Pau Castel’s research while affiliated with New York Medical College and other places

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Publications (94)


The Noncanonical RAS/MAPK Pathway and the RASopathies
  • Chapter

November 2024

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7 Reads

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Pau Castel

RASopathies are neurodevelopmental genetic disorders caused by germline variants in genes that encode critical components and/or regulators of the RAS/Mitogen-activated protein kinase (MAPK) pathway. These syndromes are characterized by overlapping clinical features, including congenital cardiac defects, distinctive facial features, cognitive impairment, and predisposition to cancer, among others. Decades of research into the function and regulation of the RAS/MAPK pathway have led to our current understanding of this critical signaling pathway. Through extensive scientific evidence, it has been possible to define the main components of the RAS/MAPK signaling and begin to draw a model for the so-called canonical RAS/MAPK pathway. This modular signaling core, nucleated by the RAS GTPases N, H, and KRAS, is triggered by extracellular cues and propagates downstream signaling through a MAPK signaling cascade formed by the kinases RAF/MEK/ERK. In addition to this canonical signaling, many studies have demonstrated that this pathway is regulated by other proteins and alternative mechanisms, highlighting the complexity and the context-specific function of RAS/MAPK pathway. Importantly, variants in the genes encoding these less-understood proteins have also been identified in different types of RASopathies. In this chapter, we discuss the role of this noncanonical RAS/MAPK pathway and its importance in the context of these disorders.



The deubiquitinase USP9X regulates RIT1 protein abundance and oncogenic phenotypes
  • Article
  • Full-text available

July 2024

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17 Reads

iScience

Amanda K. Riley

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Michael Grant

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[...]

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RIT1 is a rare and understudied oncogene in lung cancer. Despite structural similarity to other RAS GTPase proteins such as KRAS, oncogenic RIT1 activity does not appear to be tightly regulated by nucleotide exchange or hydrolysis. Instead, there is a growing understanding that the protein abundance of RIT1 is important for its regulation and function. We previously identified the deubiquitinase USP9X as a RIT1 dependency in RIT1-mutant cells. Here, we demonstrate that both wild-type and mutant forms of RIT1 are substrates of USP9X. Depletion of USP9X leads to decreased RIT1 protein stability and abundance and resensitizes cells to epidermal growth factor receptor (EGFR) tyrosine kinase inhibitors in vitro and in vivo. Our work expands upon the current understanding of RIT1 protein regulation and presents USP9X as a key regulator of RIT1-driven oncogenic phenotypes.

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Figure 1. KMT2D is methylated by SMYD2 at K1330 (A) Schematic showing the domain structure of lysine methyltransferase KMT2D and the conserved AGC consensus site containing both the S1331 phosphorylation site modified by AKT/SGK1 (previously published) and the identified methylation site at K1330. Sequence alignments for the indicated species were performed using Clustal W2. (B) In vitro screen of recombinant protein lysine methyltransferases (PKMTs) to identify the methyltransferase able to catalyze K1330me. Radiometric assays were conducted using 3 H-S-adenosyl-L-methionine (SAM) and a KMT2D peptide (amino acids [aa] 1,321-1,343)
Figure 2. SMYD2 methylation of KMT2D affects KMT2D binding genome-wide and cofactor associations in breast cancer (A) In vitro methylation assay to assess activity of immunoprecipitated full-length KMT2D
Figure 3. SMYD2 knockdown abrogates alpelisib-induced changes in gene expression, including ER-dependent transcription (A) Heatmap of RNA-seq data showing the subset of genes in MCF7 cells regulated by alpelisib treatment (3,995), with and without SMYD2 knockdown (1,248 differential, log 2 fold change >0.5 and adjusted p value <0.1). Cells were treated with 1 μM alpelisib for 24 h before collection (n = 3 technical replicates). (B) Plot of C2 pathways from gene set enrichment analysis (GSEA) in SMYD2 knockdown compared to control in DMSO and alpelisib treatment conditions. All gene sets shown were significant with a p value of <0.05. Nominal p values and false discovery rate (FDR) adjusted p values were calculated using the GSEA package. Normalized enrichment score (NES) for each gene set is indicated on the x axis. (C) GSEA plot of the Dutertre estrogen response gene set in SMYD2 knockdown compared to control in both DMSO and alpelisib treatment conditions. Nominal p values and FDR adjusted p values were calculated using the GSEA package. (D) Gene set variation analysis (GSVA) results of estrogen-responsive gene sets in ER+ patient samples from the TCGA and METABRIC datasets corresponding with median SMYD2 mRNA level. Data are shown as median with interquartile range and analyzed using a two-tailed Student's t test.
Figure 4. SMYD2 inhibition improves the therapeutic response of breast cancer to PI3K/AKT inhibitors in vitro and in vivo
Figure 5. SMYD2-mediated methylation of KMT2D affects the therapeutic response of breast cancer to PI3K/AKT inhibitors (A) PrestoBlue assays with K1330A CRISPR KI clones compared to unmodified MCF7 cells using a dose response of the PI3K inhibitor alpelisib for a treatment period of 5 days. Significance at individual doses was assessed using an unpaired, two-tailed Student's t test. p values are indicated as follows: *p < 0.05,**p < 0.01,***p < 0.001, and ****p < 0.0001 (n
Methylation of the chromatin modifier KMT2D by SMYD2 contributes to therapeutic response in hormone-dependent breast cancer

May 2024

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90 Reads

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5 Citations

Cell Reports

Activating mutations in PIK3CA are frequently found in estrogen-receptor-positive (ER+) breast cancer, and the combination of the phosphatidylinositol 3-kinase (PI3K) inhibitor alpelisib with anti-ER inhibitors is approved for therapy. We have previously demonstrated that the PI3K pathway regulates ER activity through phosphorylation of the chromatin modifier KMT2D. Here, we discovered a methylation site on KMT2D, at K1330 directly adjacent to S1331, catalyzed by the lysine methyltransferase SMYD2. SMYD2 loss attenuates alpelisib-induced KMT2D chromatin binding and alpelisib-mediated changes in gene expression, including ER-dependent transcription. Knockdown or pharmacological inhibition of SMYD2 sensitizes breast cancer cells, patient-derived organoids, and tumors to PI3K/AKT inhibition and endocrine therapy in part through KMT2D K1330 methylation. Together, our findings uncover a regulatory crosstalk between post-translational modifications that fine-tunes KMT2D function at the chromatin. This provides a rationale for the use of SMYD2 inhibitors in combination with PI3Kα/AKT inhibitors in the treatment of ER+/PIK3CA mutant breast cancer.


Abstract 1245: The histone methyltransferase KMT2D mediates subtype-specific transcriptional regulation and therapeutic response in prostate cancer

March 2024

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15 Reads

Cancer Research

Primary prostate cancer is characterized by its dependence on androgen receptor (AR) and often has hyperactive PI3K signaling, most frequently through loss of PTEN. PI3K/AKT pathway inhibitors are in late-stage clinical development in combination with anti-AR therapy. Androgen deprivation therapy is a cornerstone of treatment for advanced prostate cancer, but the development of castration-resistant prostate cancer (CRPC) eventually occurs. CRPC remains largely dependent on AR for growth with a subset of CRPC that lose AR dependence. There is an urgent need to study the molecular pathways leading to AR activation and the loss of AR reliance in order to identify therapeutic targets. KMT2D is a histone methyltransferase and an important regulator of gene expression. Here we found that KMT2D establishes the chromatin competence necessary for the recruitment of AR and FOXA1 transcription factors (TFs) to activate AR-dependent transcription in AR-high prostate models. In AR-low models such as the stem cell-like (SCL) subtype, KMT2D controls residual AR-FOXA1 gene expression programs and AP-1 TFs such as FOSL1, a driver of the SCL subtype. In SCL, single cell RNA-seq and single cell chromatin assays confirm a key role for KMT2D in the maintenance of a mixed lineage cell state through the regulation of AP-1 and FOXA1 TFs. Combined suppression of PI3K/AKT and KMT2D reduces cell proliferation in prostate cancer cells and patient derived organoids, providing a rationale for epigenetically informed combination therapies with PI3K/AKT inhibitors in PTEN-deficient prostate cancer. Together, these data establish KMT2D as a major mediator of subtype-specific chromatin accessibility and transcriptomic landscape in CRPC, required for prostate cancer growth and therapeutic response. Citation Format: Srushti Kittane, Erik Ladewig, Taibo Li, Jillian Love, Amaia Arruabarrena-Aristorena, Xinyu Guo, Mirna Sallaku, Liliana Garcia-Martinez, Ryan Blawski, Javier Carmona Sanz, Christopher Simpkins, Wanlu Chen, Bujamin Vokshi, Peihua Zhao, Nachiket Kelkar, Laura Baldino, Ingrid Kalemi, Pau Castel, Emiliano Cocco, Lluis Morey, Charles Sawyers, Hongkai Ji, Maurizio Scaltriti, Alexis Battle, Christina Leslie, Wouter Karthaus, Eneda Toska. The histone methyltransferase KMT2D mediates subtype-specific transcriptional regulation and therapeutic response in prostate 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 1245.


Abstract 3938: A novel mouse model reveals potential therapeutic strategies for RIT1-driven lung adenocarcinomas

March 2024

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10 Reads

Cancer Research

Gain-of-function oncogenic mutations in RIT1 have been identified in lung adenocarcinoma and are mutually exclusive with other reported driver mutations, highlighting the role of RIT1 as a cancer driver. RIT1 is a poorly-studied non-classical RAS GTPase and, to date, there are no reported genetically-engineered mouse models of RIT1-driven lung adenocarcinoma. Here, we have generated a conditional knock-in allele within the endogenous mouse Rit1 locus, introducing the most frequent mutation observed in lung adenocarcinomas (RIT1M90I). Upon intranasal administration of adenovirus encoding the Cre recombinase to induce somatic recombination in the mouse lungs, we observed the formation of discrete adenomas. Although Rit1M90I mutation can promote lung tumor initiation in mice, we found that this phenotype is less penetrant than the common KrasG12D mutation. Immunohistochemistry analysis of Rit1M90I and KrasG12D tumors showed expression of typical histological markers associated with lung adenocarcinoma (i.e., TTF1 and SPC), as well as increased levels of Ki67 and pERK expression. Next, to develop potential pharmacological strategies for RIT1-driven lung tumors, we first explored drugs that inhibit the RAS/MAPK pathway and are either FDA-approved or under clinical development. In vitro experiments with cell lines carrying the RIT1M90I mutation demonstrated high sensitivity to SHP2, MEK, RAF, and RAS inhibitors. In vivo, SHP2 and RAS, but not MEK, inhibitors slowed down tumor progression in both cell line and patient-derived xenografts. Our results indicate that, although RAS signaling might be needed for RIT1-dependent growth, these tumors are not sensitive to MAPK inhibition as a monotherapy and, hence, other pathways could contribute to tumor growth. Therefore, we decided to explore whether targeting RIT1 directly, could be a more beneficial strategy for these tumors. In order to inhibit oncogenic RIT1 directly, we have undertaken two complementary approaches. First, because RIT1 mutations were shown to impair its proteolysis, we explored whether restoring RIT1 proteasomal degradation could be a promising strategy. Recently, the development of Proteolysis Targeting Chimeras (PROTAC) has demonstrated that targeted degradation can be used as a pharmacological approach for the treatment of cancer. Therefore, we have tested successfully several proof-of-concept PROTAC approaches that caused degradation of oncogenic RIT1 in different cellular models. Second, we used a chemical biology approach to assess whether covalent inhibitors directed against the closely-related GTPase KRAS could cross-react and bind to oncogenic RIT1. We have identified a molecule that binds RIT1 and we are currently characterizing it in our preclinical models. Overall, our work provides comprehensive insights into the oncogenic role of RIT1 in lung cancer emphasizing its potential as a therapeutic target. Citation Format: Alessandro Mauro Mozzarelli, Antonio Cuevas-Navarro, Anatoly Urisman, Pau Castel. A novel mouse model reveals potential therapeutic strategies for RIT1-driven lung adenocarcinomas [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 3938.


Abstract 1452: A PIK3CA -driven mouse model of metaplastic breast carcinoma to assess potential therapeutic strategies

March 2024

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13 Reads

Cancer Research

Metaplastic Breast Carcinomas (MBC) are rare and aggressive breast cancers with mesenchymal or squamous components. While histologically heterogeneous, MBC are typically triple negative: they lack expression of ER, PR and HER2. Importantly, approximately 40% of MBC harbor oncogenic gain-of-function mutations in PIK3CA, the gene encoding for the catalytic subunit of PI3K. These alterations are targetable by an array of inhibitors of the PI3K-AKT-mTORC1 pathway. However, few animal models of MBC are available, and it is currently difficult to study MBC biology and response to therapy. Here, we describe the generation of a new mouse model of MBC driven by the PIK3CA H1047R mutation. We histologically and biologically characterize this model and assess potential therapeutic strategies. We used genetically-engineered mouse models to express the human PIK3CA H1047R mutation under the control of the MMTV promoter, which is mostly expressed in the mammary epithelium. Tumors developed in mice in about 1 year. We histologically characterized these tumors and generated cell lines, organoids, and tumor grafts in immunocompromised mice. The resulting model, termed MMTV-102, was evaluated for the response to PI3K-AKT-mTOR pathway inhibitors, both in vitro and in vivo. The MMTV-PIK3CAH1047R tumor was a high grade poorly differentiated MBC negative for ER, PR, and HER2. It presented squamous components, keratin depositions, mitotic figures, and vascular invasion and stained positive for the basal marker CK14. In addition, due to the presence of the PIK3CA H1047R mutation, it stained positive for phosphorylated AKT, PRAS40, and S6RP, indicating constitutive activation of the PI3K-AKT-mTORC1 pathway. MBC derived cell lines and organoids were confirmed to be triple negative and could form tumors in mice. In addition, these cellular models were sensitive to PI3Kα, pan-PI3K, and mTORC1 inhibitors in vitro and in vivo, but the mTORC1 inhibitor everolimus exhibited the strongest effect. When resistance to everolimus eventually developed in vivo, we established cell lines from these resistant tumors. In addition, we generated an everolimus resistant cell line following chronic drug exposure in vitro (MMTV-102 Par res). Both cellular models were tested for their sensitivity to the next-generation mTORC1 inhibitor RMC-5552 by proliferation and clonogenic assays. We show that our mouse model can originate MBC with an active PI3K pathway. These tumors are amenable to transplantation into immunocompromised mice and to generate cell lines and organoids, allowing us to develop a simple, versatile, and rapid model of MBC. Tumors derived from our model are sensitive to PI3Kα and mTORC1 inhibitors, but eventually develop resistance to these agents. The use of next-generation mTORC1 inhibitors can overcome resistance in some cases, providing a rationale for their implementation in the treatment of patients with MBC. Citation Format: Paola Roa, Valentina Foglizzo, Michelle R. de Marchena, Shraddha Chandthakuri, Emiliano Cocco, Pau Castel. A PIK3CA-driven mouse model of metaplastic breast carcinoma to assess potential therapeutic strategies [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 1452.


The deubiquitinase USP9X regulates RIT1 protein abundance and oncogenic phenotypes

December 2023

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30 Reads

RIT1 is a rare and understudied oncogene in lung cancer. Despite structural similarity to other RAS GTPase proteins such as KRAS, oncogenic RIT1 activity does not appear to be tightly regulated by nucleotide exchange or hydrolysis. Instead, there is a growing understanding that the protein abundance of RIT1 is important for its regulation and function. We previously identified the deubiquitinase USP9X as a RIT1 dependency in RIT1-mutant cells. Here, we demonstrate that both wild-type and mutant forms of RIT1 are substrates of USP9X. Depletion of USP9X leads to decreased RIT1 protein stability and abundance and resensitizes cells to EGFR tyrosine kinase inhibitors. Our work expands upon the current understanding of RIT1 protein regulation and presents USP9X as a key regulator of RIT1-driven oncogenic phenotypes.


The 8th International RASopathies Symposium: Expanding research and care practice through global collaboration and advocacy

November 2023

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113 Reads

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1 Citation

American Journal of Medical Genetics Part A

Germline pathogenic variants in the RAS/mitogen-activated protein kinase (MAPK) signaling pathway are the molecular cause of RASopathies, a group of clinically overlapping genetic syndromes. RASopathies constitute a wide clinical spectrum characterized by distinct facial features, short stature, predisposition to cancer, and variable anomalies in nearly all the major body systems. With increasing global recognition of these conditions, the 8th International RASopathies Symposium spotlighted global perspectives on clinical care and research, including strategies for building international collaborations and developing diverse patient cohorts in anticipation of interventional trials. This biannual meeting, organized by RASopathies Network, was held in a hybrid virtual/in-person format. The agenda featured emerging discoveries and case findings as well as progress in preclinical and therapeutic pipelines. Stakeholders including basic scientists, clinician-scientists, practitioners, industry representatives, patients, and family advocates gathered to discuss cutting edge science, recognize current gaps in knowledge, and hear from people with RASopathies about the experience of daily living. Presentations by RASopathy self-advocates and early-stage investigators were featured throughout the program to encourage a sustainable, diverse, long-term research and advocacy partnership focused on improving health and bringing treatments to people with RASopathies.


Citations (52)


... In their GTP-bound state, RAS proteins can bind to downstream effectors such as RAF, PI3K, and RalGDS to form RAS-effector complexes on the cell membrane, which initiate signal transduction predominantly (but not exclusively) via the MAPK and PI3K/AKT pathways (2). These interactions are part of larger complexes, some of which have been structurally characterized (3)(4)(5)(6)(7)(8). However, determining whether structurally defined RAS-RAS interactions play a role in this organization has been challenging due to the transient nature of these interactions in cells and other experimental systems (9). ...

Reference:

Experimental variables determine the outcome of RAS-RAS interactions
Functional and structural insights into RAS effector proteins
  • Citing Article
  • August 2024

Molecular Cell

... Rights reserved. inhibitor and may provide a valuable option for combination therapy with further optimization of SMYD2 inhibitors [56] Recent work has also linked KMT2A, a member of the same family of H3K4 methyltransferases, and its cofactor menin to the PI3K pathway. Treatment with menin-KMT2A/B inhibitors sensitizes ER positive breast cancer cell lines to the PI3K inhibitor alpelisib and leads to an increase in phospho-AKT. ...

Methylation of the chromatin modifier KMT2D by SMYD2 contributes to therapeutic response in hormone-dependent breast cancer

Cell Reports

... There are also diseasesaffecting physical makeup, spanning chronic conditions (i.e., anemia, asthma, celiac disease, inflammatory bowel disease, kidney or heart insufficiency), hormonal diseases (i.e., growth or thyroid hormone disbalances) and/or rare disorders such as achondroplasia and Down, Noonan or Turner syndromes. 1,2 For example, patients diagnosed with Friedreich ataxia tend to be underweight in young age and overweight in adulthood. 3,4 Patients with different types of mucopolysaccharidoses are known to present with a short stature. ...

The 8th International RASopathies Symposium: Expanding research and care practice through global collaboration and advocacy

American Journal of Medical Genetics Part A

... Mutations in RIT1 mainly occur in the SwitchII domain of the RIT1 protein, usually altering the functionality of the RIT1 protein. This abnormally activates classic pathways such as MAPK and PI3K/AKT and promotes tumour proliferation and survival [16]. In some malignant tumours such as lung adenocarcinoma, RIT1 mutations appear to be mutually exclusive with mutations in currently known driver genes such as KRAS and EGFR, which suggests that RIT1 may be an independent oncogenic factor in some tumours [17]. ...

RAS-dependent RAF-MAPK hyperactivation by pathogenic RIT1 is a therapeutic target in Noonan syndrome-associated cardiac hypertrophy
  • Citing Article
  • July 2023

Science Advances

... Our study showed that SPRED2 promotes autophagy by inhibiting mTORC1 in a MAPK/ERK-dependent manner. SPRED2 inhibits the activation of MAPK/ERK by interacting with rat sarcoma (RAS) [7], ribosomal S6 kinase (RSK) 1, neurofibromin [31], and RSK2 [32]. SPRED2 appears to activate autophagy by interacting with selective autophagic components, such as LC3 [5], p62, a neighbor of breast cancer susceptibility gene I (BRCA1) and cathepsin D [33], as evidenced by co-immunoprecipitation of these molecules with SPRED2. ...

The Ribosomal S6 Kinase 2 (RSK2)-SPRED2 complex regulates phosphorylation of RSK substrates and MAPK signaling

Journal of Biological Chemistry

... Like other Ras family members, RIT1 contributes to cellular signal transduction and regulation of cell proliferation, differentiation and survival. Recently, the implication of the RIT1 gene in tumours has gradually attracted attention, as the occurred mutations have been found to exhibit a close correlation with cancer occurrence and progression, such as lung adenocarcinoma [10], hepatocellular carcinoma [11], endometrial carcinoma [12] and leukaemia [13,14], among others [15]. Mutations in RIT1 mainly occur in the SwitchII domain of the RIT1 protein, usually altering the functionality of the RIT1 protein. ...

Impaired Proteolysis of Noncanonical RAS Proteins Drives Clonal Hematopoietic Transformation

Cancer Discovery

... In any case, schwannoma tumours arise from the SCs of spinal and/or peripheral nerves. Homozygous inactivation of the SPS-associated genes SMARCB1 and LZTR1 is embryonically lethal [25,26]; hence, a conditional mouse model of SPS was generated by ablating SMARCB1 and NF2 in SC lineage in specific developmental stages, further confirming the requirement of both genes in schwannoma development [26]. The availability of preclinical models is therefore crucial to develop efficient treatments. ...

Cross-species analysis of LZTR1 loss-of-function mutants demonstrates dependency to RIT1 orthologs

eLife

... This analysis allowed to establish changes in phosphorylation profiles of splicing factors upon altered PI3K signaling and highlighted a wide spectrum of so far uncharacterized phosphorylation sites. A recent report linked oncogenic PI3K signaling with splicing alterations in breast cancer on the transcriptional level 18 . Thus, we reanalyzed our RNA-seq data for differentially expressed transcripts, which were indeed associated with genes involved in cell cycle and regulation of apoptosis signaling pathways (Supplemental Figure 6C, D, Supplemental Table 2). ...

The Oncogenic PI3K-Induced Transcriptomic Landscape Reveals Key Functions in Splicing and Gene Expression Regulation

Cancer Research

... Herein, we found that both L. reuteri ZJ617 and spermidine improve whole-body energy metabolism and promote WAT browning. A previous study has confirmed that spermidine promotes WAT lipolysis in an ADRBs-dependent way 39 . Spermidine mimics the β-adrenergic stimulation activating ADRBs receptor, thereby increasing cyclic AMP levels, and driving protein kinase (PKA) signaling to promote lipolysis 39 . ...

Angiocrine polyamine production regulates adiposity

Nature Metabolism

... Vision and ocular issues, including nystagmus, strabismus, and refractive errors, commonly occur in RASopathies [130,131]. In NF1, visual changes can also occur in the presence of optic pathway gliomas [132]. ...

The seventh international RASopathies symposium: Pathways to a cure—expanding knowledge, enhancing research, and therapeutic discovery

American Journal of Medical Genetics Part A