Multiple Downstream Effectors Research Articles

RhoGDI phosphorylation by PKC promotes its interaction with death receptor p75NTR to gate axon growth and neuron survival

How receptors juggle their interactions with multiple downstream effectors remains poorly understood. Here we show that the outcome of death receptor p75NTR signaling is determined through competition of effectors for interaction with its intracellular domain, in turn dictated by the nature of the ligand. While NGF induces release of RhoGDI through recruitment of RIP2, thus decreasing RhoA activity in favor of NFkB signaling, MAG induces PKC-mediated phosphorylation of the RhoGDI N-terminus, promoting its interaction with the juxtamembrane domain of p75NTR, disengaging RIP2, and enhancing RhoA activity in detriment of NF-kB. This results in stunted neurite outgrowth and apoptosis in cerebellar granule neurons. If presented simultaneously, MAG prevails over NGF. The NMR solution structure of the complex between the RhoGDI N-terminus and p75NTR juxtamembrane domain reveals previously unknown structures of these proteins and clarifies the mechanism of p75NTR activation. These results show how ligand-directed competition between RIP2 and RhoGDI for p75NTR engagement determine axon growth and neuron survival. Similar principles are likely at work in other receptors engaging multiple effectors and signaling pathways.

Hexokinase-linked glycolytic overload and unscheduled glycolysis in hyperglycemia-induced pathogenesis of insulin resistance, beta-cell glucotoxicity, and diabetic vascular complications.

Hyperglycemia is a risk factor for the development of insulin resistance, beta-cell glucotoxicity, and vascular complications of diabetes. We propose the hypothesis, hexokinase-linked glycolytic overload and unscheduled glycolysis, in explanation. Hexokinases (HKs) catalyze the first step of glucose metabolism. Increased flux of glucose metabolism through glycolysis gated by HKs, when occurring without concomitant increased activity of glycolytic enzymes-unscheduled glycolysis-produces increased levels of glycolytic intermediates with overspill into effector pathways of cell dysfunction and pathogenesis. HK1 is saturated with glucose in euglycemia and, where it is the major HK, provides for basal glycolytic flux without glycolytic overload. HK2 has similar saturation characteristics, except that, in persistent hyperglycemia, it is stabilized to proteolysis by high intracellular glucose concentration, increasing HK activity and initiating glycolytic overload and unscheduled glycolysis. This drives the development of vascular complications of diabetes. Similar HK2-linked unscheduled glycolysis in skeletal muscle and adipose tissue in impaired fasting glucose drives the development of peripheral insulin resistance. Glucokinase (GCK or HK4)-linked glycolytic overload and unscheduled glycolysis occurs in persistent hyperglycemia in hepatocytes and beta-cells, contributing to hepatic insulin resistance and beta-cell glucotoxicity, leading to the development of type 2 diabetes. Downstream effector pathways of HK-linked unscheduled glycolysis are mitochondrial dysfunction and increased reactive oxygen species (ROS) formation; activation of hexosamine, protein kinase c, and dicarbonyl stress pathways; and increased Mlx/Mondo A signaling. Mitochondrial dysfunction and increased ROS was proposed as the initiator of metabolic dysfunction in hyperglycemia, but it is rather one of the multiple downstream effector pathways. Correction of HK2 dysregulation is proposed as a novel therapeutic target. Pharmacotherapy addressing it corrected insulin resistance in overweight and obese subjects in clinical trial. Overall, the damaging effects of hyperglycemia are a consequence of HK-gated increased flux of glucose metabolism without increased glycolytic enzyme activities to accommodate it.

O09 Targeted genetic therapy for congenital melanocytic naevi

Abstract Severe cases of congenital melanocytic naevi (CMN) are associated with substantial cutaneous and neurological morbidity and an excess childhood mortality from melanoma. Effective treatments are lacking. This mosaic disorder is caused most commonly by a heterozygous oncogenic variant NRAS c.181C>A, p.(Q61K). As NRAS has multiple downstream effectors we have sought to treat this disease by precision genetic therapy rather than downstream pathway inhibition, hypothesising that silencing of the oncogenic allele may remove cellular protection from RAS induced apoptosis. Using a lead siRNA selected for allele specificity and fewest off-target effects on RNAseq (siNRASQ61K) we show here significant selective knockdown of variant mRNA expression and knockdown of NRAS protein levels. A single treatment to primary naevus cell cultures leads to normalisation of MAPK pathway activation, reduction in proliferation rate, and importantly, triggering of apoptosis. We go on to demonstrate that receptor-targeted lipid nanoparticles (RTNPs) successfully protect siRNA from degradation, deliver cargo to naevus cells in patient skin explants and improve naevus cell targeting. A single intradermal injection of siNRASQ61K-RTNPs into transgenic mice harbouring humanised NRASQ61K(Tyr::NRASQ61K) confirms significant selective knockdown of the variant NRAS allele in vivo.Successful targeting of variant NRAS and triggering of naevus cell apoptosis now paves the way for clinical trials of this genetic therapy for CMN.

Identification of FEN1 as a prognostic marker and novel therapeutic target in gastric cancer harboring TP53 mutations.

e16057 Background: As a common tumor suppressor gene, TP53 gene mutation leads to persistent activation of multiple downstream effectors that drive the cancer phenotype. Approximately 50% of gastric cancer (GC) patients harbor TP53 mutations, which confer stronger tumor biology and shorter overall survival (OS). Given that TP53-mutated proteins have proven difficult to target directly, identifying genes that function closely with TP53 and targeting these genes appears to be a promising therapeutic strategy for TP53-mutated GC patients. Methods: TP53 functionally sensitive genes were identified by evaluating the correlation between gene-dependent scores from CRISPR knockout screens and TP53 mRNA expression in TP53 mutant GC cell lines in the Cancer Cell Line Encyclopedia (CCLE) database. If the correlation coefficient is ≥0.6, the gene was considered as TP53 functional sensitive gene. Then TP53 functionally sensitive genes associated with prognosis were screened out in The Cancer Genome Atlas (TCGA). PockDrug-Server was used to predict drug susceptibility of candidate genes. Results: The results showed that in 9 GC cell lines with TP53 mutations and 14 with TP53 wild-type, 4 genes (KLF5, FEN1, CCNA2, CCNB1) were identified as TP53 functional sensitive genes. Among the 4 genes, only FEN1 expression was significantly correlated with OS (HR=1.85, 95CI 1.06−3.26, P=0.029). Multivariate Cox regression analysis showed that high FEN1 expression was an independent predictor of prognosis in TP53-mutated GC. Pocket druggability prediction analysis presented that FEN1 had 8 drug-able pockets, four of which all have a druggability score of 0.8 or higher, including one pocket with a score of 1. Conclusions: These findings suggested that FEN1 was promising prognostic marker and therapeutic target in TP53-mutated GC.

Friend or Foe: Regulation, Downstream Effectors of RRAD in Cancer.

Ras-related associated with diabetes (RRAD), a member of the Ras-related GTPase superfamily, is primarily a cytosolic protein that actives in the plasma membrane. RRAD is highly expressed in type 2 diabetes patients and as a biomarker of congestive heart failure. Mounting evidence showed that RRAD is important for the progression and metastasis of tumor cells, which play opposite roles as an oncogene or tumor suppressor gene depending on cancer and cell type. These findings are of great significance, especially given that relevant molecular mechanisms are being discovered. Being regulated in various pathways, RRAD plays wide spectrum cellular activity including tumor cell division, motility, apoptosis, and energy metabolism by modulating tumor-related gene expression and interacting with multiple downstream effectors. Additionally, RRAD in senescence may contribute to its role in cancer. Despite the twofold characters of RRAD, targeted therapies are becoming a potential therapeutic strategy to combat cancers. This review will discuss the dual identity of RRAD in specific cancer type, provides an overview of the regulation and downstream effectors of RRAD to offer valuable insights for readers, explore the intracellular role of RRAD in cancer, and give a reference for future mechanistic studies.

The role of Rho GTPase family in cochlear hair cells and hearing.

Rho GTPases are essential regulators of the actin cytoskeleton. They are involved in various physiological and biochemical processes such as the regulation of cytoskeleton dynamics, development, proliferation, survival, and regeneration. During the development of cochlear hair cells, Rho GTPases are activated by various extracellular signals through membrane receptors to further stimulate multiple downstream effectors. Specifically, RhoA, Cdc42, and Rac1, members of the classical subfamily of the Rho GTPase family, regulate the development and maintenance of cilia by inducing the polymerization of actin monomers and stabilizing actin filaments. In addition, they also regulate the normal morphology orientation of ciliary bundles in auditory hair cells, which is an important element of cell polarity regulation. Moreover, the actin-related pathways mediated by RhoA, Cdc42, and Rac1 also play a role in the motility of outer hair cells, indicating that the function of Rho GTPases is crucial in the highly polar auditory sensory system. In this review, we focus on the expression of RhoA, Cdc42, and Rac1 in cochlear hair cells and how these small molecules participate in ciliary bundle morphogenesis and cochlear hair cell movement. We also discuss the progress of current research investigating the use of these small molecules as drug targets for deafness treatment.

Cyclophilin A associates with and regulates the activity of ZAP70 in TCR/CD3-stimulated T cells.

The ZAP70 protein tyrosine kinase (PTK) couples stimulated T cell antigen receptors (TCRs) to their downstream signal transduction pathways and is sine qua non for T cell activation and differentiation. TCR engagement leads to activation-induced post-translational modifications of ZAP70, predominantly by kinases, which modulate its conformation, leading to activation of its catalytic domain. Here, we demonstrate that ZAP70 in TCR/CD3-activated mouse spleen and thymus cells, as well as human Jurkat T cells, is regulated by the peptidyl-prolyl cis-trans isomerase (PPIase), cyclophilin A (CypA) and that this regulation is abrogated by cyclosporin A (CsA), a CypA inhibitor. We found that TCR crosslinking promoted a rapid and transient, Lck-dependent association of CypA with the interdomain B region, at the ZAP70 regulatory domain. CsA inhibited CypA binding to ZAP70 and prevented the colocalization of CypA and ZAP70 at the cell membrane. In addition, imaging analyses of antigen-specific T cells stimulated by MHC-restricted antigen-fed antigen-presenting cells revealed the recruitment of ZAP70-bound CypA to the immunological synapse. Enzymatically active CypA downregulated the catalytic activity of ZAP70 in vitro, an effect that was reversed by CsA in TCR/CD3-activated normal T cells but not in CypA-deficient T cells, and further confirmed in vivo by FRET-based studies. We suggest that CypA plays a role in determining the activity of ZAP70 in TCR-engaged T cells and impact on T cell activation by intervening with the activity of multiple downstream effector molecules.

Growth and Public Health Concerns

Seventeen scientists met for this year’s conference on Auxology held at Krobielowice castle, Poland, to discuss growth and public health concerns. The regulation of growth is complex and besides metabolic and endocrine components including hypothalamic releasing factors, growth hormone and multiple downstream effectors, comprises the full spectrum of the psychosocial, economic and emotional environment including signaling dominance, competence, prestige, or subordination and indulgence, all of this being sensitive to urban or rural lifestyle, the political climate and with marked plasticity throughout history. New statistical techniques (St. Nicolas House Analysis) are presented for analyzing anthropometric variables for public health concerns. The impact of spatial differences on developmental tempo, growth in height, and the prevalence of childhood obesity are discussed as well as the impact of social mobility on obesity, and the benefits of the biopsychosocial status when getting along with socio-economic disasters and the COVID-19 pandemic.

Hyperactivation of mTOR and AKT in a cardiac hypertrophy animal model of Friedreich ataxia

Cardiomyopathy is a primary cause of death in Friedreich ataxia (FRDA) patients with defective iron-sulfur cluster (ISC) biogenesis due to loss of functional frataxin and in rare patients with functional loss of other ISC biogenesis factors. The mechanistic target of rapamycin (mTOR) and AKT signaling cascades that coordinate eukaryotic cell growth and metabolism with environmental inputs, including nutrients and growth factors, are crucial regulators of cardiovascular growth and homeostasis. We observed increased phosphorylation of AKT and dysregulation of multiple downstream effectors of mTORC1, including S6K1, S6, ULK1 and 4EBP1, in a cardiac/skeletal muscle specific FRDA conditional knockout (cKO) mouse model and in human cell lines depleted of ISC biogenesis factors. Knockdown of several mitochondrial metabolic proteins that are downstream targets of ISC biogenesis, including lipoyl synthase and subunit B of succinate dehydrogenase, also resulted in activation of mTOR and AKT signaling, suggesting that mTOR and AKT hyperactivations are part of the metabolic stress response to ISC deficiencies. Administration of rapamycin, a specific inhibitor of mTOR signaling, enhanced the survival of the Fxn cKO mice, providing proof of concept for the potential of mTOR inhibition to ameliorate cardiac disease in patients with defective ISC biogenesis. However, AKT phosphorylation remained high in rapamycin-treated Fxn cKO hearts, suggesting that parallel mTOR and AKT inhibition might be necessary to further improve the lifespan and healthspan of ISC deficient individuals.

Argonaute 2 modulates EGFR-RAS signaling to promote mutant HRAS and NRAS-driven malignancies.

Activating mutations in RAS GTPases drive nearly 30% of all human cancers. Our prior work described an essential role for Argonaute 2 (AGO2), of the RNA-induced silencing complex, in mutant KRAS-driven cancers. Here, we identified a novel endogenous interaction between AGO2 and RAS in both wild-type (WT) and mutant HRAS/NRAS cells. This interaction was regulated through EGFR-mediated phosphorylation of Y393-AGO2, and utilizing molecular dynamic simulation, we identified a conformational change in pY393-AGO2 protein structure leading to disruption of the RAS binding site. Knockdown of AGO2 led to a profound decrease in proliferation of mutant HRAS/NRAS-driven cell lines but not WT RAS cells. These cells demonstrated oncogene-induced senescence (OIS) as evidenced by β-galactosidase staining and induction of multiple downstream senescence effectors. Mechanistically, we discovered that the senescent phenotype was mediated via induction of reactive oxygen species. Intriguingly, we further identified that loss of AGO2 promoted a novel feed forward pathway leading to inhibition of the PTP1B phosphatase and activation of EGFR–MAPK signaling, consequently resulting in OIS. Taken together, our study demonstrates that the EGFR–AGO2–RAS signaling axis is essential for maintaining mutant HRAS and NRAS-driven malignancies.

Reconstruction and analysis of a large-scale binary Ras-effector signaling network

BackgroundRas is a key cellular signaling hub that controls numerous cell fates via multiple downstream effector pathways. While pathways downstream of effectors such as Raf, PI3K and RalGDS are extensively described in the literature, how other effectors signal downstream of Ras is often still enigmatic.MethodsA comprehensive and unbiased Ras-effector network was reconstructed downstream of 43 effector proteins (converging onto 12 effector classes) using public pathway and protein–protein interaction (PPI) databases. The output is an oriented graph of pairwise interactions defining a 3-layer signaling network downstream of Ras. The 2290 proteins comprising the network were studied for their implication in signaling crosstalk and feedbacks, their subcellular localizations, and their cellular functions.ResultsThe final Ras-effector network consists of 2290 proteins that are connected via 19,080 binary PPIs, increasingly distributed across the downstream layers, with 441 PPIs in layer 1, 1660 in layer 2, and 16,979 in layer 3. We identified a high level of crosstalk among proteins of the 12 effector classes. A class-specific Ras sub-network was generated in CellDesigner (.xml file) and a functional enrichment analysis thereof shows that 58% of the processes have previously been associated to a respective effector pathway, with the remaining providing insights into novel and unexplored functions of specific effector pathways.ConclusionsOur large-scale and cell general Ras-effector network is a crucial steppingstone towards defining the network boundaries. It constitutes a ‘reference interactome’ and can be contextualized for specific conditions, e.g. different cell types or biopsy material obtained from cancer patients. Further, it can serve as a basis for elucidating systems properties, such as input–output relationships, crosstalk, and pathway redundancy.Graphical abstract3uuXA8noaAjvhTVsANJAr5Video .

Abstract MP06: The Signaling Roles Of The Adaptor Protein Nck1 In Atherosclerosis

Rational: Alterations in hemodynamic shear stress (SS) at atherosclerosis-prone sites promotes endothelial activation, characterized by nuclear factor-κB (NF-κB)-driven expression of cell adhesion molecules that mediate leukocyte homing. In addition, proinflammatory cytokines (e.g. IL-1β) promote NF-κB-dependent endothelial activation. While the recent CANTOS trial using an IL-1β antagonist highlights the potential for treating atherogenic inflammation beyond lipid lowering therapies alone, our understanding of how endothelial activation contributes to this effect remains limited. Methodology & Results: We identified the signaling adaptor Nck1 as a critical regulator of atherogenic endothelial activation. Endothelial cells deficient in Nck1 lack SS-induced NF-κB activation and proinflammatory gene expression. Additionally, we demonstrated an interaction between Nck1 and IL-1β signaling in response to SS, showing that SS activates the IL-1β receptor (IL1R1) signaling partner IRAK-1 in a Nck1-dependent manner both in vitro and in vivo . Mechanistically, point mutation analysis suggest a critical role for the Nck1 SH2 domain (phosphotyrosine binding domain) in mediating these effects. Nck1 affinity pulling down and mass spectrometry identified multiple downstream effectors of IL-1 pathway. In vivo, Global Nck1 knockout mice show reduced atherosclerosis characterized by diminished macrophage and smooth muscle incorporation and bone marrow chimeras suggest non-hematopoietic Nck1 mediates this response. Conclusions: A recent GWAS analysis linked Nck1 to coronary artery disease, underscoring its importance to human disease. Therefore, our data suggest that Nck1 mediates the formation of IL-1-related signaling complexes to induce atherogenic endothelial activation, and selective inhibition of Nck1 or its critical domains will allow efficient inhibition of atherosclerosis in humans.

GRSF1 deficiency in skeletal muscle reduces endurance in aged mice.

GRSF1 is a mitochondrial RNA-binding protein important for maintaining mitochondrial function. We found that GRSF1 is highly expressed in cultured skeletal myoblasts differentiating into myotubes. To understand the physiological function of GRSF1 in vivo, we generated mice in which GRSF1 was specifically ablated in skeletal muscle. The conditional knockout mice (Grsf1cKO) appeared normal until 7-9 months of age. Importantly, however, a reduction of muscle endurance compared to wild-type controls was observed in 16- to 18-month old Grsf1cKO mice. Transcriptomic analysis revealed more than 200 mRNAs differentially expressed in Grsf1cKO muscle at this age. Notably, mRNAs encoding proteins involved in mitochondrial function, inflammation, and ion transport, including Mgarp, Cxcl10, Nfkb2, and Sln mRNAs, were significantly elevated in aged Grsf1cKO muscle. Our findings suggest that GRSF1 deficiency exacerbates the functional decline of aged skeletal muscle, likely through multiple downstream effector proteins.

P110α and P110δ catalytic subunits of PI3 kinase regulate lysophosphatidylcholine-induced TRPC6 externalization

Lipid oxidation products, including lysophosphatidylcholine (lysoPC) inhibit endothelial cell (EC) migration in vitro and impair EC healing of arterial injuries in vivo, in part by activating phosphatidylinositol 3-kinase (PI3K), which increases the externalization of canonical transient receptor potential 6 (TRPC6) channels and the subsequent increase in intracellular calcium. Inhibition of PI3K is a potential method to decrease TRPC6 activation and restore migration, but PI3K is involved in multiple intracellular signaling pathways and has multiple downstream effectors. The goal of this study is to identify the specific p110 catalytic subunit isoforms responsible for lysoPC-induced TRPC6 externalization to identify a target for intervention while minimizing impact on alternative signaling pathways. Down-regulation of the p110α and p110δ isoforms, but not the p110β or p110γ isoforms, with small interfering RNA significantly decreased phosphatidylinositol (3,4,5)-trisphosphate production and TRPC6 externalization, and significantly improved EC migration in the presence of lysoPC. These results identify an additional role of p110α in EC and reveal for the first time a specific role of p110δ in EC, providing a foundation for subsequent in vivo studies to investigate the impact of p110 isoform inhibition on arterial healing after injury.

The RNA Binding Protein IGF2BP3 Is Required for MLL-AF4 Mediated Leukemogenesis

Chromosomal rearrangements of the mixed-lineage leukemia (MLL) gene are observed in acute lymphoblastic leukemias (ALL), acute myeloid leukemias (AML), and in rare mixed-lineage leukemia. Despite recent progress in therapeutic approaches, patients with MLL-rearranged (MLLr) leukemias still have very poor outcomes and a high risk of relapse. Of more than 90 fusion partner genes, MLL-AF4 is the most common MLL fusion protein in patients. Previously, we found that the RNA binding protein IGF2BP3 was specifically overexpressed in MLL-rearranged B-ALL, and enforced expression in vivo led to a pathologic expansion of hematopoietic stem and progenitor cells resulting in B and myeloid cell leukocytosis in the periphery. However, the requirement of IGF2BP3 in MLL-AF4 mediated leukemogenesis remains to be determined. Utilizing our previously generated list of differentially regulated targets with IGF2BP3 knockdown and a published dataset of MLL-Af4 targets, we determined that transcripts modulated by IGF2BP3 showed significant enrichment for MLL-Af4-bound genes. Furthermore, we observed that MLL-AF4 directly binds to and transcriptionally induces IGF2BP3. We performed ChIP-PCR assays on RS4;11 and SEM cell lines, human B-ALL cell lines that carry the MLL-AF4 translocation, and determined that the region in the first intron of IGF2BP3 is strongly bound by MLL-AF4. Furthermore, we observed a dose-dependent increase in luciferase reporter activity when we co-transfected a dual-luciferase reporter vector containing the promoter region of IGF2BP3 with increasing levels of MLL-AF4 expressing retroviral vector. To determine the role of Igf2bp3 in MLL-Af4 driven leukemogenesis, we generated the first Igf2bp3 KO murine model. Surprisingly, Igf2bp3 KO mice maintain normal, steady-state hematopoiesis. However, in striking contrast, deletion of Igf2bp3 in the MLL-Af4 leukemia model, significantly increases the survival of MLL-Af4 transplanted mice and greatly attenuates the disease. Furthermore, Igf2bp3 deficiency significantly reduced the tumor burden and disease severity. We observed significant decreases in WBC counts, spleen weights, and infiltrating leukemic cells visualized in histopathological analysis of hematopoietic tissues and quantified by FACS analysis. Moreover, deletion of Igf2bp3 led to a leukemia-initiating cell (LIC) disadvantage in vivo, demonstrated by significantly reduced engraftment in primary transplanted mice and reconstitution of secondary serially transplanted mice. To identify the transcripts directly regulated by Igf2bp3 in the context of MLL-Af4 driven leukemia, we carried out enhanced crosslinking and immunoprecipitation (eCLIP) transcriptome analysis of MLL-Af4 transformed early stem and progenitor cells and primary cells purified from splenic tumors of MLL-Af4 leukemic mice. We discovered an IGF2BP3-regulated post-transcriptional operon governing leukemic cell survival and proliferation, in which mRNA targets include the Hoxa locus and numerous genes within the Ras signaling pathway. In our study, we provide evidence that Igf2bp3 is required for the initiation of MLL-Af4 driven leukemia. We determined that Igf2bp3 is necessary for the development of and function of MLL-Af4 LICs. Mechanistically, we show that Igf2bp3 binds to and modulates the expression of hundreds of critical target transcripts. In summary, we demonstrate that Igf2bp3 is a positive regulator of MLLr leukemogenesis by targeting Hoxa transcripts such as Hoxa9 and numerous Ras signaling pathway transcripts, thereby controlling multiple downstream effector pathways required for disease initiation and aggressiveness. Together, our findings identify IGF2BP3 as an important, potential therapeutic target in this disease. Disclosures No relevant conflicts of interest to declare.

Discovery of a series of benzopyrimidodiazepinone TNK2 inhibitors via scaffold morphing

The protein kinase TNK2 (ACK1) is an emerging drug target for a variety of indications, in particular for cancer where it plays a key role transmitting cell survival, growth and proliferative signals via modification of multiple downstream effectors by unique tyrosine phosphorylation events. Scaffold morphing based on our previous TNK2 inhibitor XMD8-87 identified urea 17 from which we developed the potent and selective compound 32. A co-crystal structure was obtained showing 32 interacting primarily with the main chain atoms of an alanine residue of the hinge region. Additional H-bonds exist between the urea NHs and the Thr205 and Asp270 residues.

Hepatocyte-specific TAK1 deficiency drives RIPK1 kinase-dependent inflammation to promote liver fibrosis and hepatocellular carcinoma

Transforming growth factor β-activated kinase1 (TAK1) encoded by the gene MAP3K7 regulates multiple important downstream effectors involved in immune response, cell death, and carcinogenesis. Hepatocyte-specific deletion of TAK1 in Tak1ΔHEP mice promotes liver fibrosis and hepatocellular carcinoma (HCC) formation. Here, we report that genetic inactivation of RIPK1 kinase using a kinase dead knockin D138N mutation in Tak1ΔHEP mice inhibits the expression of liver tumor biomarkers, liver fibrosis, and HCC formation. Inhibition of RIPK1, however, has no or minimum effect on hepatocyte loss and compensatory proliferation, which are the recognized factors important for liver fibrosis and HCC development. Using single-cell RNA sequencing, we discovered that inhibition of RIPK1 strongly suppresses inflammation induced by hepatocyte-specific loss of TAK1. Activation of RIPK1 promotes the transcription of key proinflammatory cytokines, such as CCL2, and CCR2+ macrophage infiltration. Our study demonstrates the role and mechanism of RIPK1 kinase in promoting inflammation, both cell-autonomously and cell-nonautonomously, in the development of liver fibrosis and HCC, independent of cell death, and compensatory proliferation. We suggest the possibility of inhibiting RIPK1 kinase as a therapeutic strategy for reducing liver fibrosis and HCC development by inhibiting inflammation.

CAMKK2-CAMK4 signaling regulates transferrin trafficking, turnover, and iron\xa0homeostasis

BackgroundCirculatory iron is a hazardous biometal. Therefore, iron is transported in a redox-safe state by a serum glycoprotein - transferrin (TF). Different organs acquire iron from the systemic circulation through a tightly regulated mechanism at the blood-tissue interface which involves receptor-mediated internalization of TF. Thus, abnormal TF trafficking may lead to iron dyshomeostasis associated with several diseases including neurodegeneration. Iron -induced toxicity can cause neuronal damage to iron-sensitive brain regions. Recently, it was discovered that CAMKK2, a calcium (Ca2+)/calmodulin-activated kinase, controls receptor-mediated TF trafficking in mouse tissues, specifically in the brain. The biological function of CAMKK2 is mediated through multiple downstream effectors. Both CAMKK2 and one of its downstream kinase, CAMK4, exhibit overlapping expression in mouse brain. The role of CAMK4 in vesicular transport has been reported and loss of CAMKK2 or CAMK4 leads to cognitive defects in mouse. Therefore, it was hypothesized that CAMKK2-CAMK4 signaling regulates receptor-mediated TF trafficking and iron homeostasis which may be responsible for the neuronal malfunction observed in CAMKK2- or CAMK4-deficient mice.MethodsCAMK4−/− mouse was used to study tissue-specific turnover of TF, TF-receptor (TFRC) and iron. CRISPR/Cas9-based CAMKK2 and/or CAMK4 deleted human embryonic kidney-derived HEK293 cell clones were used to study the molecular defects in receptor-mediated TF trafficking. Further, a “zero functional G protein” condition in HEK293 cell was exploited to study CAMKK2-CAMK4 signaling-mediated regulation of intracellular Ca2+ homeostasis which was linked to calcium signaling during TF trafficking.ResultsLoss of CAMK4 leads to abnormal post-translational modifications (PTMs) and turnover of TF in mouse cerebellum and liver which was associated with iron dyshomeostasis in these tissues. The HEK293 cell-based study revealed that the absence of CAMKK2-CAMK4 signaling altered intracellular Ca2+ homeostasis and lead to abnormal calcium signaling during TF trafficking. Also, CAMKK2-CAMK4 signaling deficiency affected the molecular interaction of TF and TF-receptor-associated protein complexes which indicated a potential failure in the recruitment of interacting proteins due to differential PTMs in TF.ConclusionOverall, this study established a novel mechanistic link between intracellular Ca2+ level, receptor-mediated TF trafficking, and iron homeostasis, all regulated by CAMKK2-CAMK4 signaling.96jS1k-UhvBwj8b5MHadpoVideo Graphical abstract

SAT-124 Optimization of Experimental Conditions for Mimicking Hypoxia in Cultured Breast Cancer Cells by Using Cobalt(II) Chloride (CoCl2)

Hypoxia is a common phenomenon in solid tumor development caused by a decrease in either oxygen concentration or oxygen pressure as a result of rapid tumor cell growth. Hypoxia is characterized by stabilization of the alpha subunit of the hypoxia-inducible factor (HIF-1α) and its nuclear translocation and heterodimerization with HIF-1β. Activation of this signaling pathway involves multiple downstream effectors including carbonic anhydrase 9 (CA9, s. CAIX). A reliable method to mimic hypoxia utilizes cobalt(II) chloride (CoCl2), which directly induces the expression of HIF-1α. The aim of this study was to optimize the experimental conditions for CoCl2 treatment of breast cancer cells in vitro using three human breast cancer cell lines (MDA-MB-231, T-47D, and MCF-7 cells). We performed time- and concentration-response experiments, using various concentrations of CoCl2 (50, 100, 200, and 300 μM) for 24 and 48 hours, and measured the expression of HIF-1α and CA9 by qRT-PCR and Western blot analyses. Results demonstrated that CoCl2 downregulated HIF-1α mRNA levels but upregulated CA9 mRNA levels in a concentration- and time-dependent manner. Concomitantly, CoCl2 treatment resulted in a significant induction of HIF-1α protein levels. We further investigated the effect of the CoCl2 concentrations listed above on cell apoptosis using an in situ apoptosis detection kit. The results demonstrated that concentrations of CoCl2 up to 100 μM had no significant effect on cell apoptosis.

Abstract B14: CRAF-mediated inactivation of epigenetic repressor complexes promotes KRAS-driven tumorigenesis

Abstract Oncogenic mutations in KRAS serve as the initiating event in many human cancers, most notably pancreatic and lung cancers. While KRAS can signal through multiple downstream effector pathways, the MAPK pathway functions as one of the most critical downstream effectors promoting tumorigenesis. Despite its prominent role, inhibitors targeting the MAPK pathway have limited efficacy in KRAS mutant tumors. In the case of BRAF inhibitors, paradoxical activation is observed in KRAS-mutant tumors, while MEK and ERK inhibitors disable negative feedback loops resulting in pathway reactivation. Combinations of MEK and ERK inhibitors have been explored clinically, but a safe combination dose has not yet been established. Given this, identifying novel vulnerabilities in KRAS mutant tumors represents a significant unmet clinical need. Hence, we analyzed the dependency of KRAS-mutant tumors in the Achilles dataset and found a significant subset of KRAS and NRAS-mutant tumors that appear to depend on CRAF. Importantly, KRAS-mutant tumors were not dependent on BRAF or ARAF, the other 2 RAF isoforms. This is consistent with the studies in KRAS-mutant lung GEMMs, where CRAF and not BRAF depletion has been shown to reduce tumor incidence, suggesting that CRAF serves as the main signaling hub downstream of mutant KRAS. In this work, we investigate the kinase-dependent and -independent roles of CRAF in KRAS-mutant tumors. We demonstrate that CRAF depletion in KRAS-mutant human cancer cells inhibits colony formation on soft agar assay and mediates tumor growth inhibition in mouse xenograft models. CRAF loss resulted in activation of cell death and cell cycle arrest pathways without any alterations to downstream MAPK signaling. Interestingly, this suppression of growth upon CRAF loss in KRAS mutant cancer cells was rescued by expression of kinase-dead variants of CRAF, suggesting CRAF kinase function is not required to promote KRAS-driven tumorigenesis. In KRAS-mutant tumor cells, we observe that CRAF preferentially heterodimerizes with ARAF and upon treatment with a MEK inhibitor, the dimer preference on CRAF switches to BRAF, resulting in elevated CRAF kinase activity. To explore molecular determinants of CRAF kinase independent functions, we performed an enrichment screen to identify effectors whose loss confers a growth advantage in the CRAF-null KRAS mutant cells. We identified a class of epigenetic repressor complexes that were strongly depleted in the CRAF-null KRAS mutant cells and are in the process of validating these hits. Further, to dissect CRAF kinase-dependent versus -independent functions, we are establishing a CRAF gene signature using KRAS-mutant cancer cells and also, identifying CRAF-interacting partners that promote CRAF kinase-independent functions. Thus far, this study elucidates novel kinase-independent roles of cRAF that could be harnessed to effectively target KRAS-mutant human cancers. Citation Format: Avinashnarayan Venkatanarayan, Christiaan Klijn, Shiva Malek. CRAF-mediated inactivation of epigenetic repressor complexes promotes KRAS-driven tumorigenesis [abstract]. In: Proceedings of the AACR Special Conference on Targeting RAS-Driven Cancers; 2018 Dec 9-12; San Diego, CA. Philadelphia (PA): AACR; Mol Cancer Res 2020;18(5_Suppl):Abstract nr B14.