Activation Of Src Family Kinases Research Articles

Molecular targets of glucocorticoids that elucidate their therapeutic efficacy in aggressive lymphomas

Glucocorticoids have been used for decades to treat lymphomas without an established mechanism of action. Using functional genomic, proteomic, and chemical screens, we discover that glucocorticoids inhibit oncogenic signaling by the B cell receptor (BCR), a recurrent feature of aggressive B cell malignancies, including diffuse large B cell lymphoma and Burkitt lymphoma. Glucocorticoids induce the glucocorticoid receptor (GR) to directly transactivate genes encoding negative regulators of BCR stability (LAPTM5; KLHL14) and the PI3 kinase pathway (INPP5D; DDIT4). GR directly represses transcription of CSK, a kinase that limits the activity of BCR-proximal Src-family kinases. CSK inhibition attenuates the constitutive BCR signaling of lymphomas by hyperactivating Src-family kinases, triggering their ubiquitination and degradation. With the knowledge that glucocorticoids disable oncogenic BCR signaling, they can now be deployed rationally to treat BCR-dependent aggressive lymphomas and used to construct mechanistically sound combination regimens with inhibitors of BTK, PI3 kinase, BCL2, and CSK.

Homocysteine-induced sustained GluN2A NMDA receptor stimulation leads to mitochondrial ROS generation and neurotoxicity

Homocysteine, a sulfur-containing amino acid derived from methionine metabolism, is a known agonist of N-methyl-D-aspartate receptor (NMDAR) and is involved in neurotoxicity. Our previous findings showed that neuronal exposure to elevated homocysteine levels leads to sustained low-level increase in intracellular Ca2+, which is dependent on GluN2A subunit-containing NMDAR (GluN2A-NMDAR) stimulation. These studies further showed a role of ERK MAPK in homocysteine-GluN2A-NMDAR-mediated neuronal death. However, the intracellular mechanisms associated with such sustained GluN2A-NMDAR stimulation and subsequent Ca2+ influx have remained unexplored. Using live-cell imaging with Fluo3-AM and biochemical approaches, we show that homocysteine-GluN2A NMDAR-induced initial Ca2+ influx triggers sequential phosphorylation and subsequent activation of the proline rich tyrosine kinase 2 (Pyk2) and Src family kinases, which inturn phosphorylates GluN2A-Tyr1325 residue of GluN2A-NMDARs to maintain channel activity. The continuity of this cycle of events leads to sustained Ca2+ influx through GluN2A-NMDAR. Our findings also show that lack of activation of the regulatory tyrosine phosphatase STEP, which can limit Pyk2 and Src family kinase activity further contributes to the maintenance of this cycle. Additional studies using live-cell imaging of neurons expressing a redox-sensitive GFP targeted to the mitochondrial matrix show that treatment with homocysteine leads to a progressive increase in mitochondrial reactive oxygen species generation, which is dependent on GluN2A-NMDAR-mediated sustained ERK MAPK activation. This later findingdemonstrates a novel role of GluN2A-NMDAR in homocysteine-induced mitochondrial ROS generation and highlights the role of ERK MAPK as the intermediary signaling pathway between GluN2A-NMDAR stimulation and mitochondrial reactive oxygen species generation.

Regulation of Src family kinases by muscarinic acetylcholine receptors in heterologous cells and neurons.

Five muscarinic acetylcholine (mACh) receptor subtypes are divided into two classes: the M1 class (M1, M3, and M5) and the M2 class (M2 and M4). The former is coupled to Gq proteins, while the latter is coupled to Gi/o proteins. Accumulating evidence indicates that mACh receptors play a significant role in the regulation of the Src family kinase (SFK), a subfamily of non-receptor tyrosine kinases. mACh receptors exert their roles in a subtype-dependent fashion and preferentially target Src and Fyn, two members of SFKs that are expressed in the brain and enriched at synaptic sites. While the M1 receptor positively modulates SFK activity, the M4 receptor inhibits it. By modulating SFKs, mACh receptors are actively involved in the regulation of expression and function of a variety of receptors, structural proteins, and signaling molecules. In particular, the M4 receptor and the dopamine D1 receptor are coexpressed in striatonigral projection neurons of the striatum. Gi/o-coupled M4 and Gq-coupled D1 receptors antagonistically regulate SFK activity, thereby forming a dynamic balance controlling glutamate receptor activity, excitability of neurons, and synaptic plasticity. In summary, mACh receptors play a crucial role in regulating SFK activity in heterologous cells and neurons.

VE-cadherin junction dynamics in initial lymphatic vessels promotes lymph node metastasis.

The endothelial junction component vascular endothelial (VE)-cadherin governs junctional dynamics in the blood and lymphatic vasculature. Here, we explored how lymphatic junction stability is modulated by elevated VEGFA signaling to facilitate metastasis to sentinel lymph nodes. Zippering of VE-cadherin junctions was established in dermal initial lymphatic vessels after VEGFA injection and in tumor-proximal lymphatics in mice. Shape analysis of pan-cellular VE-cadherin fragments revealed that junctional zippering was accompanied by accumulation of small round-shaped VE-cadherin fragments in the lymphatic endothelium. In mice expressing a mutant VEGFR2 lacking the Y949 phosphosite (Vegfr2 Y949F/Y949F ) required for activation of Src family kinases, zippering of lymphatic junctions persisted, whereas accumulation of small VE-cadherin fragments was suppressed. Moreover, tumor cell entry into initial lymphatic vessels and subsequent metastatic spread to lymph nodes was reduced in mutant mice compared with WT, after challenge with B16F10 melanoma or EO771 breast cancer. We conclude that VEGFA mediates zippering of VE-cadherin junctions in initial lymphatics. Zippering is accompanied by increased VE-cadherin fragmentation through VEGFA-induced Src kinase activation, correlating with tumor dissemination to sentinel lymph nodes.

Characterization of TREM-1 Signaling in Human Neutrophils By Kinome Array and RNA-Seq

The Triggering Receptor Expressed on Myeloid Cells (TREM)-1 is a member of the Immunoglobulin superfamily and an activating receptor mainly expressed on myeloid cells. TREM-1 ligation leads to immediate activation of polymorphonuclear neutrophils (PMN) resulting in degranulation and release of reactive oxygen species as well as various effector molecules. Beyond its role in acute and chronic inflammatory processes, TREM-1 is also involved in cancer emergence and progression probably by alteration of the tumor associated neutrophils (TAN) and macrophages (TAM). Advanced information about the TREM-1 signaling cascade may reveal novel therapeutic approaches, as various kinases can specifically be targeted with kinase inhibitors. Therefore, we investigated the protein tyrosine kinome (PTK) and serine threonine kinome (STK) of human PMN after TREM-1 activation. To gain further insights beyond the signaling cascade, we performed RNAseq to validate the TREM-1 mediated activation and to reveal the TREM-1 mediated transcriptome. Purified PMN from healthy donors were stimulated with a monoclonal TREM-1 antibody compared to an isotype matched control for 20 minutes (kinome activity) or 60 minutes (RNAseq). For kinomics, PamChip arrays (PamGene) were used to quantify the kinome activity and upstream kinase analysis (UKA) was used to predict the kinase activity. The final data analysis and the UKA was performed by PamGene. A median final score (depending on the significance and the specificity of the kinase activity compared to the control group) > 1.2 was defined as specific kinase activity according to the manufacturer's recommendations. For transcriptomics, Novogene performed library preparation and sequencing, and data analysis was performed with CLC. Differentially expressed genes were defined as FDR adjusted p value ≤ 0.05 and fold change ≥2. Gene set enrichment analysis was performed with Enrichr using the libraries Reactome 2022, WikiPathway 2021 Human, and GO Biological Process 2023 and significance was defined as adjusted p value ≤ 0.05. We identified the activation of 31 PTKs and 38 STKs upon TREM-1 ligation. The kinase statistic revealed changes in the activation state of the kinases present in the TREM-1 treated cells compared to the untreated control group ranging from 2.4 to 5.5 (PTK) and from 1.2 to 1.6 (STK) suggesting more TREM-1 mediated activity alteration of the PTKs. Interestingly, there were no downregulated kinase activities after receptor ligation. Out of the PTK family, we identified ten Src family kinases as well as the Syk family member ZAP70 and other kinases such as BTK, already previously shown to abrogate TREM-1 mediated PMN activation. The RNAseq after TREM-1 ligation revealed 542 differentially expressed genes from which 102 genes were upregulated and 440 genes were downregulated. Gene set enrichment analysis revealed upregulation of genes related to nuclear events (kinase and transcription factor activaton, R-HSA-198725) and cell chemotaxis (GO:0060326) validating the activation of several kinases after TREM-1 ligation. We found increased expressions of genes involved in adipogenesis (WP236) and lung fibrosis (WP3624) suitably to the observed increased activity of Src family kinases that are described to be involved in lung fibrosis. Moreover, some upregulated genes related to NGF-stimulated transcription (R-HSA-9031628) or regulation of transcription in response to stress (GO:0043618) in which the MAPK are known to be involved. However, we did not observe clearly increased MAPK activity. Besides upregulation, TREM-1 ligation led to decreased expression of several genes involved in (positive) regulation of cytokine production involved in inflammatory response (GO:1900017 and GO:1900015) and signaling by interleukins (R-HSA-449147). Taken together, the results contribute to the knowledge about TREM-1 in inflammatory disorders and reveal approaches of its role in cancer. This study revealed various kinases possibly involved in early TREM-1 signaling of neutrophils that may be suitable targets to inhibit TREM-1 mediated cell activation. Moreover, we linked the TREM-1 mediated kinase activity to the resulting gene expression. Further studies are needed to validate the results in vitro and in vivo and and to identify suitable pharmacological inhibitors to modulate TREM-1 mediated activities in inflammatory conditions and cancer.

The Reversible BTK Inhibitor Nemtabrutinib Demonstrates Favorable Antitumor Efficacy and Enhances the Function of CAR T Cells in Mantle Cell Lymphoma

Background Covalent Bruton's tyrosine kinase (BTK) inhibitors have revolutionized the therapy of mantle cell lymphoma (MCL). In January 2023, the reversible BTK inhibitor pirtobrutinib was approved by the FDA for the treatment of relapsed or refractory MCL. Nemtabrutinib is another reversible BTK inhibitor which also targets other kinases. The objective of this study was to evaluate the efficacy of nemtabrutinib alone and in combination with anti-CD19 CAR T cells against MCL. Methods Cell viability was measured following a 72-hour treatment with nemtabrutinib in a panel of MCL cell lines using CellTiter-Glo luminescent cell viability assay (Promega). Annexin V/PI staining was utilized to determine whether nemtabrutinib induces cell death through apoptosis. Patient-derived organoids (PDOs) were generated by culturing patient primary cells in 50% Matrigel (Corning) and cultured in cytokine-containing medium. The PDOs were treated for 72 hours, and viability assays were performed. Western blotting was employed to investigate the drug impact on kinase signaling pathways. Nemtabrutinib treated Mino cells were subjected to bulk RNA sequencing to interrogate the transcriptome profiling. The anti-MCL efficacy of nemtabrutinib was tested in an ibrutinib-resistant patient derived xenograft (PDX) mouse model in vivo. Additionally, the potential synergistic effects of nemtabrutinib and anti-CD19 CAR T cells were examined using the luciferase-expressing MCL cells. Results Nemtabrutinib demonstrated comparable growth inhibitory activity to ibrutinib in MCL cell lines with IC 50 values at micromolar concentrations (IC 50 = 0.7-10.1 μM). Remarkably, in the 3D PDO screening system that mimics the tumor microenvironment for MCL, nemtabrutinib displayed superior anti-MCL efficacy compared to ibrutinib (p < 0.01). The apoptotic potential of nemtabrutinib was further investigated using Annexin V/PI staining, revealing a dose-dependent induction of apoptosis, and demonstrated a similar sensitivity profile across cell lines observed with ibrutinib. Western blotting revealed that nemtabrutinib effectively inhibited phosphorylation at both Tyr223 and Tyr551 sites of BTK, and also dramatically suppressed the activation of Src family kinases, Syk and ERK. Bulk RNA-seq was performed to examine the transcriptome profile following nemtabrutinib treatment. 449 genes were identified to be significantly upregulated and 460 genes were downregulated. Gene set enrichment analysis uncovered a significant decrease in TNFα signaling via NF-κB, inflammatory response, and IFNγ response signalings (FDR < 0.05). In an ibrutinib-resistant PDX mouse model, treatment with nemtabrutinib resulted in a significant reduction in tumor burden and effectively attenuated the tumor involvement in spleen, liver and bone marrow. Furthermore, to enhance efficacy, we investigated the combination of nemtabrutinib with anti-CD19 CAR T cells in luciferase-expressing MCL cells. The result demonstrated that nemtabrutinib enhanced the effector function and anti-MCL activity of anti-CD19 CAR T cells. Conclusion Nemtabrutinib demonstrated favorable anti-MCL efficacy in both in vitro and in vivo studies, the promising synergistic effects observed in combination with CAR T cells warrants further investigation in both preclinical and clinical settings.

Developmental roles of glomerular epithelial protein-1 in mice molar morphogenesis.

Glomerular epithelial protein-1 (Glepp1), a R3 subtype family of receptor-type protein tyrosine phosphatases, plays important role in the activation of Src family kinases and regulates cellular processes such as cell proliferation, differentiation, and apoptosis. In this study, we firstly examined the functional evaluation of Glepp1 in tooth development and morphogenesis. The precise expression level and developmental function of Glepp1 were examined by RT-qPCR, in situ hybridization, and loss and gain of functional study using a range of in vitro organ cultivation methods. Expression of Glepp1 was detected in the developing tooth germs in cap and bell stage of tooth development. Knocking down Glepp1 at E13 for 2days showed the altered expression levels of tooth development-related signaling molecules, including Bmps, Dspp, Fgf4, Lef1, and Shh. Moreover, transient knock down of Glepp1 revealed alterations in cellular physiology, examined by the localization patterns of Ki67 and E-cadherin. Similarly, knocking down of Glepp1 showed disrupted enamel rod and interrod formation in 3-week renal transplanted teeth. In addition, due to attrition of odontoblastic layers, the expression signals of Dspp and the localization of NESTIN were almost not detected after knock down of Glepp1; however, their expressions were increased after Glepp1 overexpression. Thus, our results suggested that Glepp1 plays modulating roles during odontogenesis by regulating the expression levels of signaling molecules and cellular events to achieve the proper structural formation of hard tissue matrices in mice molar development.

Activated Src kinase promotes cell cannibalism in Drosophila.

Src family kinases (SFKs) are evolutionarily conserved proteins acting downstream of receptors and regulating cellular processes including proliferation, adhesion, and migration. Elevated SFK expression and activity correlate with progression of a variety of cancers. Here, using the Drosophila melanogaster border cells as a model, we report that localized activation of a Src kinase promotes an unusual behavior: engulfment of one cell by another. By modulating Src expression and activity in the border cell cluster, we found that increased Src kinase activity, either by mutation or loss of a negative regulator, is sufficient to drive one cell to engulf another living cell. We elucidate a molecular mechanism that requires integrins, the kinases SHARK and FAK, and Rho family GTPases, but not the engulfment receptor Draper. We propose that cell cannibalism is a result of aberrant phagocytosis, where cells with dysregulated Src activity fail to differentiate between living and dead or self versus non-self, thus driving this malignant behavior.

Airway epithelium IgE-FcεRI cross-link induces epithelial barrier disruption in severe T2-high asthma

Although high-affinity immunoglobulin (Ig)E receptor (FcεRI) expression is upregulated in type 2 (T2)-high asthmatic airway epithelium, its functional role in airway epithelial dysfunction has not been elucidated. Here we report the upregulated expression of FcεRI and p-EGFR (Epidermal Growth Factor Receptor), associated with decreased expression of E-cadherin and claudin-18 in bronchial biopsies of severe T2-high asthmatics compared to mild allergic asthmatics and non-T2 asthmatics. Monomeric IgE (mIgE) decreased the expression of junction proteins, E-cadherin, claudin-18, and ZO-1, and increased alarmin messenger RNA and protein expression in cultured primary bronchial epithelial cells from T2-high asthmatics. Epithelial FcεRI ligation with mIgE decreased transepithelial electric resistance in air-liquid interface cultured epithelial cells. FcεRI ligation with mIgE or IgE- Dinitrophenyl or serum of high-level allergen-specific IgE activated EGFR and Akt via activation of Src family kinases, mediating alarmin expression, junctional protein loss, and increased epithelial permeability. Furthermore, tracheal instillation of mIgE in house dust mite-sensitized mice induced airway hyper-responsiveness, junction protein loss, epithelial cell shedding, and increased epithelial permeability. Thus, our results suggest that IgE-FcεRI cross-linking in the airway epithelium is a potential and unnoticed mechanism for impaired barrier function, increased mucosal permeability, and EGFR-mediated alarmin production in T2-high asthma.

CSK-mediated signalling by integrins in cancer.

Cancer progression and metastasis are processes heavily controlled by the integrin receptor family. Integrins are cell adhesion molecules that constitute the central components of mechanosensing complexes called focal adhesions, which connect the extracellular environment with the cell interior. Focal adhesions act as key players in cancer progression by regulating biological processes, such as cell migration, invasion, proliferation, and survival. Src family kinases (SFKs) can interplay with integrins and their downstream effectors. SFKs also integrate extracellular cues sensed by integrins and growth factor receptors (GFR), transducing them to coordinate metastasis and cell survival in cancer. The non-receptor tyrosine kinase CSK is a well-known SFK member that suppresses SFK activity by phosphorylating its specific negative regulatory loop (C-terminal Y527 residue). Consequently, CSK may play a pivotal role in tumour progression and suppression by inhibiting SFK oncogenic effects in several cancer types. Remarkably, CSK can localise near focal adhesions when SFKs are activated and even interact with focal adhesion components, such as phosphorylated FAK and Paxillin, among others, suggesting that CSK may regulate focal adhesion dynamics and structure. Even though SFK oncogenic signalling has been extensively described before, the specific role of CSK and its crosstalk with integrins in cancer progression, for example, in mechanosensing, remain veiled. Here, we review how CSK, by regulating SFKs, can regulate integrin signalling, and focus on recent discoveries of mechanotransduction. We additionally examine the cross talk of integrins and GFR as well as the membrane availability of these receptors in cancer. We also explore new pharmaceutical approaches to these signalling pathways and analyse them as future therapeutic targets.

The Src-Family Kinases SRC and BLK Contribute to the CLDN6-Adhesion Signaling.

Cell adhesion molecules, including integrins, cadherins, and claudins (CLDNs), are known to activate Src-family kinases (SFKs) that organize a variety of physiological and pathological processes; however, the underlying molecular basis remains unclear. Here, we identify the SFK members that are coupled with the CLDN6-adhesion signaling. Among SFK subtypes, BLK, FGR, HCK, and SRC were highly expressed in F9 cells and concentrated with CLDN6 along cell borders during epithelial differentiation. Immunoprecipitation assay showed that BLK and SRC, but not FGR or HCK, form a complex with CLDN6 via the C-terminal cytoplasmic domain. We also demonstrated, by pull-down assay, that recombinant BLK and SRC proteins directly bind to the C-terminal cytoplasmic domain of CLDN6 (CLDN6C). Unexpectedly, both recombinant SFK proteins recognized the CLDN6C peptide in a phosphotyrosine-independent manner. Furthermore, by comparing phenotypes of F9:Cldn6:Blk-/- and F9:Cldn6:Src-/- cells with those of wild-type F9 and F9:Cldn6 cells, we revealed that BLK and SRC are essential for CLDN6-triggered cellular events, namely epithelial differentiation and the expression of retinoid acid receptor target genes. These results indicate that selective SFK members appear to participate in the CLDN-adhesion signaling.

Regulation, targets and functions of CSK.

The Src family kinases (SFK) plays an important role in multiple signal transduction pathways. Aberrant activation of SFKs leads to diseases such as cancer, blood disorders, and bone pathologies. By phosphorylating and inactivating SFKs, the C-terminal Src kinase (CSK) serves as the key negative regulator of SFKs. Similar to Src, CSK is composed of SH3, SH2, and a catalytic kinase domain. However, while the Src kinase domain is intrinsically active, the CSK kinase domain is intrinsically inactive. Multiple lines of evidence indicate that CSK is involved in various physiological processes including DNA repair, permeability of intestinal epithelial cells (IECs), synaptic activity, astrocyte-to-neuron communication, erythropoiesis, platelet homeostasis, mast cell activation, immune and inflammation responses. As a result, dysregulation of CSK may lead to many diseases with different underlying molecular mechanisms. Furthermore, recent findings suggest that in addition to the well-established CSK-SFK axis, novel CSK-related targets and modes of CSK regulation also exist. This review focuses on the recent progress in this field for an up-to-date understanding of CSK.

LGG-08. TARGETED THERAPY IN BRAFV600E-DRIVEN PEDIATRIC GLIOMAS: UNCOVERING MECHANISMS OF RESISTANCE THROUGH A MULTI-OMIC APPROACH

Abstract BRAFV600E is a key oncogenic mutation, detected in both pediatric low-grade gliomas and high-grade gliomas. Targeting the Ras-BRAF-MAPK pathway with BRAFV600E-specific inhibitors, such as Dabrafenib, is a therapeutic strategy increasingly used in clinical practice. However, therapy failure often emerges in patients, and the mechanisms driving drug resistance remain elusive. We apply a multi-omic approach to understand the evolution of BRAFV600E glioma cell lines and ex-vivo patient-derived cultures under BRAF inhibition. To recapitulate the clinical situation and decipher mechanisms of acquired resistance, we chronically expose BRAFV600E-positive glioma cells to a sublethal dose of Dabrafenib (IC20) and generate drug-resistant cell lines. We then profile the kinome (Pamgene microarray) and the transcriptome (bulk RNAseq) of resistant versus wildtype cells: in addition to reactivation of the Ras-BRAF-MAPK pathway, we observe increased activation and expression of PDGFRB and Src family kinases (SFK) in the Dabrafenib-resistant phenotype. To identify and track the longitudinal evolution of drug-resistant states, we further assess the evolution of glioma cell subpopulations under therapeutic pressure and profile consecutively collected samples (day 0; day 2; day 10; day 30 of treatment; and 10 days post-treatment) using single-cell transcriptomics (10x Chromium system, 10x Genomics). By profiling BRAFV600E glioma cells pre-, on- and post-treatment, we find that therapeutic pressure induces changes in cell subpopulations from early stages, and these changes are selected over time. Consistent with our kinome and bulk RNAseq findings, we confirm the role of PDGFRB in driving Dabrafenib resistance in a subset of cells. Furthermore, when off-treatment, the cells recapitulate the untreated status, indicating that resistance is a temporary, drug-dependent, phenomenon relying on the kinome rewiring. By dissecting the pro-survival role of the PDGFR/SFK axis upon Dabrafenib treatment, we will provide valuable mechanistic and therapeutic insights into targeted therapies for children with BRAFV600E gliomas.

Claudin-4-adhesion signaling drives breast cancer metabolism and progression via liver X receptor β

BackgroundCell adhesion is indispensable for appropriate tissue architecture and function in multicellular organisms. Besides maintaining tissue integrity, cell adhesion molecules, including tight-junction proteins claudins (CLDNs), exhibit the signaling abilities to control a variety of physiological and pathological processes. However, it is still fragmentary how cell adhesion signaling accesses the nucleus and regulates gene expression.MethodsBy generating a number of knockout and rescued human breast cell lines and comparing their phenotypes, we determined whether and how CLDN4 affected breast cancer progression in vitro and in vivo. We also identified by RNA sequencing downstream genes whose expression was altered by CLDN4-adhesion signaling. Additionally, we analyzed by RT-qPCR the CLDN4-regulating genes by using a series of knockout and add-back cell lines. Moreover, by immunohistochemistry and semi-quantification, we verified the clinicopathological significance of CLDN4 and the nuclear receptor LXRβ (liver X receptor β) expression in breast cancer tissues from 187 patients.ResultsWe uncovered that the CLDN4-adhesion signaling accelerated breast cancer metabolism and progression via LXRβ. The second extracellular domain and the carboxy-terminal Y197 of CLDN4 were required to activate Src-family kinases (SFKs) and the downstream AKT in breast cancer cells to promote their proliferation. Knockout and rescue experiments revealed that the CLDN4 signaling targets the AKT phosphorylation site S432 in LXRβ, leading to enhanced cell proliferation, migration, and tumor growth, as well as cholesterol homeostasis and fatty acid metabolism, in breast cancer cells. In addition, RT-qPCR analysis showed the CLDN4-regulated genes are classified into at least six groups according to distinct LXRβ- and LXRβS432-dependence. Furthermore, among triple-negative breast cancer subjects, the "CLDN4-high/LXRβ-high" and "CLDN4-low and/or LXRβ-low" groups appeared to exhibit poor outcomes and relatively favorable prognoses, respectively.ConclusionsThe identification of this machinery highlights a link between cell adhesion and transcription factor signalings to promote metabolic and progressive processes of malignant tumors and possibly to coordinate diverse physiological and pathological events.

Abstract 4876: A systems pharmacology approach to discover synergistic targeted therapy combinations

Abstract The discovery of oncogenic signal transduction in cancer has enabled the development and clinical use of dozens of kinase-directed targeted therapies, but patient response is often transient and resistance invariably develops. In the setting of BRAF-mutant melanoma, for instance, a majority of patients relapse within one year of initiating targeted therapy. Combination therapies blocking compensatory signaling pathways provide a strategy for boosting treatment efficacy and delaying resistance. To find synergistic kinase inhibitor combinations, we used a multimodal systems pharmacology approach to quantify the early adaptive signaling and gene regulatory dynamics in patient-derived BRAFV600E-mutant melanoma cells under oncogene inhibition. Cells were treated with vemurafenib or vehicle and sampled longitudinally at timepoints spanning minutes to days. To measure signaling dynamics, thousands of protein phosphorylation sites covering commonly altered signaling axes in cancer were motif-enriched by immunoprecipitation, barcoded with isobaric tandem mass tags (TMT), and quantified by high-resolution mass spectrometry (MS). The resulting time-resolved phosphoproteomics data showed rapid (<15 minutes) and potent (>8-fold) downregulation of ERK1/2, confirming strong BRAF inhibition. Statistical integration of signaling measurements with time-series RNA-seq data collected at the same timepoints enabled reconstruction of multi-scale regulatory networks governing the adaptive response to oncogene inhibition. In particular, we observed early induction of SRC-family kinase (SFK) signaling and a broad cytoskeletal signaling module, implicating a compensatory prosurvival signaling program. The induction intensified over the 3-day treatment window and was fully reversed following a 6-day drug holiday, suggesting a reversible nature of drug adaptation. Immunoblotting confirmed sustained loss of ERK1/2 activity and concomitant elevated SFK phosphorylation following drug treatment. Accumulation of reactive oxygen species, a known activator of SFKs, strongly correlated with SFK activity, and both were ablated following treatment with the antioxidant precursor N-acetylcysteine. This adaptive response predicted a significant degree of synergy between vemurafenib and the pan-SFK inhibitor dasatinib, which was validated in a panel of patient-derived melanoma cell lines and in melanoma xenograft mouse models. Targeted MS of phospho-ERK1/2 and canonical SFK substrates, including CTTN, PXN, and PAG1, validated the mechanism of action of this combination. Complementary data from patient-derived non small-cell lung cancer (NSCLC) cells under tyrosine kinase inhibitor treatment demonstrates the generality of our integrative approach, and supports the notion that SFK activation may be a hallmark response to oncogenic RTK-RAS-ERK pathway inhibition. Citation Format: Cameron T. Flower, Chunmei Liu, James R. Heath, Wei Wei, Forest M. White. A systems pharmacology approach to discover synergistic targeted therapy combinations. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 4876.

Evidence That Binding of Cyclic GMP to the Extracellular Domain of NKA (Sodium-Potassium ATPase) Mediates Natriuresis.

Extracellular renal interstitial guanosine cyclic 3',5'-monophosphate (cGMP) inhibits renal proximal tubule (RPT) sodium (Na+) reabsorption via Src (Src family kinase) activation. Through which target extracellular cGMP acts to induce natriuresis is unknown. We hypothesized that cGMP binds to the extracellular α1-subunit of NKA (sodium-potassium ATPase) on RPT basolateral membranes to inhibit Na+ transport similar to ouabain-a cardiotonic steroid. Urine Na+ excretion was measured in uninephrectomized 12-week-old female Sprague-Dawley rats that received renal interstitial infusions of vehicle (5% dextrose in water), cGMP (18, 36, and 72 μg/kg per minute; 30 minutes each), or cGMP+rostafuroxin (12 ng/kg per minute) or were subjected to pressure-natriuresis±rostafuroxin infusion. Rostafuroxin is a digitoxigenin derivative that displaces ouabain from NKA. Renal interstitial cGMP and raised renal perfusion pressure induced natriuresis and increased phosphorylated SrcTyr416 and Erk 1/2 (extracellular signal-regulated protein kinase 1/2)Thr202/Tyr204; these responses were abolished with rostafuroxin coinfusion. To assess cGMP binding to NKA, we performed competitive binding studies with isolated rat RPTs using bodipy-ouabain (2 μM)+cGMP (10 µM) or rostafuroxin (10 µM) and 8-biotin-11-cGMP (2 μM)+ouabain (10 μM) or rostafuroxin (10 µM). cGMP or rostafuroxin reduced bodipy-ouabain fluorescence intensity, and ouabain or rostafuroxin reduced 8-biotin-11-cGMP staining. We cross-linked isolated rat RPTs with 4-N3-PET-8-biotin-11-cGMP (2 μM); 8-N3-6-biotin-10-cAMP served as negative control. Precipitation with streptavidin beads followed by immunoblot analysis showed that RPTs after cross-linking with 4-N3-PET-8-biotin-11-cGMP exhibited a significantly stronger signal for NKA than non-cross-linked samples and cross-linked or non-cross-linked 8-N3-6-biotin-10-cAMP RPTs. Ouabain (10 μM) reduced NKA in cross-linked 4-N3-PET-8-biotin-11-cGMP RPTs confirming fluorescence staining. 4-N3-PET-8-biotin-11-cGMP cross-linked samples were separated by SDS gel electrophoresis and slices corresponding to NKA molecular weight excised and processed for mass spectrometry. NKA was the second most abundant protein with 50 unique NKA peptides covering 47% of amino acids in NKA. Molecular modeling demonstrated a potential cGMP docking site in the ouabain-binding pocket of NKA. cGMP can bind to NKA and thereby mediate natriuresis.

Chlamydia trachomatis induces the transcriptional activity of host YAP in a Hippo-independent fashion.

The obligate intracellular pathogen Chlamydia trachomatis is the causative agent of the most common bacterial sexually transmitted disease worldwide. While the host response to infection by this pathogen has been well characterized, it remains unclear to what extent host gene expression during infection is the product of Chlamydia-directed modulation of host transcription factors. To identify transcription factors potentially modulated by Chlamydia during infection, we infected immortalized endocervical epithelial cells (End1/E6E7) with the anogenital C. trachomatis serovar L2, harvesting polyadenylated RNA for bulk RNA-sequencing. Subsequent experiments elucidating the mechanism of infection-mediated YAP activation assayed YAP target gene expression via qRT-PCR, YAP nuclear translocation via quantitative immunofluorescence, and YAP phosphorylation via Western blotting. RNA sequencing of Chlamydia-infected endocervical epithelial cells revealed gene expression consistent with activity of YAP, a transcriptional coactivator implicated in cell proliferation, wound healing, and fibrosis. After confirming induction of YAP target genes during infection, we observed an infection-dependent increase in YAP nuclear translocation sensitive to inhibition of bacterial protein synthesis. While Hippo-mediated phosphoinhibition of YAP at S127 was unaffected by C. trachomatis infection, Hippo-independent phosphorylation at Y357 was increased. Infection did not enhance nuclear translocation of Y357F mutant YAP, illustrating a requirement for phosphorylation at this residue. Pharmacological inhibition of host Src-family kinase activity attenuated YAP Y357 phosphorylation, but not nuclear translocation - which was instead sensitive to inhibition of Abl. Our results define a transcriptome-altering mechanism of pathogen-directed YAP activation that bypasses canonical inhibition by the Hippo kinase cascade, with a potential link to chlamydial fibrosis and other advanced disease sequelae. Additional study is required to determine the specific role of infection-associated Y357 phosphorylation and Abl activity in chlamydial induction of YAP.

Differences in Treatment Response in Bronchial Epithelial Cells from Idiopathic Pulmonary Fibrosis (IPF) Patients: A First Step towards Personalized Medicine?

Idiopathic pulmonary fibrosis (IPF) has a detrimental prognosis despite antifibrotic therapies to which individual responses vary. IPF pathology is associated with oxidative stress, inflammation and increased activation of SRC family kinases (SFK). This pilot study evaluates individual responses to pirfenidone, nintedanib and SFK inhibitor saracatinib, markers of redox homeostasis, fibrosis and inflammation, in IPF-derived human bronchial epithelial (HBE) cells. Differentiated HBE cells from patients with and without IPF were analyzed for potential alterations in redox and profibrotic genes and pro-inflammatory cytokine secretion. Additionally, the effects of pirfenidone, nintedanib and saracatinib on these markers were determined. HBE cells were differentiated into a bronchial epithelium containing ciliated epithelial, basal, goblet and club cells. NOX4 expression was increased in IPF-derived HBE cells but differed on an individual level. In patients with higher NOX4 expression, pirfenidone induced antioxidant gene expression. All drugs significantly decreased NOX4 expression. IL-6 (p = 0.09) and IL-8 secretion (p = 0.014) were increased in IPF-derived HBE cells and significantly reduced by saracatinib. Finally, saracatinib significantly decreased TGF-β gene expression. Our results indicate that treatment responsiveness varies between IPF patients in relation to their oxidative and inflammatory status. Interestingly, saracatinib tends to be more effective in IPF than standard antifibrotic drugs.

Regulation, targets and functions of CHK.

Src family kinases (SFKs) play pivotal roles in multiple signaling pathways (Yeatman, 2004). SFK activity is inhibited by phosphorylation at its C-terminal tyrosine, by CSK (C-terminal Src kinase) and CHK (CSK-homologous kinase). CHK expression is restricted to normal hematopoietic cells, brain, and colon tissues. Downregulation of CHK in brain and colon tumors contributes to tumorigenicity in these tissues. CHK does not phosphorylate Src efficiently, however, in contrast to CSK, CHK inhibits Src kinase activity allosterically. Although the functions of CHK are still largely unknown, potential substrates of CHK including β-synuclein, α-tubulin, α-spectrin, 14-3-3, and Hsp90 have been identified. CHK is regulated epigenetically via promoter methylation. As the unknown roles of CHK are beginning to be revealed, current knowledge of regulation, molecular targets and functions of CHK is summarized, and important topics for future CHK research are discussed.

Src Family Kinases Facilitate the Crosstalk between CGRP and Cytokines in Sensitizing Trigeminal Ganglion via Transmitting CGRP Receptor/PKA Pathway.

The communication between calcitonin gene-related peptide (CGRP) and cytokines plays a prominent role in maintaining trigeminal ganglion (TG) and trigeminovascular sensitization. However, the underlying regulatory mechanism is elusive. In this study, we explored the hypothesis that Src family kinases (SFKs) activity facilitates the crosstalk between CGRP and cytokines in sensitizing TG. Mouse TG tissue culture was performed to study CGRP release by enzyme-linked immunosorbent assay, cytokine release by multiplex assay, cytokine gene expression by quantitative polymerase chain reaction, and phosphorylated SFKs level by western blot. The results demonstrated that a SFKs activator, pYEEI (YGRKKRRQRRREPQY(PO3H2)EEIPIYL) alone, did not alter CGRP release or the inflammatory cytokine interleukin-1β (IL-1β) gene expression in the mouse TG. In contrast, a SFKs inhibitor, saracatinib, restored CGRP release, the inflammatory cytokines IL-1β, C-X-C motif ligand 1, C-C motif ligand 2 (CCL2) release, and IL-1β, CCL2 gene expression when the mouse TG was pre-sensitized with hydrogen peroxide and CGRP respectively. Consistently with this, the phosphorylated SFKs level was increased by both hydrogen peroxide and CGRP in the mouse TG, which was reduced by a CGRP receptor inhibitor BIBN4096 and a protein kinase A (PKA) inhibitor PKI (14-22) Amide. The present study demonstrates that SFKs activity plays a pivotal role in facilitating the crosstalk between CGRP and cytokines by transmitting CGRP receptor/PKA signaling to potentiate TG sensitization and ultimately trigeminovascular sensitization.