Anti-apoptotic Pathways Research Articles

Astragaloside IV- and nesfatin-1-encapsulated phosphatidylserine liposomes conjugated with wheat germ agglutinin and leptin to activate anti-apoptotic pathway and block phosphorylated tau protein expression for Parkinson's disease treatment

Heap-up of α-synuclein (α-Syn) and its association with tau protein are esteemed to trigger the onset of Parkinson's disease (PD). The purpose of this study was to develop multi-functional liposomes incorporated with 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC), cholesterol, 1,2-dimyristoyl-sn-glycero-3-phosphocholine and phosphatidylserine (PS) to load astragaloside IV (AS-IV) and nestifin-1 (NF-1), followed by grafting with wheat germ agglutinin (WGA) and leptin (Lep) (WGA-Lep-AS-IV-NF-1-PS-liposomes) to protect dopaminergic neurons from apoptosis. Experimental results showed that increasing the mole percentage of DSPC and PS enhanced the particle size, particle stability and entrapment efficiency of AS-IV and NF-1, and reduced the drug releasing rate. Strong affinity of NF-1 to PS was evidenced by nuclear magnetic resonance spectroscopy. WGA-Lep-AS-IV-NF-1-PS-liposomes diminished transendothelial electrical resistance and improved the capacity of propidium iodide, AS-IV and NF-1 to penetrate the blood-brain barrier (BBB). Immunocytochemical staining exhibited the ability of functionalized liposomes to target Lep receptor and α-Syn in MPP+-insulted SH-SY5Y cells. Western blots revealed a substantial reduction of α-Syn and phosphorylated tau protein in the anti-oxidative pathway through interaction with PS. During the course of treatment with WGA-Lep-AS-IV-NF-1-PS-liposomes, the combined activity of AS-IV and NF-1 and recognition capability simultaneously decreased the expression of Bax, and increased the expressions of Bcl-2, tyrosine hydroxylase and dopamine transporter. The liposomes carrying AS-IV and NF-1 can rescue degenerated neurons and are a promising formulation to achieve better PD management.

Der f 38 Is a Novel TLR4-Binding Allergen Related to Allergy Pathogenesis from Dermatophagoides farinae.

It is difficult to treat allergic diseases including asthma completely because its pathogenesis remains unclear. House dust mite (HDM) is a critical allergen and Toll-like receptor (TLR) 4 is a member of the toll-like receptor family, which plays an important role in allergic diseases. The purpose of this study was to characterize a novel allergen, Der f 38 binding to TLR4, and unveil its role as an inducer of allergy. Der f 38 expression was detected in the body and feces of Dermatophagoides farinae (DF). Electron microscopy revealed that it was located in the granule layer, the epithelium layer, and microvilli of the posterior midgut. The skin prick test showed that 60% of allergic subjects were Der f 38-positive. Der f 38 enhanced surface 203c expression in basophils of Der f 38-positive allergic subjects. By analysis of the model structure of Der p 38, the expected epitope sites are exposed on the exterior side. In animal experiments, Der f 38 triggered an infiltration of inflammatory cells. Intranasal (IN) administration of Der f 38 increased neutrophils in the lung. Intraperitoneal (IP) and IN injections of Der f 38 induced both eosinophils and neutrophils. Increased total IgE level and histopathological features were found in BALB/c mice treated with Der f 38 by IP and IN injections. TLR4 knockout (KO) BALB/c mice exhibited less inflammation and IgE level in the sera compared to wild type (WT) mice. Der f 38 directly binds to TLR4 using biolayer interferometry. Der f 38 suppressed the apoptosis of neutrophils and eosinophils by downregulating proteins in the proapoptotic pathway including caspase 9, caspase 3, and BAX and upregulating proteins in the anti-apoptotic pathway including BCL-2 and MCL-1. These findings might shed light on the pathogenic mechanisms of allergy to HDM.

Downregulation of miR-142a Contributes to the Enhanced Anti-Apoptotic Ability of Murine Chronic Myelogenous Leukemia Cells.

BackgroundLeukemic stem cell (LSC) is thought to be responsible for chronic myelogenous leukemia (CML) initiation and relapse. However, the inherent regulation of LSCs remains largely obscure. Herein, we integratedly analyzed miRNA and gene expression alterations in bone marrow (BM) Lin-Sca1+c-Kit+ cells (LSKs) of a tet-off inducible CML mouse model, Scl/tTA-BCR/ABL (BA).MethodsScl/tTA and TRE-BA transgenic mice were crossed in the presence of doxycycline to get double transgenic mice. Both miRNA and mRNA expression profiles were generated from BM LSKs at 0 and 3 weeks after doxycycline withdrawal. The target genes of differentially expressed miRNAs were predicted, followed by the miRNA-mRNA network construction. In vitro and in vivo experiments were further performed to elucidate their regulation and function in CML progression.ResultsAs a result of the integrated analysis and experimental validation, an anti-apoptotic pathway emerged from the fog. miR-142a was identified to be downregulated by enhanced ERK-phosphorylation in BA-harboring cells, thereby relieving its repression on Ciapin1, an apoptosis inhibitor. Moreover, miR-142a overexpression could partially rescue the abnormal anti-apoptotic phenotype and attenuate CML progression.ConclusionTaken together, this study explored the miRNA-mRNA regulatory networks in murine CML LSKs and demonstrated that ERK-miR-142a-Ciapin1 axis played an essential role in CML pathogenesis.

HOXA9 promotes MYC-mediated leukemogenesis by maintaining gene expression for multiple anti-apoptotic pathways.

HOXA9 is often highly expressed in leukemias. However, its precise roles in leukemogenesis remain elusive. Here, we show that HOXA9 maintains gene expression for multiple anti-apoptotic pathways to promote leukemogenesis. In MLL fusion-mediated leukemia, MLL fusion directly activates the expression of MYC and HOXA9. Combined expression of MYC and HOXA9 induced leukemia, whereas single gene transduction of either did not, indicating a synergy between MYC and HOXA9. HOXA9 sustained expression of the genes implicated in the hematopoietic precursor identity when expressed in hematopoietic precursors, but did not reactivate it once silenced. Among the HOXA9 target genes, BCL2 and SOX4 synergistically induced leukemia with MYC. Not only BCL2, but also SOX4 suppressed apoptosis, indicating that multiple anti-apoptotic pathways underlie cooperative leukemogenesis by HOXA9 and MYC. These results demonstrate that HOXA9 is a crucial transcriptional maintenance factor that promotes MYC-mediated leukemogenesis, potentially explaining why HOXA9 is highly expressed in many leukemias.

Dihydrocapsaicin effectively mitigates cerebral ischemia-induced pathological changes in vivo, partly via antioxidant and anti-apoptotic pathways

AimsIschemic stroke occurs when there is a sudden blockage of cerebral blood flow. This condition is a major cause of mortality, especially in low-income countries, and its incidence is dramatically increasing. Therapeutic strategies against stroke are therefore required. The present study explored the effects of dihydrocapsaicin on neuronal loss, brain infarct volume, and antioxidants in a rat model of permanent occlusion of the right middle cerebral artery (Rt.MCAO). Main methodsMale Wistar rats received dihydrocapsaicin intraperitoneally for 7 days after permanent occlusion of their right middle cerebral artery (Rt.MCAO). Then, the brain infarct volume, neuronal density, and antioxidant and anti-apoptotic activities in the cortex and hippocampus were determined at the end of the study. Key findingDihydrocapsaicin treatment was found to significantly improve neuronal density, decrease infarct volume, reduce MDA elevation, improve CAT and SOD activities, decrease the density ratio of Bax and caspase-3, and increase the density ratio of Bcl-XL to β-actin in the cerebral cortex and hippocampus. SignificanceThe present study suggests that dihydrocapsaicin effectively mitigates cerebral ischemia-induced pathological changes in vivo, partly via antioxidant and anti-apoptotic pathways.

Targeting c-IAP1, c-IAP2, and Bcl-2 Eliminates Senescent Glioblastoma Cells Following Temozolomide Treatment.

Simple SummaryDespite extensive research, malignant glioma remains the most aggressive and fatal type of brain tumor. Following resection, therapy is based on radiation concomitant with the methylating agent temozolomide (TMZ), followed by adjuvant high-dose TMZ. In previous work, we showed that following TMZ exposure, most glioma cells evade apoptosis and enter a senescent state and are thereby protected against anticancer therapy. Senescent cells may escape from senescence, contributing to the formation of recurrences or can induce the senescence-associated secretory phenotype (SASP), which may impact therapy success. Therefore, direct targeting of senescent cells might be favorable to improve the effect of TMZ-based anticancer therapy. Here we show that during TMZ-induced senescence in glioblastoma cells, the antiapoptotic factors c-IAP2 and Bcl-2 are responsible for the prevention of cell death and that inhibition of these factors by BV6 and venetoclax effectively kills senescent glioblastoma cells.Therapy of malignant glioma depends on the induction of O6-methylguanine by the methylating agent temozolomide (TMZ). However, following TMZ exposure, most glioma cells evade apoptosis and become senescent and are thereby protected against further anticancer therapy. This protection is thought to be dependent on the senescent cell anti-apoptotic pathway (SCAP). Here we analyzed the factors involved in the SCAP upon exposure to TMZ in glioblastoma cell lines (LN-229, A172, U87MG) and examined whether inhibition of these factors could enhance TMZ-based toxicity by targeting senescent cells. We observed that following TMZ treatment, c-IAP2 and Bcl-2 were upregulated. Inhibition of these SCAP factors using non-toxic concentrations of the small molecule inhibitors, BV6 and venetoclax, significantly increased cell death, as measured 144 h after TMZ exposure. Most importantly, BV6 and venetoclax treatment of senescent cells strongly increased cell death after an additional 120 h. Moreover, Combenefit analyses revealed a significant synergy combining BV6 and venetoclax. In contrast to BV6 and venetoclax, AT406, embelin, and TMZ itself, teniposide and the PARP inhibitor pamiparib did not increase cell death in senescent cells. Based on these data, we suggest that BV6 and venetoclax act as senolytic agents in glioblastoma cells upon TMZ exposure.

STAT3 but Not STAT5 Contributes to the Protective Effect of Electroacupuncture Against Myocardial Ischemia/Reperfusion Injury in Mice.

Electroacupuncture (EA) can help reduce infarct size and injury resulting from myocardial ischemia/reperfusion (I/R); however, the underlying molecular mechanism remains unknown. We previously reported that STAT5 plays a critical role in the cardioprotective effect of remote ischemic preconditioning (RIPC). Here, we assessed the effects of electroacupuncture pretreatment (EAP) on myocardial I/R injury in the presence and/or absence of Stat5 in mice and investigated whether EAP exerts its cardioprotective effects in a STAT5-dependent manner. Adult Stat5fl/fl and Stat5-cKO mice were exposed to EAP at Neiguan (PC6) for 7 days before the induction of I/R injury by left anterior descending (LAD) coronary artery ligation. The myocardial infarct size (IS), area at risk, and apoptotic rate of cardiomyocytes were detected. RT-qPCR and western blotting were used to measure gene and protein expression, respectively, in homogenized heart tissues. RNA-seq was used to identify candidate genes and pathways. Our results showed that EAP decreased IS and the rate of cardiomyocyte apoptosis. We further found that STAT5 was activated by EAP in Stat5fl/fl mice but not in Stat5-cKO mice, whereas the opposite was observed for STAT3. Following EAP, the levels of the antiapoptotic proteins Bcl-xL, Bcl-2, and p-AKT were increased in the presence of Stat5, while that of interleukin 10 (IL-10) was increased in both Stat5fl/fl and Stat5-cKO. The gene expression profile in heart tissues was different between Stat5fl/fl and the Stat5-cKO mice with EAP. Importantly, the top 30 DEGs under EAP in the Stat5-cKO mice were enriched in the IL-6/STAT3 signaling pathway. Our results revealed for the first time that the protective effect of EAP following myocardial I/R injury was attributable to, but not dependent on, STAT5. Additionally, we found that EAP could activate STAT3 signaling in the absence of the Stat5 gene, and could also activate antiapoptotic, survival, and anti-inflammatory signaling pathways.

A SNAI2-PEAK1-INHBA stromal axis drives progression and lapatinib resistance in HER2-positive breast cancer by supporting subpopulations of tumor cells positive for antiapoptotic and stress signaling markers

Intercellular mechanisms by which the stromal microenvironment contributes to solid tumor progression and targeted therapy resistance remain poorly understood, presenting significant clinical hurdles. PEAK1 (Pseudopodium-Enriched Atypical Kinase One) is an actin cytoskeleton- and focal adhesion-associated pseudokinase that promotes cell state plasticity and cancer metastasis by mediating growth factor-integrin signaling crosstalk. Here, we determined that stromal PEAK1 expression predicts poor outcomes in HER2-positive breast cancers high in SNAI2 expression and enriched for MSC content. Specifically, we identified that the fibroblastic stroma in HER2-positive breast cancer patient tissue stains positive for both nuclear SNAI2 and cytoplasmic PEAK1. Furthermore, mesenchymal stem cells (MSCs) and cancer-associated fibroblasts (CAFs) express high PEAK1 protein levels and potentiate tumorigenesis, lapatinib resistance and metastasis of HER2-positive breast cancer cells in a PEAK1-dependent manner. Analysis of PEAK1-dependent secreted factors from MSCs revealed INHBA/activin-A as a necessary factor in the conditioned media of PEAK1-expressing MSCs that promotes lapatinib resistance. Single-cell CycIF analysis of MSC-breast cancer cell co-cultures identified enrichment of p-Akthigh/p-gH2AXlow, MCL1high/p-gH2AXlow and GRP78high/VIMhigh breast cancer cell subpopulations by the presence of PEAK1-expressing MSCs and lapatinib treatment. Bioinformatic analyses on a PEAK1-centric stroma-tumor cell gene set and follow-up immunostaining of co-cultures predict targeting antiapoptotic and stress pathways as a means to improve targeted therapy responses and patient outcomes in HER2-positive breast cancer and other stroma-rich malignancies. These data provide the first evidence that PEAK1 promotes tumorigenic phenotypes through a previously unrecognized SNAI2-PEAK1-INHBA stromal cell axis.

Neuroprotective effects of Korean White ginseng and Red ginseng in an ischemic stroke mouse model

BackgroundStroke is a neurological disorder characterized by brain tissue damage following a decrease in oxygen supply to brain due to blocked blood vessels. Reportedly, 80% of all stroke cases are classified as cerebral infarction, and the incidence rate of this condition increases with age. Herein, we compared the efficacies of Korean White ginseng (WG) and Korean Red Ginseng (RG) extracts (WGex and RGex, respectively) in an ischemic stroke mouse model and confirmed the underlying mechanisms of action. MethodsMice were orally administered WGex or RGex 1 h before middle cerebral artery occlusion (MCAO), for 2 h; the size of the infarct area was measured 24 h after MCAO induction. Then, the neurological deficit score was evaluated and the efficacies of the two extracts were compared. Finally, their mechanisms of action were confirmed with tissue staining and protein quantification. ResultsIn the MCAO-induced ischemic stroke mouse model, WGex and RGex showed neuroprotective effects in the cortical region, with RGex demonstrating superior efficacy than WGex. Ginsenoside Rg1, a representative indicator substance, was not involved in mediating the effects of WGex and RGex. ConclusionWGex and RGex could alleviate the brain injury caused by ischemia/reperfusion, with RGex showing a more potent effect. At 1,000 mg/kg body weight, only RGex reduced cerebral infarction and edema, and both anti-inflammatory and anti-apoptotic pathways were involved in mediating these effects.

Emerging role of cellular senescence in the pathogenesis of oral submucous fibrosis and its malignant transformation.

Senescence is a common denominator in wound healing, fibrosis, and cancer. Although, senescence is transiently antifibrotic, when prolonged, promotes fibrosis and malignant transformation. Eligible studies indexed in MEDLINE, Embase and Web of Science were searched to understand the role of cellular senescence in the pathogenesis of oral submucous fibrosis (OSF) and its malignant transformation. The senescence-associated secretory phenotype (SASP) components like IL-1, IL-6, and GRO-α induce double-strand DNA breaks in keratinocytes and drive genetic instability. SASP derived from myofibroblasts induces epithelial-mesenchymal transition in OSF and facilitates cancer progression. The use of senolytics has been shown to eliminate senescent cells from the areas of fibrosis, thereby preventing malignancy. Naturally occurring agents such as apigenin and kaempferol inhibit SASP. Mechanistic insight into the emerging role of senescence in the pathogenesis of OSF and modalities to inhibit senescence-associated antiapoptotic pathways as a supplementary therapy to prevent malignant transformation of OSF is underlined.

WITHDRAWN: Combined therapy of adenovirus vector mediated IGF-1 gene with anti-CD20 mAbs exerts potential beneficial role on type 1 diabetes in nonobese diabetic mice

To assess the protective effects of combined treatment with anti-CD20 monoclonal antibody (mAb) and adenovirus mediated mouse insulin-like growth factor 1 (Adv-mIGF-1) gene on type 1 diabetes (T1D) in nonobese diabetic (NOD) mice at early stage. To simultaneously restore the proportion of Th cells and block the interaction of B cells, NOD model mice were assigned to four groups which received PBS, Adv-mIGF-1 gene and anti-CD20 mAbs alone or combination, respectively. After 16 weeks of therapeutic intervention, blood samples and pancreatic tissues of mice were measured via the methods of ELISA, RT-PCR, western blotting, H&E staining, TUNEL and immunohistochemistry assays. Chronic combination intervention with Adv-mIGF-1 gene and anti-CD20 mAbs reduced the T1D-related morbidity, promoted the secretion of insulin, controlled the blood glucose levels (BGLs) and alleviated insulitis of experimental mice. In addition, current combination intervention also protected the pancreatic β cells via suppressing the expression of Fas and TNF-α, inhibiting Caspase-3/8 related apoptotic pathway, and activating the Bcl-2-related antiapoptotic pathway. Furthermore, current combination therapy also increased the expression levels of PDX-1 and CK-19 genes, and finally accelerated the proliferation and differentiation of pancreatic β-cells. In addition, combination therapy could also ameliorate the pathological characteristics of diabetic nephropathy in NOD mice. Combination treatment with Adv-mIGF-1 gene and anti-CD20 mAbs may exert a potential beneficial role on T1D in NOD mice.

Molecular Hydrogen Attenuated N-methyl-N-Nitrosourea Induced Corneal Endothelial Injury by Upregulating Anti-Apoptotic Pathway.

PurposePrevious work by our group has demonstrated the value of N-methyl-N-nitrosourea (MNU)-induced corneal endothelial decompensation in animal models. The aim of this study was to investigate the effect of molecular hydrogen (H2) on MNU-induced corneal endothelial cell (CEC) injury and the underlying mechanism.MethodsMNU-induced animal models of CEC injury were washed with hydrogen-rich saline (HRS) for 14 days. Immunofluorescence staining, immunohistochemical staining, and corneal endothelial assessment were applied to determine architectural and cellular changes on the corneal endothelium following HRS treatment. MNU-induced cell models of CEC injury were co-cultured with H2. The effect of H2 was examined using morphological and functional assays.ResultsIt was shown that MNU could inhibit the proliferation and specific physiological functions of CECs by increasing apoptosis and decreasing the expression of ZO-1 and Na+/K+-ATPase, whereas H2 improved the proliferation and physiological function of CECs by anti-apoptosis. Cell experiments further confirmed that H2 could reverse MNU damage to CECs by decreasing oxidative stress injury, interfering with the NF-κB/NLRP3 pathway and the FOXO3a/p53/p21 pathway.ConclusionsThis study suggests that topical application of H2 could protect CECs against corneal damage factors through anti-apoptotic effect, reduce the incidence and severity of corneal endothelial decompensation, and maintain corneal transparency.

Abstract 2530: Differences in mRNA expression of a candidate gene, between normal cervical tissue and cervical premalignant lesions

Abstract Background: Cervical cancer is the fourth most common cancer in women worldwide, with around 85% of cases occurring in underdeveloped countries. The diagnosis of premalignant lesions is ​​the objective of cervical cancer prevention programs, aimed at increasing the chances of cure and survival time. In a previous study of our group cDNA of TMEM45A gene was detected as differentially expressed between healthy cervical tissue and a high grade squamous intraepithelial lesion (HSIL) with confirmed HPV-18 transforming infection. TMEM45A gene family is highly expressed in squamous epithelium, playing roles in differentiation, senescence and chemoresistance in some sort of epithelial carcinomas. The overexpression of this product may confer antiapoptotic pathways, contributing to the development of cervical squamous cell carcinoma. We compared the TMEM45A in situ mRNA expression in healthy cervical tissue, low grade squamous intraepithelial lesion (LSIL) and HSIL, using RNAscope chromogenic in situ hybridization (CISH). Design: TMEM45A expression was evaluated by CISH using a tissue microarray (TMA) constructed from cervical excision samples (conization and histerectomy) from 169 patients, with a mean age of 35.37 years (SD ±10.4), from a reference center for cervical pathology in the Colombian Caribbean. Stained sections from the donor blocks were reviewed by two pathologist, verifying the original sign-out diagnosis, and representative areas from normal cervix, LSIL and HSIL were selected. RNAscope 2.5HD Red TMEM45A assay was performed as a technical service in Advanced Cell Diagnostics´s laboratories (Newark, CA). The staining was visually scored by two pathologist from 0 to 4 based on the average number of dots per cell, according to a semi-quantitative analysis. The mean score values for each lesion group were compared using GraphPad Prism 7.0 software. A p< 0.05 was considered statistically significant. Results: The TMEM45A mRNA in situ expression was determined in a cervical TMA including, normal cervix: 76, LSIL: 69, and HSIL: 138, tissue cores. There was a higher mean staining score for TMEM45A mRNA in HSIL (1.51, 95% CI:1.26-1.76), compared to normal cervix (1.10, 95% CI:0.80-1.40). Mean score of TMEM45A in situ mRNA expression in HSIL was significantly higher than mean TMEM45A in situ mRNA expression in the normal cervix lining squamous epithelium (p=0.046). Conclusions: TMEM45A expression analysis identified differential expression between normal cervix lining squamous epithelium and HSIL. This finding supports the possible role of TMEM45A in carcinogenesis of well differentiated epithelial lesions. Additional studies to detect the TMEM45A expression at protein with samples of women from the Colombian Caribbean are in progress to validate these results. Citation Format: Ines Benedetti, Reinhard Rodríguez, Lia Barrios. Differences in mRNA expression of a candidate gene, between normal cervical tissue and cervical premalignant lesions [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 2530.

Inhibition of the Histone Chaperone, FACT (Facilitates Chromatin Transcription) By the Novel Small Molecule CBL0137 (Curaxin) Shows Promising Efficacy in Pre-Clinical Models of Acute Myeloid Leukemia

Introduction:Disease recurrence in patients with acute myeloid leukemia (AML) has been attributed to the self-renewal, self-protective, and quiescent characteristics of leukemia stem cells (LSC). There is currently an unmet clinical need in AML for novel targeted therapies capable of eradicating LSC to achieve long-term cure. Curaxins were identified during a cell-based screening as small molecules able to bind to DNA and alter the chromatin structure without directly causing DNA damage. Curaxin binding leads to functional inactivation of the facilitated chromatin remodeling complex (FACT) involved in transcription, replication, and DNA repair. FACT, which normally binds to DNA in a transient manner, gets trapped on the DNA following curaxin binding, leading to activation of the pro-apoptotic p53 pathway, suppression of anti-apoptotic NF-κB pathway, and FACT dependent cell death. Structurally, FACT is a histone chaperone composed of two subunits, structure specific recognition protein 1 (SSRP1) and suppressor of Ty16 (SPT16). FACT controls the assembly of nucleosomes only for cells with open dynamic chromatin states (such as embryonic stem cells and tumor cells) but not normal mature cells. FACT is an attractive candidate for leukemia therapeutics due to its high specificity for leukemia cells and LSC, with limited expression on normal hematopoietic stem cells. Here we evaluated the effects of the small molecule curaxin, CBL0137 (Cleveland Biolabs, Buffalo, NY) in vitro and in vivo in preclinical human AML models. We hypothesized that AML cells, specifically leukemia-initiating cells from primary samples, might be sensitive to apoptosis induced by curaxin treatment due to underlying FACT expression.Materials and Methods:Human AML cells (HL60 and a multi-drug resistant clone HL60/VCR) were treated in vitro with CBL0137 (doses 0.039-20μM) and assessed by WST-8 viability assays (Dojindo Molecular Technologies). Expression of both FACT subunits (SSRP1 and SPT16) was evaluated by Western blot (WB) analysis in 23 primary AML samples. A subset of primary AML cells was then plated in methocellulose culture with cytokine support. The number of leukemia colony forming unit (CFU-L) potentially reflecting leukemia initiating cell growth was quantified by microscopy after 14-15 days of incubation. In vivo treatment with CBL0137 was also assessed in SCID mice systematically engrafted with HL60/VCR-luciferase AML cells. Mice were treated with oral vehicle or CBL0137 dosed at 40 mg/kg (5 days/week for 2 weeks) or 70 mg/kg (twice a week for 5 doses). Effects on leukemia burden, toxicity, and survival were determined by whole animal bioluminescent imaging, total animal weights, and time to morbidity, respectively.Results:Expression of one or both FACT subunits was detected in 22 of the 23 primary AML samples and in HL60 cells by WB. CBL0137 resulted in dose-dependent in vitro inhibition of HL60 and HL60/VCR cell growth with IC50 values of 0.54-57 μM. CBL0137 also inhibited the colony formation ability of 9 primary AML samples at doses ranging from 0.625 - 10μM with estimated IC50 values of 1-3 μM. Of note, CBL0137 treatment in CFU-L assays resulted in similar anti-leukemic activity in patient samples characterized by low/undetectable or high/intermediate levels of FACT subunits (SSRP1 and SPT16) by WB analysis. In vivo, CBL0137 treatment in systemic HL60/VCRluciferase engrafted SCID mice was generally well tolerated with no significant weight loss. Curaxin treatment markedly decreased leukemic disease burden (as determined by bioluminescent imaging) and prolonged survival as compared with control-treated mice (p =0.0004). Animals receiving the higher dose CBL0137 regimen exhibited the greatest reduction in disease burden and longest overall survival (70 vs. 40mg/kg, p=0.04)Conclusions:Our results demonstrate that treatment with the curaxin, CBL0137, results in potent in vitro and in vivo dose-dependent anti-tumor activity in preclinical AML models. Inhibition of primary AML colony formation did not appear to correlate with baseline FACT expression in tumor cells. Taken together, these data support the future clinical development of this novel DNA interacting agent for AML therapy. Correlative studies using primary AML xenograft (PDX) models to clarify if differential FACT expression is a predictive marker for in vivo curaxin efficacy are underway. DisclosuresNo relevant conflicts of interest to declare.

PI3Kδ/AKT Inhibition Augments 2-Phenylethynesulfonamide Induced Apoptosis Via the Unfolded Protein Response in Chronic Lymphocytic Leukemia

Introduction: Chronic lymphocytic leukemia (CLL) is largely comprised of two major subsets, IGHV unmutated (U-CLL) and mutated (M-CLL) CLL, associated with progressive and indolent disease respectively. Approximately 15% patients harbour a 17p deletion and/or a TP53 mutation which is largely associated with the worst clinical outcome. The development of BCR signalosome inhibitors such as ibrutinib and idelalisib have been instrumental in the treatment of CLL, however these drugs are not curative, only supress the disease and resistance is already developing particularly in patients with a complex karyotype associated with 17p del/ TP53 mutation. Consequently continued development of novel therapies targeting pathways which enhance selective killing of tumor cells independently of p53 are urgently required. We previously demonstrated that 2-phenylacetylenesulfonamide (PAS), induced apoptosis of CLL cells in a p53-independent manner. PAS is suggested to inhibit heat shock protein (HSP) 70, a key protein involved in protein folding. Therefore in CLL cells, PAS would be predicted to cause cellular stress due to the accumulation of misfolded proteins resulting in the activation of the unfolded protein response (UPR). The UPR serves to protect cells from the consequences of protein misfolding and can function in part by activating pro-survival signaling, including the PI3K/AKT pathway. Under normal conditions the ER chaperone BiP inhibits activation of the UPR by binding the three ER transmembrane receptors; inositol-requiring enzyme 1 (IRE-1α), pancreatic ER kinase-like ER kinase (PERK) and activating transcription factor 6 (ATF6). Activation of the UPR leads to the upregulation of HSP family members including HSP70 in the cytosol and BiP/GRP78 in the endoplasmic reticulum (ER). These HSPs aid in refolding misfolded proteins or facilitate their degradation via the proteasome. If the stress is resolved, the UPR signalling pathway is switched off; however, if stress persists the UPR remains active and switches from an anti-apoptotic to a pro-apoptotic signal via p38MAPK.Aim: To determine the mechanism of PAS induced apoptosis in CLL cells.Methods: Fifty two CLL samples were treated with PAS (5-20μM) and cellular responses assessed using immunoblotting, flow cytometry and MTT assay.Results: In this study we demonstrated that the UPR is induced in CLL cells following the induction of reactive oxygen species by PAS treatment. This consequently led to the activation of both pro-apoptotic (Noxa, p<0.0001) and anti-apoptotic (AKT, p<0.05) signalling pathways. PAS treatment further increased expression of key stress proteins BiP (p=0.0005) and HSP70 (p=0.001). PAS treatment also resulted in activation of the PERK and IRE-1α arms of the UPR (cleaved XBP-1, phosphorylated eiF2α, phosphorylated p38MAPK) within 2h, which persisted up to 24h. This pathway was pivotal for CLL cell death since small molecule inhibition of either p38MAPK or PERK signalling significantly reduced PAS mediated apoptosis by approximately 50%. However PAS induced apoptosis was simultaneously antagonised by activation of the pro-survival AKT pathway. Consequently we hypothesised that PAS treatment in combination with a PI3Kδ (idelalisib) or AKT (AKTi) inhibitor may promote greater CLL cell apoptosis. Indeed our combination indices (CI) studies demonstrated that simultaneous treatment with an AKTi or the clinically approved PI3Kδ inhibitor idelalisib and PAS synergistically and significantly enhanced apoptosis of CLL cells compared with either agent alone (p<0.05).Conclusion: This data suggests that inhibiting the pro-survival arm of the UPR (PI3K/AKT), whilst simultaneously activating the pro-death arm (HSP70/p38MAPK), may be a useful strategy for the treatment of CLL and particularly samples with a 17p del or TP53 mutation and warrants further clinical investigation particularly in CLL cases that are resistant to BCR kinase or Bcl-2 family inhibitors. DisclosuresStrefford:Roche: Research Funding. Johnson:Novartis: Consultancy, Honoraria; Genmab: Consultancy, Honoraria; Carrick: Consultancy, Honoraria; Bristol-Myers Squibb: Consultancy, Honoraria; Janssen: Research Funding; Celgene: Consultancy, Honoraria; Incyte: Consultancy, Honoraria; Takeda: Consultancy, Honoraria; Karus: Consultancy, Honoraria; Boehringer Ingelheim: Consultancy, Honoraria; Zenyaku Kogyo: Consultancy, Honoraria; Epizyme: Research Funding. Forconi:AbbVie: Consultancy, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Infinity: Consultancy, Membership on an entity's Board of Directors or advisory committees; Gilead: Research Funding; Janssen-Cilag: Speakers Bureau. Steele:Gilead: Consultancy, Honoraria; Portola Pharmaceuticals: Consultancy, Honoraria, Research Funding.

Diacylglycerol Lipase-β Knockout Mice Display a Sex-Dependent Attenuation of Traumatic Brain Injury-Induced Mortality with No Impact on Memory or Other Functional Consequences.

Background: The endogenous cannabinoid system modulates inflammatory signaling in a variety of pathological states, including traumatic brain injury (TBI). The selective expression of diacylglycerol lipase-β (DAGL-β), the 2-arachidonylglycerol biosynthetic enzyme, on resident immune cells of the brain (microglia) and the role of this pathway in neuroinflammation, suggest that this enzyme may contribute to TBI-induced neuroinflammation. Accordingly, we tested whether DAGL-β-/- mice would show a protective phenotype from the deleterious consequences of TBI on cognitive and neurological motor functions. Materials and Methods: DAGL-β-/- and -β+/+ mice were subjected to the lateral fluid percussion model of TBI and assessed for learning and memory in the Morris water maze (MWM) Fixed Platform (reference memory) and Reversal (cognitive flexibility) tasks, as well as in a cued MWM task to infer potential sensorimotor/motivational deficits. In addition, subjects were assessed for motor behavior (Rotarod and the Neurological Severity Score assays) and in the light/dark box and the elevated plus maze to infer whether these manipulations affected anxiety-like behavior. Finally, we also examined whether brain injury disrupts the ceramide/sphingolipid lipid signaling system and if DAGL-β deletion offers protection. Results: TBI disrupted all measures of neurological motor function and reduced body weight, but did not affect body temperature or performance in common assays used to infer anxiety. TBI also impaired performance in MWM Fixed Platform and Reversal tasks, but did not affect cued MWM performance. Although no differences were found between DAGL-β-/- and -β+/+ mice in any of these measures, male DAGL-β-/- mice displayed an unexpected survival-protective phenotype, which persisted at increased injury severities. In contrast, TBI did not elicit mortality in female mice regardless of genotype. TBI also produced significant changes in sphingolipid profiles (a family of lipids, members of which have been linked to both apoptotic and antiapoptotic pathways), in which DAGL-β deletion modestly altered levels of select species. Conclusions: These findings indicate that although DAGL-β does not play a necessary role in TBI-induced cognitive and neurological function, it appears to contribute to the increased vulnerability of male mice to TBI-induced mortality, whereas female mice show high survival rates irrespective of DAGL-β expression.

HSP70-mediated neuroprotection by combined treatment of valproic acid with hypothermia in a rat asphyxial cardiac arrest model.

It has been reported that valproic acid (VPA) combined with therapeutic hypothermia can improve survival and neurologic outcomes in a rat asphyxial cardiac arrest model. However, neuroprotective mechanisms of such combined treatment of valproic acid with hypothermia remains unclear. We hypothesized that epigenetic regulation of HSP70 by histone acetylation could increase HSP70-mediated neuroprotection suppressed under hypothermia. Male Sprague-Dawley rats that achieved return of spontaneous circulation (ROSC) from asphyxial cardiac arrest were randomized to four groups: normothermia (37°C ± 1°C), hypothermia (33°C ± 1°C), normothermia + VPA (300 mg/kg IV initiated 5 minutes post-ROSC and infused over 20 min), and hypothermia + VPA. Three hours after ROSC, acetyl-histone H3 was highly expressed in VPA-administered groups (normothermia + VPA, hypothermia + VPA). Four hours after ROSC, HSP70 mRNA expression levels were significantly higher in normothermic groups (normothermia, normothermia + VPA) than in hypothermic groups (hypothermia, hypothermia + VPA). The hypothermia + VPA group showed significantly higher HSP70 mRNA expression than the hypothermia group. Similarly, at five hours after ROSC, HSP70 protein levels were significantly higher in normothermic groups than in hypothermic groups. HSP70 levels were significantly higher in the hypothermia + VPA group than in the hypothermia group. Only the hypothermia + VPA group showed significantly attenuated cleaved caspase-9 levels than the normothermia group. Hypothermia can attenuate the expression of HSP70 at transcriptional level. However, VPA administration can induce hyperacetylation of histone H3, leading to epigenetic transcriptional activation of HSP70 even in a hypothermic status. Combining VPA treatment with hypothermia may compensate for reduced activation of HSP70-mediated anti-apoptotic pathway.

Acupuncture for Parkinson's Disease: Efficacy Evaluation and Mechanisms in the Dopaminergic Neural Circuit.

Parkinson's disease (PD) is a chronic and progressive neurodegenerative disease caused by degeneration of dopaminergic neurons in the substantia nigra. Existing pharmaceutical treatments offer alleviation of symptoms but cannot delay disease progression and are often associated with significant side effects. Clinical studies have demonstrated that acupuncture may be beneficial for PD treatment, particularly in terms of ameliorating PD symptoms when combined with anti-PD medication, reducing the required dose of medication and associated side effects. During early stages of PD, acupuncture may even be used to replace medication. It has also been found that acupuncture can protect dopaminergic neurons from degeneration via antioxidative stress, anti-inflammatory, and antiapoptotic pathways as well as modulating the neurotransmitter balance in the basal ganglia circuit. Here, we review current studies and reflect on the potential of acupuncture as a novel and effective treatment strategy for PD. We found that particularly during the early stages, acupuncture may reduce neurodegeneration of dopaminergic neurons and regulate the balance of the dopaminergic circuit, thus delaying the progression of the disease. The benefits of acupuncture will need to be further verified through basic and clinical studies.

Potential Biomarkers for Treatment Response to the BCL-2 Inhibitor Venetoclax: State of the Art and Future Directions.

Simple SummaryApoptosis dysergulation is vital to oncogenesis. Efforts to mitigate this cancer hallmark have been ongoing for decades, focused mostly on inhibiting BCL-2, a key anti-apoptosis effector. The approval of venetoclax, a selective BCL-2 inhibitor, for clinical use has been a turning point in the field of oncology. While resulting in impressive improvement in objective outcomes, particularly for patients with chronic lymphocytic leukemia/small lymphocytic lymphoma and acute myeloid leukemia, the use of venetoclax has exposed a variety of resistance mechanisms to BCL-2 inhibition. As the field continues to move forward, improved understanding of such mechanisms and the potential biomarkers that could be harnessed to optimize patient selection for therapies that include venetoclax and next-generation BCL-2 inhibitors are gaining increased importance.Intrinsic apoptotic pathway dysregulation plays an essential role in all cancers, particularly hematologic malignancies. This role has led to the development of multiple therapeutic agents targeting this pathway. Venetoclax is a selective BCL-2 inhibitor that has been approved for the treatment of chronic lymphoid leukemia and acute myeloid leukemia. Given the reported resistance to venetoclax, understanding the mechanisms of resistance and the potential biomarkers of response is crucial to ensure optimal drug usage and improved patient outcomes. Mechanisms of resistance to venetoclax include alterations involving the BH3-binding groove, BCL2 gene mutations affecting venetoclax binding, and activation of alternative anti-apoptotic pathways. Moreover, various potential genetic biomarkers of venetoclax resistance have been proposed, including chromosome 17p deletion, trisomy 12, and TP53 loss or mutation. This manuscript provides an overview of biomarkers that could predict treatment response to venetoclax.

Ceramide Kinase Inhibition Blocks IGF-1-Mediated Survival of Otic Neurosensory Progenitors by Impairing AKT Phosphorylation.

Sphingolipids are bioactive lipid components of cell membranes with important signal transduction functions in health and disease. Ceramide is the central building block for sphingolipid biosynthesis and is processed to form structurally and functionally distinct sphingolipids. Ceramide can be phosphorylated by ceramide kinase (CERK) to generate ceramide-1-phosphate, a cytoprotective signaling molecule that has been widely studied in multiple tissues and organs, including the developing otocyst. However, little is known about ceramide kinase regulation during inner ear development. Using chicken otocysts, we show that genes for CERK and other enzymes of ceramide metabolism are expressed during the early stages of inner ear development and that CERK is developmentally regulated at the otic vesicle stage. To explore its role in inner ear morphogenesis, we blocked CERK activity in organotypic cultures of otic vesicles with a specific inhibitor. Inhibition of CERK activity impaired proliferation and promoted apoptosis of epithelial otic progenitors. CERK inhibition also compromised neurogenesis of the acoustic-vestibular ganglion. Insulin-like growth factor-1 (IGF-1) is a key factor for proliferation, survival and differentiation in the chicken otocyst. CERK inhibition decreased IGF-1-induced AKT phosphorylation and blocked IGF-1-induced cell survival. Overall, our data suggest that CERK is activated as a central element in the network of anti-apoptotic pro-survival pathways elicited by IGF-1 during early inner ear development.