Changes In Phosphorylation Research Articles

Abstract C053: Multi-omics guided design of lymphoid organoids to study cooperative cell signaling in lymphomas

Abstract Our collaborative research (Cell1) used 1001 patients of diffuse large B-cell lymphoma (DLBCL) to show that genetic alterations interact with clinical factors to impact overall survival (OS) in DLBCL. B cell receptor (BCR) signaling-driven activated B cell-like (ABC) DLBCL subtypes is the most chemo-resistant subtype to the frontline therapy R-CHOP, with 40% of patients experiencing no response or relapse. We have systematically examined disparities in lymphoma outcomes and identified African-American (AA) patients as a poor-risk population in DLBCL, with a diagnosis age 10 years younger than other racial groups. We found that AA-DLBCL patients experienced inferior 5-year OS compared to non-Hispanic whites (38% vs 46%)2,3. We performed the first-ever characterization of genetic alterations among AA-DLBCL patients and demonstrated distinct mutation patterns across DLBCL arising in AA ancestry groups4. While some of the most frequent genetic mutations in AA-DLBCL are related to chromatin and epigenetics, few studies have investigated racial disparities in patients with aggressive ABC DLBCLs. The mechanisms that determine this racial inequality are unknown but may lie in the particular spectrum of mutations that act in concert with intricate survival signals imparted by the lymph node tumor microenvironment (Ly-TME). The pathogenesis of ABC DLBCLs has been linked to constitutive activation of and cooperation between the BCR and Toll-like receptor (TLR) pathways. These pathways are emerging as novel therapeutic targets, with a particular interest in MALT1 (mucosa-associated lymphoid tissue translocation protein 1). However, the genetic heterogeneity of ABC DLBCLs and the cooperative signaling mediated by the Ly-TME have led to discrepancies in drug responses. As a first step towards understanding Ly-TME in DLBCLs (Nature Materials5), we used DLBCL patient samples from multiple cohorts, covering >1100 samples and imaging analysis to determine immune and ECM cues in the lymphoid TME. These samples were not classified into racial categories. We designed a biomaterials-based modular lymphoma organoid that presents specific integrin ligands at controlled densities to DLBCLs. Human ABC-DLBCLs responded to MALT1 inhibition in an integrin-ligand-dependent manner. TLR activation with TLR agonists rescued cells from MALT1 inhibitors, possibly due to increased MALT1 expression. The inclusion of T-cell signals increased MALT1 expression and rescued cells from MALT1 inhibitor-mediated killing in cell lines and human PDX organoids. Changes in RNA-seq and phosphorylation in the TLR & BCR pathways were used to inform effective combination therapy approaches with MALT1 inhibitors in vitro and in vivo5. Ongoing work is determining the racial disparities in AA lymphomas through complex interactions between Ly-TME and DLBCLs.

Ultra High‐Plex Spatial Proteogenomic Investigation of Giant Cell Glioblastoma Multiforme Immune Infiltrates Reveals Distinct Protein and RNA Expression Profiles

AbstractBackgroundThe advancement of spatially resolved, multiplex proteomic and transcriptomic technologies has revolutionized and redefined the approaches to complex biological questions pertaining to tissue heterogeneity, tumor microenvironments, cellular interactions, cellular diversity, and therapeutic response. Most spatial technologies yield single analyte proteomic or transcriptomic datasets from separate formalin‐fixed paraffin‐embedded (FFPE) tissues sections. Multiple studies have demonstrated a poor correlation between RNA expression and protein abundance owing to transcriptional and translational regulation, target turnover, and post‐translational protein modifications. Therefore, a workflow that accurately measures RNA and protein simultaneously within a single tissue section with distinct spatial context is critical to a more complete biological understanding of cellular interactions and activities. Such multimodal omic datasets of protein and DNA or RNA have been termed “spatial proteogenomics”.MethodHere we present a novel spatial proteogenomic (SPG) assay on the GeoMx® Digital Spatial Profiler platform with NGS readout that enables ultra high‐plex digital quantitation of proteins (147‐plex) and RNA (whole transcriptome,>18,000‐plex) from a single FFPE sample. We demonstrated high concordance, R>0.85, and minor change in sensitivity (<11%) between the SPG assay and the single analyte GeoMx Whole Transcriptome Atlas and GeoMx NGS Protein assays. We used the SPG assay to interrogate 23 different glioblastoma multiforme samples across 4 pathologies.ResultsWe observed clustering of both RNA and protein based on cancer pathology and anatomic location. The in‐depth investigation of giant cell glioblastoma multiforme (gcGBM) revealed distinct protein and RNA expression profiles compared to that of glioblastoma multiforme (GBM). Spatial proteogenomics allowed simultaneous interrogation of critical protein post‐translational modifications alongside whole transcriptomic profiles within the same distinct cellular neighborhoods.ConclusionsWithin our dataset, we observed >2‐fold higher protein expression levels of phospho‐GSK3β (Ser9) in gcGBM compared to GBM. Inactivation of GSK3β through phosphorylation has been shown to enhance proliferation of GBM cells. We also observed differential protein expression phosphorylated Tau variants. Phospho‐Thr231 Tau was >2‐fold higher in GBM compared to gcGBM. Associated with neurodegenerative Alzheimer’s disease, changes in Tau expression and phosphorylation have also been observed in glioblastoma. Our study exemplifies the utility of the SPG assay in expanding our understanding of glioblastoma multiforme molecular pathology.

APOEε4 potentiates the effects of Aβ pathology on the deposition of neurofibrillary tangles via tau phosphorylation

AbstractBackgroundThe mechanisms by which the apolipoprotein E ε4 (APOEε4) allele influences Alzheimer’s disease (AD) pathophysiological progression are poorly understood. Here, we tested the association of APOEε4 carriership and amyloid‐β (Aβ) burden with longitudinal tau pathology progression.MethodWe studied 104 individuals across the aging and AD clinical spectrum from the McGill TRIAD cohort. Study participants underwent clinical assessments, APOE genotyping, magnetic resonance imaging, positron emission tomography (PET) for Aβ ([18F]AZD4694) and tau ([18F]MK6240) at baseline, as well as an additional follow‐up tau‐PET scan (mean follow‐up, 2.4 years). We further assessed longitudinal changes in tau phosphorylation (plasma phosphorylated tau at threonine 217 [p‐tau217+]), brain atrophy (gray matter density), and clinical function (clinical dementia rating scale sum of boxes).ResultWe found that APOEε4 carriership potentiates Aβ effects on longitudinal tau tangle accumulation over two years (Figure 1). Interestingly, the APOEε4‐potentiated Aβ effects on tangles were mediated by longitudinal plasma p‐tau217+ increase (Figure 2). In addition, this longitudinal tau accumulation as measured by PET was accompanied by brain atrophy and clinical decline during the follow‐up period (Figure 3).ConclusionOur results support a model in which the APOEε4 allele plays a key role in Aβ downstream effects on the aggregation of phosphorylated tau in the form of neurofibrillary tangles in the living human brain, which is a key factor in the development of dementia. These observations have important implications for the design of future trials by suggesting that the combination of therapies targeting both ApoeE4 and Aβ pathology might have the potential to synergistically halt tau progression in AD.

Synaptic vs Extrasynaptic NMDA receptors distribution in Alzheimer’s human brain

AbstractBackgroundThe malfunction of NMDA receptors (NMDARs) is believed to play a key role in Alzheimer’s Disease (AD), the most prevalent type of dementia. NMDARs are activated in different ways: activation at synapses leads to synaptic plasticity, while activation outside of synapses causes excitotoxicity, harm to mitochondria, and cell death. The objective of our research is to better understand the distribution of NMDARs in the brain of individuals with AD, specifically focusing on the distinction between synaptically and extrasynaptically activated NMDARs.MethodOur research involved separating the synaptic and extrasynaptic membranes from the frontal cortex of both Alzheimer’s Disease (AD) patients and individuals without dementia. We examined the quantity of NMDAR subunits GluN1, GluN2B, and GluN2A. Additionally, we investigated the phosphorylation of GluN2B at Y1472 and Y1336, which are linked to synaptic and extrasynaptic membranes, respectively. To achieve our goal, we employed two distinct mouse models of AD: TAUP301S and APPswe/PS1dE9.ResultOur findings revealed that in comparison to non‐demented individuals, the levels of GluN1, GluN2B, GluN2A, and pY1472‐GluN2B at synaptic membranes were lower in Alzheimer’s Disease (AD) patients. At extrasynaptic membranes, we observed an elevation in GluN2B levels and a decline in Y1336‐GluN2B phosphorylation. We also found that the changes in GluN2B phosphorylation were consistent with the APPswe/PS1dE9 mouse models of AD.ConclusionOur research discovered that levels of GluN1, GluN2B, GluN2A, and pY1472‐GluN2B were decreased at the synaptic membranes of Alzheimer’s Disease patients compared to individuals without dementia. Additionally, an increase of GluN2B levels and a decrease in Y1336‐GluN2B phosphorylation were seen at extrasynaptic membranes. Furthermore, the alterations in GluN2B phosphorylation were also mirrored in the APPswe/PS1dE9 mouse models for AD.

Influence of Insulin Resistance and Metabolic Dysregulation on Synaptic Protein Profile in SH‐SY5Y Neuroblastoma Cells

AbstractBackgroundBrain insulin resistance and metabolic dysregulation are significantly associated with Alzheimer’s disease development[1,2]. Metformin is the most prescribed type‐2‐diabetic drug and has been used to treat peripheral insulin resistance (IR) driven metabolic dysfunction through the activation of AMP‐kinase (AMPK)[3,4], but whether metformin is effective in improving brain metabolism is unknown. We have previously shown that chronic AMPK activation negatively impacts neuronal morphology and impairs synaptic protein content in healthy neurons[5]. However, whether AMPK activation under an IR state will exacerbate neuronal impairments is not known. Thus, we examined the effect of metformin‐driven AMPK activation on SH‐SY5Y cells with and without high‐insulin induced IR.MethodsRetinoic acid‐differentiated SH‐SY5Y neuroblastoma cells were treated for 48h with either 1) vehicle (Ctl), 2) 100 M insulin (INS, mimicking hyperinsulinemia), 3) 2 mM metformin (MET), or 4) a combination of INS/MET.ResultsINS‐treated cells demonstrated higher Akt S473, mTOR S2448, and p70s6k T389 phosphorylation, indicative of hyperactive mTORC1 signaling associated with brain IR. INS cells had lower insulin receptor‐ (IR‐) content and higher IRS‐1 S636 phosphorylation, further demonstrating insulin signaling abnormalities. MET increased AMPK T172 phosphorylation, and increased phosphorylation of its downstream effectors ULK S555 and raptor S792, which, collectively, is indicative of mTORC1 inhibition. These phosphorylation increases occurred alongside higher IRS‐1 and insulin receptor‐ content and reduced IR‐ content. INS/MET‐treated cells produced no change in AMPK T172 or raptor S792 phosphorylation but produced increased ULK S555 phosphorylation. INS/MET did not recover increased Akt S473, mTOR S2448, p70s6k T389, or IRS‐1 S636 phosphorlyation nor did it recover IR‐ content. No differences were seen between groups for pre‐/post‐synaptic protein content. INS and MET treatments showed reduced NeuN and synaptophysin content. INS/MET treatment showed reductions in synaptophysin. Morphologically, cell size & area, and neurite length & area all increased with INS and MET treatments, however, these increases were absent with the INS/MET treatment.ConclusionThese findings indicate that IR and MET impact neuronal morphology and reduce the development of mature neurons. These impairments are likely driven through Akt and AMPK signaling seperately and suggest that MET is not effective in treating IR‐driven impairments in the brian.

APOEε4 potentiates the effects of Aβ pathology on the deposition of neurofibrillary tangles via tau phosphorylation

AbstractBackgroundThe mechanisms by which the apolipoprotein E e4 (APOEe4) allele influences Alzheimer’s disease (AD) pathophysiological progression are poorly understood. Here, we tested the association of APOEe4 carriership and amyloid‐ß (Aß) burden with longitudinal tau pathology progression.MethodWe studied 104 individuals across the aging and AD clinical spectrum from the McGill TRIAD cohort. Study participants underwent clinical assessments, APOE genotyping, magnetic resonance imaging, positron emission tomography (PET) for Aß ([18F]AZD4694) and tau ([18F]MK6240) at baseline, as well as an additional follow‐up tau‐PET scan (mean follow‐up, 2.4 years). We further assessed longitudinal changes in tau phosphorylation (plasma phosphorylated tau at threonine 217 [p‐tau217+]), brain atrophy (gray matter density), and clinical function (clinical dementia rating scale sum of boxes).ResultWe found that APOEe4 carriership potentiates Aß effects on longitudinal tau tangle accumulation over two years (Figure 1). Interestingly, the APOEe4‐potentiated Aß effects on tangles were mediated by longitudinal plasma p‐tau217+ increase (Figure 2). In addition, this longitudinal tau accumulation as measured by PET was accompanied by brain atrophy and clinical decline during the follow‐up period (Figure 3).ConclusionOur results support a model in which the APOEe4 allele plays a key role in Aß downstream effects on the aggregation of phosphorylated tau in the form of neurofibrillary tangles in the living human brain, which is a key factor in the development of dementia. These observations have important implications for the design of future trials by suggesting that the combination of therapies targeting both ApoeE4 and Aß pathology might have the potential to synergistically halt tau progression in AD.

Integrated proteomic analysis reveals interactions between phosphorylation and ubiquitination in rose response to Botrytis infection.

As two of the most abundant post-translational modifications, phosphorylation and ubiquitination play a significant role in modulating plant-pathogen interactions and increasing evidence indicates their crosstalk in plant immunity. Rose (Rosa sp.) is one of the most important ornamental plants and can be seriously infected by Botrytis cinerea. Here, integrated proteomics analysis was performed to detect global proteome, phosphorylation, and ubiquitination changes in rose upon B. cinerea infection and investigate the possible phosphorylation and ubiquitination crosstalk. A total of 6165 proteins, 11 774 phosphorylation and 10 582 ubiquitination sites, and 77 phosphorylation and 13 ubiquitination motifs were identified. Botrytis cinerea infection resulted in 169 up-regulated and 122 down-regulated proteins, 291 up-regulated and 404 down-regulated phosphorylation sites, and 250 up-regulated and 634 down-regulated ubiquitination sites. There were 12 up-regulated PR10 proteins and half of them also showed reduced ubiquitination. A lot of kinases probably involved in plant pattern-triggered immunity signaling were up-regulated phosphoproteins. Noticeably, numerous kinases and ubiquitination-related proteins also showed a significant change in ubiquitination and phosphorylation, respectively. A cross-comparison of phosphoproteome and ubiquitylome indicated that both of two post-translational modifications of 104 proteins were dynamically regulated, and many putative pattern-triggered immunity signaling components in the plant plasma membrane were co-regulated. Moreover, five selected proteins, including four PR10 proteins and a plasma membrane aquaporin, were proven to be involved in rose resistance to B. cinerea. Our study provides insights into the molecular mechanisms underlying rose resistance to B. cinerea and also increases the database of phosphorylation and ubiquitination sites in plants.

Abstract 17307: Pre-Existing Endoplasmic Reticulum (ER) Stress Inhibits Sars-Co-V2 Entry Into Host Cells Through Modulation of AKT Signaling and Unfolded Protein Response (UPR)

Introduction: It is well known that SARS-Co-V2 infection can induce ER stress-associated activation of unfolded protein response (UPR) in various host cells which may contribute to the pathogenesis of COVID-19. However, the interplay between SARS-Co-V2 infection and UPR signaling in pre-existing ER stress associated pathological conditions has not been well elucidated. Hypothesis: We hypothesized that modulation of a pre-existing ER stress in host cells could attenuate susceptibility to SARS-CoV-2 infection by activating a range of cellular defense. Methods: Increasing concentrations of Tunicamycin (Tm) and Thapsigargin (Tg) have been used to induce ER stress in Huh-7 cells. After 6h treatment, cells were infected with SARS-CoV-2 pseudotyped particles (SARS-CoV-2pp) for 48 p.i. SARS-Co-V2-2pp entry was measured using Bright Glo TM luciferase assay. Cell viability was measured by cell titer Glo ® luminescent cell viability assay. The mRNA and protein expression of UPR markers were evaluated using RT-qPCR and Western blot methods. Results: Tm (5 μg/ml) and Tg (1 μM) efficiently inhibited SARS-CoV-2pp entry into cells without any cytotoxic effect. Chemical ER stress-induced inhibition of SARS-CoV-2pp entry was associated with significant reduction of ACE2 expression in Tg-infected cells but not with Tm. Strikingly, Tm and Tg revealed differential effects in modulating the expression of ER stress genes in infected cells. Both Tm and Tg significantly reduced the expression of stress-inducible ER chaperone GRP78/BIP in infected cells. In contrast, the IRE1-XBP1s and PERK-eIF2α-ATF4-CHOP signaling pathways were downregulated in Tg-infected cells only. Additionally, insulin-mediated glucose uptake, phosphorylation of Ser 307 IRS-1 and downstream p-AKT were enhanced in Tg-infected cells without any change in ERK phosphorylation. Conclusions: These findings suggest that pre-existing ER stress could modulate a specific UPR response in infected cells capable of counteracting the stress-inducible elements signaling, thereby depriving SARS-Co-V2 of essential components for their entry and replication. Pharmacological manipulation of ER stress in host cells might provide new therapeutic strategies to alleviate SARS-CoV-2 infection.

Sustained CREB phosphorylation by lipid-peptide liquid crystalline nanoassemblies

Cyclic-AMP-response element-binding protein (CREB) is a leucine zipper class transcription factor that is activated through phosphorylation. Ample CREB phosphorylation is required for neurotrophin expression, which is of key importance for preventing and regenerating neurological disorders, including the sequelae of long COVID syndrome. Here we created lipid-peptide nanoassemblies with different liquid crystalline structural organizations (cubosomes, hexosomes, and vesicles) as innovative nanomedicine delivery systems of bioactive PUFA-plasmalogens (vinyl ether phospholipids with polyunsaturated fatty acid chains) and a neurotrophic pituitary adenylate cyclase-activating polypeptide (PACAP). Considering that plasmalogen deficiency is a potentially causative factor for neurodegeneration, we examined the impact of nanoassemblies type and incubation time in an in vitro Parkinson’s disease (PD) model as critical parameters for the induction of CREB phosphorylation. The determined kinetic changes in CREB, AKT, and ERK-protein phosphorylation reveal that non-lamellar PUFA-plasmalogen-loaded liquid crystalline lipid nanoparticles significantly prolong CREB activation in the neurodegeneration model, an effect unattainable with free drugs, and this effect can be further enhanced by the cell-penetrating peptide PACAP. Understanding the sustained CREB activation response to neurotrophic nanoassemblies might lead to more efficient use of nanomedicines in neuroregeneration.

Targeting Covalent and Non-Covalent Btki-Resistant CLL Using the Dual Irreversible/Reversible 4 th Generation BTK Inhibitor LP-168

Background: Treatment of chronic lymphocytic leukemia (CLL) has been transformed with targeted therapies including inhibitors of Bruton's tyrosine kinase (BTKi). Currently three covalent BTK inhibitors (cBTKi) are approved for CLL, but most patients eventually relapse, commonly through acquisition of the C481S BTK mutation (Woyach et al. 2014). Pirtobrutinib and nemtabrutinib are non-covalent BTK inhibitors (ncBTKi) developed to target and inhibit C481S mutant BTK. However novel secondary site mutations in BTK, namely T474I and L528W, have been found to confer resistance to both cBTKi and ncBTKi (Wang et al. 2022). Regardless of these mutations, BCR signaling remains intact, suggesting that inhibition of BTK maintains its therapeutic importance. LP-168 is a novel ultra-selective 4 th generation BTKi with an active warhead capable of covalent interaction with WT BTK or non-covalent binding when a BTK C481 mutation is present. Methods: To determine the target selectivity of LP-168 we screened the compound against 468 kinases in the scanMAX Kinase Assay. For validation of covalent binding, dialysis against WT BTK was performed. Primary CLL B cells were isolated by negative selection and treated with LP-168 for all experiments. BCR signaling alterations were assessed via immunoblot to observe changes in target protein phosphorylation. Changes in CLL migration towards CXCL12 or CXCL13 were interrogated by transwell migration. Cytotoxicity towards primary CLL cells was measured via Annexin V/PI flow cytometry with and without HS-5 stromal cell support. Experiments using TMD8 cells were performed following CRISPR modification to insert WT, C481S, or T474I BTK. In vivo survival studies were performed using both the Eμ-TCL1 and Eμ-MTCP1 mouse engraftment models. Results: LP-168 demonstrated selectivity towards BTK with roughly 700-fold selectivity for BTK vs its next off-target. In an enzymatic assay, LP-168 displayed nanomolar potency towards both WT BTK (IC 50=0.11 nM) and C481S BTK (IC 50=1.0 nM). Following 2-hour drugging, LP-168 inhibited BCR signaling in primary CLL B cells at increasing concentrations, demonstrated by reduced phosphorylation of BTK (92%, p<0.0001, n=8) and its immediate downstream target PLCγ2 (41%, p=0.0013, n=8). Migration capacity of primary CLL cells towards CXCL12 and CXCL14 was also found to decrease after 2-hours of treatment with LP-168 (52%, p<0.001, n=10 and 51%, p<0.001, n=10, respectively). After 48 hours of exposure, LP-168 was able to induce cytotoxicity of primary CLL cells alone and in co-culture with HS-5 stromal cells in a dose-dependent manner, with similar or improved efficacy to other BTKi. LP-168 decreased CLL cell production of CCL3 and CCL4 chemokines (CCL3: 93%, p<0.001, n=8; CCL4: 93%, p<0.001, n=8). LP-168 also demonstrated modest cytotoxicity towards TMD8 cells harboring either C481S BTK (24%, p=0.0008 , n=3) or T474I BTK (13%, p=0.0690, n=3), as well as decreased production of CCL3 (C481S: 95%, p<0.0001, n=3; T474I: 99%, p<0.0001, n=3) and CCL4 (C481S: 97%, p<0.0001, n=3; T474I: 98%, p<0.0001, n=3) (Figure 1A). Further, we observed inhibition of downstream BCR signaling in primary CLL B cells harboring C481S via immunoblotting and cytokine production. Finally, to evaluate the in vivo efficacy of LP-168 we tested this compound in both the Eµ-TCL1 and Eµ-MTCP1 models. We treated mice daily via oral gavage and found that 50 mg/kg of LP-168 significantly improved survival in the Eµ-TCL1 model when compared to vehicle (median 51 vs 44 days; p=0.0018) or ibrutinib at 50 mg/kg (median 51 vs 45 days; p=0.0098) and the Eµ-MTCP1 model when compared with vehicle (median 122 vs 62 days; p<0.0001) or ibrutinib at 50 mg/kg (median 122 vs 96 days; p=0.0162) (Figure 1B). Conclusions: Collectively, our data demonstrate that LP-168 is a potent and selective inhibitor of BTK with activity against CLL, even in the presence of mutations that mediate resistance to cBTKi and ncBTKi. These data support the continued preclinical and clinical investigation of LP-168, which is currently being studied in the phase 1 setting of CLL (NCT04775745) and NHL (NCT04993690).

The PKC-β Inhibitor MS-553 Displays Preclinical Efficacy in BTK Inhibitor Resistant Chronic Lymphocytic Leukemia

Background: Treatment of chronic lymphocytic leukemia (CLL) has been revolutionized by usage of targeted therapies against the B-cell receptor (BCR) signaling cascade, specifically Bruton's Tyrosine Kinase (BTK). However, resistance to BTK inhibition occurs through acquisition of various mutations in BTK or its immediate downstream signaling partner, PLCγ2. Clinical outcomes for these patients are poor, emphasizing the need for alternative therapeutics. In the presence of these mutations, BCR signaling remains intact, suggesting that targeting molecules downstream of BTK may be effective. Protein kinase C-β (PKCβ) is a downstream component of the BCR pathway that has been shown to be over-expressed in CLL. Moreover, PKCβ is essential to the development of CLL in the Eμ-TCL1 mouse model and its expression in stromal cells is required for the survival of leukemic B-cells (Holler et al. 2009). This suggests that signaling through PKCβ, in both CLL cells and cells in the microenvironment, is essential to promote leukemogenesis. MS-553 is a potent, ATP competitive, reversible inhibitor of multiple PKC isoforms including PKCβ, with the potential to overcome BTKi mediated resistance. Methods: Primary CLL cells were isolated by negative selection and treated with up to 5 μM MS-553. Cell viability was assessed via Annexin V/PI with and without HS-5 stromal cell co-culture. BCR and canonical WNT signaling changes were evaluated by change in target protein phosphorylation or total protein by immunoblot following drugging. Changes in downstream NFκB and WNT signaling were interrogated by RT-qPCR. Using genetically modified TMD8 cells that express only C481S or T474I mutated BTK, we tested MS-553's effect on cell-viability and BCR signaling. In vivo survival studies were performed using the Eμ-MTCP1 (Walker et al. 2021) adoptive transfer mouse model, an aggressive model of CLL, with subsequent randomization into vehicle or MS-553 treatment arms. Results: Following 72 hours of drugging, 5 μM MS-553 displayed modest cytotoxicity against primary CLL cells both alone (50%, p<0.0001, n=8) and in the presence of HS-5 stromal cells (25%, p=0.0008, n=8). In primary CLL cells harboring the C481S BTK mutation, we observed retained cytotoxicity following 72 hours of drugging (29%, p=0.00029, n=6). At 24 hours, 5 μM MS-553 inhibited downstream BCR and WNT signaling in primary CLL cells, demonstrated by reduced phosphorylation of PKC-β (43%, p=0.0024, n=15) and several of its downstream targets including pGSK3β (45%, p<0.0001, n=15), pIκBα (66%, p<0.0001, n=15), and pERK (17%, p=0.0301, n=15), and reduced total protein of β-Catenin (82%, p<0.0001, n=9), Cyclin D2 (71%, p=0.0011, n=9), and cMyc (85%, p<0.0001, n=9) compared to vehicle treated, stimulated samples. In both treatment naïve and C481S patient samples, mRNA expression of NFκB and WNT pathway targets BCL-2, OCT-2, BFL-1, MYC, CD40, Cyclin D1, and Cyclin D2 were all decreased after 48 hours of treatment with 1 or 5 μM MS-553. MS-553 also showed efficacy in the presence of BTK mutations: viability in TMD8 cells was reduced following 72-hour drugging with MS-553 regardless of BTK mutational status (WT: 22%, p=0.0006, n=3; C481S BTK: 18%, p=0.0032, n=3; T474I BTK: 19%, p=0.0015, n=3). Inflammatory cytokine production is inhibited by MS-553 through its ability to decrease CCL3 and CCL4 cytokine expression in WT and mutant BTK-harboring TMD8 cells (p<0.0001, n=3) (Figure 1A). To validate inhibition of BCR signaling in the C481S BTK setting, we treated primary CLL cells harboring this mutation and observed a reduction in both CCL3 and CCL4 production (61%, p=0.001229, n=5). As proof of concept, we examined MS-553's efficacy in vivo in the Eμ-MTCP1 mouse engraftment model, and found a significant reduction in disease progression and increase in survival (median 117 days) when compared to vehicle control (median 56 days, p=0.0003) (Figure 1B). Conclusion: Our data demonstrate the efficacy of the PKCβ inhibitor MS-553 in preclinical models of CLL including that with BTK mutations conferring BTKi resistance. These findings support continued preclinical and clinical investigation of MS-553 in CLL, and the phase 1 trial of this agent is currently underway (NCT03492125) (Blachly et al. 2022).

Doxorubicin-induced modulation of TGF-β signaling cascade in mouse fibroblasts: insights into cardiotoxicity mechanisms

Doxorubicin (DOX)-induced cardiotoxicity has been widely observed, yet the specific impact on cardiac fibroblasts is not fully understood. Additionally, the modulation of the transforming growth factor beta (TGF-β) signaling pathway by DOX remains to be fully elucidated. This study investigated DOX’s ability to modulate the expression of genes and proteins involved in the TGF-β signaling cascade in mouse fibroblasts from two sources by assessing the impact of DOX treatment on TGF-β inducible expression of pivotal genes and proteins within fibroblasts. Mouse embryonic fibroblasts (NIH3T3) and mouse primary cardiac fibroblasts (CFs) were treated with DOX in the presence of TGF-β1 to assess changes in protein levels by western blot and changes in mRNA levels by quantitative reverse transcriptase polymerase chain reaction (qRT-PCR). Our results revealed a dose-dependent reduction in cellular communication network factor 2 (CCN2) protein levels upon DOX treatment in both NIH3T3 and CFs, suggesting an antifibrotic activity by DOX in these fibroblasts. However, DOX only inhibited the TGF-β1 induced expression of COL1 in NIH3T3 cells but not in CFs. In addition, we observed that DOX treatment reduced the expression of BMP1 in NIH3T3 but not primary cardiac fibroblasts. No significant changes in SMAD2 protein expression and phosphorylation in either cells were observed after DOX treatment. Finally, DOX inhibited the expression of Atf4 gene and increased the expression of Cdkn1a, Id1, Id2, Runx1, Tgfb1, Inhba, Thbs1, Bmp1, and Stat1 genes in NIH3T3 cells but not CFs, indicating the potential for cell-specific responses to DOX and its modulation of the TGF-β signaling pathway.

Inhibition of HSP90 distinctively modulates the global phosphoproteome of Leishmania mexicana developmental stages.

In the unicellular parasites Leishmania spp., the etiological agents of leishmaniasis, a complex infectious disease that affects 98 countries in 5 continents, chemical inhibition of HSP90 protein leads to differentiation from promastigote to amastigote stage. Recent studies indicate potential role for protein phosphorylation in the life cycle control of Leishmania. Also, recent studies suggest a fundamentally important role of RNA-binding proteins (RBPs) in regulating the downstream effects of the HSP90 inhibition in Leishmania. Phosphorylation-dephosphorylation dynamics of RBPs in higher eukaryotes serves as an important on/off switch to regulate RNA processing and decay in response to extracellular signals and cell cycle check points. In the current study, using a combination of highly sensitive TMT labeling-based quantitative proteomic MS and robust phosphoproteome enrichment, we show for the first time that HSP90 inhibition distinctively modulates global protein phosphorylation landscapes in the different life cycle stages of Leishmania, shedding light into a crucial role of the posttranslational modification in the differentiation of the parasite under HSP90 inhibition stress. We measured changes in phosphorylation of many RBPs and signaling proteins including protein kinases upon HSP90 inhibition in the therapeutically relevant amastigote stage. This work provides insights into the importance of HSP90-mediated protein cross-talks and regulation of phosphorylation in Leishmania, thus significantly expanding our knowledge of the posttranslational modification in Leishmania biology.

Epigenetic Profiling of PTPN11 Mutant JMML Hematopoietic Stem and Progenitor Cells Reveals an Aberrant Histone Landscape.

Juvenile myelomonocytic leukemia (JMML) is a deadly pediatric leukemia driven by RAS pathway mutations, of which >35% are gain-of-function in PTPN11. Although DNA hypermethylation portends severe clinical phenotypes, the landscape of histone modifications and chromatin profiles in JMML patient cells have not been explored. Using global mass cytometry, Epigenetic Time of Flight (EpiTOF), we analyzed hematopoietic stem and progenitor cells (HSPCs) from five JMML patients with PTPN11 mutations. These data revealed statistically significant changes in histone methylation, phosphorylation, and acetylation marks that were unique to JMML HSPCs when compared with healthy controls. Consistent with these data, assay for transposase-accessible chromatin with sequencing (ATAC-seq) analysis revealed significant alterations in chromatin profiles at loci encoding post-translational modification enzymes, strongly suggesting their mis-regulated expression. Collectively, this study reveals histone modification pathways as an additional epigenetic abnormality in JMML patient HSPCs, thereby uncovering a new family of potential druggable targets for the treatment of JMML.

Proteomics, Transcriptomics, and Phosphoproteomics Reveal the Mechanism of Talaroconvolutin-A Suppressing Bladder Cancer via Blocking Cell Cycle and Triggering Ferroptosis

Talaroconvolutin-A (TalaA) is a compound from the endophytic fungus T. convolutispora of the Chinese herbal medicine Panax notoginseng. Whether TalaA exerts anticancer activity in bladder cancer remains unknown. Using CCK8 assay, EdU staining, crystal violet staining, flow cytometry, living/dead cell staining, and Western blotting, we studied the anticancer activity of TalaA in vitro. Moreover, we performed xenograft tumor implantation. The antitumor effects were evaluated through H&E and immunohistochemistry staining. Proteomics was conducted to detect changes in the protein profile; transcriptomics was performed to detect changes in mRNA abundance; phosphoproteomics was used to detect changes in protein phosphorylation. TalaA inhibited tumor cell proliferation, DNA replication, and colony formation in a dose-dependent manner in bladder cancer cells. The IC50 values of TalaA on SW780 and UM-UC-3 cells were 5.7 and 8.2 μM, respectively. TalaA (6.0 mg/kg) significantly repressed the growth of xenografted tumors and did not affect the body weight nor cause obvious hepatorenal toxicity. TalaA arrested the cell cycle by downregulating cyclinA2, cyclinB1, and AURKB and upregulating p21/CIP. TalaA also elevated intracellular reactive oxygen species and upregulated transferrin and heme oxygenase 1 to induce ferroptosis. Moreover, TalaA was able to bind to MAPKs (MAPK1, MAPK8, and MAPK14) to inhibit the phosphorylation of ∗SP∗ motif of transcription regulators. This study revealed that TalaA inhibited bladder cancer by arresting cell cycle to suppress proliferation and triggering ferroptosis to cause cell death. Conclusively, TalaA would be a potential candidate for treating bladder cancer by targeting MAPKs, suppressing the cell cycle, and inducing ferroptosis.

AXL receptor tyrosine kinase modulates gonadotropin-releasing hormone receptor signaling

BackgroundGonadotropin-releasing hormone (GnRH) receptors are essential for reproduction and are expressed in numerous urogenital, reproductive, and non-reproductive cancers. In addition to canonical G protein-coupled receptor signaling, GnRH receptors functionally interact with several receptor tyrosine kinases. AXL is a receptor tyrosine kinase expressed in numerous tissues as well as multiple tumors. Here we tested the hypothesis that AXL, along with its endogenous ligand Gas6, impacts GnRH receptor signaling.MethodsWe used clonal murine pituitary αT3-1 and LβT2 gonadotrope cell lines to examine the effect of AXL activation on GnRH receptor-dependent signaling outcomes. ELISA and immunofluorescence were used to observe AXL and GnRH receptor expression in αT3-1 and LβT2 cells, as well as in murine and human pituitary sections. We also used ELISA to measure changes in ERK phosphorylation, pro-MMP9 production, and release of LHβ. Digital droplet PCR was used to measure the abundance of Egr-1 transcripts. A transwell migration assay was used to measure αT3-1 and LβT2 migration responses to GnRH and AXL.ResultsWe observed AXL, along with the GnRH receptor, expression in αT3-1 and LβT2 gonadotrope cell lines, as well as in murine and human pituitary sections. Consistent with a potentiating role of AXL, Gas6 enhanced GnRH-dependent ERK phosphorylation in αT3-1 and LβT2 cells. Further, and consistent with enhanced post-transcriptional GnRH receptor responses, we found that Gas6 increased the abundance of Egr-1 transcripts. Suggesting functional significance, in LβT2 cells, Gas6/AXL signaling stimulated LHβ production and enhanced GnRH receptor-dependent generation of pro-MMP9 protein and promoted cell migration.ConclusionsAltogether, these data describe a novel role for AXL as a modulator of GnRH receptor signaling.5LYCavpivq5eiBQqEyV1BjVideo

Phosphoproteomic insight into the changes in structural proteins of muscle architecture associated with texture softening of grass carp (Ctenopharyngodon idella) fillets during chilling storage.

Texture is an important sensory attribute of fish affected by modifications of structural proteins in muscle architecture. To investigate the changes in protein phosphorylation during texture softening of fish, the proteins of grass carp muscle after chilling storage of 0day and 6days were compared by phosphoproteomics, and their association with texture was analyzed. Totally 1026 unique phosphopeptides on 656 phosphoproteins were identified as differential. They were mainly classified as intracellular myofibril and cytoskeleton, and extracellular matrix, of which the molecular function and biological process were binding into supramolecular assembly and myofilament contraction. The concomitant dephosphorylation of kinases and assembly regulators indicated dephosphorylation and disassembly tendency of sarcomeric architecture. Correlation analysis defined the relation between texture and dephosphorylation of myosin light chain, actin, collagen and cytoskeleton. This study revealed that protein phosphorylation may affect the texture of fish muscle through regulating sarcomeric assembly of structural proteins in muscle architecture.

New Insights into Radio-Resistance Mechanism Revealed by (Phospho)Proteome Analysis of Deinococcus Radiodurans after Heavy Ion Irradiation.

Deinococcus radiodurans (D. radiodurans) can tolerate various extreme environments including radiation. Protein phosphorylation plays an important role in radiation resistance mechanisms; however, there is currently a lack of systematic research on this topic in D. radiodurans. Based on label-free (phospho)proteomics, we explored the dynamic changes of D. radiodurans under various doses of heavy ion irradiation and at different time points. In total, 2359 proteins and 1110 high-confidence phosphosites were identified, of which 66% and 23% showed significant changes, respectively, with the majority being upregulated. The upregulated proteins at different states (different doses or time points) were distinct, indicating that the radio-resistance mechanism is dose- and stage-dependent. The protein phosphorylation level has a much higher upregulation than protein abundance, suggesting phosphorylation is more sensitive to irradiation. There were four distinct dynamic changing patterns of phosphorylation, most of which were inconsistent with protein levels. Further analysis revealed that pathways related to RNA metabolism and antioxidation were activated after irradiation, indicating their importance in radiation response. We also screened some key hub phosphoproteins and radiation-responsive kinases for further study. Overall, this study provides a landscape of the radiation-induced dynamic change of protein expression and phosphorylation, which provides a basis for subsequent functional and applied studies.

Phosphatases modified by LH signaling in ovarian follicles: testing their role in regulating the NPR2 guanylyl cyclase†.

In response to luteinizing hormone (LH), multiple proteins in rat and mouse granulosa cells are rapidly dephosphorylated, but the responsible phosphatases remain to be identified. Because the phosphorylation state of phosphatases can regulate their interaction with substrates, we searched for phosphatases that might function in LH signaling by using quantitative mass spectrometry. We identified all proteins in rat ovarian follicles whose phosphorylation state changed detectably in response to a 30-min exposure to LH, and within this list, identified protein phosphatases or phosphatase regulatory subunits that showed changes in phosphorylation. Phosphatases in the phosphoprotein phosphatase (PPP) family were of particular interest because of their requirement for dephosphorylating the natriuretic peptide receptor 2 (NPR2) guanylyl cyclase in the granulosa cells, which triggers oocyte meiotic resumption. Among the PPP family regulatory subunits, PPP1R12A and PPP2R5D showed the largest increases in phosphorylation, with 4-10 fold increases in signal intensity on several sites. Although follicles from mice in which these phosphorylations were prevented by serine-to-alanine mutations in either Ppp1r12a or Ppp2r5d showed normal LH-induced NPR2 dephosphorylation, these regulatory subunits and others could act redundantly to dephosphorylate NPR2. Our identification of phosphatases and other proteins whose phosphorylation state is rapidly modified by LH provides clues about multiple signaling pathways in ovarian follicles.

Extrapyramidal Side Effects with Chronic Atypical Antipsychotic Can Be Predicted by Labeling Pattern of FosB and phosphoThr34-DARPP-32 in Nucleus Accumbens.

Extrapyramidal side effects (EPS) can be induced by neuroleptics that regulate the expression of transcription factor FosB and dopaminergic mediator DARPP-32 in the striatum. However, the long-term neurobiological changes in striatal projection neurons resulting from a cumulative dosage of typical and atypical antipsychotics are poorly understood. The present study aimed to determine the differential and long-lasting changes in FosB distribution and DARPP-32 phosphorylation in the striatum and nucleus accumbens (NAc) associated with chronic antipsychotic-induced EPS. Male C57Bl/6J mice received daily injections of Olanzapine (Olz, 15 mg/kg), Clozapine (Clz, 20 mg/kg), or Haloperidol (Hal, 1 mg/kg), for a period of 11 weeks with a 4-day withdrawal period before the last dosage. Catalepsy for detection of EPS, along with open-field and rotarod tests, were assessed as behavioral correlates of motor responses. Additionally, FosB and phosphorylated-DARPP-32 immunohistochemistry were examined in striatal regions after treatment. All antipsychotics produced catalepsy and reduced open-field exploration, such as impaired rota-rod performance after Olz and Hal. The washout period was critical for Clz-induced side effects reduction. Both Olz and Clz increased FosB in NAc Shell-region, and phosphoThr34-DARPP-32 in NAc. Only Clz reduced phosphoThr75-DARPP-32 in the dorsal striatum and showed FosB/phosphoThr34-Darpp-32-ir in the NAc Core region. This study provides evidence that atypical antipsychotics such as Olz and Clz also give rise to EPS effects frequently associated with a cumulative dosage of typical neuroleptics such as Hal. Nevertheless, FosB/phosphoThr34-Darpp-32-ir in the NAc Core region is associated with hypokinetic movements inhibition.