Ras Signal Transduction Research Articles

Ras superfamily GTPases and signal transduction in Euglena gracilis

Biological complexity is challenging to define, but can be considered through one or more features, including overall genome size, number of genes, morphological features, multicellularity, number of life cycle stages and the ability to adapt to different environments. Euglena gracilis meets several of these criteria, with a large genome of ∼38,000 protein coding genes and a considerable ability to survive under many different conditions, some of which can be described as challenging or harsh. Potential molecular exemplars of complexity tying these aspects together are signalling pathways, including GTPases, kinases and ubiquitylation, which increase the functionality of the gene-encoded proteome manyfold. Each of these examples can modulate both protein activity and gene expression. To address the connection between genome size and complexity I have undertaken a brief, and somewhat qualitative, survey of the small ras-like GTPase superfamily of E. gracilis. Unexpectedly, apart from Rab-GTPases which control intracellular transport and organelle identify, the size of the GTPase cohort is modest, and, for example, has not scaled with gene number when compared to the close relatives, trypanosomatids. I suggest that understanding the functions of this protein family will be vital to uncovering the complexity of E. gracilis biology.

Hyperinsulinism Gateway Between Diabetes and Cancer: New Roles for Bifunctional Antidiabetic - Anticancer Active Somatostatins, IGF - 1 Inhibitors and Insulin Sensitizers

Hyperinsulinism refers to a condition of abnormally high concentration of circulating insulin in the body commonly seen in obese and type 2 diabetic patients due to insulin resistance. Elevated circulating insulin is the main factor linking obesity, diabetes and cancer and an important mechanism of cancer therapeutic resistance and failure. The insulin receptor subfamily which is made up of the insulin receptor (IR), the type 1 IGF receptor (IGF-1) which binds insulin-like growth factors I and II, and the orphan insulin receptor-related receptor (IRRR) have been identified and linked to the process of carcinogenesis, as cancer cells require insulin for growth. IR receptors are overexpressed in various cancers and insulin activates growth mostly through these receptors and their interplays through mainly the PI3K/AKT/mTOR, MAPK and RAS signal transduction pathways. Estrogens and androgens synergistic effects worsen hyperinsulinism and accelerates its carcinogenesis properties while progesterone and Sex Hormone Binding Globulins exert modulatory effects. This review examined the various factor interplays in hyperinsulinism that links diabetes with cancer and the biomarkers involved. It equally identified the potentials and therapeutic roles for Somatostatins, IGF – 1 inhibitors and Insulin sensitizing antidiabetics in preventing and reversing the cancer linked trends.

Targeting RAS mutants in malignancies: successes, failures, and reasons for hope.

RAS genes are the most frequently mutated oncogenes and play critical roles in the development and progression of malignancies. The mutation, isoform (KRAS, HRAS, and NRAS), position, and type of substitution vary depending on the tissue types. Despite decades of developing RAS-targeted therapies, only small subsets of these inhibitors are clinically effective, such as the allele-specific inhibitors against KRASG12C . Targeting the remaining RAS mutants would require further experimental elucidation of RAS signal transduction, RAS-altered metabolism, and the associated immune microenvironment. This study reviews the mechanisms and efficacy of novel targeted therapies for different RAS mutants, including KRAS allele-specific inhibitors, combination therapies, immunotherapies, and metabolism-associated therapies.

97 (PB087) - The ULK1 inhibitor ENV-201 impairs tumor growth in KRAS-driven flank xenografts as a single agent and in combination with the KRAS inhibitor adagrasib

Background: Lung cancer is the leading cancer cause of death in the US, and non-small cell lung cancers (NSCLCs) account for 85% of those cases. Among NSCLCs, mutations resulting in ectopic activation of the RAS signal transduction pathway account for at least a quarter of cases, and the activating KRASG12Cmutation is found in 11% of all NSCLC patient samples. When treated with RAS pathway-inhibiting therapeutics, KRAS-driven tumors have been shown to become dependent on autophagy for continued survival. Simultaneous targeting of both the RAS pathway and autophagy therefore presents an opportunity to block multiple mechanisms of tumor growth and survival. Materials and methods: We have demonstrated the added benefit of KRAS inhibition and autophagy inhibition in an animal model, using the NCI-H2122 cell line (homozygous for the activating KRASG12C mutation) as flank xenografts in NOD/SCID mice. ENV-201 is a selective and potent antagonist of the autophagy-initiating kinase, ULK1, one which also inhibits the related ULK2. Mice were treated via daily oral dosing of (1) vehicle, (2) ENV-201 (20 mg/kg), (3) the KRASG12C-specific inhibitor adagrasib (Mirati Therapeutics, 25 mg/kg), or (4) combination of both ENV-201 and adagrasib at the same doses. Treatment lasted 30 days, body weight was monitored daily, and tumor size was measured three times a week. All animal work was carried out at and met necessary approvals at Crown Bioscience. Results: Both ENV-201 and adagrasib treatments resulted in significant tumor growth inhibition when either drug was used as a single agent (32% or 55% TGI, respectively). Significant additive tumor growth inhibition (78%) was also observed when animals were treated with both drugs in combination. Conclusions: ENV-201-mediated ULK1 inhibition thus shows promise as both a single agent or in combination with a growth factor/RAS/MAPK pathway-targeting cancer treatment. Conflict of interest: Corporate-sponsored Research: All authors are employees of Endeavor BioMedicines.

PSIV-B-20 Genome-Wide Association Analyses Revealed Potential Molecular Mechanisms Affecting Milk Fever in Dairy Cattle

Abstract Milk fever, or hypocalcemia, has a significant economic impact on the dairy industry and increases the risk of other costly disorders including ketosis and displaced abomasum. The objective of this study was to identify genetic variants, genes and molecular mechanisms causing genetic variation in the development of milk fever. A genomic based mixed linear animal model was fitted to include single nucleotide polymorphisms (SNPs), breed, and pen as fixed effects and implemented in GCTA software. A total of 436 Jersey and Holstein cows with varying milk fever history were genotyped for high density SNP chip (782,139 SNP). A total of 401 animals with 617,527 SNPs passed the quality control and were included in the association analyses. Two SNPs located on chromosome 1 were found significant at 5% genome-wide false discovery rate. The 100 most significant SNPs were mapped to corresponding genes using the Ensemble database. A total of 24 genes were found to overlap with the significant SNPs and analyzed using the in-silico functional analyses implemented in DAVID software. The genomic heritability estimated in this population was 0.11. The study identified potential biological pathways associated with milk fever including regulation of Ras and GTPase signal transduction, mammary gland development and ossification. The identified genomic regions and genes will be validated in a larger population. These results could enable us to develop genomic testing tools based on the identified genes to select for cows with low incidence of milk fever and provide insights on which animals within the herd will likely require dietary or management adjustments to prevent and treat milk fever.

Abstract 6051: Longitudinal single-cell sequencing of high-risk neuroblastoma tumors reveals intrinsic and extrinsic mechanisms of therapy resistance

Abstract Neuroblastoma is a childhood cancer originating from embryonic neuronal progenitor cells and is the most common cancer diagnosed in infants. Although the majority of patients respond to initial chemotherapy, most high-risk patients suffer relapse due to therapy resistance. Here, we generated single-cell transcriptome and bulk whole-genome sequencing data of diagnosis and post-therapy samples from 23 patients diagnosed with high-risk neuroblastoma. We found two distinct adrenergic populations in the patient samples. Most tumor cells showed a mature sympathoblast phenotype whereas a small fraction was in an undifferentiated state with activation of protein translation, unfolded protein, and oxidative phosphorylation pathways. We found that therapy-resistant tumor cells in these two subpopulations had distinct characteristics. The more mature resistant subpopulation upregulated genes associated with epithelial-to-mesenchymal transition, including TWIST1, PLCB1 and CD276. The undifferentiated resistant subpopulation upregulated Ras signal transduction and TP53 pathway genes. Analysis of the immune microenvironment revealed that most tumor-associated macrophages became more immunosuppressive post-therapy via multiple newly gained signaling interactions including the complement signaling pathway. We discovered a limited infiltration of T lymphocytes in the tumor microenvironment, and chemotherapy induced an effector state with upregulated mTOR signaling and metabolism. Overall, our study revealed subpopulations of tumor cells in neuroblastoma that responded differently to induction chemotherapy. Our findings uncovered distinct molecular signatures of the resistant cells and their interactions with the immune microenvironment, paving the way for developing novel therapies for high-risk neuroblastoma. Citation Format: Yasin Uzun, Liron D. Grossmann, Chia-Hui Chen, Anusha Thadi, Chi-Yun Wu, Peng Gao, Dinh Diep, Lea Surrey, Daniel Martinez, Tasleema Patel, Qi Qiu, Sarah Johnson, Wenbao Yu, Shane Drabings, Changya Chen, Yuxuan Hu, Gregory Chen, Derek A. Oldridge, Kun Zhang, Hao Wu, Kathrin Bernt, Nancy Zhang, John M. Maris, Kai Tan. Longitudinal single-cell sequencing of high-risk neuroblastoma tumors reveals intrinsic and extrinsic mechanisms of therapy resistance [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 6051.

Genomic Analysis and Biochemical Investigation Reveal Mechanisms of Schwann Cell Transformation and Treatment Resistance in Malignant Peripheral Nerve Sheath Tumors (MPNSTs)

<h3>Purpose/Objective(s)</h3> In contrast to benign Schwann cell derived tumors such as neurofibromas and schwannomas, malignant peripheral nerve sheath tumors (MPNSTs) metastasize, and have poor outcomes. MPNSTs are associated with loss of <b>NF1</b>, a tumor suppressor that inhibits Ras signaling, and loss of Polycomb Repressive Complex 2 (PRC2) which resulting in epigenetic dysregulation. Translationally, MPNSTs are resistant to Ras/MEK inhibition with selumetinib and radiation therapy yet the mechanisms underlying how PRC2 loss cooperates with Ras misactivation to drive transformation and treatment resistance remains unclear. Here, we performed DNA methylation profiling and RNA-sequencing of Schwann cell tumors and cell lines as well as investigated <b>in vitro</b> responses to radiotherapy and selumetinib. <h3>Materials/Methods</h3> We identified 119 Schwann cell tumor resection specimens with sufficient tissue for DNA methylation profiling (66 schwannoma, 13 neurofibroma, 40 MPNSTs) and RNA-sequencing (23 schwannoma, 9 neurofibroma, 9 MPNSTs). To study cell signaling and treatment responses, we utilized primary human cell lines generated from <b>NF1</b> intact peripheral nerve, <b>NF1</b> mutant neurofibromas, and MPNSTs (n = 6). We assessed Ras signal transduction by immunoblot/immunoprecipitation for downstream effectors and performed cell viability assays following selumetinib or radiotherapy. <h3>Results</h3> Principal component analysis (PCA) of DNA methylation and RNA-sequencing data demonstrated a gradient of epigenetic signatures across Schwann cell tumors, with schwannomas comprising a distinct cluster from neurofibromas and MPNST. Moreover, differential DNA methylation and RNA-sequencing analyses both revealed enrichment for PRC2 target genes and repression of Schwann cell differentiation genes in MPNSTs. RNA-sequencing of MPNST versus neurofibroma cell lines demonstrated a PRC2 mediated signature enriched for cell differentiation genes and negative feedback regulators of Ras signaling, suggesting PRC2 dysregulation mediates de-differentiation and increased Ras pathway output in MPNSTs. In contrast to neurofibroma cells, MPNST cells were deficient in PRC2 and <b>NF1</b> while exhibiting increased basal active Ras-GTP levels insensitive to serum starvation. PRC2 deficient MPNST cell lines were more resistant to selumetinib and radiation therapy when compared to <b>NF1</b> deficient neurofibroma cells. <h3>Conclusion</h3> PRC2 loss causes epigenetic dysregulation that may mediate Schwann cell de-differentiation programs and altered Ras signaling in MPNSTs compared to neurofibromas. Translationally, these epigenetic changes may contribute to increased resistance to targeted therapy and radiation. The mechanisms underlying transformation and treatment resistance upon PRC2 loss are under further investigation.

Sulfarotene, a synthetic retinoid, overcomes stemness and sorafenib resistance of hepatocellular carcinoma via suppressing SOS2-RAS pathway

BackgroundRecurrent hepatocellular carcinoma (HCC) shows strong resistance to sorafenib, and the tumor-repopulating cells (TRCs) with cancer stem cell-like properties are considered a driver for its high recurrent rate and drug resistance.MethodsSuppression of TRCs may thus be an effective therapeutic strategy for treating this fatal disease. We evaluated the pharmacology and mechanism of sulfarotene, a new type of synthetic retinoid, on the cancer stem cell-like properties of HCC TRCs, and assessed its preclinical efficacy in models of HCC patient-derived xenografts (PDXs).ResultsSulfarotene selectively inhibited the growth of HCC TRCs in vitro and significantly deterred TRC-mediated tumor formation and lung metastasis in vivo without apparent toxicity, with an IC50 superior to that of acyclic retinoid and sorafenib, to which the recurrent HCC exhibits significant resistance at advanced stage. Sulfarotene promoted the expression and activation of RARα, which down-regulated SOS2, a key signal mediator associated with RAS activation and signal transduction involved in multiple downstream pathways. Moreover, sulfarotene selectively inhibited tumorigenesis of HCC PDXs with high expression for SOS2.ConclusionsOur study identified sulfarotene as a selective inhibitor for the TRCs of HCC, which targets a novel RARα-SOS2-RAS signal nexus, shedding light on a new, promising strategy of target therapy for advanced liver cancer.

Interaction between Ras and Bcl2L12 in B cells suppresses IL-10 expression

The pathogenesis of recurrent tonsillitis is to be further investigated. B cell-derived interleukin (IL)-10 plays a critical role in immune regulation. Ras activation plays an important role in cancer and many immune disorders. This study aims to investigate the role of Ras activation in down regulating IL-10 expression in tonsillar B cells. Surgically removed tonsil tissues were collected from patients with recurrent acute tonsillar inflammation; B cells were isolated from the tonsillar tissues by flow cytometry sorting to be analyzed by the Ras-specific enzyme-linked immunosorbent assay and pertinent immunological approaches. We found that, compared to peripheral B cells (pBC), B cells isolated from the tonsillar tissues with recurrent inflammation (tBC) showed higher Ras activation, lower IL-10 expression and higher Bcl2L12 expression. Bcl2L12 formed a complex with GAP (GTPase activating protein) to prevent Ras from deactivating. The Ras activation triggered the MAPK/Sp1 pathway to promote the Bcl2L12 expression in B cells. Bcl2L12 prevented the IL-10 expression in tBCs, that was counteracted by inhibition of Ras or the Ras signal transduction pathway. In conclusion, Bcl2L12 interacts with Ras activation to compromise immune tolerance in the tonsils by inhibiting the IL-10 expression in tBCs. Inhibition of Bcl2L12 can restore the IL-10 expression in tBCs.

The ERK mitogen-activated protein kinase signaling network: the final frontier in RAS signal transduction.

The RAF-MEK-ERK mitogen-activated protein kinase (MAPK) cascade is aberrantly activated in a diverse set of human cancers and the RASopathy group of genetic developmental disorders. This protein kinase cascade is one of the most intensely studied cellular signaling networks and has been frequently targeted by the pharmaceutical industry, with more than 30 inhibitors either approved or under clinical evaluation. The ERK-MAPK cascade was originally depicted as a serial and linear, unidirectional pathway that relays extracellular signals, such as mitogenic stimuli, through the cytoplasm to the nucleus. However, we now appreciate that this three-tiered protein kinase cascade is a central core of a complex network with dynamic signaling inputs and outputs and autoregulatory loops. Despite our considerable advances in understanding the ERK-MAPK network, the ability of cancer cells to adapt to the inhibition of key nodes reveals a level of complexity that remains to be fully understood. In this review, we summarize important developments in our understanding of the ERK-MAPK network and identify unresolved issues for ongoing and future study.

Danhong Injection Alleviates Cardiac Fibrosis via Preventing the Hypermethylation of Rasal1 and Rassf1 in TAC Mice.

Background/Aim Danhong injection (DHI) is a Chinese patent drug used for relieving cardiovascular diseases. Recent studies have suggested that DNA methylation plays a pivotal role in the maintenance of cardiac fibrosis (CF) in cardiovascular diseases. This study was aimed at identifying the effect and the underlying mechanism of DHI on CF, especially the DNA methylation. Methods A CF murine model was established by thoracic aortic constriction (TAC). A 28-day daily treatment with or without DHI via intraperitoneal injection was carried out immediately following TAC surgery. The changes in cardiac function, pathology, and fibrosis following TAC were measured by echocardiography and immunostaining. We used methyl-seq analysis to assess the DNA methylation changes in whole genes and identified the methylation changes of two Ras signaling-related genes in TAC mice, including Ras protein activator like-1 (Rasal1) and Ras-association domain family 1 (Rassf1). Next, the methylation status and expression levels of Rasal1 and Rassf1 genes were consolidated by bisulfite sequencing, quantitative reverse transcription polymerase chain reaction (RT-qPCR), and Western blotting, respectively. To determine the underlying molecular mechanism, the expressions of DNA methyltransferases (DNMTs), Tet methylcytosine dioxygenase 3 (TET3), fibrosis-related genes, and the activity of Ras/ERK were measured by RT-qPCR and Western blotting. Results DHI treatment alleviated CF and significantly improved cardiac function on day 28 of TAC. The methyl-seq analysis identified 42,606 differential methylated sites (DMSs), including 19,618 hypermethylated DMSs and 22,988 hypomethylated DMSs between TAC and sham-operated mice. The enrichment analysis of these DMSs suggested that the methylated regulation of Ras signal transduction and focal adhesion-related genes would be involved in the TAC-induced CF development. The results of bisulfite sequencing revealed that the TAC-induced methylation affected the CpG site in both of Rasal1 and Rassf1 genes, and DHI treatment remarkably downregulated the promoter methylation of Rasal1 and Rassf1 in CF hearts. Furthermore, DHI treatment upregulated the expressions of Rasal1 and Rassf1, inhibited the hyperactivity of Ras/ERK, and decreased the expressions of fibrosis-related genes. Notably, we found that DHI treatment markedly downregulated the expression of DNMT3B in CF hearts, while it did not affect the expressions of DNMT1, DNMT3A, and TET3. Conclusion Aberrant DNA methylation of Rasal1 and Rassf1 genes was involved in the CF development. DHI treatment alleviated CF, prevented the hypermethylation of Rasal1 and Rassf1, and downregulated DNMT3B expression in CF hearts.

Combined Inhibition of SHP2 and MEK Is Effective in Models of NF1-Deficient Malignant Peripheral Nerve Sheath Tumors

Loss of the RAS GTPase-activating protein (RAS-GAP) NF1 drives aberrant activation of RAS/MEK/ERK signaling and other effector pathways in the majority of malignant peripheral nerve sheath tumors (MPNST). These dysregulated pathways represent potential targets for therapeutic intervention. However, studies of novel single agents including MEK inhibitors (MEKi) have demonstrated limited efficacy both preclinically and clinically, with little advancement in overall patient survival. By interrogation of kinome activity through an unbiased screen and targeted evaluation of the signaling response to MEK inhibition, we have identified global activation of upstream receptor tyrosine kinases (RTK) that converges on activation of RAS as a mechanism to limit sensitivity to MEK inhibition. As no direct inhibitors of pan-RAS were available, an inhibitor of the protein tyrosine phosphatase SHP2, a critical mediator of RAS signal transduction downstream of multiple RTK, represented an alternate strategy. The combination of MEKi plus SHP099 was superior to MEKi alone in models of NF1-MPNST, including those with acquired resistance to MEKi. Our findings have immediate translational implications and may inform future clinical trials for patients with MPNST harboring alterations in NF1. SIGNIFICANCE: Combined inhibition of MEK and SHP2 is effective in models of NF1-MPNST, both those naïve to and those resistant to MEKi, as well as in the MPNST precursor lesion plexiform neurofibroma.

NF1 Tumor Suppressor Gene Inactivation in Juvenile Myelomonocytic Leukemia: New Genetic Evidence and Consequences for Diagnostic Work-up

Neurofibromatosis type 1 (NF-1) predisposes to juvenile myelomonocytic leukemia (JMML) via loss of function of the NF1 tumor suppressor gene and consecutive deregulation of Ras signal transduction. Affected individuals usually carry one defective NF1 allele in the germline; somatic inactivation of the second NF1 allele in hematopoietic cells is associated with transformation to leukemia. We previously demonstrated that a major mechanism for biallelic loss of NF1 function in patients with JMML/NF-1 is mitotic recombination leading to uniparental disomy (UPD) of the 17q chromosome arm (Flotho, 2007; Steinemann, 2010). Using contemporary resequencing and microarray technology, we have now revisited the genetics of NF1 inactivation in JMML. Specifically, we addressed two questions: 1) Are genetic findings in leukemic cells of JMML/NF-1 patients consistent with the clinical diagnosis and the two-hit concept? 2) Does the quintuple-negative (QN) group of JMML (patients without clinical evidence of NF-1 and negative for mutations in PTPN11, KRAS, NRAS, or CBL) contain unrecognized cases likely driven by NF1? We investigated 156 children with JMML registered in studies EWOG-MDS 98 or 2006 and tested for mutations in PTPN11, KRAS, NRAS, and CBL. Twenty-five children (16%) were clinically diagnosed as NF-1 based on &amp;gt;=6 café-au-lait spots (CALS) or family history plus CALS. Family history was positive in 9 JMML/NF-1 patients; &amp;gt;=6, 4, and 1 CALS were described in 23, 1, and 1 patients, respectively. The median age at diagnosis of JMML in the NF-1 group was 35.9 months (range, 4.2 to 71.4). Sixteen children (10%) were grouped as JMML-QN. Granulocyte DNA from bone marrow or peripheral blood collected at time of diagnosis was used for next-generation sequencing of the entire NF1 coding sequence (custom Ampliseq enrichment and Miseq, Illumina). Pathogenicity of NF1 variants was assessed according to ACMG criteria. Affymetrix Cytoscan HD array analysis was applied to detect segmental deletions or copy number-neutral loss of heterozygosity (LOH). Among 25 JMML/NF-1 cases, 8 exhibited an NF1 loss-of-function mutation at near-100% variant allelic frequency (VAF) in combination with UPD involving almost the entire 17q arm, suggesting single mitotic recombination as the leukemic driver. One case had an NF1 mutation at near-100% VAF and segmental 17q UPD, suggesting the unusual occurrence of double mitotic recombination. Nine cases carried two independent pathogenic NF1 mutations at near-50% VAF each; here, germline and somatic events could not be distinguished due to unavailability of non-hematopoietic or parental DNA. Four cases exhibited an NF1 microdeletion in combination with a pathogenic NF1 mutation at near-100% VAF; non-hematopoietic tissue available in 2 of these 4 cases failed to display the mutation, indicating the microdeletion as the constitutional event. A deleterious mutation at 71% VAF but no LOH was revealed in one sample. Only monoallelic evidence of NF1 deficiency was found in 2 cases. In the JMML-QN group, 9/16 cases had previously unrecognized NF1 alterations. Compound-heterozygous pathogenic NF1 mutations were found in 2 and homozygous pathogenic NF1 mutations combined with focal LOH in 3 patients, strongly suggesting NF1 as the JMML driver. In the absence of clinical NF-1 features, the findings in these 5 children may be explained by postzygotic NF1 mosaicism, onset of JMML before clinical manifestation of NF-1, or double somatic NF1 hits in the hematopoietic lineage. Four cases carried monoallelic pathogenic or likely pathogenic NF1 mutations with VAF at ~50% or less, providing inconclusive evidence of driver function as it is also possible that these are secondary hits in a myeloproliferative neoplasm (MPN) driven by an unidentified event. There was no genetic evidence of NF1 involvement in the remaining 7 JMML-QN cases. Several important conclusions can be drawn from our study: The clinical diagnosis is reliable in children with JMML/NF-1; we propose that it can be made on the basis of CALS and JMML alone as the patients are generally too young to display the conventional spectrum of NF-1 features. Children without features of NF-1 should only be assigned to JMML-QN after genetic work-up of NF1 because this will unmask involvement of NF1 in a significant number of cases. In suspected JMML-QN without identifiable NF1 lesion, other forms of MPN should be considered. Disclosures Locatelli: Jazz Pharmaceeutical: Speakers Bureau; Medac: Speakers Bureau; Miltenyi: Speakers Bureau; Bellicum Pharmaceutical: Membership on an entity's Board of Directors or advisory committees; Novartis: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Amgen: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau. Niemeyer:Celgene: Consultancy; Novartis: Consultancy.

Dynamic palmitoylation events following T-cell receptor signaling

Palmitoylation is the reversible addition of palmitate to cysteine via a thioester linkage. The reversible nature of this modification makes it a prime candidate as a mechanism for regulating signal transduction in T-cell receptor signaling. Following stimulation of the T-cell receptor we find a number of proteins are newly palmitoylated, including those involved in vesicle-mediated transport and Ras signal transduction. Among these stimulation-dependent palmitoylation targets are the v-SNARE VAMP7, important for docking of vesicular LAT during TCR signaling, and the largely undescribed palmitoyl acyltransferase DHHC18 that is expressed in two isoforms in T cells. Using our newly developed On-Plate Palmitoylation Assay (OPPA), we show DHHC18 is capable of palmitoylating VAMP7 at Cys183. Cellular imaging shows that the palmitoylation-deficient protein fails to be retained at the Golgi and to localize to the immune synapse upon T cell activation.

Proteomic Profiling of Colon Cancer Tissues: Discovery of New Candidate Biomarkers.

Colon cancer is an aggressive tumor form with a poor prognosis. This study reports a comparative proteomic analysis performed by using two-dimensional differential in-gel electrophoresis (2D-DIGE) between 26 pooled colon cancer surgical tissues and adjacent non-tumoral tissues, to identify potential target proteins correlated with carcinogenesis. The DAVID functional classification tool revealed that most of the differentially regulated proteins, acting both intracellularly and extracellularly, concur across multiple cancer steps. The identified protein classes include proteins involved in cell proliferation, apoptosis, metabolic pathways, oxidative stress, cell motility, Ras signal transduction, and cytoskeleton. Interestingly, networks and pathways analysis showed that the identified proteins could be biologically inter-connected to the tumor-host microenvironment, including innate immune response, platelet and neutrophil degranulation, and hemostasis. Finally, transgelin (TAGL), here identified for the first time with four different protein species, collectively down-regulated in colon cancer tissues, emerged as a top-ranked biomarker for colorectal cancer (CRC). In conclusion, our findings revealed a different proteomic profiling in colon cancer tissues characterized by the deregulation of specific pathways involved in hallmarks of cancer. All of these proteins may represent promising novel colon cancer biomarkers and potential therapeutic targets, if validated in larger cohorts of patients.

Conformation-specific inhibitors of activated Ras GTPases reveal limited Ras dependency of patient-derived cancer organoids

The small GTPases H, K, and NRAS are molecular switches indispensable for proper regulation of cellular proliferation and growth. Several mutations in the genes encoding members of this protein family are associated with cancer and result in aberrant activation of signaling processes caused by a deregulated recruitment of downstream effector proteins. In this study, we engineered variants of the Ras-binding domain (RBD) of the C-Raf proto-oncogene, Ser/Thr kinase (CRAF). These variants bound with high affinity with the effector-binding site of Ras in an active conformation. Structural characterization disclosed how the newly identified RBD mutations cooperate and thereby enhance affinity with the effector-binding site in Ras compared with WT RBD. The engineered RBD variants closely mimicked the interaction mode of naturally occurring Ras effectors and acted as dominant-negative affinity reagents that block Ras signal transduction. Experiments with cancer cells showed that expression of these RBD variants inhibits Ras signaling, reducing cell growth and inducing apoptosis. Using these optimized RBD variants, we stratified patient-derived colorectal cancer organoids with known Ras mutational status according to their response to Ras inhibition. These results revealed that the presence of Ras mutations was insufficient to predict sensitivity to Ras inhibition, suggesting that not all of these tumors required Ras signaling for proliferation. In summary, by engineering the Ras/Raf interface of the CRAF-RBD, we identified potent and selective inhibitors of Ras in its active conformation that outcompete binding of Ras-signaling effectors.

Collagen VI (CVI) antibodies regulate macrophages differentiation through FcgammaRIIb signal transduction pathway

We previously reported that monoclonal antibody against collagen VI (CVI mAb) regressing atherosclerotic plaque through inducing the shift of macrophage differentiation, namely from M1 to M2, and ensuing the macrophages engulf the plaque lipids and transfer the lipids to HDL through restoring lipid transfer protein ABCA1 expression. Here we report the cellular signal transduction on the CVI mAb inhibition of oxLDL‐induced M1 differentiation. Using purified human CD14+ cells, we find that oxLDL induces M1 differentiation is JNK and ERK MAPKs dependent. And CVI mAb inhibits M1, while activates M2 differentiation, though oxLDL also activates M2 differentiation in some way, but the M1/M2 ratio are significantly reversed by CVI mAb. The molecular mechanism behind these is that CVI mAb activates macrophage FcgammaRIIb and its downstream SHIP and Dok phosphatases, and inhibits Syk and Ras signal transduction pathway, which are MAPKs upstream kinases, so that CVI mAb inhibits ERK, JNK, and p38 MAPKs activation, and ensuing M1 differentiation. Thus, we found the signal transduction pathway of CVI mAb inhibition of oxLDL‐induced M1 differentiation: CVI mAb→FcgammaRIIb→SHIP→Dok→Syk and Ras→ERK and JNK MAPKs→M1. These results might give a new clue on control the lipids‐induced inflammatory reaction on the large blood vessel and therapeutic method to prevent atherosclerosis.Support or Funding InformationThis work was supported by the National Natural Science Foundation of China (grants 81772074, 81974045), the Natural Science Foundation of Guangdong, China (grant 2012B050600002), the Guangdong Province Talent Recruitment Foundation and the Guangdong Innovative Research Team Program (No. 201001Y0104675344), Project of Guangzhou city scientific plan (201804020002).

Pediatric Acute Myeloid Leukemia (AML): From Genes to Models Toward Targeted Therapeutic Intervention.

This review aims to provide an overview of the current knowledge of the genetic lesions driving pediatric acute myeloid leukemia (AML), emerging biological concepts, and strategies for therapeutic intervention. Hereby, we focus on lesions that preferentially or exclusively occur in pediatric patients and molecular markers of aggressive disease with often poor outcome including fusion oncogenes that involve epigenetic regulators like KMT2A, NUP98, or CBFA2T3, respectively. Functional studies were able to demonstrate cooperation with signaling mutations leading to constitutive activation of FLT3 or the RAS signal transduction pathways. We discuss the issues faced to faithfully model pediatric acute leukemia in mice. Emerging experimental evidence suggests that the disease phenotype is dependent on the appropriate expression and activity of the driver fusion oncogenes during a particular window of opportunity during fetal development. We also highlight biochemical studies that deciphered some molecular mechanisms of malignant transformation by KMT2A, NUP98, and CBFA2T3 fusions, which, in some instances, allowed the development of small molecules with potent anti-leukemic activities in preclinical models (e.g., inhibitors of the KMT2A–MENIN interaction). Finally, we discuss other potential therapeutic strategies that not only target driver fusion-controlled signals but also interfere with the transformed cell state either by exploiting the primed apoptosis or vulnerable metabolic states or by increasing tumor cell recognition and elimination by the immune system.

JMML genomics and decisions.

Juvenile myelomonocytic leukemia (JMML) is a unique clonal hematopoietic disorder of early childhood characterized by hyperactivation of the RAS signal transduction pathway. Approximately 90% of patients harbor molecular alteration in 1 of 5 genes (PTPN11, NRAS, KRAS, NF1, CBL), which define genetically and clinically distinct JMML subtypes. Three subtypes, PTPN11- , NRAS-, and KRAS-mutated JMML, are characterized by heterozygous somatic gain-of-function mutations in non syndromic children, while two subtypes, JMML in neurofibromatosis type 1 and in JMML in children with CBL syndrome, are characterized by germ line RAS disease and acquired biallelic inactivation of the respective tumor suppressor genes in hematopoietic cells. In addition to the initiating RAS pathway lesion, secondary genetic alterations within and outside of the RAS pathway are detected in about half the patients. Most recently, genome-wide DNA methylation profiles identified distinct methylation signatures correlating with clinical and genetic features and highly predictive of outcome. JMML is a stem cell disorder, and most JMML patients require allogeneic stem cell transplantation for long-term survival. However, spontaneous disease regression is noted in the majority of children with CBL-mutated JMML and in some NRAS-mutated cases. In the absence of 1 of the 5 canonical RAS pathway alteration, rare mutations in other RAS genes and non-JMML myeloproliferative disorders need to be excluded. Understanding the genetic basis of myeloproliferative disorders in early childhood will greatly improve clinical decision making.

Seven‐In‐Absentia (SINA) Family E3 Ligases in Development and Growth

The RAS signal transduction cascade is a pivotal signaling pathway that controls numerous fundamental cellular processes, such as cell proliferation, differentiation, motility, and apoptosis. Oncogenic K‐RAS pathway activation is detected in approximately 30% of all human cancers. Seven‐IN‐Absentia (SINA) is an evolutionarily conserved E3 ubiquitin ligase that is the most downstream signaling module identified in the RAS signaling pathway. Underscoring the importance of SINA is its high evolutionary conservation with over 83% amino acid identity shared between Drosophila SINA and its human SINA homologs (SIAHs). As a major signaling hub and the downstream signaling “gatekeeper” in the RAS pathway, SIAHs are uniquely positioned to inhibit “undruggable” oncogenic K‐RAS activation that is prevalent in high‐grade metastatic cancer. Thus, it is important to delineate the enzymatic activity, molecular regulation, and substrate targeting mechanism(s) of this highly conserved family of SINA/SIAH E3 ligases. By deploying the elegant and powerful Drosophila photoreceptor cell development system, we conducted a genetic modifier screen and identified 28 new sina mutant alleles that exhibit a range of mutant phenotypes. For example, sina complete loss of function (null alleles) is cell lethal, suggesting that SINA is an essential gene in development. All other sina mutant alleles exhibit stronger phenotypes than the previously published sina2 and sina3 alleles, providing us with an opportunity to study sinaloss‐of‐function mutant phenotypes. Sequencing analysis of these newly identified mutant alleles reveals the critical roles of several immutatable amino acid residues essential for SINA function. To demonstrate the functional conservation of SINA/SIAH proteins, we have generated a collection of transgenic fly lines that carry either wild‐type (WT) or dominant negative (DN) DmSINA and human SIAH. The altered DmSINA/hSIAH expression under the control of sev‐, GMR‐, and dpp‐ and salivary gland‐GAL4 drivers revealed the biological consequences of gain of function and/or loss of function of DmSINA/hSIAH1/2 in transmitting RAS signaling in central nervous system (CNS) and peripheral nervous system (PNS) development. Immunofluorescent staining of developing imaginal discs and electron micrographs of adult tissues show that ectopic expression of SINAWT/DN/SIAHWT/DN in neurons resulted in dramatic changes in neuronal cell fate in the developing eye and notum. Furthermore, immunofluorescent staining of larval salivary glands revealed a critical role for SINA family E3 ligases in regulation of cell shape, focal adhesions, and cellular junctions. The results from these transgenic animals suggested that Drosophila SINA and human SIAH1/SIAH2 are evolutionarily conserved and functionally interchangeable. Through this developmental neurobiology and cellular biology‐based study, we aim to dissect the molecular action of SINA/SIAH1/2 in RAS signaling. Furthermore, we hope to translate these findings to demonstrate the anticancer efficacy of the next‐generation anti‐SIAH‐based anti‐K‐RAS strategy against high‐grade metastatic human cancer in the future.Support or Funding InformationExtramural funding support provided by NIH NIGMS R01 GM069922Z‐05S1 and NCI R01 CA140550.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.