SOS1 Mutations Research Articles

Deciphering the toxicity of polyhexamethylene guanidine phosphate in lung carcinogenesis: Mutational profiles and molecular mechanisms

Polyhexamethylene guanidine (PHMG) is widely utilized in personal hygiene products due to its bactericidal, non-volatile, and hydrophilic properties. However, the long-term toxic effects and underlying mechanisms associated with respiratory exposure to the commonly used form, PHMG phosphate (PHMG-p), are still insufficiently understood. This study aims to elucidate the types of pulmonary lesions and the incidence of lung cancer associated with varying concentrations of PHMG-p and observation periods, along with the molecular mechanisms underlying this relationship. To assess these effects, CT scans and pathological analyses were conducted for up to 54 weeks following initial exposure to PHMG-p. Furthermore, to investigate the underlying causes of pulmonary toxicity, TGF-beta-activated kinase 1 was identified as a PHMG-p-binding protein, and its associated signaling pathways, including necroptosis, apoptosis, and MKK7, were explored. Somatic mutational signature, and gene ontology (GO) analyses were performed to investigate the genetic characteristics of PHMG-p-induced lung carcinogenesis. PHMG-p exposure led to somatic mutations in lung cancer-related genes, including TP53, SOS1, KMT2D, MDM2, ERBB2, SETD2, MET, ARID1A, RBM10, and CDKN2A as well as in genes such as RAB31, WASHC1, DDX11, ECD, STAB2, MUC2, and MUC5AC. The mutated genes were primarily associated with impaired DNA repair mechanisms. GO analysis highlighted the activation of pathways related to cell cycle checkpoints, necroptosis, MAPK, and idiopathic pulmonary fibrosis, while also revealing the suppression of signaling pathways associated with natural killer cells, GADD45, LXR/RXR activation, and IL-15 production. Gain-of-function experiments confirmed the oncogenic roles of PLAU and HMGA2, as well as the tumor-suppressive functions of TBX4 and GPX3. These findings suggest that PHMG-p activates necroptosis and MAPK signaling, increases the frequency of somatic mutations, and inhibits apoptosis, thus fostering an environment conducive to carcinogenesis. This underscores the importance of understanding the potential health risks associated with PHMG-p exposure and provides insights for future research and regulatory considerations regarding the safety of personal hygiene products.

Characterization of the BH1406 non-small cell lung cancer (NSCLC) cell line carrying an activating SOS1 mutation

Characterization of the BH1406 non-small cell lung cancer (NSCLC) cell line carrying an activating SOS1 mutation

Abstract 2080: SOS1 inhibition is an effective therapeutic strategy in SOS1-mutant cancer

Abstract SOS1 is a principal guanine nucleotide exchange factor for canonical RAS GTPases and activates RAS signaling. Mutations in SOS1 are found in several prominent cancer types, including 2-4% of NSCLC, CRC, and melanoma. The recent development of novel SOS1 inhibitors (SOS1i), including BI-3406, that block interaction between SOS1 and RAS, presents a potential therapy for SOS1-driven cancers. Here we show that the majority of SOS1 mutations observed in patients enhance RAS guanine nucleotide exchange in cells and activate RAS-MAPK signaling. We treated 11 SOS1 mutant cancer cell lines without RAS co-mutation with a SOS1i and found that 10 were sensitive to SOS1 inhibition in 2D and/or 3D cell culture. In contrast, cell lines with SOS1 and RAS co-mutations were resistant to SOS1 inhibition. In vitro treatment of SOS1 mutant cells with a SHP2 inhibitor, which is presumed to inhibit the activity of both SOS1 and SOS2, revealed a subset of four cell lines that were more sensitive to SHP2 inhibition than SOS1 inhibition. This implied that, in certain contexts, SOS2 can compensate for the loss of SOS1 activity. Accordingly, these cells exhibited a SOS2-dependent rebound in RAS-MAPK signaling after treatment with SOS1i. SOS2 knock-down further sensitized these cells to SOS1 inhibition, resulting in responses equivalent to SHP2 inhibition. Thus, upon SOS1 inhibition relief of ERK1/2-dependent negative regulation likely contributes to receptor tyrosine kinase-driven SOS2 activation that may mediate SOS1 inhibitor resistance in some contexts. In vivo, SOS1i efficiently inhibited tumor growth in four SOS1-mutant NSCLC, CRC, and AML xenograft models. The tumor-suppressive effects of SOS1i in these models were comparable to or stronger than the effects of SHP2i or MEKi. Thus, our results suggest that SOS1 inhibition could be an effective therapeutic approach in RAS wild-type cancer patients with SOS1 mutations. Citation Format: Matthew J. Sale, Neal Mukherjee, Martín Koch, Regina Ruzicka, Robin Jacob, Christoph Albrecht, Teresa Zanin, Thomas Madensky, Robyn L. Schenk, Anke Baum, Kaja Kostyrko, Daniel Gerlach, Marco H. Hofmann, Frank McCormick. SOS1 inhibition is an effective therapeutic strategy in SOS1-mutant cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 2080.

Prominent corneal nerves in pure mucosal neuroma syndrome, a clinical phenotype distinct from multiple endocrine neoplasia type 2B

BackgroundPure mucosal neuroma syndrome (MNS), an autosomal dominant neurocutaneous disorder, is a rare discrete subgroup in multiple endocrine neoplasia (MEN) type 2B, which present without associated endocrinopathies of MEN2B but with typical physical features such as prominent corneal nerves.Case presentationThis report describes a 41-year-old patient with complaint of itchy eyes and irritation, presenting with blocked gland orifices in the upper and lower eyelids, light conjunctival hyperemia, a semitransparent neoplasm measuring 2 mm*2 mm on the nasal limbus suggestive of neuromas, and prominent corneal nerves. In vivo confocal microscopy (IVCM) revealed structural alterations—namely a prominent hyperreflective, thickened nerve plexus and a normal endothelium—in both eyes. Testing for SOS1 mutation was positive. This patient may represent a discrete subgroup termed pure mucosal neuroma syndrome (MNS), which presents with the characteristic appearance of MEN2B but without RET gene mutations.ConclusionProminent corneal nerves have been described in some diseases, such as multiple endocrine neoplasia (MEN) type 1 and type 2A and 2B, congenital ichthyosis, Refsum’s disease, leprosy, etc. Ophthalmic assessment including prominent corneal nerves has proven valuable in asymptomatic individuals of MEN2B. Our case illustrates the importance of recognizing the ocular features of MNS, a rare presentation of MEN2B, in order to prevent prophylactic thyroidectomy in these patients for prophylactic thyroidectomy is not mandatory in MNS. However, regular monitoring and genetic counseling are still necessary.

Abstract 1715: SOS1 inhibition is an effective therapeutic strategy in SOS1-mutant cancer

Abstract SOS1 is a principal guanine nucleotide exchange factor for canonical RAS GTPases and activates RAS signaling. Mutations in SOS1 are found in 3-5% of non-small cell lung cancers (NSCLC), colorectal cancers (CRC) and melanomas. The recent development of novel SOS1 inhibitors, including BI-3406 and BI-1701963, that block interaction between SOS1 and RAS, presents a potential therapy for SOS1-driven cancers. Here we show that the majority of SOS1 mutations observed in patients enhance RAS guanine nucleotide exchange in cells and activate RAS-MAPK signaling. Of the 11 available SOS1-mutant cancer cell lines without RAS co-mutation, 10 were sensitive to SOS1 inhibition in 2D and/or 3D cell culture, while cells with SOS1 and RAS co-mutations were resistant to SOS1 inhibition. Treatment with a SHP2 inhibitor, which is presumed to inhibit the activity of both SOS1 and SOS2, revealed a subset of SOS1-mutant cell lines that were more sensitive to SHP2 inhibition than SOS1 inhibition. This implied that, in certain settings, SOS2 can compensate for the loss of SOS1 activity. Accordingly, these cells exhibited a rebound in RAS signaling after treatment with SOS1i that was dependent on SOS2. SOS2 knock-down further sensitized these cells to SOS1 inhibition, resulting in responses equivalent to SHP2 inhibition. In addition, we show that, as with SOS1, SOS2 is a target for ERK1/2-dependent feedback phosphorylation. Thus, relief of ERK1/2-dependent negative regulation of SOS2, following SOS1 inhibition, likely contributes to receptor tyrosine kinase (RTK)-SOS2 activation that may mediate SOS1 inhibitor resistance in some contexts. In vivo, SOS1i inhibited tumor growth in xenograft models of SOS1-mutant AML and NSCLC. Tumor-suppressive effects of SOS1i in these models were comparable to or stronger than the effects of SHP2i or MEKi. Thus, our results suggest that SOS1 inhibition could be an effective therapeutic approach in RAS wild-type cancer patients with SOS1 mutations. Citation Format: Matthew J. Sale, Neal Mukherjee, Regina Ruzicka, Robin Jacob, Christoph Albrecht, Teresa Zanin, Tom Madensky, Kaja Kostyrko, Robyn L. Schenk, Anke Baum, Daniel Gerlach, Marco H. Hofmann, Frank McCormick. SOS1 inhibition is an effective therapeutic strategy in SOS1-mutant cancer [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 1715.

LACK OF EMBRYONIC HOMOZYGOUS OR ADULT HETEROZYGOUS LYMPHATIC PHENOTYPES FOR A Sos1 MUTATION AND LACK OF LYMPHATIC EMBRYONIC PHENOTYPES FOR A HOMOZYGOUS Cx47 MUTATION IN MICE

We have studied the lymphatic phenotypes of 2 mutations, known to cause abnormalities of lymphatics in humans, in mice. The Cx47 R260C mutation (variably penetrant in humans heterozygous for it and causing limb lymphedema) had an adult mouse phenotype of hyperplasia and increased lymph nodes only in homozygous condition but we did not find any anatomical phenotype in day 16.5 homozygous embryos. Mice harboring the Sos1 mutation E846K (causing Noonan's in man which occasionally shows lymphatic dysplasia) had no adult heterozygous phenotype in lymphatic vessel appearance and drainage (homozygotes are early embryonic lethals) while day 16.5 heterozygous embryos also had no detectable anatomical phenotype.

Molecular characterization using exome sequencing of two probands with the undiagnosed developmental delay from Central Brazil

Noonan syndrome (NS) is a heterogeneous autosomal dominant disorder caused by germline mutations in genes belonging RAS-MAPK pathway. Herein, we described two patients with developmental delay and syndromic features from Central Brazil diagnosed with NS using an exome sequencing target gene panel. Germline mutation in genes that participates in the RAS-MAPK signaling pathway are associated with developmental disorders that share particular clinical features such as craniofacial dysmorphisms, congenital heart defects, musculoskeletal and ocular abnormalities, and neurocognitive impairment. The exome sequencing through the intellectual disability gene panel was an effective approach to identify de novo pathogenic mutations in SOS1 and PTPN11 genes that are responsible for Noonan syndrome and was an efficient method to direct the adequate clinical management and better follow-up of the probands and their families.

Cardiac features of Noonan syndrome in Japanese patients.

Cardiovascular disease is one of the most important problems in long-term follow-up for Noonan syndrome. We examined cardiovascular issues and clinical manifestations, with a focus on the cardiovascular disease and prognosis of patients with Noonan syndrome. This single-centre study evaluated patients who were clinically and genetically diagnosed with Noonan syndrome. Forty-three patients diagnosed with Noonan syndrome were analysed. The most prevalent responsible mutation was found in PTPN11 (25/43). The second and third most prevalent causative genes were SOS1 (6/43) and RIT1 (5/43), respectively, and 67.4% of genetically diagnosed patients with Noonan syndrome had structural cardiovascular abnormalities. Pulmonary valve stenosis was prevalent in patients with mutations in PTPN11 (8/25), SOS1 (4/6), and RIT1 (4/5). Hypertrophic cardiomyopathy was found in two of three patients with mutations in RAF1. There was no difference in the cardiovascular events or cardiovascular disease prevalence in patients with or without PTPN11 mutations. The proportion of RIT1 mutation-positive patients who underwent intervention due to cardiovascular disease was significantly higher than that of patients with PTPN11 mutations. Patients who underwent any intervention for pulmonary valve stenosis exhibited significantly higher pulmonary flow velocity than patients who did not undergo intervention, when they visited our hospital for the first time. All patients who underwent intervention for pulmonary valve stenosis had a pulmonary flow velocity of more than 3.0 m/s at first visit. These findings suggest that genetic information can provide a clinical prognosis for cardiovascular disease and may be part of genotype-based follow-up in Noonan syndrome.

Treatment of severe Kaposiform lymphangiomatosis positive for NRAS mutation by MEK inhibition.

Kaposiform lymphangiomatosis (KLA) is a complex lymphatic anomaly involving most commonly the mediastinum, lung, skin and bones with few effective treatments. In recent years, RAS-MAPK pathway mutations were shown to underlie the pathogenesis of several complex lymphatic anomalies. Specifically, an activating NRAS mutation (p.Q61R) was found in the majority of KLA patients. Recent reports demonstrated promising results of treatment with the MEK inhibitor, Trametinib, in patients with complex lymphatic anomalies harboring gain of function mutations in ARAF and SOS1, as well as loss of function mutation in the CBL gene, a negative regulator of the RAS-MAPK pathway. We present a 9-year-old child with a severe case of KLA harboring the typical NRAS (p.Q61R) mutation detected by plasma-derived cell free DNA, responsive to trametinib therapy. The NRAS somatic mutation was detected from plasma cfDNA using droplet digital PCR. Concurrent in-vitro studies of trametinib activity on mutant NRAS affected lymphatic endothelial cells were performed using a three-dimensional spheroid sprouting assay. Trametinib treatment lead to resolution of lifelong thrombocytopenia, improvement of pulmonary function tests and wellbeing, as well as weaning from prolonged systemic steroid treatment. Concurrent studies of mutant NRAS-expressing cells showed enhanced lymphangiogenic capacity along with over activation of the RAS-MAPK and PI3K-AKT-mTOR pathways, both reversed by trametinib. Trametinib treatment can substantially change the prognosis of patients with RAS pathway associated lymphatic anomalies. This is the first description of successful trametinib treatment of a patient with KLA harboring the most characteristic NRAS p.Q61R mutation. Treatment can significantly change the prognosis of patients with RAS pathway-associated lymphatic anomalies. We devised an in vitro model of KLA enabling a reproducible method for the continued study of disease pathogenesis. Mutated NRAS p.Q61R cells demonstrated increased lymphangiogenic capacity.

Joint involvement in Noonan syndrome. A retrospective paediatric descriptive study.

Noonan syndrome is a rare genetic disorder characterized mainly by congenital heart disease, occasional intellectual disability, and varied orthopaedic, rheumatological and haematologic anomalies. Despite potentially serious functional consequences, joint involvement has been rarely studied in the literature. Our objective was to perform a retrospective study evaluating the prevalence and characteristics of joint involvement in Noonan syndrome. We recorded articular symptoms, including their type and frequency, in patients with Noonan syndrome followed up in French hospitals. Patients were included if the diagnosis was confirmed before the age of 20 based on the van der Burgt criteria or genetic analysis. Data are presented as frequencies or medians (ranges), and patient groups were compared using chi-square or Fisher tests. Seventy-one patients were included from 4 centres. The average age was 12.5 years (range: 2-36). Musculoskeletal pain was found in 18 patients (25%) and joint stiffness in 10 (14%) located in the wrists, elbows, ankles, knees and hips, which was usually bilateral. Only one destructive form was described (multiple villonodular synovitis and a giant cell lesion of the jaw). There were no cases of systemic lupus erythaematosus (SLE) or other autoimmune arthritis. Raynaud's phenomenon was observed in 3 patients. Only 50% of joint complaints led to additional exploration. SOS1 mutations (P<0.05) and treatment with growth hormone (GH) (P<0.05) were the only factors significantly related to musculoskeletal pain. Patients treated with GH did not have more SOS1 mutations. Patients experiencing pain were not more likely to experience stiffness, joint hypermobility, or coagulation abnormalities. Joint manifestations were frequent in Noonan syndrome, predominant in large joints, and rarely explored. Multiple villonodular synovitis is characteristic but rare. Auto-immune disorders were not described in this cohort. A more multidisciplinary approach could be recommended for the early detection of possibly disabling rheumatologic manifestations.

PATH-25. H3 K27M-MUTANT DIFFUSE NON-MIDLINE GLIOMA: REPORT OF A CASE WITH EXTENDED FOLLOW-UP

Abstract H3 K27M-mutant diffuse gliomas without midline involvement are extremely rare and their clinical behavior remains elusive due to limited reported follow-up data. We describe an H3 K27M-mutant diffuse non-midline glioma patient with extended follow-up. A 34-year-old woman presented with headache, memory loss, periods of changes in taste and smell, and confusion. Imaging studies revealed an 2.3 cm expansile T2/flair hyperintensity with patchy postcontrast enhancement centered in the left amygdala without associated restricted diffusion and no involvement of the midline structures. The tumor was debulked and consisted of a mitotically active infiltrating astrocytic glioma without tumor necrosis or microvascular proliferation, consistent with anaplastic astrocytoma. Targeted 187-gene neuro-oncology NGS testing detected an H3F3A K27M mutation along with a PTPN11 and a PPM1D mutation. FISH 1p/19q-codeletion testing was negative. MGMT promoter was unmethylated. The patient was treated with chemoradiation with temozolomide for 6 weeks followed by 12 cycles of temozolomide. Four years after initial resection, an area of increased post-contrast enhancement indicating tumor recurrence/progression was noted. Partial resection of the recurrent tumor revealed a mitotically active infiltrating astrocytic glioma with tumor necrosis consistent with glioblastoma. Molecular profiling of the recurrent tumor by the neuro-oncology NGS panel detected similar mutational profile (identical H3F3A K27M and PTPN11 mutation; different PPM1D mutation) with an additional SOS1 mutation. The patient completed 4 cycles of Lomustine and although clinically stable, imaging studies showed slight increase in residual tumor size concerning for tumor progression. Lomustine was discontinued, Bevacizumab therapy was initiated and patient was enrolled in clinical trial (NCT02525692). Despite tumor progression, this patient has had a relatively long disease course (5 years) suggesting that H3 K27M-mutant non-midline diffuse gliomas although molecularly similar, may follow a more favorable clinical course than their midline counterparts possibly due to the hemispheric location, which is more amenable for surgical resection.

In-Silico and In-Vitro Analysis of Human SOS1 Protein Causing Noonan Syndrome - A Novel Approach to Explore the Molecular Pathways.

AimsPerform in-silico analysis of human SOS1 mutations to elucidate their pathogenic role in Noonan syndrome (NS).BackgroundNS is an autosomal dominant genetic disorder caused by single nucleotide mutation in PTPN11, SOS1, RAF1, and KRAS genes. NS is thought to affect approximately 1 in 1000. NS patients suffer different pathogenic effects depending on the mutations they carry. Analysis of the mutations would be a promising predictor in identifying the pathogenic effect of NS.MethodsWe performed computational analysis of the SOS1 gene to identify the pathogenic nonsynonymous single nucleotide polymorphisms (nsSNPs) th a t cause NS. SOS1 variants were retrieved from the SNP database (dbSNP) and analyzed by in-silico tools I-Mutant, iPTREESTAB, and MutPred to elucidate their structural and functional characteristics.ResultsWe found that 11 nsSNPs of SOS1 that were linked to NS. 3D modeling of the wild-type and the 11 nsSNPs of SOS1 showed that SOS1 interacts with cardiac proteins GATA4, TNNT2, and ACTN2. We also found that GRB2 and HRAS act as intermediate molecules between SOS1 and cardiac proteins. Our in-silico analysis findings were further validated using induced cardiomyocytes (iCMCs) derived from NS patients carrying SOS1 gene variant c.1654A>G (NSiCMCs) and compared to control human skin fibroblast-derived iCMCs (C-iCMCs). Our in vitro data confirmed that the SOS1, GRB2 and HRAS gene expressions as well as the activated ERK protein, were significantly decreased in NS-iCMCs when compared to C-iCMCs.ConclusionThis is the first in-silico and in vitro study demonstrating that 11 nsSNPs of SOS1 play deleterious pathogenic roles in causing NS.

Genotype-cardiac phenotype correlations in a large single-center cohort of patients affected by RASopathies: Clinical implications and literature review.

Congenital heart disease (CHD) and hypertrophic cardiomyopathy (HCM) are common features in patients affected by RASopathies. The aim of this study was to assess genotype- phenotype correlations, focusing on the cardiac features and outcomes of interventions for cardiac conditions, in a single-center cohort of 116 patients with molecularly confirmed diagnosis of RASopathy, and compare these findings with previously published data. All enrolled patients underwent a comprehensive echocardiographic examination. Relevant information was also retrospectively collected through the analysis of clinical records. As expected, significant associations were found between PTPN11 mutations and pulmonary stenosis (both valvular and supravalvular) and pulmonary valve dysplasia, and between SOS1 mutations and valvular defects. Similarly, HRAS mutations were significantly associated with HCM. Potential associations between less prevalent mutations and cardiac defects were also observed, including RIT1 mutations and HCM, SOS2 mutations and septal defects, and SHOC2 mutations and septal and valve abnormalities. Patients with PTPN11 mutations were the most likely to require both a primary treatment (transcatheter or surgical) and surgical reintervention. Other cardiac anomalies less reported until recently in this population, such as isolated functional and structural mitral valve diseases, as well as a sigmoid-shaped interventricular septum in the absence of HCM, were also reported. In conclusion, our study confirms previous data but also provides new insights on cardiac involvement in RASopathies. Further research concerning genotype/phenotype associations in RASopathies could lead to a more rational approach to surgery and the consideration of drug therapy in patients at higher risk due to age, severity, anatomy, and comorbidities.

Hypomagnesemia and seizures in a patient with an SOS1 mutation

Noonan syndrome (NS) is an autosomal dominant multisystem disorder. The son of sevenless homolog 1 (symbolized SOS1) gene encodes a guanine nucleotide exchange factor (GEF) for Ras proteins.[1] Genetic disorders in which the SOS1 mutation has been confirmed include NS. Missense mutations in SOS1 account for roughly 10% of NS.[2]

TBIO-27. RASOPATHIES AND BRAIN TUMOROGENESIS: ARE SOS1 MUTATIONS ARE CONCERNED?

Germ line gain-of-function mutations in several members of the RAS/MAPK pathway, including PTPN11 are associated with signalopathies named Rasopathies and known as Noonan syndrome and closely related conditions. Patients harboring Rasopathies are at increased risk of myeloproliferative diseases and solid tumors, such as neuroblastoma. Mutations of SOS1, the gene encoding a guanine nucleotide exchange factor for Ras, represent the second most frequent genetic defect in Rasopathies. However, SOS1 mutations are rare in human malignancies and patients with germline SOS1 mutations may not be at increased risk of developing cancer. Here, we report a SOS1 variant found to segregate in a Tunisian pedigree with many members affected by brain tumors as well as epileptic disorder. During our genetic counselling for congenital heart diseases, a 9-year-old female born at Sfax from a consanguineous couple and having pulmonic valvular stenosis, has been investigated at the molecular level. Screening of mutations in the entire coding sequence of PTPN11, Braf and SOS1, was conducted using HRM analysis and bidirectional sequencing. Heterozygous single nucleotide substitution of SOS1 gene: c.1655 G>A was confirmed. This mutation affected the PH‐REM linker domain with substitution of residue Arg552 to Lys: p.Arg552Lys. This mutation accounts for one‐third of all mutations reported in SOS1 during Rasopathies. Although no other molecular exploration was done, family history revealed other affected children by neurodevelopmental and epileptic conditions as well as recurrent brain malignancies in the paternal family. Two aunts developed blindness and then died subsequently to tumor progression.

Severe Lymphatic Disorder Resolved With MEK Inhibition in a Patient With Noonan Syndrome and SOS1 Mutation.

Noonan syndrome is a multiorgan system disorder mediated by genetic defects along the RASknown as RASopathies. It is the second most common syndromic cause of congenital heart disease and, in ∼20% of the cases, is associated with severe lymphatic disorders, including chylothorax and protein-losing enteropathy. Recently, we reported on the use of mitogen-activated protein kinase inhibition in a patient with an ARAF mutation and severe lymphatic disorder leading to an abrupt improvement in symptoms and complete remodeling of the central lymphatic system. Here, we present a patient with Noonan syndrome and severe lymphatic abnormality, leading to transfusion-dependent upper gastrointestinal bleeding and protein-losing enteropathy. The patient stopped responding to medical therapy and underwent several lymphatic interventional procedures, which led only to a temporary improvement in symptoms. Because of a lack of other treatment options, an expanded access approval was obtained, and the patient initiated treatment by mitogen-activated protein kinase inhibition using trametinib. This led to resolution of her symptoms, with complete normalization of her electrolyte levels, hemoglobin, and albumin within 3 months of starting the drug. Similar to the previously reported case, she also had complete and generalized remodeling of her lymphatic system. In patients with RAS pathway defects complicated by a severe lymphatic disorder, inhibition of the RAS-MAPK pathway should be considered as a possible treatment option in patients who failed conventional treatment and might be a first-line treatment in the future.

Abstract 489: Identification And In-silico Analysis Of Pathogenic Non-synonymous Snps Of Human Sos1 Protein In Noonan Syndrome

Introduction: Noonan syndrome is a genetic disorder (autosomal dominant) characterized by short stature, congenital heart disease, bleeding problems, developmental delays, and skeletal malformation. It is mainly caused by a single nucleotide alteration in four genes PTPN11, SOS1, RAF1, and KRAS . In this study, we computationally analyzed the SOS1 gene to identify the pathogenic non-synonymous single nucleotide polymorphisms (nsSNPs), which is known to cause Noonan syndrome. Hypothesis: We hypothesize that in-silico analysis of human SOS1 mutations in Noonan syndrome would be a promising predictor to study the post-translational modifications. Methods and Results: The variant information of SOS1 was collected from the dbSNP database and the literature review on Noonan syndrome. They were further analyzed by in-silico tools such as I-Mutant, iPTREE-STAB, and MutPred for their structural and functional properties. We found that 11 nsSNPs are more pathogenic for Noonan syndrome. The 3D comparative protein of 11 nsSNPs with its wild-type SOS1 was modeled by using I-Tasser and validated via ERRAT and RAMPAGE. The protein-protein interactions of SOS1, GATA4, TNNT2, and ACTN2 were analyzed using STRING, which showed that HRAS was intermediate between SOS1 and ACTN2 (Fig. 1) . Conclusion: This is the first in-silico study of the SOS1 variant with Noonan syndrome. We proposed that this 11 nsSNPs are the most pathogenic variant of SOS1 , which helps to screen the Noonan patient. Furthermore, our results are promising to study the gain/loss of post-translational modification (PTM) by mutation in cardiac genes and helps to explore the novel molecular pathways.$graphic_{DB5B0E7D-4DA6-4569-A16F-E05B2C9C4D2F}$$

Genetic Examination for Fetuses with Increased Fetal Nuchal Translucency by Genomic Technology

This study aims to investigate the value of chromosomal microarray analysis (CMA) and whole exome sequencing (WES) in fetuses with increased nuchal translucency (defined as NT above the 95th centile for the crown-rump length). A total of 374 singleton pregnancies with gestational ages ranging from 11 to 13 + 6 weeks were investigated. Ultrasound displayed increased NT and no detectable structural malformations in these fetuses. Pregnancies were divided into 4 groups according to the NT values: 95th centile-3.4 mm (114 cases); 3.5-4.4 mm (150 cases); 4.5-5.4 mm (55 cases); and ≥5.5 mm (55 cases). The possible chromosomal anomalies were all analyzed by CMA first. Furthermore, 24 cases with increased NT but negative CMA results were investigated by WES, and the outcomes were followed up. Among all the 374 cases, causative genetic defects were detected in 100/374 (26.7%) of the cases along with 9 variants of unknown significance (VOUS) by CMA. CMA testing yielded 30 pathogenic variants (30/55), accounting for a detection rate of 54.5%, and 1 VOUS in the group of NT ≥5.5 mm, indicating the highest detection rate in the 4 groups. The 24 cases of the CMA negative sub-cohort with WES analysis further yielded 2 VOUS and 3 likely pathogenic variants, including 2 dominant de novo mutations in SOS1 and ECE1 and 1 recessive inherited compound heterozygous mutation in PIGN, which are associated with cardiac defects. All 3 cases opted for termination of pregnancy (TOP). In addition, 2 cases with increased NT were negative by both CMA and WES analysis, and fetal demise occurred. In conclusion, for the investigation of fetuses with increased NT exome sequencing is suggested to be considered in cases with negative CMA findings. However, appropriate genetic counseling should be given to optimizing its utilization in prenatal diagnosis.

The plasma-membrane polyamine transporter PUT3 is regulated by the Na+ /H+ antiporter SOS1 and protein kinase SOS2.

In Arabidopsis, the plasma membrane transporter PUT3 is important to maintain the cellular homeostasis of polyamines and plays a role in stabilizing mRNAs of some heat-inducible genes. The plasma membrane Na+ /H+ transporter SOS1 and the protein kinase SOS2 are two salt-tolerance determinants crucial for maintaining intracellular Na+ and K+ homeostasis. Here, we report that PUT3 genetically and physically interacts with SOS1 and SOS2, and these interactions modulate PUT3 transport activity. Overexpression of PUT3 (PUT3OE) results in hypersensitivity of the transgenic plants to polyamine and paraquat. The hypersensitivity of PUT3OE is inhibited by the sos1 and sos2 mutations, which indicates that SOS1 and SOS2 are required for PUT3 transport activity. A protein interaction assay revealed that PUT3 physically interacts with SOS1 and SOS2 in yeast and plant cells. SOS2 phosphorylates PUT3 both in vitro and in vivo. SOS1 and SOS2 synergistically activate the polyamine transport activity of PUT3, and PUT3 also modulates SOS1 activity by activating SOS2 in yeast cells. Overall, our findings suggest that both plasma-membrane proteins PUT3 and SOS1 could form a complex with the protein kinase SOS2 in response to stress conditions and modulate the transport activity of each other through protein interactions and phosphorylation.

Combination of the MEK Inhibitor Trametinib and Pyrvinium Pamoate Efficiently Targets RAS Pathway-Mutated Acute Myeloid Leukemia in Preclinical Models

While acute myeloid leukemia (AML) is still associated with a low cure rate, recent advances in understanding its molecular complexity have significantly improved therapy for subgroups of patients, including those harboring FLT3, IDH1 or IDH2 mutations1. However, more than half of AML cases still lack a druggable oncogenic target. Human cancers frequently harbor mutations in RAS oncogene family members, including NRAS, KRAS and HRAS, which drive oncogenesis by augmenting cellular proliferation and survival. These are small protein GTPases, regulated by a switch between active GTP-linked and inactive GDP-bound states governed by a complex network of guanine exchange factors (GEFs, favoring RAS-GTP) and GTPase activating factors (GAPs, favoring RAS-GDP). RAS activation - due to either extrinsic recruitment by transmembrane tyrosine kinase receptors or intrinsic mutations - propagates through the downstream RAF/MEK/ERK and PI3K/AKT signaling pathways. Besides RAS-activating mutations conferring independence from physiological regulators, human cancers harbor mutations in other RAS network genes such as NF1 (encoding neurofibromin, a RAS GAP), BRAF or PTPN11 (encoding the SHP2 tyrosine phosphatase involved in RAS activation). Somatic alterations of RAS pathway genes, notably NRAS, KRAS, PTPN11 (missense mutations) and NF1 (mutations and deletions), are reported in up to 20% of AML cases2. Generally arising as late driver events, RAS pathway mutations participate in leukemogenesis through mitogen activated protein kinase (MAPK) activation. The anti-tumor activity of MEK inhibitors in Nras-mutated AML in mice and in some NRAS or KRAS-mutated AML patients suggests that deregulated RAS pathway signaling may represent a bona fide therapeutic target. However, strategies to inhibit RAS - indirectly in most cases - have been hampered by signaling feedback, redundancy and tumor heterogeneity 3. We identified 127 cases of AML with unmet therapeutic need within a cohort from which we excluded those with European leukemia network (ELN) favorable prognosis or FLT3-ITD mutations. Targeted next-generation sequencing revealed RAS pathway alterations in 50 patients (39.3%) and NF1 mutations and deletions, mostly large cytogenetically detected deletions, in 17 (14.8%). NRAS, KRAS, PTPN11, CBL and BRAF variants were detected in 13 (10.4%), 10 (7.9%), 9 (7.2%), 5 (3.9%) and 2 (1.6%) cases, respectively. Mutations in RAF1, RASA1, SOS1 and MAP2K2 were observed in a single case each. RAS pathway alterations appeared in the putative main leukemic clone as well as in subclones inferred from variant allele frequencies. Concurrent RAS pathway mutations were observed in nine cases. Among 79 patients homogeneously treated with intensive induction chemotherapy, RAS pathway alterations correlated with higher clinical proliferation markers (elevated white blood cell count, blast cell percentage and LDH levels) and reduced survival probability, particularly within the ELN intermediate-risk subgroup. We established robust models of RAS/MAPK activation through genetic NF1 disruption or expression of NRASG12D or PTPN11D61Y in growth factor (GF)-dependent cell lines. We assessed oncogenic addiction to the RAS pathway in these cells through GF-independence, increased RAS activity, faster propagation in immunocompromised mice and an exquisite sensitivity to pharmacological MEK inhibition in vitro and in vivo. High-content pharmacological screens with FDA-approved molecules identified pyrvinium pamoate, an anti-helminthic agent, as preferentially active in RAS-activated cells. This compound significantly impaired cell viability and colony formation in primary AML samples with RAS pathway alterations. Moreover, the combination of trametinib and pyrvinium pamoate demonstrated synergy in cell line models and even primary samples. While pyrvinium pamoate strongly inhibited mitochondrial respiration and induced metabolic reprograming towards increased glycolysis, trametinib impaired glycolysis and mitochondrial respiratory capacity, suggesting a mechanistic basis for the synergy observed. These data highlight the translational opportunity in developing pyrvinium pamoate for RAS pathway mutated AML.