Efficacy Of Targeted Therapeutics Research Articles

Genetic assessment of efficacy and safety profiles of coagulation cascade proteins identifies Factors II and XI as actionable anticoagulant targets.

Anticoagulants are routinely used by millions of patients worldwide to prevent blood clots. Yet, problems with anticoagulant therapy remain, including a persistent and cumulative bleeding risk in patients undergoing prolonged anticoagulation. New safer anticoagulant targets are needed. To prioritize anticoagulant targets with the strongest efficacy [venous thromboembolism (VTE) prevention] and safety (low bleeding risk) profiles, we performed two-sample Mendelian randomization and genetic colocalization. We leveraged three large-scale plasma protein data sets (deCODE as discovery data set and Fenland and Atherosclerosis Risk in Communities as replication data sets] and one liver gene expression data set (Institut Universitaire de Cardiologie et de Pneumologie de Québec bariatric biobank) to evaluate evidence for a causal effect of 26 coagulation cascade proteins on VTE from a new genome-wide association meta-analysis of 44 232 VTE cases and 847 152 controls, stroke subtypes, bleeding outcomes, and parental lifespan as an overall measure of efficacy/safety ratio. A 1 SD genetically predicted reduction in F2 blood levels was associated with lower risk of VTE [odds ratio (OR) = 0.44, 95% confidence interval (CI) = 0.38-0.51, P = 2.6e-28] and cardioembolic stroke risk (OR = 0.55, 95% CI = 0.39-0.76, P = 4.2e-04) but not with bleeding (OR = 1.13, 95% CI = 0.93-1.36, P = 2.2e-01). Genetically predicted F11 reduction was associated with lower risk of VTE (OR = 0.61, 95% CI = 0.58-0.64, P = 4.1e-85) and cardioembolic stroke (OR = 0.77, 95% CI = 0.69-0.86, P = 4.1e-06) but not with bleeding (OR = 1.01, 95% CI = 0.95-1.08, P = 7.5e-01). These Mendelian randomization associations were concordant across the three blood protein data sets and the hepatic gene expression data set as well as colocalization analyses. These results provide strong genetic evidence that F2 and F11 may represent safe and efficacious therapeutic targets to prevent VTE and cardioembolic strokes without substantially increasing bleeding risk.

Single-cell transcriptome analysis profiling lymphatic invasion-related TME in colorectal cancer.

Lymphatic invasion (LI) is extremely aggressive and induces worse prognosis among patients with colorectal cancer (CRC). Thus, it is critical to characterize the cellular and molecular mechanisms underlying LI in order to establish novel and efficacious therapeutic targets that enhance the prognosis of CRC patients. RNA-seq data, clinical and survival information of colon adenocarcinoma (COAD) patients were obtained from the TCGA database. In addition, three scRNA-seq datasets of CRC patients were acquired from the GEO database. Data analyses were conducted with the R packages. We assessed the tumor microenvironment (TME) differences between LI+ and LI- based scRNA-seq data, LI+ cells exhibited augmented abundance of immunosuppression and invasive subset. Marked extracellular matrix network activation was also observed in LI+ cells within SPP1+ macrophages. We revealed that an immunosuppressive and pro-angiogenic TME strongly enhanced LI, as was evidenced by the CD4+ Tregs, CD8+ GZMK+, SPP1+ macrophages, e-myCAFs, and w-myCAFs subcluster infiltrations. Furthermore, we identified potential LI targets that influenced tumor development, metastasis, and immunotherapeutic response. Finally, a novel LIRS model was established based on the expression of 14 LI-related signatures, and in the two testing cohorts, LIRS was also proved to have accurate prognostic predictive ability. In this report, we provided a valuable resource and extensive insights into the LI of CRC. Our conclusions can potentially benefit the establishment of highly efficacious therapeutic targets as well as diagnostic biomarkers that improve patient outcomes.

Restoring glucose balance: Conditional HMGB1 knockdown mitigates hyperglycemia in a Streptozotocin induced mouse model

Diabetes mellitus (DM) poses a significant global health burden, with hyperglycemia being a primary contributor to complications and high morbidity associated with this disorder. Existing glucose management strategies have shown suboptimal effectiveness, necessitating alternative approaches. In this study, we explored the role of high mobility group box 1 (HMGB1) in hyperglycemia, a protein implicated in initiating inflammation and strongly correlated with DM onset and progression. We hypothesized that HMGB1 knockdown will mitigate hyperglycemia severity and enhance glucose tolerance. To test this hypothesis, we utilized a novel inducible HMGB1 knockout (iHMGB1 KO) mouse model exhibiting systemic HMGB1 knockdown. Hyperglycemic phenotype was induced using low dose streptozotocin (STZ) injections, followed by longitudinal glucose measurements and oral glucose tolerance tests to evaluate the effect of HMGB1 knockdown on glucose metabolism. Our findings showed a substantial reduction in glucose levels and enhanced glucose tolerance in HMGB1 knockdown mice. Additionally, we performed RNA sequencing analyses, which identified potential alternations in genes and molecular pathways within the liver and skeletal muscle tissue that may account for the in vivo phenotypic changes observed in hyperglycemic mice following HMGB1 knockdown. In conclusion, our present study delivers the first direct evidence of a causal relationship between systemic HMGB1 knockdown and hyperglycemia in vivo, an association that had remained unexamined prior to this research. This discovery positions HMGB1 knockdown as a potentially efficacious therapeutic target for addressing hyperglycemia and, by extension, the DM epidemic. Furthermore, we have revealed potential underlying mechanisms, establishing the essential groundwork for subsequent in-depth mechanistic investigations focused on further elucidating and harnessing the promising therapeutic potential of HMGB1 in DM management.

Prognostic value and immune landscapes of TERT promoter methylation in triple negative breast cancer.

Treatment options for patients with triple-negative breast cancer (TNBC) remain limited to mainstay therapies owing to a lack of efficacious therapeutic targets. Accordingly, there is an urgent need to discover and identify novel molecular targets for the treatment and diagnosis of this disease. In this study, we analyzed the correlation of telomerase reverse transcriptase (TERT) methylation status with TERT expression, prognosis, and immune infiltration in TNBC and identified the role of TERT methylation in the regulation TNBC prognosis and immunotherapy. Data relating to the transcriptome, clinicopathological characteristics and methylation of TNBC patients were obtained from The Cancer Genome Atlas (TCGA) database. TERT expression levels and differential methylation sites (DMSs) were detected. The correlations between TERT expression and DMSs were calculated. Kaplan-Meier curves was plotted to analyze the relationship between the survival of TNBC patients and the DMSs. The correlations of DMSs and TERT expression with several immunological characteristics of immune microenvironment (immune cell infiltration, immunomodulators, immune-related biological pathways, and immune checkpoints) were assessed. The results were validated using 40 TNBC patients from Sun Yat-sen University Cancer Center (SYSUCC). Six DMSs were identified. Among them, four sites (cg11625005, cg07380026, cg17166338, and cg26006951) were within the TERT promoter, in which two sites (cg07380026 and cg26006951) were significantly related to the prognosis of patients with TNBC. Further validation using 40 TNBC samples from SYSUCC showed that the high methylation of the cg26006951 CpG site was associated with poor survival prognosis (P=0.0022). TERT expression was significantly correlated with pathological N stage and clinical stage, and cg07380026 were significantly associated with pathological T and N stages in the TCGA cohort. Moreover, the methylation site cg26006951, cg07380026 and TERT expression were significantly correlated with immune cell infiltration, common immunomodulators, and the level of the immune checkpoint receptor lymphocyte activation gene 3 (LAG-3) in TNBC patients. TERT promotertypermethylation plays an important role in TERT expression regulation and tumor microenvironment in TNBC. It is associated with overall survival and LAG-3 expression. TERT promoter hypermethylation may be a potential molecular biomarker for predicting response to the TERT inhibitors and immune checkpoint inhibitors in TNBC.

Ameliorating the Role of Aripiprazole in Memory Deficits Induced by Intracerebroventricular Streptozotocin-Induced Dementia of Alzheimer's Type.

Alzheimer's disease (AD) is a progressive neurodegenerative disorder causing immense suffering for the patients. Dopamine D2 and 5-hydroxytryptamine receptor 1A (5-HT1A) receptors' activation has been reported to play a crucial role in managing neurological outcomes in the brain and other health disorders. This study aimed to investigate the role of aripiprazole, a dopamine D2 and 5-HT1A selective receptors' activator, in the restoration of memory deficit induced by streptozotocin in mice. The cognitive functions of animals were determined using the Morris water maze. Brain sections were stained with hematoxylin and eosin and Congo red to examine the structural deviations. Brain oxidative stress (thiobarbituric acid reactive substance and glutathione), acetylcholinesterase activity, IL-6, and IL-10 were measured to assess biochemical alterations. Activation of D2 and 5-HT1A with aripiprazole attenuated STZ-induced cognitive deficit, increased brain GSH levels, reduced TBARS levels, AChE activity, IL-6 levels, and IL-10 levels and prevented STZ-induced brain anomalies in mice. Hence, the present study concluded that aripiprazole mitigated STZ-induced memory impairment and can be used as an efficacious therapeutic target for the management of AD.

Tumour necrosis factor receptor 1 inhibition and cardiovascular disease: a cis-Mendelian randomization study

Abstract Funding Acknowledgements Type of funding sources: Public grant(s) – National budget only. Main funding source(s): National Institute for Health and Care research (NIHR). Background Tumour necrosis factor receptor 1 (TNFR1) is a newly proposed drug target that has emerged from a growing understanding of the inflammatory basis of cardiovascular disease[1]. A range of pre-clinical models and observational data in humans support the potential efficacy of TNFR1 inhibition. However, pre-clinical models are poor predictors of therapeutic efficacy in humans and observational data is inherently vulnerable to confounding and reverse causation[2,3]. Mendelian randomization (MR) is a genetic epidemiological approach that overcome these limitations and are capable of robust causal inference[3], rendering them better able to investigate the potential efficacy of novel drug targets[2]. Purpose To leverage MR to investigate the causal effect of TNFR1 inhibition on the risk of developing coronary artery disease (CAD), ischaemic stroke (IS), atrial fibrillation (AF), and heart failure (HF) (see Figure 1). Methods cis-Mendelian randomization was performed to estimate the causal effect of TNFR1 inhibition (weighted by serum C-reactive protein (CRP) levels) on liability to coronary artery disease (CAD), ischaemic stroke (IS), atrial fibrillation (AF), and heart failure (HF). Anti-TNF therapies are a cornerstone treatment modality for Crohn’s disease and so Crohn’s was included as a positive control to validate the genetic instrument for TNFR1 inhibition. Furthermore, Bayesian colocalization (coloc) was performed to assess the likelihood that a shared causal variant underlies both TNFR1 signalling and each of these four cardiovascular diseases, thus allowing for interrogation of the risk of confounding by linkage disequilibrium in MR estimates[4]. Genetic association estimates were leveraged from the largest genome-wide association studies (GWAS) for which full publicly available summary data were available. Results As expected, a significant cis-MR effect for genetically proxied TNFR1 inhibition was observed for Crohn’s disease (odds ratio (OR) per 1 standard deviation (SD) reduction in CRP = 0.05, 95% CI = 0.01 to 0.28, P = 5.32 x 10-4). However, no evidence of a causal association was observed between genetically proxied TNFR1 inhibition and the odds of developing CAD (OR per SD reduction in CRP = 0.99, 95% CI = 0.55 to 1.76), IS (OR per SD reduction in CRP = 1.55, 95% CI = 0.30 to 8.03), AF (OR per SD reduction in CRP = 1.11, 95% CI = 0.54 to 2.29), or HF (OR per SD reduction in CRP = 1.02, 95% CI = 0.46 to 2.29) (see Figure 2). No evidence of colocalization was observed between TNFR1 signalling and any of these four cardiovascular diseases. Conclusions These findings do not support TNFR1 signalling inhibition as likely to be an efficacious therapeutic target for coronary artery disease, ischaemic stroke, atrial fibrillation, or heart failure.

Leveraging genetic predictors of factor XI levels to anticipate results from clinical trials.

Factor XI (FXI) is a promising therapeutic target for the prevention of thrombotic disease without increasing bleeding risk. We performed Mendelian randomization (MR) analyses to investigate the association of genetically predicted reductions in FXI levels with risk of venous thromboembolism, ischemic stroke, bleeding outcomes, and lifespan. Genetically predicted reductions in FXI levels were associated with lower risk of ischemic stroke (odds ratio per 1 standard deviation (SD) lower serum FXI 0.90, 95% confidence interval 0.87-0.93, p = 1.59 × 10-11 ), and venous thromboembolism (0.54, 0.49-0.59, p = 2.13 × 10-39 ) but did not associate with increased bleeding risk (p > 0.16). Genetically predicted reductions in serum FXI levels associated with longer lifespan (0.37 years per 1 SD lower serum FXI, 0.13-0.61, p = 0.003). These genetic data support FXI as a potentially efficacious and safe therapeutic target and anticipate positive results from ongoing phase 3 clinical trials.

Psoriatic Arthritis: Pathogenesis and Targeted Therapies.

Psoriatic arthritis (PsA), a heterogeneous chronic inflammatory immune-mediated disease characterized by musculoskeletal inflammation (arthritis, enthesitis, spondylitis, and dactylitis), generally occurs in patients with psoriasis. PsA is also associated with uveitis and inflammatory bowel disease (Crohn's disease and ulcerative colitis). To capture these manifestations as well as the associated comorbidities, and to recognize their underlining common pathogenesis, the name of psoriatic disease was coined. The pathogenesis of PsA is complex and multifaceted, with an interplay of genetic predisposition, triggering environmental factors, and activation of the innate and adaptive immune system, although autoinflammation has also been implicated. Research has identified several immune-inflammatory pathways defined by cytokines (IL-23/IL-17, TNF), leading to the development of efficacious therapeutic targets. However, heterogeneous responses to these drugs occur in different patients and in the different tissues involved, resulting in a challenge to the global management of the disease. Therefore, more translational research is necessary in order to identify new targets and improve current disease outcomes. Hopefully, this may become a reality through the integration of different omics technologies that allow better understanding of the relevant cellular and molecular players of the different tissues and manifestations of the disease. In this narrative review, we aim to provide an updated overview of the pathophysiology, including the latest findings from multiomics studies, and to describe current targeted therapies.

Glucosylceramide in T cells regulates the pathology of inflammatory bowel disease

Inflammatory bowel disease (IBD) is a chronic inflammatory disease in the colon characterized by excessive activation of T cells. Glycosphingolipids (GSLs) are composed of lipid rafts in cellular membranes, and their content is linked to immune cell function. In the present study, we investigated the involvement of GSLs in IBD. Microarray data showed that in IBD patients, the expression of only UDP-glucose ceramide glucosyltransferase (UGCG) decreased among the GSLs synthases. Ad libitum access to dextran sulfate sodium (DSS) resulted in decreased UGCG and glucosylceramide (GlcCer) content in mesenteric lymph nodes and T cells from the spleen. Furthermore, the knockdown of Ugcg in T cells exacerbated the pathogenesis of colitis, which was accompanied by a decrease in Treg levels. Treatment with GlcCer nanoparticles prevented DSS-induced colitis. These results suggested that GlcCer in T cells is involved in the pathogenesis of IBD. Furthermore, GlcCer nanoparticles are a potential efficacious therapeutic target for IBD patients.

Dysfunctional lactate metabolism in human alveolar type II cells from idiopathic pulmonary fibrosis lung explant tissue

BackgroundIdiopathic Pulmonary Fibrosis (IPF) is the most common and progressive form of the interstitial lung diseases, leading most patients to require lung transplants to survive. Despite the relatively well-defined role of the fibroblast in the progression of IPF, it is the alveolar type II epithelial cell (AEC2) that is now considered the initiation site of damage, driver of disease, and the most efficacious therapeutic target for long-term resolution. Based on our previous studies, we hypothesize that altered lactate metabolism in AEC2 plays a pivotal role in IPF development and progression, affecting key cellular and molecular interactions within the pulmonary microenvironment.MethodsAEC2s isolated from human patient specimens of non-fibrotic and IPF lungs were used for metabolic measurements, lactate dehydrogenase (LDH) analyses and siRNA-mediated knockdown experiments.ResultsAEC2s isolated from human IPF lung explant tissues had lower rates of oxidative metabolism and were more glycolytic lactate-producing cells than were AEC2 from control, non-fibrotic lung explant tissues. Consistent with this shift in metabolism, patient-derived IPF AEC2s exhibited LDH tetramers that have higher ratios of LDHA:LDHB (i.e., favoring pyruvate to lactate conversion) than control AEC2s. Experimental manipulation of LDHA subunit expression in IPF AEC2s restored the bioenergetic profile characteristic of AEC2 from non-fibrotic lungs.ConclusionsThese results are consistent with the concept that altered lactate metabolism may be an underlying feature of AEC2 dysfunction in IPF and may be a novel and important target for therapeutic treatment.

Periplogenin suppresses the growth of esophageal squamous cell carcinoma in vitro and in vivo by targeting STAT3.

The mortality rate of esophageal squamous cell carcinoma (ESCC) is higher than that of other cancers worldwide owing to a lack of therapeutic targets and related drugs. This study aimed to find new drugs by targeting an efficacious therapeutic target in ESCC patients. Signal transducer and activator of transcription 3 (STAT3) is hyperactive in ESCC. Herein, we identified a novel STAT3 inhibitor, periplogenin, which strongly inhibited phosphorylation of STAT3 at Tyr705. Docking models and pull-down assays revealed that periplogenin bound directly and specifically to STAT3, leading to significant suppression of subsequent dimerization, nuclear import, and transcription activities. In addition, STAT3 knockdown cell lines were insensitive to periplogenin, whereas in contrast, STAT3-overexpressing cells were more sensitive to periplogenin, indicating that STAT3 was a target of periplogenin. Intraperitoneally administered periplogenin exhibited efficacious therapeutic effects in ESCC patient-derived xenograft models and dramatically impaired the phosphorylation of STAT3 and expression levels of STAT3-mediated downstream genes. Thus, our study demonstrated that periplogenin acted as a new STAT3 inhibitor, suppressing the growth of ESCC in vitro and in vivo, providing a basis for its potential application in ESCC treatment and prevention.

High levels of human epididymis protein 4 mRNA and protein expression are associated with chemoresistance and a poor prognosis in pancreatic cancer.

Pancreatic cancer is associated with an exceedingly poor prognosis, warranting the development of novel therapeutic strategies and discovery of prognostic predictors. Given that chemoresistance-related molecules are reportedly associated with the poor prognosis of pancreatic cancer, the present study aimed to identify molecules that could be efficacious therapeutic targets for pancreatic cancer. First, 10 patient-derived xenografts (PDXs) were established from patients with pancreatic cancer. Subsequently, after treating tumor tissue generated from the PDXs with standard drugs, next-generation sequencing (NGS) was performed using these tissues. The results of NGS analysis and immunohistochemical analysis on 80 pancreatic cancer tissues revealed that human epididymis protein 4 (HE4) expression in the anticancer drug-treated PDX group was higher than that in the untreated PDXs. In addition, chemoresistance ability was observed in tumor cell lines overexpressing HE4. Furthermore, Kaplan-Meier analysis of tumor tissues from 80 patients with pancreatic cancer was performed and it was found that patients with a high HE4 expression level had a poor survival rate compared with those who had a low HE4 expression level. Multivariate analysis also indicated the high expression level of HE4 was an independent poor prognostic biomarker. Thus, it was concluded that high gene and protein expression levels of HE4 mediate chemoresistance and are independent prognostic factors for pancreatic cancer.

Therapeutic targeting of nitroglycerin-mediated trigeminovascular neuronal hypersensitivity predicts clinical outcomes of migraine abortives.

Cranial hypersensitivity is a prominent symptom of migraine, exhibited as migraine headache exacerbated with physical activity, and cutaneous facial allodynia and hyperalgesia. The underlying mechanism is believed to be, in part, activation and sensitization of dural-responsive trigeminocervical neurons. Validated preclinical models that exhibit this phenotype have great utility for understanding putative mechanisms and as a tool to screen therapeutics. We have previously shown that nitroglycerin triggers cranial allodynia in association with migraine-like headache, and this translates to neuronal cranial hypersensitivity in rats. Furthermore, responses in both humans and rats are aborted by triptan administration, similar to responses in spontaneous migraine. Here, our objective was to study the nitroglycerin model examining the effects on therapeutic targets with newly approved treatments, specifically gepants and ditans, for the acute treatment of migraine. Using electrophysiological methods, we determined changes to ongoing firing and somatosensory-evoked cranial sensitivity, in response to nitroglycerin, followed by treatment with a calcitonin gene-related peptide receptor antagonist, gepant (olcegepant), a 5-HT1F receptor agonist, ditan (LY344864), and an NK1 receptor antagonist (GR205171). Nitroglycerin induced activation of migraine-like central trigeminocervical neurons, and intracranial and extracranial neuronal hypersensitivity. These responses were aborted by olcegepant and LY344864. However, GR205171, which failed in clinical trial for both abortive and preventive treatment of migraine, had no effect. These data support the nitroglycerin model as a valid approach to study cranial hypersensitivity and putative mechanisms involved in migraine and as a screen to dissect potentially efficacious migraine therapeutic targets.

Periostin: A Potential Therapeutic Target For Pulmonary Hypertension?

POSTN (Periostin) is an ECM (extracellular matrix) protein involved in tissue remodeling in response to injury and a contributing factor in tumorigenesis, suggesting that POSTN plays a role in the pathogenesis of pulmonary hypertension (PH). We aimed to gain insight into the mechanistic contribution of POSTN in experimental mouse models of PH and correlate these findings with PH in humans. We used genetic epistasis approaches in human pulmonary artery endothelial cells (hPAECs), human pulmonary artery smooth muscle cells, and experimental mouse models of PH (Sugen 5416/hypoxia or chronic hypoxia) to discern the role of POSTN and its relationship to HIF (hypoxia-inducible factor)-1α signaling. We found that POSTN expression was correlated with the extent of PH in mouse models and in humans. Decreasing POSTN improved hemodynamic and cardiac responses in PH mice, blunted the release of growth factors and HIF-1α, and reversed the downregulated BMPR (bone morphogenetic protein receptor)-2 expression in hPAECs from patients with PH, whereas increasing POSTIN had the opposite effects and induced a hyperproliferative and promigratory phenotype in both hPAECs and human pulmonary artery smooth muscle cells. Overexpression of POSTN-induced activation of HIFs and increased the production of ET (endothelin)-1 and VEGF (vascular endothelial growth factor) in hPAECs. SiRNA-mediated knockdown of HIF-1α abolished the proangiogenic effect of POSTN. Blockade of TrkB (tyrosine kinase receptor B) attenuated the effect of POSTN on HIF-1α expression, while inhibition of HIF-1α reduced the expression of POSTN and TrkB. These results suggest that hPAECs produce POSTN via a HIF-1α-dependent mechanism. Our study reveals that POSTN expression is increased in human and animal models of PH and fosters PH development via a positive feedback loop between HIF-1α and POSTN during hypoxia. We propose that manipulating POSTIN expression may be an efficacious therapeutic target in the treatment of PH. Our results also suggest that POSTN may serve as a biomarker to estimate the severity of PH.

CGAS-STING pathway in oncogenesis and cancer therapeutics.

The host innate immunity offers the first line of defense against infection. However, recent evidence shows that the host innate immunity is also critical in sensing the presence of cytoplasmic DNA derived from genomic instability events, such as DNA damage and defective cell cycle progression. This is achieved through the cyclic GMP-AMP synthase (cGAS)/Stimulator of interferon (IFN) genes (STING) pathway. Here we discuss recent insights into the regulation of this pathway in cancer immunosurveillance, and the downstream signaling cascades that coordinate immune cell recruitment to the tumor microenvironment to destroy transformed cells through cellular senescence or cell death programs. Its central role in immunosurveillance positions the cGAS-STING pathway as an attractive anti-cancer immunotherapeutic drug target for chemical agonists or vaccine adjuvants and suggests a key node to be targeted in a synthetic lethal approach. We also discuss adaptive mechanisms used by cancer cells to circumvent cGAS-STING signaling and present evidence linking chronic cGAS-STING activation to inflammation-induced carcinogenesis, cautioning against the use of activating the cGAS-STING pathway as an anti-tumor immunotherapy. A deeper mechanistic understanding of the cGAS-STING pathway will aid in the identification of potentially efficacious anti-cancer therapeutic targets.

DJ-1 exerts anti-inflammatory effects and regulates NLRX1-TRAF6 via SHP-1 in stroke

BackgroundAcute inflammation induced by reactive astrocytes after cerebral ischemia/reperfusion (I/R) injury is important for protecting the resultant lesion. Our previous study demonstrated that DJ-1 is abundantly expressed in reactive astrocytes after cerebral I/R injury. Here, we show that DJ-1 negatively regulates the inflammatory response by facilitating the interaction between SHP-1 and TRAF6, thereby inducing the dissociation of NLRX1 from TRAF6.MethodsWe used oxygen-glucose deprivation/reoxygenation (OGD/R) in vitro in primary astrocyte cultures and transient middle cerebral artery occlusion/reperfusion (MCAO/R) in vivo to mimic I/R insult.ResultsThe inhibition of DJ-1 expression increased the expression of the inflammatory cytokines TNF-α, IL-1β, and IL-6. DJ-1 knockdown facilitated the interaction between NLRX1 and TRAF6. However, the loss of DJ-1 attenuated the interaction between SHP-1 and TRAF6. In subsequent experiments, a SHP-1 inhibitor altered the interaction between SHP-1 and TRAF6 and facilitated the interaction between NLRX1 and TRAF6 in DJ-1-overexpressing astrocytes.ConclusionThese findings suggest that DJ-1 exerts an SHP-1-dependent anti-inflammatory effect and induces the dissociation of NLRX1 from TRAF6 during cerebral I/R injury. Thus, DJ-1 may be an efficacious therapeutic target for the treatment of I/R injury.

Targeting NMNAT1 in Acute Myeloid Leukemia

Acute myeloid leukemia (AML) is primarily a disease of older adults with poor treatment outcomes. Despite years of intensive research, the standard induction therapy for AML has remained largely unchanged for decades. Thus, the development of new and efficacious therapeutic targets for AML is urgently needed. Leukemia cells exhibit multiple metabolic aberrations that may be therapeutically targeted. Here, we show that nicotinamide adenine dinucleotide (NAD+) promotes leukemogenesis and causes chemotherapy treatment resistance through fueling energetic metabolism, and pinpoints nicotinamide nucleotide adenylyltransferase 1 (NMNAT1) is a novel therapeutic target for AML. To identify novel genes essential for AML, we performed a whole genome CRISPR dropout screen by using MOLM13 cell line and identified 1,951 essential genes (Fig. A). By searching druggable targets among these genes, we narrowed down to 345 genes, among which we found two genes, NMNAT1 (nicotinamide nucleotide adenylyltransferase 1) and NAMPT (nicotinamide phosphoribosyltransferase), both involved in key steps in NAD+ biosynthesis. We comprehensively analyzed dependency scores for all genes involved in the NAD+ biosynthetic pathways (de novo synthesis pathway, the Preiss-Handler pathway and the salvage pathway) across a broad panel of cancer cell lines from the Dependency Map database (https://depmap.org/portal/). The results showed that NMNAT1 and NAMPT are both strongly selective and uniquely required for hematological malignancies compared to other cancers (Fig. B). Since little success has been achieved for NAMPT inhibitors in clinical trials, our attention was drawn to NMNAT1, which encodes a nuclear localized enzyme catalyzing the final step in NAD+ biosynthesis. We confirmed that deletion of NMNAT1 in AML cells significantly reduced nuclear NAD+ level and cell viability over time while sparing normal hematopoietic progenitor cells, suggesting that NMNAT1 is targetable to AML. Overexpression of wild-type Nmnat1 but not the enzymatically inactive forms rescued NMNAT1-KO AML, indicating that the catalytic activity of NMNAT1 is required for AML. To study the role of NAD+ in AML, we first measured NAD+ levels in leukemic and normal cells, and found higher NAD+ levels in leukemia-initiating cells from a murine MLL-AF9-induced AML model compared to normal cells. Supplementation of NAD+ metabolites (NMN, NAM and NR) increased AML proliferation, enhanced glycolysis (lactate production) and oxidative phosphorylation (ATP production), resulting in chemotherapy resistance (Fig. C). Deletion of NMNAT1 sensitized AML cell to chemotherapy treatment. To study the role of NMNAT1 in leukemogenesis in vivo, we genetically deleted NMNAT1 in murine or human leukemia cells, transplanted them into recipient mice, and found that deletion of NMNAT1 reduced leukemic burden and extended leukemia-free survival (Fig. D). Finally, to reveal the molecular mechanisms underlying NMNAT1 KO-mediated cell death (increased levels of gamma-H2AX), RNA-seq and functional assay of NAD+ dependent enzymes were performed. We found that the reduction of nuclear NAD+ resulting from NMNAT1 deletion upregulated genes involved in DNA repair pathway, which may be linked to impaired PARPs and Sirtuins activity. Our findings reveal the important function of NAD+ in leukemogenesis and chemoresistance, and identify NMANT1 as a novel therapeutic target for AML. Figure Disclosures No relevant conflicts of interest to declare.

Targeted Therapeutics for Transthyretin amyloid cardiomyopathy: Time to Move Forward

Introduction Deposition of wild type or mutant transthyretin (TTR) amyloid fibrils causes TTR cardiac amyloidosis (ATTR-CA). Targeted therapeutics (TT) for ATTR-CA include TTR stabilizers (tafamidis, dilfunisal) and oligonucleotide drugs (Revusiran, Patisiran and Inotersen).TTR stabilizers prevent dissociation of amyloid tetramers and thereby limit the deposition of amyloid fibrils. Oligonucleotide drugs inhibit hepatic synthesis of transthyretin. Recent studies have demonstrated improved outcomes with TT in patients with ATTR-CA. Hypothesis We performed a pooled analysis to study the safety and efficacy of TT in patients with ATTR-CA. Methods We searched PubMed for all studies comparing the safety and efficacy of targeted therapeutics (TT) with placebo in patients with ATTR-CA. Primary outcomes were 1) all-cause mortality, 2) cardiac hospitalizations, 3) serious adverse events and 4) change in global longitudinal strain. Random effects model with Mantel-Haenszel method was used to analyze the data. Results We identified six studies which reported at least one of the outcomes comparing TT and placebo. One study was stopped prematurely due to increased mortality in the TT arm. Pooled analysis included 1164 patients, of which 691 patients received TT and 473 patients received placebo. When compared to placebo, TT significantly reduced all-cause mortality [OR 0.39 (95% CI: 0.16 - 0.97, P =0.04)]. Pooled analysis after exclusion of aforementioned prematurely terminated study also showed decrease in all-cause mortality with TT[OR 0.27 (95% CI: 0.10 - 0.71, P =0.008)]. There was no difference in serious adverse events with TT [OR 1.06 (95% CI: 0.78 - 1.44, P =0.72)]. A trend towards improvement in global longitudinal strain with TT (mean difference -0.69 (95% CI: -1.44 - 0.05, P =0.07) was noted. Only two studies reported the effect on cardiac hospitalizations. Conclusions In patients with ATTR-CA, TT reduced all-cause mortality and were well-tolerated as compared to placebo.

Systemic Inhibition of Soluble Tumor Necrosis Factor with XPro1595 Exacerbates a Post-Spinal Cord Injury Depressive Phenotype in Female Rats.

Spinal cord injury (SCI) is associated with a three-fold risk of major depressive disorder compared with the general population. Current antidepressant therapy is often not as effective in this patient population, suggesting the need for a more efficacious therapeutic target. The goal of this study was to elucidate the role of inflammatory cytokine tumor necrosis factor (TNF) in the dorsal raphe nucleus (DRN, the principle source of serotonin to the brain) in the development and possible treatment of depression after SCI. A depressive phenotype following moderate T9 contusion was identified in adult female rats using a battery of behavioral tests (forced swim test, sucrose preference test, novel object recognition test, open field locomotion, and social exploration). Data revealed two clusters of injured rats (58%) that exhibit increased immobility in the forced swim test, indicating depressive phenotype or a melancholic-depressive phenotype with concomitant decrease in sucrose preference. ElevatedTNF levels in the DRN of these two clusters correlated with increased immobility in the forced swim test. We then tested the efficacy of soluble TNF inhibition with XPro1595 treatment to prevent the depressive phenotype after SCI. Subcutaneous (s.c.) delivery of XPro1595 caused an exacerbation of depressive phenotype, with all treated clusters exhibiting increased forced swim immobility compared with saline-treated non-depressed rats. Intracerebroventricular (i.c.v.) administration of the drug did not prevent or enhance the development of depression after injury. These results suggest a complex role for TNF-based neuroinflammation in SCI-induced depression that needs to be further explored, perhaps in conjunction with a broader targeting of additional post-SCI inflammatory cytokines.

Deciphering the Allosteric Effect of Antagonist Vismodegib on Smoothened Receptor Deactivation Using Metadynamics Simulation.

The smoothened receptor (Smo) plays a key role in Hedgehog (Hh) signaling pathway and it has been regarded as an efficacious therapeutic target for basal cell carcinoma (BCC) and medulloblastoma (MB). Nevertheless, the resistance mutation and active mutants of Smo have put forward the requirement of finding more effective inhibitors. Herein, we performed metadynamics simulations on Smo bound with vismodegib (Smo-Vismod) and with cholesterol (Smo-CLR), respectively, to explore the inhibition mechanism of vismodegib. The simulation results indicated that vismodegib-induced shifts of TM5, TM6, and TM7, which permitted the extracellular extension of TM6 and extracellular loop3 (ECL3) to enter the extracellular cysteine-rich domain (CRD) groove. Therefore, an open CRD groove that has not been noticed previously was observed in Smo-Vismod complex. As a consequence, the occupied CRD groove prevents the binding of cholesterol. In addition, the HD and ECLs play crucial roles in the interaction of CRD and TMD. These results reveal that TM5, TM6, and TM7 play important roles in allosteric inhibition the activation of Smo and disrupting cholesterol binding by vismodegib binding. Our results are expected to contribute to understanding the allosteric inhibition mechanism of Smo by vismodegib. Moreover, the detailed conformational changes contribute to the development of novel Smo inhibitors against resistance mutation and active mutants of Smo.