Activation Of Signal Transduction Pathways Research Articles

MAPK Cascade Signaling Is Involved in α-MMC Induced Growth Inhibition of Multiple Myeloma MM.1S Cells via G2 Arrest and Mitochondrial-Pathway-Dependent Apoptosis In Vitro

Multiple myeloma is a hematological malignancy characterized by the unrestricted proliferation of plasma cells that secrete monoclonal immunoglobulins in the bone marrow. Alpha-momorcharin (α-MMC) is a type I ribosome-inactivating protein extracted from the seeds of the edible plant Momordica charantia L., which has a variety of biological activities. This study aimed to investigate the inhibitory effect of α-MMC on the proliferation of multiple myeloma MM.1S cells and the molecular mechanism of MM.1S cell death induced through the activation of cell signal transduction pathways. The cell counting kit-8 (CCK-8) assay was used to determine the inhibitory effect of α-MMC on the proliferation of MM.1S cells and its toxic effect on normal human peripheral blood mononuclear cells (PBMCs). The effect of α-MMC on the MM.1S cells' morphology was observed via inverted microscope imaging. The effects of α-MMC on the MM.1S cell cycle, mitochondrial membrane potential (MMP), and apoptosis were explored using propidium iodide, JC-1, annexin V- fluorescein isothiocyanate/propidium iodide fluorescence staining, and flow cytometry (FCM) analysis. Western blot was used to detect the expressions levels of apoptosis-related proteins and MAPK-signaling-pathway-related proteins in MM.1S cells induced by α-MMC. The results of the CCK-8 showed that in the concentration range of no significant toxicity to PBMCs, α-MMC inhibited the proliferation of MM.1S cells in a time-dependent and concentration-dependent manner, and the IC50 value was 13.04 and 7.518 μg/mL for 24 and 48 h, respectively. Through inverted microscope imaging, it was observed that α-MMC induced a typical apoptotic morphology in MM.1S cells. The results of the FCM detection and analysis showed that α-MMC could arrest the MM.1S cells cycle at the G2 phase, decrease the MMP, and induce cell apoptosis. Western blot analysis found that α-MMC upregulated the expression levels of Bax, Bid, cleaved caspase-3, and cleaved PARP, and downregulated the expression levels of Mcl-1. At the same time, α-MMC decreased the expression levels of p-c-Raf, p-MEK1/2, p-ERK1/2, p-MSK1, and p-P90RSK, and increased the expression levels of p-p38, p-SPAK/JNK, p-c-Jun, and p-ATF2. The above results suggest that α-MMC can inhibit the proliferation of multiple myeloma MM.1S cells. MAPK cascade signaling is involved in the growth inhibition effect of α-MMC on MM.1S cells via cycle arrest and mitochondrial-pathway-dependent apoptosis.

Low-Temperature Signaling Pathways and Their Signaling Factors in Plant

Low temperature as abiotic stress adversely impacts plant growth and development, and limits the ecological distribution of plants as well. Throughout their long evolutionary history, plants have developed a range of complicated and precise molecular regulatory mechanisms to deal with low-temperature stress, involving the activation of signal transduction pathways and the regulation of related genes. In this review, we provide a systematic summary of the most recent research findings regarding three hypotheses of cellular perception of low-temperature signals and two major intracellular low-temperature signaling pathways, including CBF-dependent signaling pathways and CBF-independent signaling pathways. Focus is placed on the functions of each component of the ICE-CBF-COR signaling cascade as well as their interrelationships. This review concludes that although some progress has been made in the identification, function, and mechanism of low-temperature response genes, their roles in the low-temperature regulatory network and molecular mechanisms still need to be studied in detail, which will be of great significance for improving the low-temperature tolerance of plants and adapting to climate change.

Regulation of enolase activation to promote neural protection and regeneration in spinal cord injury.

Spinal cord injury (SCI) is a debilitating condition characterized by damage to the spinal cord resulting in loss of function, mobility, and sensation with no U.S. Food and Drug Administration-approved cure. Enolase, a multifunctional glycolytic enzyme upregulated after SCI, promotes pro- and anti-inflammatory events and regulates functional recovery in SCI. Enolase is normally expressed in the cytosol, but the expression is upregulated at the cell surface following cellular injury, promoting glial cell activation and signal transduction pathway activation. SCI-induced microglia activation triggers pro-inflammatory mediators at the injury site, activating other immune cells and metabolic events, i.e., Rho-associated kinase, contributing to the neuroinflammation found in SCI. Enolase surface expression also activates cathepsin X, resulting in cleavage of the C-terminal end of neuron-specific enolase (NSE) and non-neuronal enolase (NNE). Fully functional enolase is necessary as NSE/NNE C-terminal proteins activate many neurotrophic processes, i.e., the plasminogen activation system, phosphatidylinositol-4,5-bisphosphate 3-kinase/protein kinase B, and mitogen-activated protein kinase/extracellular signal-regulated kinase. Studies here suggest an enolase inhibitor, ENOblock, attenuates the activation of Rho-associated kinase, which may decrease glial cell activation and promote functional recovery following SCI. Also, ENOblock inhibits cathepsin X, which may help prevent the cleavage of the neurotrophic C-terminal protein allowing full plasminogen activation and phosphatidylinositol-4,5-bisphosphate 3-kinase/mitogen-activated protein kinase activity. The combined NSE/cathepsin X inhibition may serve as a potential therapeutic strategy for preventing neuroinflammation/degeneration and promoting neural cell regeneration and recovery following SCI. The role of cell membrane-expressed enolase and associated metabolic events should be investigated to determine if the same strategies can be applied to other neurodegenerative diseases. Hence, this review discusses the importance of enolase activation and inhibition as a potential therapeutic target following SCI to promote neuronal survival and regeneration.

Trafficking of hormones and trophic factors to secretory and extracellular vesicles: a historical perspective and new hypothesis.

It is well known that peptide hormones and neurotrophic factors are intercellular messengers that are packaged into secretory vesicles in endocrine cells and neurons and released by exocytosis upon the stimulation of the cells in a calcium-dependent manner. These secreted molecules bind to membrane receptors, which then activate signal transduction pathways to mediate various endocrine/trophic functions. Recently, there is evidence that these molecules are also in extracellular vesicles, including small extracellular vesicles (sEVs), which appear to be taken up by recipient cells. This finding raised the hypothesis that they may have functions differentiated from their classical secretory hormone/neurotrophic factor actions. In this article, the historical perspective and updated mechanisms for the sorting and packaging of hormones and neurotrophic factors into secretory vesicles and their transport in these organelles for release at the plasma membrane are reviewed. In contrast, little is known about the packaging of hormones and neurotrophic factors into extracellular vesicles. One proposal is that these molecules could be sorted at the trans-Golgi network, which then buds to form Golgi-derived vesicles that can fuse to endosomes and subsequently form intraluminal vesicles. They are then taken up by multivesicular bodies to form extracellular vesicles, which are subsequently released. Other possible mechanisms for packaging RSP proteins into sEVs are discussed. We highlight some studies in the literature that suggest the dual vesicular pathways for the release of hormones and neurotrophic factors from the cell may have some physiological significance in intercellular communication.

Function-oriented synthesis of Imidazo[1,2-a]pyrazine and Imidazo[1,2-b]pyridazine derivatives as potent PI3K/mTOR dual inhibitors

The PI3K-Akt-mTOR signaling pathway is a highly frequently activated signal transduction pathway in human malignancies, which has been a hot target for anti-tumoral drug discovery. Based on our previous research, a function-oriented synthesis (FOS) of imidazo[1,2-a]pyrazines and imidazo[1,2-b]pyridazines was conducted, and their anticancer activities in vitro and in vivo were evaluated. Among them, compound 42 exhibited excellent dual PI3K/mTOR inhibitory activity, with IC50 values on PI3Kα and mTOR of 0.06 nM and 3.12 nM, respectively, much better than our previous reported compound 15a. Furthermore, compound 42 exhibited significant in vitro and in vivo anti-tumoral activities, great kinase selectivity, low hepatotoxicity, modest plasma clearance and acceptable oral bioavailability, which is a promising PI3K/mTOR targeted anti-cancer drug candidate.

The Anti-Inflammatory Effects of Iberin on TNF-α-Stimulated Human Oral Epithelial Cells: In Vitro Research.

Iberin is a bioactive chemical found in cruciferous plants that has been demonstrated to have anticancer properties. However, there have been no reports on its effects on periodontal resident cells, and many questions remain unanswered. The aim of this study was to examine whether iberin had anti-inflammatory effects on human oral epithelial cells, including influences on signal transduction pathway activation in TNF-α-στιμυλατεd χελλσ. Iberin inhibited the production of interleukin (IL)-6 and C-X-C motif chemokine ligand 10 (CXCL10), as well as the expression of vascular cell adhesion molecule (VCAM)-1, inducible nitric oxide synthase (iNOS), and cyclooxygenase (COX)-2 in tumor necrosis factor (TNF)-α-stimulated TR146 cells, a human oral epithelial cell line. Moreover, iberin administration increased the expression of antioxidant signaling pathways, such as Heme Oxygenase (HO)-1 and NAD(P)H quinone dehydrogenase 1 (NQO1). Furthermore, we found that iberin could inhibit the activation of the nuclear factor (NF)-κB, signal transducer and activator of transcription (STAT)3, and p70S6 kinase (p70S6K)-S6 ribosomal protein (S6) pathways in TNF-α-stimulated TR146 cells. In conclusion, iberin reduced inflammatory mediator expression in human oral epithelial cells by preventing the activation of particular signal transduction pathways.

Targeting feedback activation of signaling transduction pathways to overcome drug resistance in cancer.

Dysregulation or aberrant signaling transduction contributes to tumorigenesis. Targeting these abnormal signaling pathways becomes an effective anticancer strategy. However, feedback activation or crosstalk between signaling pathways drives adaptive drug resistance which causes failure of cancer therapy. In this review article, we summarized treatments that cause feedback activation of AKT, ERK, STAT3, EGFR, FGFR, and HER2/3 signaling pathways and the combination therapy to enhance anti-tumor effect or to overcome drug resistance, to explore the underlying mechanisms that define the protein molecules participated or regulated the feedback activation. In addition, we reviewed clinical trials that employ combination treatments to suppress feedback activation and improve therapeutic efficacy of cancer treatments.

Functions of Nutrient-Sensing Nuclear Receptors in Health.

Nutrients play important roles in the regulation of physiological and pathophysiological events in higher animals. Nuclear receptors (NRs) share a common modular functional structure and constitute a transcription factor superfamily consisting of 48 members in humans. Some NRs are activated by the binding of small lipophilic molecules such as food components including fat-soluble vitamins (vitamins A, K, and D) or lipids (phosphatidylcholine, oleoylethanolamide, or fatty acids). NRs contribute to cell growth, differentiation, or metabolic regulation. Generally, NRs bound to their ligands function as a transcription factors targeting specific DNA sequences in genes. Additionally, ligand-bound NRs mediate the activation of specific intracellular signal transduction pathways. On the other hand, some NRs are functional without binding a ligand. Information on the roles and functions of nutrient-sensing NRs in physiological or pathophysiological events not only leads to an understanding of the need for nutrients, but also contributes to the prevention and amelioration of nutrition-related diseases.

Path Sampling Simulations Reveal How the Q61L Mutation Alters the Dynamics of KRas.

Flexibility is essential for many proteins to function, but can be difficult to characterize. Experiments lack resolution in space and time, while the time scales involved are prohibitively long for straightforward molecular dynamics simulations. In this work, we present a multiple state transition path sampling simulation study of a protein that has been notoriously difficult to characterize in its active state. The GTPase enzyme KRas is a signal transduction protein in pathways for cell differentiation, growth, and division. When active, KRas tightly binds guanosine triphosphate (GTP) in a rigid state. The protein-GTP complex can also visit more flexible states, in which it is not active. KRas mutations can affect the conversion between these rigid and flexible states, thus prolonging the activation of signal transduction pathways, which may result in tumor formation. In this work, we apply path sampling simulations to investigate the dynamic behavior of KRas-4B (wild type, WT) and the oncogenic mutant Q61L (Q61L). Our results show that KRas visits several conformational states, which are the same for WT and Q61L. The multiple state transition path sampling (MSTPS) method samples transitions between the different states in a single calculation. Tracking which transitions occur shows large differences between WT and Q61L. The MSTPS results further reveal that for Q61L, a route to a more flexible state is inaccessible, thus shifting the equilibrium to more rigid states. The methodology presented here enables a detailed characterization of protein flexibility on time scales not accessible with brute-force molecular dynamics simulations.

Small G protein RAC-2 regulates forgetting via the JNK-1 signalling pathway in Caenorhabditis elegans.

Although forgetting was once regarded as a passive decline in memory and an occasional source of embarrassment, recent research suggests that it is an active biological process of removing outdated or irrelevant memories via activation of specific genes and signal transduction pathways. Rho family G proteins are known to have a role in synaptic plasticity mediated by the actin cytoskeleton. However, the current study reveals that another Rho guanosine triphosphate enzyme (GTPase), RAC-2, facilitates the occurrence of forgetting in Caenorhabditis elegans independent of actin dynamics. Functioning downstream of RAC-2 in the same signalling pathway, JNK-1 and its phosphorylated protein are required to positively regulate forgetting. The pan-neuronal rescue of RAC-2 or JNK-1, instead of AWC neuron-specific expression, reverses the delayed forgetting caused by the rac-2 mutation, which indicates that the involvement of RAC-2/JNK-1 in more than AWCs must be required. In summary, our work elucidates the action of the Rho GTPase RAC-2 and downstream JNK-1 as a potential novel pathway in forgetting in C. elegans.

Crosslinking of membrane CD13 in human neutrophils mediates phagocytosis and production of reactive oxygen species, neutrophil extracellular traps and proinflammatory cytokines.

Aminopeptidase N, or CD13, is a cell membrane ectopeptidase highly expressed in myeloid cells. Through its enzymatic activity, CD13 regulates the activity of several bioactive peptides, such as endorphins and enkephalins, chemotactic peptides like MCP-1 and IL-8, angiotensin III, bradikinin, etc. In recent years, it has been appreciated that independently of its peptidase activity, CD13 can activate signal transduction pathways and mediate effector functions such as phagocytosis and cytokine secretion in monocytes and macrophages. Although neutrophils are known to express CD13 on its membrane, it is currently unknown if CD13 can mediate effector functions in these cells. Here, we show that in human neutrophils CD13 can mediate phagocytosis, which is dependent on a signaling pathway that involves Syk, and PI3-K. Phagocytosis mediated by CD13 is associated with production of reactive oxygen species (ROS). The level of phagocytosis and ROS production mediated by CD13 are similar to those through FcγRIII (CD16b), a widely studied receptor of human neutrophils. Also, CD13 ligation induces the release of neutrophil extracellular traps (NETs) as well as cytokine secretion from neutrophils. These results support the hypothesis that CD13 is a membrane receptor able to activate effector functions in human neutrophils.

Infection by Endophytic Epichloë sibirica Was Associated with Activation of Defense Hormone Signal Transduction Pathways and Enhanced Pathogen Resistance in the Grass Achnatherum sibiricum.

Epichloë endophytes can improve the resistance of host grasses to pathogenic fungi, but the underlying mechanisms remain largely unknown. Here, we used phytohormone quantifications, gene expression analysis, and pathogenicity experiments to investigate the effect of Epichloë sibirica on the resistance of Achnatherum sibiricum to Curvularia lunata pathogens. Comparison of gene expression patterns between endophyte-infected and endophyte-free leaves revealed that endophyte infection was associated with significant induction of 1,758 and 765 differentially expressed genes in the host before and after pathogen inoculation, respectively. Functional analysis of the differentially expressed genes suggested that endophyte infection could activate the constitutive resistance of the host by increasing photosynthesis, enhancing the ability to scavenge reactive oxygen species, and actively regulating the expression of genes with function related to disease resistance. We found that endophyte infection was associated with induction of the expression of genes involved in the biosynthesis pathways of jasmonic acid, ethylene, and pipecolic acid and amplified the defense response of the jasmonic acid/ethylene co-regulated EIN/ERF1 transduction pathway and Pip-mediated TGA transduction pathway. Phytohormone quantifications showed that endophyte infection was associated with significant accumulation of jasmonic acid, ethylene, and pipecolic acid after pathogen inoculation. Exogenous phytohormone treatments confirmed that the disease index of plants was negatively related to both jasmonic acid and ethylene concentrations. Our results demonstrate that endophyte infection can not only improve the constitutive resistance of the host to phytopathogens before pathogen inoculation but also be associated with enhanced systemic resistance of the host to necrotrophs after C. lunata inoculation.

Anticancer Perspectives of Sanguinarine: A Review

Background: Sanguinarine, a benzophenanthridine alkaloid extracted from Sanguinaria canadensis has been found to show an inhibitory role on proapoptotic and growth inhibitory activities in various tumor cells of experimental subjects. Objective: This review highlights and summarizes the effects of sanguinarine against cancers alongside the possible mechanisms involved as depicted in various in vitro and in vivo models. Results: Sanguinarine shows anticancer activities via various mechanisms like inhibiting cancer cell proliferation, aberrantly activated signal transduction pathways, sang-mediated angiogenesis, and induction of cell death and tumor suppressors’ expressions. It also enhances the cytotoxic effects of chemotherapeutics via sensitizing cancer cells against these drugs, hence improving their pharmacological attributes. Conclusion: Due to the significant role of sanguinarine against various types of cancer, it can be explored for its potential functions in cancer treatment.

Transcriptional regulation mechanism of wheat varieties with different nitrogen use efficiencies in response to nitrogen deficiency stress

BackgroundAs one of the microelements, nitrogen play essential roles in cereal production. Although the use of chemical fertilizers has significantly improved the yield of wheat, it has also caused increasingly adverse environmental pollution. Revealing the molecular mechanism manipulating wheat nitrogen use efficiency (NUE), and cultivating wheat germplasms with high nitrogen use efficiency has become important goals for wheat researchers. In this study, we investigated the physiological and transcriptional differences of three wheat cultivars with different NUE under low nitrogen stress.ResultsThe results showed that, under low nitrogen conditions, the activities of nitrogen metabolism-related enzymes (GS, NR, GDH), antioxidant enzymes (SOD, POD, CAT) and soluble protein contents of ZM366 (high NUE cultivar) were higher than those of JD8 (low NUE cultivar). The hybrid cultivar of ZM366 and JD8 showed mid-parent or over-parent heterosis. Transcriptome analysis revealed that ‘alanine, aspartate and glutamate metabolism’, ‘terpenoid backbone biosynthesis’ and ‘vitamin B6 metabolism’ pathways play key roles in nitrogen use efficiency in wheat. The significant enhancement of the ‘Calvin cycle’ and ‘photorespiration’ in ZM366 contributed to its higher level of carbon metabolism under low nitrogen stress, which is an important attribute differs from the other two varieties. In addition, the activation of ABA signal transduction and biosynthesis pathways also helps to maintain NUE under low- nitrogen conditions. Moreover, bHLH transcription factors were also found to play a positive role in wheat NUE.ConclusionsIn conclusion, these results enriched our knowledge of the mechanism of wheat NUE, and provided a theoretical basis for improving wheat NUE and breeding new cultivars.

Comparative proteomic analysis identifies potential pathogenicity/virulence factors in Fusarium oxysporum f.sp. ricini inciting vascular wilt disease in castor

Fusarium oxysporum f.sp. ricini (FOR), the causal agent of castor wilt, is the major pathogen of castor. However, there is meagre information regarding FOR pathogenesis and virulence. Development of effective disease management strategies requires a deep understanding of the pathogenicity and virulence factors deployed by the pathogen to infect its host. We have compared the mycelial proteomes of FOR isolates showing differential virulence, to identify potential pathogenicity and virulence factors. Differentially accumulated/upregulated protein spots in the virulent isolate were further identified by MALDI-TOF/TOF. The identified proteins showed homology with the fungal proteins that play crucial roles in pathogen penetration, colonization, invasion, degradation of plant cutin, detoxification of host-generated ROS, activation of signal transduction pathways, stress response, post-translational modification and toxin production. Our study suggested that the identified proteins might contribute to the high degree of virulence of the virulent FOR isolates.

Intracellular Molecular Targets and Signaling Pathways Involved in Antioxidative and Neuroprotective Effects of Cannabinoids in Neurodegenerative Conditions.

In the last few decades, endocannabinoids, plant-derived cannabinoids and synthetic cannabinoids have received growing interest as treatment options in neurodegenerative conditions. In various experimental settings, they have displayed antioxidative, anti-inflammatory, antiapoptotic, immunomodulatory, and neuroprotective effects. However, due to numerous targets and downstream effectors of their action, the cellular and molecular mechanisms underlying these effects are rather complex and still under discussion. Cannabinoids are able to neutralize free radicals and modulate the production of reactive oxygen species and the activity of antioxidative systems acting on CB1 and CB2 cannabinoid receptors. The activation of CB1 receptors stimulates signaling pathways involved in antioxidative defense and survival (such as the phosphoinositide 3-kinase (PI3K)/Akt, mitogen-activated protein kinase (MAPK), and Nrf2 pathways) and regulates glutamatergic signaling, the activation of N-methyl-D-aspartate (NMDA) receptors, calcium influx, and the induction of Ca2+-regulated signaling cascades, whereas the neuroprotective effects mediated by CB2 receptors are due to the suppression of microglial activation and the release of prooxidative and proinflammatory mediators. This review summarizes the main molecular mechanisms and new advances in understanding the antioxidative and neuroprotective effects of cannabinoids. Because of the plethora of possible pharmacological interventions related to oxidative stress and cannabinoid-mediated neuroprotection, future research should be directed towards a better understanding of the interplay between activated signal transduction pathways and molecular targets with the aim to improve treatment options and efficacy by targeting the endocannabinoid system.

Improved diagnosis of inflammatory bowel disease and prediction and monitoring of response to anti-TNF alpha treatment based on measurement of signal transduction pathway activity.

Objective: Ulcerative colitis (UC) and Crohn's disease (CD) are two subtypes of chronic inflammatory bowel disease (IBD). Differential diagnosis remains a challenge. Anti-TNFα treatment is an important treatment for IBD, yet resistance frequently occurs and cannot be predicted. Consequently, many patients receive ineffective therapy with potentially adverse effects. Novel assays are needed to improve diagnosis, and predict and monitor response to anti-TNF-α compounds. Design: Signal transduction pathway (STP) technology was used to quantify activity of STPs (androgen and estrogen receptor, PI3K, MAPK, TGFβ, Notch, Hedgehog, Wnt, NFκB, JAK-STAT1/2, and JAK-STAT3 pathways) in colon mucosa samples of CD and UC patients, based on transcriptome analysis. Previously described STP assay technology is based on computational inference of STP activity from mRNA levels of target genes of the STP transcription factor. Results: Results show that NFκB, JAK-STAT3, Wnt, MAPK, and androgen receptor pathways were abnormally active in CD and UC. Colon and ileum-localized CD differed with respect to STP activity, the JAK-STAT1/2 pathway being abnormally active in ileal CD. High activity of NFκB, JAK-STAT3, and TGFβ pathways was associated with resistance to anti-TNFα treatment in UC and colon-located CD, but not in ileal CD. Abnormal STP activity decreased with successful treatment. Conclusion: We believe that measuring mucosal STP activity provides clinically relevant information to improve differential diagnosis of IBD and prediction of resistance to anti-TNFα treatment in patients with colon-localized IBD, and provides new targets for treatment and overcoming anti-TNFα resistance.

Characterization of S100A12 from nile tilapia (Oreochromis niloticus) and its roles on inflammatory responses

S100A12 is a member of S100 proteins family that induces pro-inflammatory response via ligating with the receptor for advanced glycation end products (RAGE) and subsequent activation of intracellular signal transduction pathways. But information about fish S100A12 remain largely unclear. In this study, the S100A12 homolog (On-S100A12) was identified from Nile tilapia (Oreochromis niloticus). On-S100A12 was mainly expressed in liver and intestine. After Streptococcus agalactiae infection in vivo, S100A12 significantly increased in brain, intestine, liver and head kidney, suggesting S100A12 might played roles in immune response. The further in vitro experiments found that recombinant protein of S100A12 (rOn-S100A12) upregulated the expression of IL1-β, TLR2, TNF-α and inhibited the expression of IL-10, indicating On-S100A12 promoted inflammatory response and activation of M1 macrophages. The present data lay a foundation to further explore the roles of fish S100 during immune defense and will also be beneficial for better understanding of fish immune-regulatory network.

A Temporal Comparative RNA Transcriptome Profile of the Annexin Gene Family in the Salivary versus Lacrimal Glands of the Sjögren's Syndrome-Susceptible C57BL/6.NOD-Aec1Aec2 Mouse.

A generally accepted hypothesis for the initial activation of an immune or autoimmune response argues that alarmins are released from injured, dying and/or activated immune cells, and these products complex with receptors that activate signal transduction pathways and recruit immune cells to the site of injury where the recruited cells are stimulated to initiate immune and/or cellular repair responses. While there are multiple diverse families of alarmins such as interleukins (IL), heat-shock proteins (HSP), Toll-like receptors (TLR), plus individual molecular entities such as Galectin-3, Calreticulin, Thymosin, alpha-Defensin-1, RAGE, and Interferon-1, one phylogenetically conserved family are the Annexin proteins known to promote an extensive range of biomolecular and cellular products that can directly and indirectly regulate inflammation and immune activities. For the present report, we examined the temporal expression profiles of the 12 mammalian annexin genes (Anxa1-11 and Anxa13), applying our temporal genome-wide transcriptome analyses of ex vivo salivary and lacrimal glands from our C57BL/6.NOD-Aec1Aec2 mouse model of Sjögren’s Syndrome (SS), a human autoimmune disease characterized primarily by severe dry mouth and dry eye symptoms. Results indicate that annexin genes Anax1-7 and -11 exhibited upregulated expressions and the initial timing for these upregulations occurred as early as 8 weeks of age and prior to any covert signs of a SS-like disease. While the profiles of the two glands were similar, they were not identical, suggesting the possibility that the SS-like disease may not be uniform in the two glands. Nevertheless, this early pre-clinical and concomitant upregulated expression of this specific set of alarmins within the immune-targeted organs represents a potential target for identifying the pre-clinical stage in human SS as well, a fact that would clearly impact future interventions and therapeutic strategies.

97 (PB087) - The ULK1 inhibitor ENV-201 impairs tumor growth in KRAS-driven flank xenografts as a single agent and in combination with the KRAS inhibitor adagrasib

Background: Lung cancer is the leading cancer cause of death in the US, and non-small cell lung cancers (NSCLCs) account for 85% of those cases. Among NSCLCs, mutations resulting in ectopic activation of the RAS signal transduction pathway account for at least a quarter of cases, and the activating KRASG12Cmutation is found in 11% of all NSCLC patient samples. When treated with RAS pathway-inhibiting therapeutics, KRAS-driven tumors have been shown to become dependent on autophagy for continued survival. Simultaneous targeting of both the RAS pathway and autophagy therefore presents an opportunity to block multiple mechanisms of tumor growth and survival. Materials and methods: We have demonstrated the added benefit of KRAS inhibition and autophagy inhibition in an animal model, using the NCI-H2122 cell line (homozygous for the activating KRASG12C mutation) as flank xenografts in NOD/SCID mice. ENV-201 is a selective and potent antagonist of the autophagy-initiating kinase, ULK1, one which also inhibits the related ULK2. Mice were treated via daily oral dosing of (1) vehicle, (2) ENV-201 (20 mg/kg), (3) the KRASG12C-specific inhibitor adagrasib (Mirati Therapeutics, 25 mg/kg), or (4) combination of both ENV-201 and adagrasib at the same doses. Treatment lasted 30 days, body weight was monitored daily, and tumor size was measured three times a week. All animal work was carried out at and met necessary approvals at Crown Bioscience. Results: Both ENV-201 and adagrasib treatments resulted in significant tumor growth inhibition when either drug was used as a single agent (32% or 55% TGI, respectively). Significant additive tumor growth inhibition (78%) was also observed when animals were treated with both drugs in combination. Conclusions: ENV-201-mediated ULK1 inhibition thus shows promise as both a single agent or in combination with a growth factor/RAS/MAPK pathway-targeting cancer treatment. Conflict of interest: Corporate-sponsored Research: All authors are employees of Endeavor BioMedicines.