Serine-threonine Research Articles

Abstract 2751: Functional assessment of STK11 C-terminal domain variants

Abstract STK11 mutations in KRAS-driven lung adenocarcinomas (LUADs) are associated with aggressive tumor phenotypes characterized by increased risk of metastasis and lower overall and progression free survival. STK11 is a serine-threonine kinase that is comprised of an N-terminal domain containing a nuclear localization signal, a kinase domain, and a C-terminal domain (CTD). In LUAD, STK11 loss of function impacts many aspects of coordinated cell motility and promotes alterations characteristic of metastasis including cell polarity and adhesion. Both kinase activity and subcellular localization are important for STK11 tumor suppressor function. Our objective was to develop a reliable method to identify pathogenic STK11 mutations. Using archival data from the University of Vermont Medical Center, we identified 28 STK11 missense variants that had not yet been functionally characterized. We evaluated the functional impact of these variants on STK11 kinase activity through detection of autophosphorylation using an in-vitro kinase assay, as well as their ability to induce p53 activity through a luciferase reporter. Of the variants studied, 5 were CTD mutations. These variants retained kinase activity and the ability to bind STK11’s partners STRAD-alpha and MO25 in co-immunoprecipitation assays. Although the CTD of STK11 has no catalytic activity, it is thought to be integral for translocation to the cytoplasm where it can colocalize with actin and the cell membrane. STK11 dependent regulation of directional migration requires both proper localization to the actin cytoskeleton and kinase activity. Moreover, it has been reported that the C-terminal polybasic motif of STK11 (aa403-426), consisting of 3 Lysine and 5 Arginine residues is key for its localization and activation of AMPK at the plasma membrane. Therefore, when ascertaining functional status of STK11 in vivo, it is important to consider kinase independent STK11 functions. In LUAD cell lines, we show through immunofluorescent microscopy that expression of a truncated construct encoding STK11 with a CTD deletion results in nuclear sequestration. Additionally, point mutants in 3 of the polybasic motif residues (R409W, K416E and K423E) retain kinase activity and STRAD-alpha binding, but also lead to nuclear sequestration - highlighting the importance of a multipronged approach when assessing STK11 function. Future studies will assess the ability of these C-terminal domain mutants to phosphorylate cytoplasmic substrates, and their impact on cell migration and invasion. Additionally, we will utilize the TurboID proximity ligation system to assess the effect of C-terminal domain point mutations on binding partner accessibility. Citation Format: Gopika Nandagopal, Sean Lenahan, Hannah Ross, Hailey Sarausky, David Seward, Paula Deming. Functional assessment of STK11 C-terminal domain variants [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 2751.

Abstract 1153: Using spatial transcriptomics to dissect cell to cell cooperation in lung adenocarcinoma

Abstract Lung cancer is the second most common cancer in the United States and the leading cause of cancer related deaths in this country. Unfortunately, efficacious treatments for lung cancer remain suboptimal. Lung cancer is characterized by several distinct subtypes of which lung adenocarcinoma is the most common, comprising about 40% of this malignancy. The hallmark gene mutations of lung adenocarcinoma include TP53, RAS, and STK11. LKB1 is a serine-threonine kinase (coded by the gene STK11) that largely functions as a tumor suppressor, and is mutated in 20-30% of non-small cell lung cancers (NSCLCs). The diversity between and within lung cancer subtypes, as well as within a patient (i.e. metastases vs primary tumor), makes treating this cancer very challenging. Lung adenocarcinoma, in particular, has collective invasion packs of cells adjacent to the primary tumor that correlate with metastatic disease in mouse models. We hypothesize that the transcriptomic profile of the collective invasion packs in lung adenocarcinoma patients varies significantly from the adjacent primary tumor and represents a targetable metastatic sub-population. This work will help to identify specific cell signaling pathways that have the translational potential to develop novel therapeutics for metastatic disease, ultimately improving patient outcomes through precision medicine. Utilizing patient lung adenocarcinoma samples with KRAS and KRAS+LKB1 mutations, we identified regions of interest including bulk tumor and surrounding invasion packs. Then, using GeoMx digital spatial profiling technology (by Nanostring) and next-gen sequencing, we generated transcriptomic profiles from bulk tumor and invasion packs. Preliminary results indicate region specific transcriptomic differences, highlighting the heterogeneity of these cell populations. More specifically, collective invasion packs exhibit upregulation of gene networks involving immune cell differentiation, function, oxidative phosphorylation, tumor invasiveness, and mitochondrial structure. To discern the metastatic potential, transcriptomic profiles and biological functions will be compared between invasion packs, tumor bulk vs invasion packs, and finally, inter-patient differences. Successful completion of this project will characterize transcriptomes of metastatic lung cancer and has the potential to ultimately identify biomarkers of aggressive disease. Lastly, this foundational study will pave the way for future studies on the transcriptomic landscape of metastatic pediatric cancers and cancer predisposition syndromes. Citation Format: Raehannah Jamshidi, Lyra Griffiths, Rich Johnston, Vaunita Parihar, Frank Schneider, Adam Marcus. Using spatial transcriptomics to dissect cell to cell cooperation in lung adenocarcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 1153.

Abstract 3298: Unlocking the potential of OPM-383: A novel LRRK2 inhibitor in cancer therapy

Abstract Background: Leucine-rich repeat kinase 2 (LRRK2) plays a pivotal role in regulating various cellular processes, such as cell proliferation, survival, and inflammation. LRRK2 exhibits dual functionality as a serine-threonine kinase and as a GTPase. It is involved in the modulation of multiple signaling pathways, including WNT, MAPK, NF-kB and mTOR. Germline mutations in LRRK2 are associated with an increased risk of cancer, particularly hormone-related and colorectal cancers. LRRK2 also promotes tumor cell growth and survival in papillary renal and thyroid carcinomas, DLBCL and cholangiocarcinoma cells. OPM has designed and developed a novel oral LRRK2 inhibitor, OPM383, using its proprietary Nanocyclix® technology. In this study, we have evaluated the pharmacokinetic (PK) properties, efficacy and tolerability of OPM383 in a colon carcinoma model and a panel of patient-derived organoids. Materials and Methods: In vitro potency of our Type I inhibitor, OPM383, against full-length wild-type LRRK2 and the G2019S pathogenic LRRK2 mutant was assessed in in a Lanthascreen cell assay. For PK studies, we analyzed OPM383 plasma levels using UPLVC/MS-MS. Evaluation of inhibition of LRRK2 phosphorylation (Ser935) was assessed by western blot. MC-38 cells were inoculated into C57BL/6 mice. When tumor masses reached 75 mm³, mice were randomized to receive OPM383 (50 mg/kg, orally, daily), anti-PD1 antibody (10 mg/kg, ip, twice weekly), or their combination. OPM383 was also evaluated in patient-derived organoids using the SEngine-Paris® platform. Results: OPM-383 inhibited wild-type LRRK2 and LRRK2[G2019S] with an IC50 of 42 nM and 33 nM, respectively. OPM383 is highly selective among a panel of >400 protein kinases (only 6.7% cross-reactivity at 100 nM). The compound is permeable, metabolically stable and possesses drug-like properties. OPM-383 exhibited a favorable pharmacokinetic profile in mice (AUC of 11858 h*ng/mL and T max of 3 hours at a dose of 30 mg/kg via oral administration). Preliminary e-ADME results indicated moderate inhibition of CYP3A4BFC but no significant inhibition of CYPs 1A2, 2D6, 3A4BQ, 2C19, and 2C9 by OPM-383. After 90 minutes of oral administration in CD1 mice, we observed the inhibition of LRRK2 phosphorylation (Ser935) in the brain, kidney, and PBMCs. In assessing its potential antitumor effects, OPM383 was tested on a panel of patient-derived organoids. Notably, OPM383 inhibited the proliferation of 7 out of 29 patient-derived organoids after 6 days of exposure at various concentrations (<10 µM). In mice bearing subcutaneous MC38 tumors, OPM383 significantly inhibited tumor growth compared to the vehicle-treated group. Conclusion: This study characterizes a potent first-in-class LRRK2 inhibitor in cancer models. The findings suggest that OPM383 could serve as an attractive lead scaffold for designing and synthesizing a new class of kinase inhibitors for potential applications in cancer therapy. Citation Format: Maria Eugenia Riveiro, Petra Blom, Kenji Shoji, Jan Hoflack. Unlocking the potential of OPM-383: A novel LRRK2 inhibitor in cancer therapy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 3298.

Abstract 4278: Role of WNK1 in extracellular matrix modification in breast cancer

Abstract Introduction: Breast cancer (BCa) is the most common cancer and the second leading cause of cancer related deaths in women in the United States. Metastatic dissemination of cancer cells remains the leading cause of mortality in BCa. Therefore, determination of the mechanisms that promote metastasis is important for development of new therapeutic strategies. The extracellular matrix (ECM), which is the main non-cellular component of the tumor microenvironment (TME), plays an important role in BCa progression and metastasis. The cancer-associated fibroblasts (CAFs) are the principal cell types that together with BCa cells drive the modification of ECM during cancer development. Lysine deficient receptor protein kinase 1 (WNK1), a widely expressed serine threonine kinase that regulates blood pressure and electrolyte balance in the body, and is highly overexpressed in BCa microenvironment. The aim of the present study was to investigate the role of WNK1 in modification of ECM in BCa. Methods: The clinical relevance of WNK1 overexpression in BCa was determined by immunohistochemically staining human BCa sections, analyzing data from publicly available databases and creating orthotopic mouse models of BCa using 4T1 mouse BCa cells followed by treatment of the tumor bearing mice with WNK1 inhibitors, WNK-IN-11 and WNK463 (10 mg/kg , oral for 10 days). In vitro assays were conducted using 4T1 cells and mouse embryonic fibroblasts (MEFs) to determine the regulatory role of WNK1 on cancer cells and fibroblasts. Results and Conclusion: Our results indicated that WNK1 is strongly expressed in BCa cells and fibroblasts, the two most abundant cell types in the BCa microenvironment. Furthermore, WNK1 expression strongly correlated with expressions of matrix regulatory and cross-linking genes. Our results demonstrated that WNK1 inhibitors significantly reduced orthotopic 4T1 growth and metastasis. WNK1 inhibition also significantly inhibited collagen deposition in tumor tissues, which is a negative prognostic indicator in BCa. In vitro proliferation and migration assays using 4T1 cells and MEFs identified that WNK1 inhibitors (1-100nM) can significantly inhibit the proliferation and migration of 4T1 cells and MEFs. Results from co-culture studies with 4T1 cells and MEFs further indicated that WNK1 inhibition in MEFs could significantly reduce the invasive properties of 4T1 cells. Taken together, our results suggest that blocking the actions of WNK1 in BCa may be a promising therapeutic approach. Citation Format: Prabhat Suman, Sooraj Kakkat, Suhas S. Patil, Veronica Ramirez, Elba A. Turbat-Herrera, Seema Singh, Chandrani Sarkar, Debanjan Chakroborty. Role of WNK1 in extracellular matrix modification in breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 4278.

Abstract 5584: Experimental ‘loss-of function’ annotation of STK11 mutations with prognostic and therapeutic implications (TNG260mutationfinder.com)

Abstract Loss of function mutations in Serine Threonine Kinase 11 (STK11) occur in 15% of lung adenocarcinoma and have been shown to drive resistance to immune checkpoint blockade clinically, as well as in preclinical models. Though STK11 is commonly inactivated in human cancer with strong implications for treatment outcomes, few STK11 mutations identified from tumor samples have been functionally characterized. TNG260 is an inhibitor of CoREST that is currently being investigated in combination with pembrolizumab for the treatment of STK11-mutant cancer (NCT05887492). Patients are eligible for enrollment in the TNG260 phase 1/2 trial if their tumor contains a deleterious STK11 mutation. To begin classifying non-annotated variants, over 2,000 distinct mutations in the STK11 gene were identified from STK11 literature or public repositories of tumor sequencing data such as AACR Project GENIE and ClinVar. Where possible, loss-of-function annotations were captured from literature or predictive tools such as PolyPhen-2. However, many STK11 variants, particularly missense mutations, have never been functionally characterized. We developed a functional screening approach to characterize STK11 alterations using the lung adenocarcinoma cell line A549. A549 cells contain homozygous loss of STK11 via a truncation mutation at Q37, and re-expression of wild-type STK11 in these cells strongly impairs their growth in vitro and in vivo. We created a library of STK11 variant cDNAs, each containing a unique barcode. This library was expressed in A549, and cells were maintained in vitro or in vivo to allow for positive selection of STK11 loss-of-function variants and depletion of variants that behave like wild-type STK11. At the end of the screen, variants were quantified by NGS using each mutant cDNA’s unique barcode and compared to well-annotated controls. These data were assembled to generate TNG260mutationfinder.com – the first website to curate STK11 variants with functional annotations. Citation Format: Leanne G. Ahronian, Preksha Shahagadkar, Lauren Flynn, Lauren Grove, Shangtao Liu, Samuel R. Meier, Binzheng Shen, Hannah Stowe, Hsin-Jung Wu, Yi Yu, Andre Mignault, Iga Sienczylo, Heather DiBenedetto, Silvia Fenoglio, Teng. Experimental ‘loss-of function’ annotation of STK11 mutations with prognostic and therapeutic implications (TNG260mutationfinder.com) [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 5584.

Abstract 3916: TNG260, a small molecule CoREST inhibitor, sensitizes STK11-mutant NSCLC to anti-PD1 immunotherapy

Abstract Loss of function (LOF) of the serine-threonine kinase STK11/LKB1, which has been reported in ~15% of non-small cell lung cancer (NSCLC), drives resistance to immune checkpoint therapies such as PD1. In vivo CRISPR screens in tumor-bearing immune competent mice identified the Corepressor of Repressor Element 1 Silencing Transcription (CoREST) complex as a synthetic lethal immune evasion target for STK11- mutant tumors. Here, we demonstrate that TNG260, a small molecule drug specifically targeting the CoREST complex, can sensitize STK11 mutant lung cancers to anti-PD1 immunotherapy. Combination treatment with TNG260 and anti-PD1 arrested KRAS/STK11 mutant NSCLC tumor growth with no major toxicity observed, as confirmed in both allograft and genetically engineered mouse models (GEMMs) of STK11 mutant NSCLC. Further transcriptional analysis via bulk RNA-seq revealed that treatment of KRAS/STK11 mutant lung tumors with TNG260 promotes genes related to inflammatory responses and suppresses genes related to cell cycle and mitotic checkpoint(s). This phenotype was not observed in STK11 wildtype KRAS/P53 mutant lung tumors. Furthermore, combinational treatment of TNG260 with PD1 antibody showed increased macrophages, neutrophils and dendritic cells signatures in vivo. Our data suggests that epigenetic reprogramming via TNG260 sensitizes STK11 mutant NSCLC tumors to anti- PD1 treatment via targeting of the CoREST complex. This work identifies new vulnerabilities in STK11 LOF cancers that can be exploited therapeutically. TNG260 is currently being investigated in combination with pembrolizumab for the treatment of STK11-mutant cancer (NCT05887492), including NSCLC. Citation Format: Ayushi Patel, Soumyadip Sahu, Ke Geng, Salman Punekar, Janaye Stephens, Jiehui Deng, Ting Chen, Leanne Ahronian, Minjie Zhang, Jannik Andersen, Brian Haines, Kwok-Kin Wong. TNG260, a small molecule CoREST inhibitor, sensitizes STK11-mutant NSCLC to anti-PD1 immunotherapy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 3916.

Abstract 5714: Synergistic effect of the CDC7 inhibitor, monzosertib (AS-0141) with current therapies in AML models

Abstract Introduction: Cell division cycle 7 (CDC7) is a highly conserved serine-threonine kinase that plays an important role in the initiation of DNA replication and cell cycle progression. Aberrant expression of CDC7 have been reported to cause uncontrolled proliferation of many cancer types, suggesting that CDC7 inhibitors may provide a great potential for the development of novel therapy for cancers. Monzosertib (AS-0141) is a potent, selective, orally bioavailable small molecule inhibitor of CDC7, and is currently being evaluated in Phase I study for the treatment of solid tumors. In a preclinical cancer cell panel study, monzosertib showed strong antiproliferative activities against a variety of cancer types, and acute myeloid lymphoma (AML) cell lines were found to be the most sensitive to monzosertib. As a single agent, treatment with monzosertib accumulates DNA damage in cancer cells and induces cell death. In this study, we aimed to investigate the antitumor effects of monzosertib alone and in combination with other anticancer drugs in AML models. Method: Antiproliferative activity of monzosertib was examined against a panel of 35 human cancer cell lines of various cancer types. DNA metyltransferase (DNMT) inhibitors (azacitidine or decitabine) and BCL2 inhibitor (venetoclax) were evaluated for their synergistic/antagonistic effects in combination with monzosertib against human AML cell lines (THP-1, HL-60, MV4-11, MOLM-14, TF-1, U-937 and NOMO-1). The combination index (CI) was calculated using the Chou-Talalay method. Flow cytometry assay was used to analyze apoptosis. To evaluate the in vivo efficacy, tumor-bearing mice were treated with monzosertib alone or in combination with venetoclax. Results: The combination of monzosertib with azacitidine, decitabine, or venetoclax resulted in a significant synergistic antiproliferative effects against AML cell lines in vitro. The flow cytometry assay indicated that azacitidine combination induced apoptosis and increased cell death in THP-1 cells. In vivo, oral administration of monzosertib demonstrated robust in vivo antitumor efficacy in a MV4-11 tumor bearing xenograft mouse model, both as a single agent and in combination with venetoclax. Conclusions: Monzosertib, a selective CDC7 inhibitor, demonstrated strong antiproliferative activity against human AML cell lines, both as a single agent and in combination with standard therapies. Monzosertib exerts synergistic antitumor effect with venetoclax in a human AML xenograft mouse model. These results suggest that monzosertib has a potential to enhance the antitumor efficacy of standard of care agents for AML patients. Citation Format: Hiroko Endo, Hatsuo Furuichi, Akinori Arimura, Yu Nishioka, Masaaki Sawa. Synergistic effect of the CDC7 inhibitor, monzosertib (AS-0141) with current therapies in AML models [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 5714.

Abstract 3373: Protein kinase D1 alleviate etoposide induced DNA damage through up regulation of alpha-catenin

Abstract Interaction between E-cadherin and β-catenin at cell-cell adherens junctions is pivotal in cell adhesion, and dysregulation of these genes is associated with disease progression in various solid tumors, notably in prostate cancer. α-catenin acts as a physical linker between the cadherin/β-catenin complex and actin cytoskeleton, through its mechanosensory properties has potential to modify interactions between cadherins and cytoskeleton and results in sensitivity of cells to DNA damage and toxicity. Membranous β-catenin interacts with the cellular cytoskeleton through threonine 120 (T120) phosphorylation and its association with α-catenin. We want to investigate the role of protein Kinase D1 (PrKD1) in DNA damage response, using various prostate cancer cell lines; PrKD1-silenced cells (LNCaP sh-PrKD1) and overexpression of PrKD1 (C4-2 PrKD1). PrKD1 silence and overexpress cells were treated with PrKD1 inhibitor and Etoposide to observe the effect of DNA damage repair altering various genes in prostate cancer cells. Silencing α-Catenin increased p-γH2AX (ser-139) expression in C4-2 PrKD1 cells. Conversely silenced PrKD1 (LNCaP sh-PrKD1) cells represented decreased α-Catenin expression, and increased p-γH2AX (ser-139). Overexpression of PrKD1 in prostate cancer cells increased α-catenin levels and conferred resistance to etoposide-induced DNA damage. Silencing PrKD1 compromised DNA damage protective effect. Furthermore, we elucidate the role of Protein kinase D1 (PrKD1), a unique serine-threonine kinase that phosphorylates β-catenin at T120, in this interaction. We treated C4-2 and C4-2 PrKD1 cells with PrKD1 inhibitor and immuno-precipitated β-Catenin complex represented decrease in β-Catenin (T-120) and α-Catenin than untreated C4-2 PrKD1 cells. We observed crosstalk between α-catenin and PrKD1 in DNA damage repair. In-silico analysis affirmed that the loss of T120 phosphorylation disrupts β- and α-catenin interaction. PrKD1 inhibitor decreased cell viability in dose response fashion, and also decrease the p-γH2AX (ser-139) in C4-2 PrKD1 cells than C4-2 cells. In vitro findings were corroborated in a mouse prostate cancer PDX xenograft model by inhibiting PrKD1 kinase activity, resulting in reduced β-catenin T120 phosphorylation and α-catenin levels. In vivo experiments with PrKD1 inhibitor represented tolerability in mice and its efficacy in modulating α-catenin and PrKD1 expression to inhibit tumor progression. Study for the first time provides a mechanistic basis of the protective role of PrKD1 against DNA damage. Citation Format: Sanjeev Shukla, Samuel Serrano, Mohammed Al-Toubat, Teruko Osumi, K.C. Balaji. Protein kinase D1 alleviate etoposide induced DNA damage through up regulation of alpha-catenin [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 3373.

Dietary Zn Deficiency Inhibits Cell Proliferation via the GPR39-Mediated Suppression of the PI3K/AKT/mTOR Signaling Pathway in the Jejunum of Broilers.

A prior investigation revealed that a lack of Zinc (Zn) could hinder intestinal cell proliferation in broiler chickens; however, the mechanisms responsible for this effect remain unclear. We aimed to investigate the possible mechanisms of dietary Zn deficiency in inhibiting the jejunal cell proliferation of broilers. For this study, a total of 112 chickens (21 days old) were randomly divided into two treatments (seven replicate cages per treatment, eight chickens per replicate cage): the control group (CON) and the Zn deficiency group. The duration of feeding was 21 d. Chickens in the control group were provided with a basal diet containing an extra addition of 40 mg Zn/kg in the form of Zn sulfate, whereas chickens in the Zn deficiency group were given the basal diet with no Zn supplementation. The results indicated that, in comparison to the CON, Zn deficiency increased (p < 0.05) the duodenal and jejunal crypt depth (CD) of broilers on d 28 and jejunal and ileal CD on d 35, and decreased (p < 0.05) the duodenal, jejunal, and ileal villus height/crypt depth (VH/CD) on d 28 and the jejunal VH, jejunal and ileal villus surface area, and VH/CD on d 35. Furthermore, Zn deficiency decreased (p < 0.0001) the number of proliferating cell nuclear antigen-positive cells and downregulated (p < 0.01) the mRNA or protein expression levels of phosphatidylinositol 3-kinase (PI3K), phosphorylated PI3K, phosphorylated serine-threonine kinase (AKT), phosphorylated mechanistic target of rapamycin (mTOR), G protein-coupled receptor 39 (GPR39), and extracellular-regulated protein kinase, but upregulated (p < 0.05) the mRNA or protein expression levels of P38 mitogen-activated protein kinase, c-jun N-terminal kinase (JNK) 1 and JNK2, and phosphorylated protein kinase C in the jejunum of the broilers on d 42. It was concluded that dietary Zn deficiency inhibited cell proliferation possibly via the GPR39-mediated suppression of the PI3K/AKT/mTOR signaling pathway in the jejunum of broilers.

Wheat powdery mildew resistance gene Pm13 encodes a mixed lineage kinase domain-like protein

Wheat powdery mildew is one of the most destructive diseases threatening global wheat production. The wild relatives of wheat constitute rich sources of diversity for powdery mildew resistance. Here, we report the map-based cloning of the powdery mildew resistance gene Pm13 from the wild wheat species Aegilops longissima. Pm13 encodes a mixed lineage kinase domain-like (MLKL) protein that contains an N-terminal-domain of MLKL (MLKL_NTD) domain in its N-terminus and a C-terminal serine/threonine kinase (STK) domain. The resistance function of Pm13 is validated by mutagenesis, gene silencing, transgenic assay, and allelic association analyses. The development of introgression lines with significantly reduced chromosome segments of Ae. longissima encompassing Pm13 enables widespread deployment of this gene into wheat cultivars. The cloning of Pm13 may provide valuable insights into the molecular mechanisms underlying Pm13-mediated powdery mildew resistance and highlight the important roles of kinase fusion proteins (KFPs) in wheat immunity.

Integrative Kinase Activity Profiling and Phosphoproteomics of rd10 Mouse Retina during cGMP-Dependent Retinal Degeneration.

Inherited retinal degenerative diseases (IRDs) are a group of rare diseases that lead to a progressive loss of photoreceptor cells and, ultimately, blindness. The overactivation of cGMP-dependent protein kinase G (PKG), one of the key effectors of cGMP-signaling, was previously found to be involved in photoreceptor cell death and was studied in murine IRD models to elucidate the pathophysiology of retinal degeneration. However, PKG is a serine/threonine kinase (STK) with several hundred potential phosphorylation targets and, so far, little is known about the specificity of the target interaction and downstream effects of PKG activation. Here, we carried out both the kinome activity and phosphoproteomic profiling of organotypic retinal explant cultures derived from the rd10 mouse model for IRD. After treating the explants with the PKG inhibitor CN03, an overall decrease in peptide phosphorylation was observed, with the most significant decrease occurring in seven peptides, including those from the known PKG substrate cyclic-AMP-response-element-binding CREB, but also Ca2+/calmodulin-dependent kinase (CaMK) peptides and TOP2A. The phosphoproteomic data, in turn, revealed proteins with decreased phosphorylation, as well as proteins with increased phosphorylation. The integration of both datasets identified common biological networks altered by PKG inhibition, which included kinases predominantly from the so-called AGC and CaMK families of kinases (e.g., PKG1, PKG2, PKA, CaMKs, RSKs, and AKTs). A pathway analysis confirmed the role of CREB, Calmodulin, mitogen-activated protein kinase (MAPK) and CREB modulation. Among the peptides and pathways that showed reduced phosphorylation activity, the substrates CREB, CaMK2, and CaMK4 were validated for their retinal localization and activity, using immunostaining and immunoblotting in the rd10 retina. In summary, the integrative analysis of the kinome activity and phosphoproteomic data revealed both known and novel PKG substrates in a murine IRD model. This data establishes a basis for an improved understanding of the biological pathways involved in cGMP-mediated photoreceptor degeneration. Moreover, validated PKG targets like CREB and CaMKs merit exploration as novel (surrogate) biomarkers to determine the effects of a clinical PKG-targeted treatment for IRDs.

PIM Kinase Inhibition Sensitizes Neuroblastoma to Doxorubicin

BackgroundChemoresistance contributes to relapse in high-risk neuroblastoma. Cancer cells acquire resistance through multiple mechanisms, including drug efflux pumps. In neuroblastoma, multidrug resistance-associated protein-1 (MRP1/ABCC1) efflux pump expression correlates with worse outcomes. These pumps are regulated by PIM kinases, a family of serine–threonine kinases, overexpressed in neuroblastoma. We hypothesized PIM kinase inhibition would sensitize neuroblastoma cells by modulating MRP1. MethodsKocak database query evaluated ABCC1, PIM1, PIM2, and PIM3 expression in neuroblastoma patients. SK-N-AS and SK-N-BE(2) cells were treated with doxorubicin or the pan-PIM kinase inhibitor, AZD1208. Flow cytometry assessed intracellular doxorubicin accumulation. AlamarBlue assay measured viability. The lethal dose 50% (LD50) of each drug and combination indices (CI) were calculated and isobolograms constructed to determine synergy. ResultsKocak database query demonstrated positive correlation between PIM genes and ABCC1. PIM kinase inhibition increased intracellular doxorubicin accumulation in both cell lines, suggesting PIM kinase regulation of MRP1. Isobolograms showed synergy between AZD1208 and doxorubicin. ConclusionsThe correlation between PIM and ABCC1 gene expression suggests PIM kinases may contribute to neuroblastoma chemotherapeutic resistance. PIM kinase inhibition increased intracellular doxorubicin accumulation. Combination treatment with AZD1208 and doxorubicin decreased neuroblastoma cell viability in a synergistic fashion. These findings support further investigations of PIM kinase inhibition in neuroblastoma. Type of StudyBasic Science Research. Level of EvidenceNA.

SUMOylation of OsPSTOL1 is essential for regulating phosphate starvation responses in rice and Arabidopsis.

Although rice is one of the main sources of calories for most of the world, nearly 60% of rice is grown in soils that are low in phosphorus especially in Asia and Africa. Given the limitations of bioavailable inorganic phosphate (Pi) in soils, it is important to develop crops tolerant to low phosphate in order to boost food security. Due to the immobile nature of Pi, plants have developed complex molecular signalling pathways that allow them to discern changes in Pi concentrations in the environment and adapt their growth and development. Recently, in rice, it was shown that a specific serine-threonine kinase known as Phosphorus-starvation tolerance 1 (PSTOL1) is important for conferring low phosphate tolerance in rice. Nonetheless, knowledge about the mechanism underpinning PSTOL1 activity in conferring low Pi tolerance is very limited in rice. Post-translation modifications (PTMs) play an important role in plants in providing a conduit to detect changes in the environment and influence molecular signalling pathways to adapt growth and development. In recent years, the PTM SUMOylation has been shown to be critical for plant growth and development. It is known that plants experience hyperSUMOylation of target proteins during phosphate starvation. Here, we demonstrate that PSTOL1 is SUMOylated in planta, and this affects its phosphorylation activity. Furthermore, we also provide new evidence for the role of SUMOylation in regulating PSTOL1 activity in plant responses to Pi starvation in rice and Arabidopsis. Our data indicated that overexpression of the non-SUMOylatable version of OsPSTOL1 negatively impacts total root length and total root surface area of rice grown under low Pi. Interestingly, our data also showed that overexpression of OsPSTOL1 in a non-cereal species, Arabidopsis, also positively impacts overall plant growth under low Pi by modulating root development. Taken together our data provide new evidence for the role of PSTOL1 SUMOylation in mediating enhanced root development for tolerating phosphate-limiting conditions.

(013) Glycated Serum Albumin Decreases Connexin 43 Phosphorylation in the Corpus Cavernosum

Abstract Introduction Glycated serum albumin (GSA) is an early glycosylation product that participates in diabetic vascular complications, including retinopathy, nephropathy, neuropathy, and coronary artery disease. Elevated GSA levels and the associated damage to the corpus cavernosum might be limited to not only diabetic patients but also non-diabetic patients and even individuals with normal glucose levels. Objective This study aimed to examine the role of GSA in early damage to the corpus cavernosum and identify the pathways involved in GSA-induced endothelium damage. Methods Nine 8-week-old male Sprague-Dawley (SD) rats (250–300 g) were divided into the control (saline vehicle, n=3) and GSA (200 μg/kg, n=6) groups. The corpus cavernosum tissues were harvested. Phosphorylated and non-phosphorylated connexin 43 (Cx43), endothelial nitric oxide synthase (eNOS), phosphatidylinositol 3-kinase (PI3K), and serine-threonine kinase (Akt) were tested by immunohistochemistry and western blotting. Human umbilical vein endothelial cells (HUVECs) overexpressing Cx43 were used to analyze the Cx43 phosphorylation sites (S368, S262, Y265, S255, and S279/282) using western blotting. Results The expression of phosphorylated Cx43 in the penis was significantly lower in GSA-treated rats than in controls. The expression levels of p-Cx43, p-eNOS, p-PI3K, and p-Akt were significantly decreased in HUVECs exposed to GSA in dose- and time-dependent manners. The most significant impact on all four proteins was observed with 1 μg/mL of GSA for 12 h. Phosphorylation at the S368, S262, Y265, S255, and S279/282 sites of Cx43 was downregulated by GSA, and S368 was the most significantly suppressed phosphorylation site compared with the other sites. Conclusions GSA decreases the expression of p-Cx43 in the corpus cavernosum of rats. This effect might be also related to the decreased phosphorylation of p-eNOS, p-PI3K, and p-Akt, as well as by the downregulation of phosphorylation at the S368 site. Disclosure No.

CDKL5 Deficiency Disorder: Some Lessons Learned 20 Years After the First Description.

Loss of function variants in the Cyclin-dependent kinase-like 5 gene (CDKL5) causes CDKL5 deficiency disorder (CDD). Most cases of CDD are due to a de novo missense or truncating variants. The CDKL5 gene was discovered in 1998 as part of the genomic mapping of the chromosome Xp22 region that led to the discovery of the serine-threonine kinases STK9. Since then, there have been significant advancements in the description of the disease in humans, the understanding of the pathophysiology, and the management of the disease. There have been many lessons learned since the initial description of the condition in humans in 2003. In this article, we will focus on pathophysiology, clinical manifestations, with particular focus on seizures because of its relevance to the medical practitioners and researchers and guidelines for management. We finalize the manuscript with the voice of the parents and caregivers, as discussed with the 2019 meeting with the Food and Drug Administration.

Coconut (Cocos nucifera (L.)) Water Improves Glucose Uptake with Concomitant Modulation of Antioxidant and Purinergic Activities in Isolated Rat Psoas Muscles.

The present study investigated the effect of coconut water on glucose uptake and utilization, and metabolic activities linked to hyperglycemia in isolated rat psoas muscles. Coconut water was subjected to in vitro antioxidant and antidiabetic assays, which cover 2,2'-diphenyl-1-picrylhydrazyl (DPPH) scavenging activity, ferric reducing antioxidant power (FRAP), and inhibition of α-glucosidase and α-amylase activities. Psoas muscles were isolated from male Sprague Dawley rats and incubated with coconut water in the presence of glucose. Control consisted of muscles incubated with glucose only, while normal control consisted of muscles not incubated in coconut water and/or glucose. The standard antidiabetic drug was metformin. Incubation with coconut water led to a significant increase in muscle glucose uptake, with concomitant exacerbation of glutathione level, and SOD and catalase activities, while suppressing malondialdehyde level, and ATPase and E-NTDase activities. Coconut water showed significant scavenging activity against DPPH, and significantly inhibited α-glucosidase and α-amylase activities. LC-MS analysis of coconut water revealed the presence of ellagic acid, butin, quercetin, protocatechuic acid, baicalin, and silibinin. Molecular docking analysis revealed potent molecular interactions between the LC-MS-identified compounds, and AKT-2 serine and PI-3 kinase. These results indicate the potential of coconut water to enhance glucose uptake, while concomitantly improving antioxidative and purinergic activities. They also indicate the potential of coconut water to suppress postprandial hyperglycemia. These activities may be attributed to the synergistic effects of the LC-MS-identified compounds.

Tepotinib and tivantinib as potential inhibitors for the serine/threonine kinase of the mpox virus: insights from structural bioinformatics analysis

The serine/threonine kinase (STK) plays a central role as the primary kinase in poxviruses, directing phosphoryl transfer reactions. Such reactions are pivotal for the activation of certain proteins during viral replication, assembly, and maturation. Therefore, targeting this key protein is anticipated to impede virus replication. In this work, a structural bioinformatics approach was employed to evaluate the potential of drug-like kinase inhibitors in binding to the ATP-binding pocket on the STK of the Mpox virus. Virtual screening of known kinase inhibitors revealed that the top 10 inhibitors exhibited binding affinities ranging from −8.59 to −12.05 kcal/mol. The rescoring of compounds using the deep-learning default model in GNINA was performed to predict accurate binding poses. Subsequently, the top three inhibitors underwent unbiased molecular dynamics (MD) simulations for 100 ns. Molecular Mechanics/Generalized Born Surface Area (MM/GBSA) analysis and Principal Component Analysis (PCA) suggested tepotinib as a competitive inhibitor for Mpox virus STK as evidenced by its binding free energy and the induction of similar conformational behavior of the enzyme. Nevertheless, it is sensible to experimentally test all top 10 compounds, as scoring functions and energy calculations may not consistently align with experimental findings. These insights are poised to provide an attempt to identify an effective inhibitor for the Mpox virus. Communicated by Ramaswamy H. Sarma

Identification of Ossnrk1a-1 Regulated Genes Associated with Rice Immunity and Seed Set.

Sucrose non-fermenting-1-related protein kinase-1 (SnRK1) is a highly conserved serine-threonine kinase complex regulating plants' energy metabolisms and resistance to various types of stresses. However, the downstream genes regulated by SnRK1 in these plant physiological processes still need to be explored. In this study, we found that the knockout of OsSnRK1a resulted in no obvious defects in rice growth but notably decreased the seed setting rate. The ossnrk1a mutants were more sensitive to blast fungus (Magnaporthe oryzae) infection and showed compromised immune responses. Transcriptome analyses revealed that SnRK1a was an important intermediate in the energy metabolism and response to biotic stress. Further investigation confirmed that the transcription levels of OsNADH-GOGAT2, which positively controls rice yield, and the defense-related gene pathogenesis-related protein 1b (OsPR1b) were remarkably decreased in the ossnrk1a mutant. Moreover, we found that OsSnRK1a directly interacted with the regulatory subunits OsSnRK1β1 and OsSnRK1β3, which responded specifically to blast fungus infection and starvation stresses, respectively. Taken together, our findings provide an insight into the mechanism of OsSnRK1a, which forms a complex with specific β subunits, contributing to rice seed set and resistance by regulating the transcription of related genes.

The Nitro Group Reshapes the Effects of Pyrido[3,4-g]quinazoline Derivatives on DYRK/CLK Activity and RNA Splicing in Glioblastoma Cells.

Serine-threonine protein kinases of the DYRK and CLK families regulate a variety of vital cellular functions. In particular, these enzymes phosphorylate proteins involved in pre-mRNA splicing. Targeting splicing with pharmacological DYRK/CLK inhibitors emerged as a promising anticancer strategy. Investigation of the pyrido[3,4-g]quinazoline scaffold led to the discovery of DYRK/CLK binders with differential potency against individual enzyme isoforms. Exploring the structure-activity relationship within this chemotype, we demonstrated that two structurally close compounds, pyrido[3,4-g]quinazoline-2,10-diamine 1 and 10-nitro pyrido[3,4-g]quinazoline-2-amine 2, differentially inhibited DYRK1-4 and CLK1-3 protein kinases in vitro. Unlike compound 1, compound 2 efficiently inhibited DYRK3 and CLK4 isoenzymes at nanomolar concentrations. Quantum chemical calculations, docking and molecular dynamic simulations of complexes of 1 and 2 with DYRK3 and CLK4 identified a dramatic difference in electron donor-acceptor properties critical for preferential interaction of 2 with these targets. Subsequent transcriptome and proteome analyses of patient-derived glioblastoma (GBM) neurospheres treated with 2 revealed that this compound impaired CLK4 interactions with spliceosomal proteins, thereby altering RNA splicing. Importantly, 2 affected the genes that perform critical functions for cancer cells including DNA damage response, p53 signaling and transcription. Altogether, these results provide a mechanistic basis for the therapeutic efficacy of 2 previously demonstrated in in vivo GBM models.

CircPCNXL2 promotes tumor growth and metastasis by interacting with STRAP to regulate ERK signaling in intrahepatic cholangiocarcinoma

Backgroundcircular RNAs (circRNAs) have been reported to exert important effects in the progression of numerous cancers. However, the functions of circRNAs in intrahepatic cholangiocarcinoma (ICC) are still unclear.MethodscircPCNXL2 (has_circ_0016956) were identified in paired ICC by circRNA microarray. Then, we assessed the biological functions of circPCNXL2 by CCK8, EdU, clone formation, transwell, wound healing assays, and xenograft models. RNA pull-down, mass spectrometry, and RNA immunoprecipitation (RIP) were applied to explore the interaction between cirrcPCNXL2 and serine-threonine kinase receptor-associated protein (STRAP). RNA pull-down, RIP and luciferase reporter assays were used to investigate the sponge functions of circPCNXL2. In the end, we explore the effects of circPCNXL2 and trametinib (a MEK1/2 inhibitor) in vivo.ResultscircPCNXL2 was upregulated in ICC tissues and cell lines, which promoted the proliferation and metastasis of ICC in vitro and in vivo. In terms of the mechanisms, circPCNXL2 could directly bind to STRAP and induce the interaction between STRAP and MEK1/2, resulting in the tumor promotion in ICC by activation of ERK/MAPK pathways. Besides, circPCNXL2 could regulate the expression of SRSF1 by sponging miR-766-3p and subsequently facilitated the growth of ICC. Finally, circPCNXL2 could partially inhibit the anti-tumor activity of trametinib in vivo.ConclusioncircPCNXL2 played a crucial role in the progression of ICC by interacting with STRAP to activate the ERK signaling pathway, as well as by modulating the miR-766-3p/SRSF1 axis. These findings suggest that circPCNXL2 may be a promising biomarker and therapeutic target for ICC.