Machine learning–driven multiscale modeling reveals lipid-dependent dynamics of RAS signaling proteins

RAS is a signaling protein associated with the cell membrane that is mutated in up to 30% of human cancers. RAS signaling has been proposed to be regulated by dynamic heterogeneity of the cell membrane. Investigating such a mechanism requires near-atomistic detail at macroscopic temporal and spatial scales, which is not possible with conventional computational or experimental techniques. We demonstrate here a multiscale simulation infrastructure that uses machine learning to create a scale-bridging ensemble of over 100,000 simulations of active wild-type KRAS on a complex, asymmetric membrane. Initialized and validated with experimental data (including a new structure of active wild-type KRAS), these simulations represent a substantial advance in the ability to characterize RAS-membrane biology. We report distinctive patterns of local lipid composition that correlate with interfacially promiscuous RAS multimerization. These lipid fingerprints are coupled to RAS dynamics, predicted to influence effector binding, and therefore may be a mechanism for regulating cell signaling cascades.

Network analysis of Dengue NS1 interacting core human proteins driving dengue pathogenesis

Aim: We aimed to identify critical human proteins involved in cathepsin L regulation Background: It has been shown that Dengue Virus (DENV) NS1 activates cathepsin L (CTSL). The CTSL activates heparanase, which cleaves heparan sulfate proteoglycans and causes dengue pathogenesis. NS1 directly interacts with PTBP1 and Gab proteins. Gab protein activates the Ras signaling pathway. Still, no known direct interaction partners are linking GAB1 to cathepsin L. Objective: Our objective includes three main points.1-Network analysis of NS1 interacting human proteins 2- Identification of protein-drug and protein-disease interactions 3- Identification of core proteins involved in cathepsin L regulation. Method: We collected NS1 interacting Human proteins from DenHunt, Int-Act Molecular Interaction Database, Virus Mentha, Virus Pathogen Database and Analysis Resource (ViPR), and Virus MINT. We employed Pesca, cytohubba, and centiscape as the significant plug-ins in Cytoscape for network analysis. To study protein-diseases and protein-drugs interaction, we used NetworkAnalyst. Result: Based on the prior knowledge on the interaction of NS1 with GAB1 and PTBP1 human proteins, we found several core proteins that drive dengue pathogenesis. The proteins EED, NXF1, and MOV10, are the mediators between PTBP1 and CTSL. Similarly, DNM2, GRB2, PXN, PTPRC, and NTRK1 mediate GAB1 and PTBP1. The common first neighbors of MOV10, NXF1, and EED were identified, and the common primary pathways in all three subnetworks were mRNA processing and protein translation. The common interaction partners were considered for drug and disease network analysis. These proteins were; PARP1, NFKB2, HDAC2, SLC25A4, ATP5A1, EPN1, CTSL, UBR4, CLK3, and ARPC4. PARP1 was the highly connected node in the protein-drug network. The highest degree protein, LMNA, was associated with many diseases. The NXF1 is connected with LMNA. Here, we reported one essential protein, namely, NXF1 protein, which links PTBP1 with CTSL. The NXF1 is also connected with TPM3, which is connected to CTSL. Conclusion: We listed functionally important proteins which are involved in cathepsin L activation. Based on network properties, we proposed, NXF1 and TPM3 are the important high centrality proteins in dengue infection.

An RNA-binding switch drives ribosome biogenesis and tumorigenesis downstream of RAS oncogene

Oncogenic RAS signaling reprograms gene expression through both transcriptional and post-transcriptional mechanisms. While transcriptional regulation downstream of RAS is relatively well-characterized, how RAS post-transcriptionally modulates gene expression to promote malignancy is unclear. Using quantitative RNA Interactome Capture analysis, we reveal that oncogenic RAS signaling reshapes the RNA-bound proteomic landscape of cancer cells, with a network of nuclear proteins centered around Nucleolin displaying enhanced RNA-binding activity. We show that Nucleolin is phosphorylated downstream of RAS, which increases its binding to pre-ribosomal-RNA (rRNA), boosts rRNA production, and promotes ribosome biogenesis. This Nucleolin-dependent enhancement of ribosome biogenesis is crucial for RAS-induced cancer cell proliferation, and can be targeted therapeutically to inhibit tumor growth. Our results reveal that oncogenic RAS signaling drives ribosome biogenesis by regulating the RNA-binding activity of Nucleolin, and highlights the crucial role of this process in RAS-mediated tumorigenesis.

Cytoplasmic innate immune sensing by the caspase-4 non-canonical inflammasome promotes cellular senescence

Cytoplasmic recognition of microbial lipopolysaccharides (LPS) in human cells is elicited by the caspase-4 and caspase-5 noncanonical inflammasomes, which induce a form of inflammatory cell death termed pyroptosis. Here we show that LPS-mediated activation of caspase-4 also induces a stress response promoting cellular senescence, which is dependent on the caspase-4 substrate gasdermin-D and the tumor suppressor p53. Furthermore, we found that the caspase-4 noncanonical inflammasome is induced and assembled in response to oncogenic RAS signaling during oncogene-induced senescence (OIS). Moreover, targeting caspase-4 expression in OIS showed its critical role in the senescence-associated secretory phenotype and the cell cycle arrest induced in cellular senescence. Finally, we observed that caspase-4 induction occurs in vivo in mouse models of tumor suppression and ageing. Altogether, we are showing that cellular senescence is induced by cytoplasmic LPS recognition by the noncanonical inflammasome and that this pathway is conserved in the cellular response to oncogenic stress.

A systems approach to investigate GPCR-mediated Ras signaling network in chemoattractant sensing

A GPCR-mediated signaling network enables a chemotactic cell to generate adaptative Ras signaling in response to a large range of concentrations of a chemoattractant. To explore potential regulatory mechanisms of GPCR-controlled Ras signaling in chemosensing, we applied a software package, Simmune, to construct detailed spatiotemporal models simulating responses of the cAR1-mediated Ras signaling network. We first determined dynamics of G-protein activation and Ras signaling in Dictyostelium cells in response to cAMP stimulations using live-cell imaging and then constructed computation models by incorporating potential mechanisms. Using simulations, we validated the dynamics of signaling events and predicted the dynamic profiles of those events in the cAR1-mediated Ras signaling networks with defective Ras inhibitory mechanisms, such as without RasGAP, with RasGAP overexpression, or RasGAP hyperactivation. We described a method of using Simmune to construct spatiotemporal models of a signaling network and run computational simulations without writing mathematical equations. This approach will help biologists to develop and analyze computational models that parallel live-cell experiments.

Ras signaling and its effector RREB1 are required for the dissociation of MEE cells in palatogenesis

Cleft palate is one of the major congenital craniofacial birth defects. The etiology underlying the pathogenesis of cleft palate has largely remained unelucidated. Dissociation of the medial edge epithelium (MEE) at the contacting region of palatal shelves and subsequent migration or apoptosis of MEE cells is required for the proper MEE removal. Ras Responsive Element Binding Protein 1 (RREB1), a RAS transcriptional effector, has recently been shown to play a crucial role in developmental EMT, in which loss of epithelial characteristics is an initial step, during mid-gastrulation of embryonic development. Interestingly, the involvement of RREB1 in cleft palate has been indicated in humans. Here, we demonstrated that pan-Ras inhibitor prevents the dissociation of MEE during palatal fusion. Rreb1 is expressed in the palatal epithelium during palatal fusion, and knockdown of Rreb1 in palatal organ culture resulted in palatal fusion defects by inhibiting the dissociation of MEE cells. Our present findings provide evidence that RREB1-mediated Ras signaling is required during palatal fusion. Aberrant RREB1-mediated Ras signaling might be involved in the pathogenesis of cleft palate.

Evaluating the effects of RAS and CK2 Inhibitors in ESR1 Mutated Breast Cancer

Background and Hypothesis: Estrogen Receptor alpha (ESR1) is rarely mutated in primary breast cancers but is frequently mutated in metastasis that can appear after many years of anti-estrogen therapy. Mutations to ESR1 can result in estrogen-independent activity of ESR1 causing anti-estrogen to become ineffective. Previous work on this project has led to the hypothesis that RAS pathway activation in metastatic cancer cells as a result of ESR1 mutation leads to elevated CK2 activity which ultimately results in metastatic progression. Therefore, we hypothesize that the use of RAS signaling inhibitors or CK2 inhibitors have efficacy in blocking or reducing the metastatic progression of metastatic breast cancers with hyperactive RAS pathways. Experimental Design or Project Methods: The estrogen receptor positive, anti-estrogen sensitive breast cancer cell line MCF-7 and the same cell line genomically modified to replace wild type ESR1 to breast cancer metastasis enriched Y537S or D538G ESR1 mutation were used in this study. Cells were treated with various concentrations of the RAS pathway inhibitor Salirasib or CK2 inhibitor Silmitasertib and cell proliferation rates were measured using bromodeoxyuridine incorporation ELISA. Results: Thus far, the use of RAS signaling inhibitors or CK2 inhibitors have not shown efficacy in decreasing the proliferation rates of modified ESR1 MCF-7 cells. While there is a general trend of growth inhibition by these inhibitors at a higher concentration, there is no significant difference between the ESR1 mutant expressing cells and their respective controls. Conclusion and Potential Impact: This study will establish the feasibility of using RAS signaling inhibitors or CK2 inhibitors in the treatment of metastatic estrogen receptor-positive breast cancer. Future studies testing the effects of these drugs either alone or in combination with the clinically used anti-estrogen Fulvestrant for not only primary tumor growth but also metastasis in clinically relevant in vivo models may ultimately lead to clinical translation. Finally, demonstrating efficacy in these types of drugs may fuel the further refinement of drugs targeting these pathways to treat metastatic breast cancer.

Juvenile Myelomonocytic Leukemia and other myelodysplastic/myeloproliferative neoplasms

Juvenile Myelomonocytic Leukemia (JMML) is a clonal hematopoietic disorder that usually occurs in early childhood, characterized by hyperactivation of the RAS signaling pathway. About 90% of patients have mutations in 1 of 5 genes (PTPN11, NRAS, KRAS, NF1, CBL) that define genetically and clinically distinct subtypes of the disease, with a highly variable clinical course. Allogeneic hematopoietic stem cell transplantation (HSCT) remains the therapy of choice for most patients with JMML, although children with CBL mutations and few of those with N-RAS mutations may have spontaneous resolution of hematologic abnormalities. The results of HSCT in patients with JMML have progressively improved over time, but relapse is still an important cause of treatment failure.

Oncogenic RAS commandeers amino acid sensing machinery to aberrantly activate mTORC1 in multiple myeloma

Oncogenic mutations within the RAS pathway are common in multiple myeloma (MM), an incurable malignancy of plasma cells. However, the mechanisms of pathogenic RAS signaling in this disease remain enigmatic and difficult to inhibit therapeutically. We employed an unbiased proteogenomic approach to dissect RAS signaling in MM by combining genome-wide CRISPR-Cas9 screening with quantitative mass spectrometry focused on RAS biology. We discovered that mutant isoforms of RAS organized a signaling complex with the amino acid transporter, SLC3A2, and MTOR on endolysosomes, which directly activated mTORC1 by co-opting amino acid sensing pathways. MM tumors with high expression of mTORC1-dependent genes were more aggressive and enriched in RAS mutations, and we detected interactions between RAS and MTOR in MM patient tumors harboring mutant RAS isoforms. Inhibition of RAS-dependent mTORC1 activity synergized with MEK and ERK inhibitors to quench pathogenic RAS signaling in MM cells. This study redefines the RAS pathway in MM and provides a mechanistic and rational basis to target this novel mode of RAS signaling.

Re-entry into mitosis and regeneration of intestinal stem cells through enteroblast dedifferentiation in Drosophila midguts

Many adult tissues and organs including the intestine rely on resident stem cells to maintain homeostasis. In mammalian intestines, upon ablation of resident stem cells, the progenies of intestinal stem cells (ISCs) such as secretory cells and tuft cells can dedifferentiate to generate ISCs to drive epithelial regeneration, but whether and how the ISC progenies dedifferentiate to generate ISCs under physiological conditions remains unknown. Here we show that infection of pathogenic bacteria induces enteroblasts (EBs) as one type of ISC progenies to re-enter the mitotic cycle in the Drosophila intestine. The re-entry into mitosis is dependent on epithermal growth factor receptor (EGFR)-Ras signaling and ectopic activation of EGFR-Ras signaling in EBs is sufficient to drive EBs cell-autonomously to re-enter into mitosis. In addition, we examined whether EBs gain ISC identity as a prerequisite to divide, but the immunostaining with stem cell marker Delta shows that these dividing EBs do not gain ISC identity. After employing lineage tracing experiments, we further demonstrate that EBs dedifferentiate to generate functional ISCs after symmetric divisions of EBs. Together, our study in Drosophila intestines uncovers a new role of EGFR-Ras signaling in regulating re-entry into mitosis and dedifferentiation during regeneration and reveals a novel mechanism by which ISC progenies undergo dedifferentiation through a mitotic division, which has important implication to mammalian tissue homeostasis and tumorigenesis.