activities, including suppressing proliferation, inducing apoptosis, arresting the cell cycle in G0/G1 phase, and inhibiting the migration and invasion abilities of MGC-803 gastric cancer cells. In addition to this, a total of 5861 proteins were identified by quantitative proteomics research strategy of TMT labeling, of which 19 differential proteins (two upregulated and 17 downregulated) were screened out. Combining bioinformatics analyses and the reported roles in cancer progression of the 19 proteins, we speculated that isoprenyl carboxyl methyltransferase (ICMT) was the most likely target of licoricidin. Western blot assays and IHC assays subsequently proved that licoricidin significantly downregulated the expression of ICMT, both in MGC-803 cells and in xenograft tumors. Moreover, licoricidin effectively reduced the level of active Ras-GTP and blocked the phosphorylation of Raf and Erk, which may be involved in its anti-gastric cancer effects. In summary, we first demonstrated that licoricidin exerted favorable anti-gastric cancer activities via the ICMT/Ras pathway, which suggests that licoricidin, as a natural product, could be a novel candidate for the management of gastric cancer.

Salvianolic Acid A Protects against Lipopolysaccharide-Induced Acute Lung Injury by Inhibiting Neutrophil NETosis.

Salvianolic acid A (SAA) is one of bioactive polyphenol extracted from a Salvia miltiorrhiza (Danshen), which was widely used to treat cardiovascular disease in traditional Chinese medicine. SAA has been reported to be protective in cardiovascular disease and ischemia injury, with anti-inflammatory and antioxidative effect, but its role in acute lung injury (ALI) is still unknown. In this study, we sought to investigate the therapeutic effects of SAA in a murine model of lipopolysaccharide- (LPS-) induced ALI. The optimal dose of SAA was determined by comparing the attenuation of lung injury score after administration of SAA at three different doses (low, 5 mg/kg; medium, 10 mg/kg; and, high 15 mg/kg). Dexamethasone (DEX) was used as a positive control for SAA. Here, we showed that the therapeutic effect of SAA (10 mg/kg) against LPS-induced pathologic injury in the lungs was comparable to DEX. SAA and DEX attenuated the increased W/D ratio and the protein level, counts of total cells and neutrophils, and cytokine levels in the BALF of ALI mice similarly. The oxidative stress was also relieved by SAA and DEX according to the superoxide dismutase and malondialdehyde. NET level in the lungs was elevated in the injured lung while SAA and DEX reduced it significantly. LPS induced phosphorylation of Src, Raf, MEK, and ERK in the lungs, which was inhibited by SAA and DEX. NET level and phosphorylation level of Src/Raf/MEK/ERK pathway in the neutrophils from acute respiratory distress syndrome (ARDS) patients were also inhibited by SAA and DEX in vitro, but the YEEI peptide reversed the protective effect of SAA completely. The inhibition of NET release by SAA was also reversed by YEEI peptide in LPS-challenged neutrophils from healthy volunteers. Our data demonstrated that SAA ameliorated ALI via attenuating inflammation, oxidative stress, and neutrophil NETosis. The mechanism of such protective effect might involve the inhibition of Src activation.

Molecular target therapeutics of EGF-TKI and downstream signaling pathways in non-small cell lung cancers.

Lung carcinoma (LC) is the third most common cancer diagnosis and accounted for the most cancer-related mortality worldwide in 2018. Based on the type of cells from which it originates, LC is commonly classified into non-small cell lung cancers (NSCLC) and small cell lung cancers (SCLC). NSCLC account for the majority of LC and can be further categories into adenocarcinoma, large cell carcinoma, and squamous cell carcinoma. Accurate classification of LC is critical for its adequate treatment and therapeutic outcome. Since NSCLC express more epidermal growth factor receptor (EGFR) with activation mutations, targeted therapy EGFR-tyrosine kinase inhibitors (TKIs) have been considered as primary option of NSCLC patients with activation EGFR mutation. In this review, we present the genetic alterations, reported mutations in EGFR, and TKIs treatment in NSCLC patients with an emphasis on the downstream signaling pathways in NSCLC progression. Among the signaling pathways identified, mitogen activation protein kinase (MAPK), known also as extracellular signal-regulated protein kinase (Erk) pathway, is the most investigated among the related pathways. EGFR activation leads to the autophosphorylation of its kinase domain and subsequent activation of Ras, phosphorylation of Raf and MEK1/2, and the activation of ERK1/2. Phosphatidylinositol 3-kinase (PI3K)/Akt is another signal pathway that regulates cell cycle and has been linked to NSCLC progression. Currently, three generations of EGFR TKIs have been developed as a first-line treatment of NSCLC patients with EGFR activation and mutation in which these treatment options will be further discussed in this review. The Supplementary Appendix for this article is available at http://links.lww.com/JCMA/A138.

METTL3 Facilitates Tumor Progression by COL12A1/MAPK Signaling Pathway in Esophageal Squamous Cell Carcinoma.

Background: Esophageal squamous cell carcinoma (ESCC) is one of the most common aggressive tumors in the world. m6A modification has been implicated to play an important role in many biological progressions. METTL3 as the main methyltransferase has been found in many cancers, including ESCC. Here, we investigated the underlying mechanism of METTL3 in the development of ESCC.Methods: Quantitative real-time PCR (qRT-PCR), immunohistochemical (IHC) and western blot were used to detect METTL3 expression. To evaluate the function of METTL3, MTS, colony formation, scratch wound healing assay, and transwell and invasion assays were performed. To find out the downstream target of METTL3, mRNA sequencing (mRNA-seq) was conducted. GO and KEGG functional enrichment analyses were carried out to predict possible biological processes and signaling pathways. qRT-PCR and western blot were performed to identify the expression of COL12A1 and the phosphorylation status of RAF, MRK and ERK. Cotransfection of small interfering RNA (for METTL3 silence) with plasmid (for overexpression of COL12A1) and the following gain- and loss-of-function experiments were performed to detect the target gene function of COL12A1 in progression of ESCC mediated by METTL3.Results: Using TCGA database, higher METTL3 expression was found in ESCC tissues. Moreover, we found that METTL3 was significantly increased in ESCC patient tissues compared with normal tissues and correlated with poor prognosis. The expression of METTL3 in ESCC cell lines was assessed. The gain- and loss-of-function indicates that METTL3 promotes cell proliferation, migration and invasion. Additionally, we confirmed that METTL3 can promote the expression of COL12A1 and upregulate the phosphorylation of RAF, MER and ERK, and moreover COL12A1 can restrain siMETTL3-mediated inhibition of proliferation, migration and invasion in ESCC.Conclusion: Our study revealed that METTL3 may have an oncogenic role, facilitating the ESCC progression and metastasis by COL12A1/MAPK signaling pathway.

MiR-155-5p suppresses SOX1 to promote proliferation of cholangiocarcinoma via RAF/MEK/ERK pathway

BackgroundAccumulating evidence has demonstrated the close relation of SOX1 with tumorigenesis and tumor progression. Upregulation of SOX1 was recently shown to suppress growth of human cancers. However, the expression and role of SOX1 in cholangiocarcinoma (CCA) is not well characterized.MethodsExpression levels of SOX1 in CCA tissues and normal bile duct tissues were examined using public GEO database. Western blot and immunohistochemistry were used to confirm the expression levels. Cell proliferation assay (CCK-8) and colony formation assay were performed to assess proliferation of CCA cells. A mouse model of subcutaneous transplantable tumors was used to evaluated proliferation of CCA in vivo. The putative regulating factor of SOX1 were determined using Targetscan and dual-luciferase reporter assay.ResultsSOX1 was downregulated in CCA tissues. Overexpression of SOX1 significantly inhibited cell proliferation in vitro and suppressed tumor growth in vivo. miR-155-5p directly targeted the 3′-untranslated region (3′UTR) of SOX1 and inhibited expression of SOX1, resulting in the activation of RAF, MEK and ERK phosphorylation, and thus CCA proliferation. However, restoration of SOX1 expression in miR-155-5p overexpressing cell lines decreased the phosphorylation level of RAF, MEK and ERK, as well as the proliferation of CCA cells.ConclusionMiR-155-5p decreased the expression of SOX1 by binding to its 3′UTR, which activated the RAF/MEK/ERK signaling pathway and promoted CCA progression.

Abstract P046: ENGOT-ov60/GOG3052/RAMP 201: A phase 2 study of VS-6766 (dual RAF/MEK inhibitor) alone and in combination with defactinib (FAK inhibitor) in recurrent low-grade serous ovarian cancer (LGSOC)

Abstract Background: VS-6766 is a unique small molecule inhibitor that blocks MEK kinase activity and RAF phosphorylation of MEK. This mechanism of blockade has been shown to limit compensatory MEK activation, thereby potentially enhancing efficacy of MEK inhibition. Defactinib, (VS-6063), an orally active small molecule, is a potent adenosine 5'- triphosphate (ATP) competitive, reversible inhibitor of focal adhesion kinase (FAK). Defactinib has shown synergistic activity with BRAF and MEK inhibitors in both in vitro and in vivo solid tumor models. Prior molecularly unselected studies with single agent MEK inhibitors have shown response rates up to 26% in recurrent LGSOC. A third of patients with recurrent LGSOC harbor somatic KRAS mutations. FAK inhibition has been shown to induce tumor regression when combined with RAF, MEK or RAF/MEK inhibitors in in vivo models of KRAS mutant ovarian cancer. The combination of VS-6766 and defactinib is currently being evaluated in the ongoing Investigator Sponsored FRAME study (NCT03875820). In this proof of concept study, durable objective responses have been reported in recurrent LGSOC patients, particularly those with KRAS mutations including patients who have had a prior MEK inhibitor (Banerji et al AACR 2020). Based on preclinical studies demonstrating efficacy of both VS-6766 and the VS-6766/defactinib combination and preliminary results of the FRAME study, the phase II ENGOT-ov60/GOG3052 has been developed in recurrent LGSOC. Methods: This is a Phase II, adaptive, two-part, multicenter, parallel cohort, randomized, open label study designed to evaluate the efficacy and safety of VS-6766 versus VS-6766 in combination with defactinib (NCT04625270). The study will be conducted in two parts. Part A will determine the optimal regimen based on confirmed overall response rate (independent radiology review) in KRAS-mutated LGSOC. Part B will determine the efficacy of the optimal regimen identified in Part A in KRAS-mutated and KRAS wild-type LGSOC. The minimum expected enrollment is 52 subjects with KRAS-mutated tumors (32 subjects in Part A and 20 in Part B) and 36 with KRAS wild-type tumors in Part B. Patients will be randomized to receive VS-6766 (4.0 mg PO, twice weekly 3 weeks on, 1 week off) or VS6766 with defactinib (VS-6766 3.2 mg PO, twice weekly + defactinib 200 mg PO BID 3 weeks on, 1 week off) till progression. Key inclusion criteria include histologically confirmed LGSOC, presence of KRAS mutation (Part A), prior systemic therapy for metastatic disease and up to 1 prior line of MEK/RAF inhibitor therapy permitted. This international study is open to enrollment. Citation Format: Susana N. Banerjee, Bradley J. Monk, Els Van Nieuwenhuysen, Kathleen N. Moore, Ana Oaknin, Michel Fabbro, Nicoletta Columbo, David M. O’Malley, Robert L. Coleman, Jonathan Pachter, Andrew Koustenis, Gloria Patrick, Lorna Leonard, Rachel Grisham. ENGOT-ov60/GOG3052/RAMP 201: A phase 2 study of VS-6766 (dual RAF/MEK inhibitor) alone and in combination with defactinib (FAK inhibitor) in recurrent low-grade serous ovarian cancer (LGSOC) [abstract]. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2021 Oct 7-10. Philadelphia (PA): AACR; Mol Cancer Ther 2021;20(12 Suppl):Abstract nr P046.

Abstract P048: A phase 2 study of VS-6766 (dual RAF/MEK inhibitor) RAMP 202, as a single agent and in combination with defactinib (FAK inhibitor) in recurrent KRAS-mutant (KRAS-MT) non-small cell lung cancer (NSCLC)

Abstract Background: VS-6766 is a unique small molecule inhibitor of both RAF and MEK. In contrast to several other MEK inhibitors available, VS-6766 blocks both MEK kinase activity and RAF phosphorylation of MEK. This sequential blockade mechanism enables VS-6766 to block MEK signaling more consistently without the compensatory and paradoxical activation of MEK that reduces the efficacy of other small molecule inhibitors of MEK and RAF. Defactinib (VS-6063), an orally active small molecule, is a potent adenosine 5'- triphosphate (ATP) competitive, reversible inhibitor of focal adhesion kinase (FAK). Defactinib has shown synergistic activity with BRAF and MEK inhibitors in both in vitro and in vivo preclinical solid tumor models. Specifically, in several human tumor cell lines with mutations in RAS or BRAF or wildtype RAS and BRAF, defactinib has shown synergy with MEK inhibitors or VS-6766. In mouse models of KRAS mutant ovarian cancer, BRAF-mutant melanoma or uveal melanoma, FAK inhibition has been shown to induce tumor regression when combined with RAF, MEK or RAF/MEK inhibitors, while the single agents have only induced tumor stasis. The combination of VS-6766 and defactinib is currently being evaluated in the Investigator Sponsored FRAME study. Preliminary efficacy results are available for a small number of subjects with KRAS mutated NSCLC treated with a combination of VS-6766 and defactinib. Of the 10 subjects with NSCLC, 1 subject with KRAS-G12V- mutated cancer achieved PR while 3/10 subjects received treatment for ≥24 weeks. In an updated analysis of response in 15 subjects with KRAS-mutant NSCLC, there were 2 PRs and 7 SDs (ORR: 13%). The two subjects with KRAS G12V- mutated NSCLC both achieved PR (ORR: 100%). Methods: This is an adaptive, two-part, Phase 2, multicenter, parallel cohort, randomized, open label study designed to evaluate the efficacy and safety of VS-6766 versus VS-6766 in combination with defactinib in subjects with recurrent NSCLC(NCT04620330). The study will be conducted in two parts. Part A will determine the optimal regimen, either VS-6766 monotherapy or VS-6766 in combination with defactinib based on confirmed overall response rate as assessed by independent radiology review. Part A will consist of three arms in KRAS mutated NSCLC Arm 1 monotherapy of VS-6766, Arm 2 the combination of VS-6766 and defactinib in KRAS G12V mutated and Arm 3 the combination in other KRAS mutated. Part B will determine the efficacy of the optimal regimen identified in Part A in KRAS mutated NSCLC. Subjects enrolled must have histologic or cytologic evidence of NSCLC, measurable disease according to RECIST V1.1 and known KRAS mutation. The study will enroll up to 377 subjects globally with 57 subjects (32 KRAS G12V & 25 KRAS other) in Part A and an additional 144 KRAS G12V and 176 KRAS other in Part B. This study is open for enrollment. Citation Format: D. Ross Camidge, Jonathan Pachter, Andrew Koustenis, Gloria Patrick, David R. Spigel. A phase 2 study of VS-6766 (dual RAF/MEK inhibitor) RAMP 202, as a single agent and in combination with defactinib (FAK inhibitor) in recurrent KRAS-mutant (KRAS-MT) non-small cell lung cancer (NSCLC) [abstract]. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2021 Oct 7-10. Philadelphia (PA): AACR; Mol Cancer Ther 2021;20(12 Suppl):Abstract nr P048.

STOML2 interacts with PHB through activating MAPK signaling pathway to promote colorectal Cancer proliferation

BackgroundHighly expressed STOML2 has been reported in a variety of cancers, yet few have detailed its function and regulatory mechanism. This research aims to reveal regulatory mechanism of STOML2 and to provide evidence for clinical therapeutics, via exploration of its role in colorectal cancer, and identification of its interacting protein.MethodsExpression level of STOML2 in normal colon and CRC tissue from biobank in Nanfang Hospital was detected by pathologic methods. The malignant proliferation of CRC induced by STOML2 was validated via gain-of-function and loss-of-function experiments, with novel techniques applied, such as organoid culture, orthotopic model and endoscopy monitoring. Yeast two-hybrid assay screened interacting proteins of STOML2, followed by bioinformatics analysis to predict biological function and signaling pathway of candidate proteins. Target protein with most functional similarity to STOML2 was validated with co-immunoprecipitation, and immunofluorescence were conducted to co-localize STOML2 and PHB. Pathway regulated by STOML2 was detected with immunoblotting, and subsequent experimental therapy was conducted with RAF inhibitor Sorafenib.ResultsSTOML2 was significantly overexpressed in colorectal cancer and its elevation was associated with unfavorable prognosis. Knockdown of STOML2 suppressed proliferation of colorectal cancer, thus attenuated subcutaneous and orthotopic tumor growth, while overexpressed STOML2 promoted proliferation in cell lines and organoids. A list of 13 interacting proteins was screened out by yeast two-hybrid assay. DTYMK and PHB were identified to be most similar to STOML2 according to bioinformatics in terms of biological process and signaling pathways; however, co-immunoprecipitation confirmed interaction between STOML2 and PHB, rather than DTYMK, despite its highest rank in previous analysis. Co-localization between STOML2 and PHB was confirmed in cell lines and tissue level. Furthermore, knockdown of STOML2 downregulated phosphorylation of RAF1, MEK1/2, and ERK1/2 on the MAPK signaling pathway, indicating common pathway activated by STOML2 and PHB in colorectal cancer proliferation.ConclusionsThis study demonstrated that in colorectal cancer, STOML2 expression is elevated and interacts with PHB through activating MAPK signaling pathway, to promote proliferation both in vitro and in vivo. In addition, combination of screening assay and bioinformatics marks great significance in methodology to explore regulatory mechanism of protein of interest.

RNA-binding protein CELF1 promotes cardiac hypertrophy via interaction with PEBP1 in cardiomyocytes.

Cardiac hypertrophy is considered as a common pathophysiological process in various cardiovascular diseases. CUG triplet repeat-binding protein 1 (CELF1) is an RNA-binding protein that has been shown to be an important post-transcription regulator and involved in several types of cancer, whereas its role in cardiac remodeling remains unclear. Herein, we found that the expression of CELF1 was significantly increased in pressure overload-induced hypertrophic hearts and angiotensin II (Ang II)-induced neonatal cardiomyocytes. Based on transverse aortic constriction-induced cardiac hypertrophy model, CELF1 deficiency markedly ameliorated cardiac hypertrophy, cardiac fibrosis, oxidative stress, and apoptosis. Accordingly, CELF1 deficiency alleviated the production of reactive oxygen species (ROS) and apoptosis of neonatal cardiomyocytes via inhibition of Raf1, TAK1, ERK1/2, and p38 phosphorylation. Mechanistically, depletion or overexpression of CELF1 negatively regulated the protein expression of phosphatidylethanolamine-binding protein 1 (PEBP1), while the mRNA expression of PEBP1 remained unchanged. RNA immunoprecipitation revealed that CELF1 directly interacted with PEBP1 mRNA. Biotin pull-down analysis and dual-luciferase assay showed that CELF1 directly bound to the fragment 1 within 3'UTR of PEBP1. Moreover, knockdown of PEBP1 partially enhanced the production of ROS and apoptosis of neonatal cardiomyocytes inhibited by CELF1 deficiency. In conclusion, CELF1 binds to the 3'UTR of PEBP1 and acts as an endogenous activator of MAPK signaling pathway. Inhibition of CELF1 attenuates pathological cardiac hypertrophy, oxidative stress, and apoptosis, thus could be a potential therapeutic strategy of pathological cardiac hypertrophy.

SPRED2 loss-of-function causes a recessive Noonan syndrome-like phenotype

Upregulated signal flow through RAS and the mitogen-associated protein kinase (MAPK) cascade is the unifying mechanistic theme of the RASopathies, a family of disorders affecting development and growth. Pathogenic variants in more than 20 genes have been causally linked to RASopathies, the majority having a dominant role in promoting enhanced signaling. Here, we report that SPRED2 loss of function is causally linked to a recessive phenotype evocative of Noonan syndrome. Homozygosity for three different variants-c.187C>T (p.Arg63∗), c.299T>C (p.Leu100Pro), and c.1142_1143delTT (p.Leu381Hisfs∗95)-were identified in four subjects from three families. All variants severely affected protein stability, causing accelerated degradation, and variably perturbed SPRED2 functional behavior. When overexpressed in cells, all variants were unable to negatively modulate EGF-promoted RAF1, MEK, and ERK phosphorylation, and time-course experiments in primary fibroblasts (p.Leu100Pro and p.Leu381Hisfs∗95) documented an increased and prolonged activation of the MAPK cascade in response to EGF stimulation. Morpholino-mediated knockdown of spred2a and spred2b in zebrafish induced defects in convergence and extension cell movements indicating upregulated RAS-MAPK signaling, which were rescued by expressing wild-type SPRED2 but not the SPRED2Leu381Hisfs∗95 protein. The clinical phenotype of the four affected individuals included developmental delay, intellectual disability, cardiac defects, short stature, skeletal anomalies, and a typical facial gestalt as major features, without the occurrence of the distinctive skin signs characterizing Legius syndrome. These features, in part, characterize the phenotype of Spred2-/- mice. Our findings identify the second recessive form of Noonan syndrome and document pleiotropic consequences of SPRED2 loss of function in development.

ENGOT-ov60/GOG3052/RAMP 201: A phase 2 study of VS-6766 (dual RAF/MEK inhibitor) alone and in combination with defactinib (FAK inhibitor) in recurrent low-grade serous ovarian cancer (LGSOC).

TPS5603 Background: VS-6766 is a unique small molecule inhibitor that blocks MEK kinase activity and RAF phosphorylation of MEK. This mechanism of blockade has been shown to limit compensatory MEK activation, thereby potentially enhancing efficacy of MEK inhibition. Defactinib, (VS-6063), an orally active small molecule, is a potent adenosine 5'- triphosphate (ATP) competitive, reversible inhibitor of focal adhesion kinase (FAK). Defactinib has shown synergistic activity with BRAF and MEK inhibitors in both in vitro and in vivo solid tumor models. Prior molecularly unselected studies with single agent MEK inhibitors have shown response rates up to 26% in recurrent LGSOC. A third of patients with recurrent LGSOC harbor somatic KRAS mutations. FAK inhibition has been shown to induce tumor regression when combined with RAF, MEK or RAF/MEK inhibitors in in vivo models of KRAS mutant ovarian cancer. The combination of VS-6766 and defactinib is currently being evaluated in the ongoing Investigator Sponsored FRAME study (NCT03875820). In this proof of concept study, durable objective responses have been reported in recurrent LGSOC patients, particularly those with KRAS mutations including patients who have had a prior MEK inhibitor (Banerji et al AACR 2020). Based on preclinical studies demonstrating efficacy of both VS-6766 and the VS-6766/defactinib combination and preliminary results of the FRAME study, the phase II ENGOT-ov60/GOG3052 has been developed in recurrent LGSOC. Methods: This is a Phase II, adaptive, two-part, multicenter, parallel cohort, randomized, open label study designed to evaluate the efficacy and safety of VS-6766 versus VS-6766 in combination with defactinib (NCT04625270). The study will be conducted in two parts. Part A will determine the optimal regimen based on confirmed overall response rate (independent radiology review) in KRAS-mutated LGSOC. Part B will determine the efficacy of the optimal regimen identified in Part A in KRAS-mutated and KRAS wild-type LGSOC. The minimum expected enrollment is 52 subjects with KRAS-mutated tumors (32 subjects in Part A and 20 in Part B) and 36 with KRAS wild-type tumors in Part B. Patients will be randomized to receive VS-6766 (4.0 mg PO, twice weekly 3 weeks on, 1 week off) or VS6766 with defactinib (VS-6766 3.2 mg PO, twice weekly + defactinib 200 mg PO BID 3 weeks on, 1 week off) till progression. Key inclusion criteria include histologically confirmed LGSOC, presence of KRAS mutation (Part A), prior systemic therapy for metastatic disease and up to 1 prior line of MEK/RAF inhibitor therapy permitted. This international study is open to enrollment. Clinical trial information: NCT04625270.