Concomitant Apoptosis Research Articles

The divergent outcome of IL-4Rα signalling on Foxp3 T regulatory cells in listeriosis and tuberculosis.

Forkhead box P3 (Foxp3) T regulatory cells are critical for maintaining self-tolerance, immune homeostasis, and regulating the immune system. We investigated interleukin-4 receptor alpha (IL-4Rα) signalling on T regulatory cells (Tregs) during Listeria monocytogenes (L. monocytogenes) infection using a mouse model on a BALB/c background, specifically with IL-4Rα knockdown in Tregs (Foxp3creIL-4Rα-/lox). We showed an impairment of Treg responses, along with a decreased bacterial burden and diminished tissue pathology in the liver and spleen, which translated into better survival. Mechanistically, we observed an enhancement of the Th1 signature, characterised by increased expression of the T-bet transcription factor and a greater number of effector T cells producing IFN-γ, IL-2 following ex-vivo stimulation with heat-killed L. monocytogenes in Foxp3creIL-4Rα-/lox mice. Furthermore, CD8 T cells from Foxp3creIL-4Rα-/lox mice displayed increased cytotoxicity (Granzyme-B) with higher proliferation capacity (Ki-67), better survival (Bcl-2) with concomitant reduced apoptosis (activated caspase 3). In contrast to L. monocytogenes, Foxp3creIL-4Rα-/lox mice displayed similar bacterial burdens, lung pathology and survival during Mycobacterium tuberculosis (M. tuberculosis) infection, despite increased T cell numbers and IFN-γ, TNF and IL-17 production. Our results demonstrated that the diminished IL-4Rα signalling on Foxp3+ T regulatory cells resulted in a loss of their functionality, leading to survival benefits in listeriosis but not in tuberculosis.

Averrhoa carambola Leaf Extract Induces Apoptosis-Like Death with Increased ROS Generation in Leishmania donovani.

The parasitic disease leishmaniasis is responsible for high mortality and morbidity rates worldwide. The visceral form is the most severe form of leishmaniasis (or leishmaniosis), which is caused predominantly by Leishmania donovani. Currently, clinically recommended antileishmanial drugs are not convenient because of several medical complications and resistance issues. Phytocompounds are the best candidates in this regard. The present study aimed to evaluate the antileishmanial activity of Averrhoa carambola leaf extract. The antipromastigote activity and cytotoxicity were assessed using the MTT assay. Morphological distortions were determined using phase contrast microscopy and scanning electron microscopy (SEM). Reactive oxygen species (ROS) production, nonprotein thiol depletion and apoptotic death in promastigotes were determined via flow cytometry. UV-visible spectroscopy and energy dispersive X-ray (EDX) spectroscopy was performed for elemental analysis. Fourier-transform infrared spectroscopy (FTIR) and liquid chromatography‒mass spectrometry (LCMS) were used to characterize the phytocomponent(s) present in the extract. The chloroform extract of Averrhoa carambola leaf (ACCEX) (IC50 = 50.76 ± 1.7µg/mL) exhibited the highest activity, followed by the ethyl acetate, hexane, and methanol extracts. ACCEX has also exhibited lower toxicity towards host macrophages. ACCEX also induced morphological distortions in promastigotes, with significant generation of ROS and the concomitant apoptosis initiation followed by a decrease in the nonprotein thiol level. The major phytometabolites present in ACCEX were identified from the National Institute of Standards and Technology (NIST) database and from a literature review. This study suggested that Averrhoa carambola leaf extracts are rich in some classes of biologically active phytocompounds and exhibit good antileishmanial activity.

Elevated temperature as a dominant driver to aggravate cadmium toxicity: Investigations through toxicokinetics and omics

Despite the great interest in the consequences of global change stressors on marine organisms, their interactive effects on cadmium (Cd) bioaccumulation/biotoxicity are very poorly explored, particularly in combination with the toxicokinetic model and molecular mechanism. According to the projections for 2100, this study investigated the impact of elevated pCO2 and increased temperature (isolated or joint) on Cd uptake dynamics and transcriptomic response in the marine copepod Tigriopus japonicus. Toxicokinetic results showed significantly higher Cd uptake in copepods under increased temperature and its combination with elevated pCO2 relative to the ambient condition, linking to enhanced Cd bioaccumulation. Transcriptome analysis revealed that, under increased temperature and its combination with elevated pCO2, up-regulated expression of Cd uptake-related genes but down-regulation of Cd exclusion-related genes might cause increased cellular Cd level, which not only activated detoxification and stress response but also induced oxidative stress and concomitant apoptosis, demonstrating aggravated Cd biotoxicity. However, these were less pronouncedly affected by elevated pCO2 exposure. Therefore, temperature seems to be a primary factor in increasing Cd accumulation and its toxicity in the future ocean. Our findings suggest that we should refocus the interactive effects between climate change stressors and Cd pollution, especially considering temperature as a dominant driver.

Discovery of Clinically Used Octenidine as NRAS Repressor That Effectively Inhibits NRAS-Mutant Melanoma.

Mutations in NRAS promote tumorigenesis and drug resistance. As this protein is often considered an undruggable target, it is urgent to develop novel strategies to suppress NRAS for anticancer therapy. Recent reports indicated that a G-quadruplex (G4) structure formed in the untranslated region of NRAS mRNA can downregulate NRAS translation, suggesting a potential NRAS suppression strategy. Here, we developed a novel cell-based method for large-scale screening of NRAS G4 ligand using the G-quadruplex-triggered fluorogenic hybridization probe and successfully identified the clinically used agent Octenidine as a potent NRAS repressor. This compound suppressed NRAS translation, blocked the MAPK and PI3K-AKT signaling, and caused concomitant cell cycle arrest, apoptosis, and autophagy. It exhibited better antiproliferation effects over clinical antimelanoma agents and could inhibit the growth of NRAS-mutant melanoma in a xenograft mouse model. Our results suggest that Octenidine may be a prominent anti-NRAS-mutant melanoma agent and represent a new NRAS-mutant melanoma therapy option.

Cytotoxic n-Hexane Fraction of the Egyptian Pteris vittata Functions as Anti-breast Cancer Through Coordinated Actions on Apoptotic and Autophagic Pathways.

We investigated the possible anticancer mechanisms of Pteris vittata [PV] n-hexane extract on MCF-7 [breast cancer cell line]. Cultured cell lines were treated with various concentrations of this extract ± Baf-A1 [autophagic inhibitor]. Cells' viability, apoptotic markers [caspase-7, Bax, and Bcl-2], autophagic markers [light chain 3 [LC-3] and P62/SQSTM1]], and the tumor suppressor P53 and its mRNA were checked by their corresponding methods. Treated cell lines showed significant concentration and time-dependent reductions in cell viability in response to PV-n-hexane extract and also exhibited a concomitant induction of apoptosis [increased chromatin condensation, nuclear fragmentation, and pro-apoptotic Bax, and cleaved caspase-7 levels while decreased Bcl-2 levels] and autophagy [increased autophagosomes vacuoles, and LC3B II levels while decreased P62/SQSTM1 levels]. Moreover, PV-n-hexane extract-treated cells showed significant increases in the P53 and its mRNA levels. The addition of Baf-A1 reversed the PV-n-hexane extract autophagic effects and increased apoptotic cell percentage with a much increase in the cleaved caspase-7 and P53 protein and its mRNA levels. We concluded that the PV-n-hexane extract exhibits cytotoxic effects on the MCF-7 cell line with significant reductions in cell viability and concomitant autophagy and apoptosis induction. Inhibition of autophagy in the PV-treated MCF-7 cells enhances apoptosis via a p35-dependent pathway.

Targeting BAP1 with small compound inhibitor for colon cancer treatment

BRCA1-associated protein-1 (BAP1) is a ubiquitin C-terminal hydrolase domain-containing deubiquitinase. The gene encoding BAP1 is mutated in various human cancers, including mesothelioma, uveal melanoma and renal cell carcinoma. BAP1 plays roles in many cancer-related cellular functions, including cell proliferation, cell death, and nuclear processes crucial for genome stability, such as DNA repair and replication. While these findings suggest that BAP1 functions as a tumor suppressor, recent data also suggest that BAP1 might play tumor-promoting roles in certain cancers, such as breast cancer and hematopoietic malignancies. Here, we show that BAP1 is upregulated in colon cancer cells and tissues and that BAP1 depletion reduces colon cancer cell proliferation and tumor growth. BAP1 contributes to colon cancer cell proliferation by accelerating DNA replication and suppressing replication stress and concomitant apoptosis. A recently identified BAP1 inhibitor, TG2-179-1, which seems to covalently bind to the active site of BAP1, exhibits potent cytotoxic activity against colon cancer cells, with half-maximal inhibitory concentrations of less than 10 μM, and inhibits colon tumor growth. TG2-179-1 exerts cytotoxic activity by targeting BAP1, leading to defective replication and increased apoptosis. This work therefore shows that BAP1 acts oncogenically in colon cancer and is a potential therapeutic target for this cancer. Our work also suggests that TG2-179-1 can be developed as a potential therapeutic agent for colon cancer.

The Gut-Ex-Vivo System (GEVS) Is a Dynamic and Versatile Tool for the Study of DNBS-Induced IBD in BALB/C and C57BL/6 Mice, Highlighting the Protective Role of Probiotics.

IBD is a spectrum of pathologies characterized by dysregulated immune activation leading to uncontrolled response against the intestine, thus resulting in chronic gut inflammation and tissue damage. Due to its complexity, the molecular mechanisms responsible for disease onset and progression are still elusive, thus requiring intense research effort. In this context, the development of models replicating the etiopathology of IBD and allowing the testing of new potential therapies is critical. Colon from C57BL/6 or BALB/c mice was cultivated in a Gut-Ex-Vivo System (GEVS), exposed for 5 h to DNBS 1.5 or 2.5 mg/mL, in presence or absence of two probiotic formulations (P1 = Bifidobacterium breve BR03 (DSM16604) and B632 (DSM24706); P2 = Lacticaseibacillus rhamnosus LR04 (DSM16605), Lactiplantibacillus plantarum LP14 (DSM33401) and Lacticaseibacillus paracasei LPC09), and the main hallmarks of IBD were evaluated. Gene expression analysis revealed the following DNBS-induced effects: (i) compromised tight junction organization, responsible for tissue permeability dysregulation; (ii) induction of ER stress, and (iii) tissue inflammation in colon of C57BL/6 mice. Moreover, the concomitant DNBS-induced apoptosis and ferroptosis pathways were evident in colon from both BALB/c and C57BL/6 mice. Finally, the co-administration of probiotics completely prevented the detrimental effects of DNBS. Overall, we have provided results demonstrating that GEVS is a consistent, reliable, and cost-effective system for modeling DNBS-induced IBD, useful for studying the onset and progression of human disease at the molecular level, while also reducing animal suffering. Moreover, we have confirmed the beneficial effect of probiotics administration in promoting the remission of IBD.

Rhabdomyolysis-induced acute kidney injury and concomitant apoptosis induction via ROS-mediated ER stress is efficaciously counteracted by epigallocatechin gallate

Rhabdomyolysis induced acute kidney injury (RIAKI) is a life-threatening condition responsible for approximately 19-58% of AKI cases worldwide. We performed an intramuscular injection of glycerol (10 mL/kg) in male wistar rats to induce AKI. Epigallocatechin gallate (EGCG) was administered for 3 consecutive days to evaluate its protective effects. We observed significant downregulation in serum creatinine, blood urea nitrogen (BUN) and LDH at different time points on EGCG treatment groups in a dose-dependent manner. Similarly, H&E staining also revealed that EGCG was able to reduce the formation of damaged tubules and tubular necrosis which was prominently spread throughout the kidney tissue of glycerol treatment group. Concomitantly, we observed upregulated inflammation, ER stress and elevated oxidative stress in the glycerol treated group only, which was significantly normalized upon EGCG treatment in both in vitro and in vivo studies. The occurrence of apoptosis in kidney tubules was found to be relatively higher in glycerol treated group and H2O2 treated HEK-293 cells. The results obtained after EGCG treatment revealed a significant decrease in apoptotic cell population, which was further validated by immunofluorescence staining against p53 and comet assay in HEK-293 cells and p53 IHC in kidney tissues. Western blotting also revealed a systemic downregulation of intrinsic mitochondrial apoptotic pathway markers such as bax, bcl-2, pro and cleaved caspase 3, caspase 9 and PARP1. Additionally, the results for flow cytometry analysis and TUNEL assay corroborated apoptotic equilibrium. Conclusively, we reckon EGCG as a multi-therapeutic natural product that can be used the for treatment of AKI.

Persistent activation of Nrf2 in a p62-dependent non-canonical manner aggravates lead-induced kidney injury by promoting apoptosis and inhibiting autophagy

IntroductionLead (Pb) is an environmental toxicant that poses severe health risks to humans and animals, especially renal disorders. Pb-induced nephrotoxicity has been attributed to oxidative stress, in which apoptosis and autophagy are core events. ObjectivesNuclear factor erythroid 2-related factor 2 (Nrf2) acts as a major contributor to counteract oxidative damage, while hyperactivation or depletion of Nrf2 pathway can cause the redox imbalance to induce tissue injury. This study was performed to clarify the function and mechanism of Nrf2 in Pb-triggered kidney injury. Methods and resultsFirst, data showed that Pb exposure activates Nrf2 pathway in primary rat proximal tubular cells. Next, Pb-induced Nrf2 activation was effectively regulated by pharmacological modulation or siRNA-mediated knockdown in vitro and in vivo assays. Notably, Pb-triggered cytotoxicity, renal injury and concomitant apoptosis were improved by Nrf2 downregulation, confirming that Pb-induced persistent Nrf2 activation contributes to nephrotoxicity. Additionally, Pb-triggered autophagy blockage was relieved by Nrf2 downregulation. Mechanistically, we found that Pb-induced persistent Nrf2 activation is attributed to reduced Nrf2 ubiquitination and nuclear-cytoplasmic loss of Keap1 in a p62-dependent manner. ConclusionsIn conclusion, these findings highlight the dark side of persistent Nrf2 activation and potential crosstalk among Pb-induced persistent Nrf2 activation, apoptosis and autophagy blockage in Pb-triggered nephrotoxicity.

Neurons die with heightened but functional macro- and chaperone mediated autophagy upon increased amyloid-ß induced toxicity with region-specific protection in prolonged intermittent fasting

Alzheimer's disease (AD) is a devastating neurodegenerative condition with significant socio-economic impact that is exacerbated by the rapid increase in population aging, particularly impacting already burdened health care systems of poorly resourced countries. Accumulation of the amyloid-β (Aβ) peptide, generated through amyloid precursor protein (APP) processing, manifesting in senile plaques, is a well-established neuropathological feature. Aβ plays a key role in driving synaptic dysfunction, neuronal cell loss, glial cell activation and oxidative stress associated with the pathogenesis of AD. Thus, the enhanced clearance of Aβ peptide though modulation of the mechanisms that regulate intracellular Aβ metabolism and clearance during AD progression have received major attention. Autophagy, a lysosome-based major proteolytic pathway, plays a crucial role in intracellular protein quality control and has been shown to contribute to the clearance of Aβ peptide. However, to what extent autophagy activity remains upregulated and functional in the process of increasing Aβ neurotoxicity is largely unclear. Here, we investigated the extent of neuronal toxicity in vitro by characterising autophagic flux, the expression profile of key amyloidogenic proteins, and proteins associated with prominent subtypes of the autophagy pathway to dissect the interplay between the engagement of proteolytic pathways and cell death onset in the context of APP overexpression. Moreover, we assessed the neuroprotective effects of a caloric restriction regime in vivo on the modulation of autophagy in specific brain regions. Our results reveal that autophagy is upregulated in the presence of high levels of APP and Aβ and remains heightened and functional despite concomitant apoptosis induction, suggestive of a mismatch between autophagy cargo generation and clearance capacity. These findings were confirmed when implementing a prolonged intermittent fasting (IF) intervention in a model of paraquat-induced neuronal toxicity, where markers of autophagic activity were increased, while apoptosis onset and lipid peroxidation were robustly decreased in brain regions associated with neurodegeneration. This work highlights that especially caloric restriction mimetics and controlled prolonged IF may indeed be a highly promising therapeutic strategy at all stages of AD-associated pathology progression, for a cell-inherent and cell specific augmentation of Aβ clearance through the powerful engagement of autophagy and thereby robustly contributing to neuronal protection.

Concomitant High Apoptosis Inhibitor of Macrophage (AIM) and Low Prostate-Specific Antigen (PSA) Indicates Activated T Cell-Mediated Anticancer Immunity, Enhance Sensitivity to Pembrolizumab, and Elicit Good Prognosis in Prostate Cancer.

Background: Despite its widespread use, the use of prostate-specific antigen (PSA) alone as a screening biomarker for prostate cancer (PCa) leads often to unwarranted prostate biopsy, over-diagnosis, and consequently, over-treatment, because of its limited specificity. There are reports that the apoptosis inhibitor of macrophage (AIM), secreted mainly by macrophages and epithelial cells, is upregulated during inflammation and facilitates immune recognition of cancerous cells by blocking human regulator of complement activation. Objective: These controversies around the PSA utility necessitate a reexamination of its use as a screening tool. More so, despite the suggested implication of AIM in anticancer immunosurveillance, there is a dearth of information on its role in therapy response, disease progression, and clinical outcomes of patients with PCa. These inform the present study to probe the nature and role of AIM/PSA signaling in anticancer immunity and prognosis in PCa. Methods: A combination of bioinformatics-aided statistical analyses, gene function annotation, and immune infiltrate analyses, coupled with tissue staining, and function assays, namely migration, invasion, and clonogenicity assays, we employed. Results: We demonstrated that AIM and PSA expression levels are inversely correlated in PCa clinical samples and cell lines, with AIMlowPSAhigh defining PCa, compared to AIMhighPSAlow in normal samples. Concomitant aberrant PSA and significantly suppressed AIM expression levels positively correlated with high-grade disease and characterized by advanced stage prostate cancer, regardless of mutation status. We found that a high PSA/AIM ratio is associated with disease recurrence in patients with prostate cancer but is equivocal for overall survival. In addition, PSA-associated AIM suppression is implicated in the enhanced ‘metastability’ of PCa and a high AIM/PSA ratio is associated with strong castration-induced regression. CRISPR-mediated AIM knockout was associated with higher PSA expression while ectopic expression of AIM significantly attenuated the migration and invasive capability of PC3 and DU145 cells. Interestingly, compared to normal samples, we observed that AIM, biomarkers of T-cell activation and M1 phenotype markers are co-suppressed in PCa samples. Conclusion: Herein, we demonstrate that AIM/CD5L binds to PSA and that a high PSA/AIM ratio defines advanced stage PCa (regardless of mutation status), is implicated in enhanced metastability, and associated with disease recurrence, while a high AIM/PSA ratio is associated with strong castration-induced regression. More so, the ectopic expression of AIM significantly enhances the anticancer effect of Pembrolizumab and elicits an increased CD8+ T-cell count in AIMhiPSAloPDL1+ PCa cases that are respondent to Pembrolizumab treatment.

A point mutation in the Pdia6 gene results in loss of pancreatic β-cell identity causing overt diabetes

ObjectiveProtein disulfide isomerases (PDIs) are oxidoreductases that are involved in catalyzing the formation and rearrangement of disulfide bonds during protein folding. One of the PDI members is the PDI-associated 6 (PDIA6) protein, which has been shown to play a vital role in β-cell dysfunction and diabetes. However, very little is known about the function of this protein in β-cells in vivo. This study aimed to describe the consequences of a point mutation in Pdia6 on β-cell development and function. MethodsWe generated an ENU mouse model carrying a missense mutation (Phe175Ser) in the second thioredoxin domain of the Pdia6 gene. Using biochemical and molecular tools, we determined the effects of the mutation on the β-cell development at embryonic day (E)18.5 and β-cell identity as well as function at postnatal stages. ResultsMice homozygous for the Phe175Ser (F175S) mutation were mildly hyperglycemic at weaning and subsequently became hypoinsulinemic and overtly diabetic at the adult stage. Although no developmental phenotype was detected during embryogenesis, mutant mice displayed reduced insulin-expressing β-cells at P14 and P21 without any changes in the rate of cell death and proliferation. Further analysis revealed an increase in BiP and the PDI family member PDIA4, but without any concomitant apoptosis and cell death. Instead, the expression of prominent markers of β-cell maturation and function, such as Ins2, Mafa, and Slc2a2, along with increased expression of α-cell markers, Mafb, and glucagon was observed in adult mice, suggesting loss of β-cell identity. ConclusionsThe results demonstrate that a global Pdia6 mutation renders mice hypoinsulinemic and hyperglycemic. This occurs due to the loss of pancreatic β-cell function and identity, suggesting a critical role of PDIA6 specifically for β-cells.

Synthesis, Anticancer Assessment, and Molecular Docking of Novel Chalcone-Thienopyrimidine Derivatives in HepG2 and MCF-7 Cell Lines.

Heterocycles containing thienopyrimidine moieties have attracted attention due to their interesting biological and pharmacological activities. In this research article, we reported the synthesis of a series of new hybrid molecules through merging the structural features of chalcones and pyridothienopyrimidinones. Our results indicated that the synthesis of chalcone-thienopyrimidine derivatives from the corresponding thienopyrimidine and chalcones proceeded in a relatively short reaction time with good yields and high purity. Most of these novel compounds exhibited moderate to robust cytotoxicity against HepG2 and MCF-7 cancer cells similar to that of 5-fluorouracil (5-FU). The results indicated that IC50 of the two compounds (3b and 3g) showed more potent anticancer activities against HepG2 and MCF-7 than 5-FU. An MTT assay and flow cytometry showed that only 3b and 3g had anticancer activity and antiproliferative activities at the G1 phase against MCF-7 cells, while six compounds (3a-e and 3g) had cytotoxicity and cell cycle arrest at different phases against HepG2 cells. Their cytotoxicity was achieved through downregulation of Bcl-2 and upregulation of Bax, caspase-3, and caspase-9. Although all tested compounds increased oxidative stress via increment of MDA levels and decrement of glutathione reductase (GR) activities compared to control, the 3a, 3b, and 3g in HepG2 and 3b and 3g in MCF-7 achieved the target results. Moreover, there was a positive correlation between cytotoxic efficacy of the compound and apoptosis in both HepG2 (R2 = 0.531; P = 0.001) and MCF-7 (R2 = 0.219; P = 0.349) cell lines. The results of molecular docking analysis of 3a-g into the binding groove of Bcl-2 revealed relatively moderate binding free energies compared to the selective Bcl-2 inhibitor, DRO. Like venetoclax, compounds 3a-g showed 2 violations from Lipinski's rule. However, the results of the ADME study also revealed higher drug-likeness scores for compounds 3a-g than for venetoclax. In conclusion, the tested newly synthesized chalcone-pyridothienopyrimidinone derivatives showed promising antiproliferative and apoptotic effects. Mechanistically, the compounds increased ROS production with concomitant cell cycle arrest and apoptosis. Therefore, regulation of the cell cycle and apoptosis are possible targets for anticancer therapy. The tested compounds could be potent anticancer agents to be tested in future clinical trials after extensive pharmacodynamic, pharmacokinetic, and toxicity profile investigations.

Neuroprotective Effects of the Sonic Hedgehog Signaling Pathway in Ischemic Injury through Promotion of Synaptic and Neuronal Health.

Cerebral ischemia is a common cerebrovascular condition which often induces neuronal apoptosis, leading to brain damage. The sonic hedgehog (Shh) signaling pathway has been reported to be involved in ischemic stroke, but the underlying mechanisms have not been fully elucidated. In the present study, we demonstrated that expressions of Shh, Ptch, and Gli-1 were significantly downregulated at 24 h following oxygen-glucose deprivation (OGD) injury in neurons in vitro, effects which were associated with increasing numbers of apoptotic cells and reactive oxygen species generation. In addition, expressions of synaptic proteins (neuroligin and neurexin) were significantly downregulated at 8 h following OGD, also associated with concomitant neuronal apoptosis. Treatment with purmorphamine, a Shh agonist, increased Gli-1 in the nucleus of neurons and protected against OGD injury, whereas the Shh inhibitor, cyclopamine, produced the opposite effects. Activation of Shh signals promoted CREB and Akt phosphorylation; upregulated the expressions of BDNF, neuroligin, and neurexin; and decreased NF-κB phosphorylation following OGD. Notably, this activation of Shh signals was accompanied by improved neurobehavioral responses along with attenuations in edema and apoptosis at 48 h postischemic insult in rats. Taken together, these results demonstrate that activation of the Shh signaling pathway played a neuroprotective role in response to ischemic exposure via promotion of synaptic and neuronal health.

Abstract A47: BMI1 constitutes a novel therapeutic vulnerability in fusion-positive rhabdomyosarcoma

Abstract Background: Despite intense efforts within pediatric oncology, novel, effective therapy for alveolar rhabdomyosarcoma, known as fusion-positive rhabdomyosarcoma (FP RMS) given the PAX-FOXO1 fusions characteristic of the disease, remains unrealized. Like many pediatric tumors, FP RMS displays a quiet genomic landscape, when focusing on the coding genome. However, the epigenome plays key roles in shaping tumor aggression, and previous studies have demonstrated that FP RMS is specifically enriched for methylation of Polycomb target genes, suggesting that Polycomb complexes may be deregulated and could constitute novel therapeutic targets. We hypothesized that BMI1, a key member of the Polycomb family and a tractable therapeutic target, represents a novel therapeutic vulnerability in FP RMS. Methods: We analyzed RNA and protein expression in FP RMS cell lines, patient-derived xenografts (PDXs), and human tumor specimens. We used genetic and pharmacologic approaches to manipulate BMI1 in FP RMS cells and measured effects on proliferation, cell cycle, apoptosis, and signal transduction. To examine the effect of in vivo inhibition of BMI1, we utilized xenograft models of FP RMS. Results: We examined RNA-Seq tumor datasets and tumor microarrays and demonstrated that BMI1 is robustly expressed in FP RMS tumors, PDXs, and cell line models. Next, in 2 cell line models, we depleted BMI-1 using shRNAs and siRNAs, and found that this led to striking (~70%) decreases in cell growth secondary to both G1/S phase arrest and apoptosis. Given these findings, we asked whether small-molecule inhibitors of BMI-1 mediated similar effects. We treated 4 independent FP RMS cell line models with both PTC-209 (first-generation inhibitor) and PTC-028 (orally available second-generation inhibitor with higher potency). Both compounds inhibited BMI-1 function and greatly reduced cell proliferation in FP rhabdomyosarcoma cell line models. Similar to genetically mediated depletion, pharmacologic inhibition of BMI1 led to G1/S phase arrest and apoptosis, as demonstrated by Annexin V staining and PARP cleavage. Finally, in a xenograft model of an aggressive FP RMS, PTC-028 treatment decreased tumor growth (p=0.0005) and significantly prolonged survival by 17 days (p=0.0002). Importantly, treatment was well tolerated without evidence of toxicity or weight loss. Conclusions: BMI1 is robustly expressed in FP RMS and both genetic and pharmacologic inhibition of BMI1 lead to striking decreases in cell proliferation, with concomitant cell cycle arrest and apoptosis. BMI1 inhibition significantly decreases tumor growth, prolongs survival, and is well tolerated. Currently, we are further investigating combining BMI1 inhibition with standard chemotherapy and novel agents, as well as defining molecular mechanisms by which BMI1 exerts molecular functions in FP RMS. Targeting BMI-1 could provide a novel therapeutic option for patients with FP RMS, with potential broader implications for additional aggressive sarcomas. Citation Format: Cara E. Shields, Selma M. Cuya, Sarah Chappell, Komal Rathi, Shiv Patel, Sindhu Potlapalli, Robert W. Schnepp. BMI1 constitutes a novel therapeutic vulnerability in fusion-positive rhabdomyosarcoma [abstract]. In: Proceedings of the AACR Special Conference on the Advances in Pediatric Cancer Research; 2019 Sep 17-20; Montreal, QC, Canada. Philadelphia (PA): AACR; Cancer Res 2020;80(14 Suppl):Abstract nr A47.

Proteome-wide identification of arginine methylation in colorectal cancer tissues from patients

BackgroundProtein arginine methylation reaction is catalyzed by protein arginine methyltransferase (PRMT) and the modification is implicated in various diseases including cancer. Currently, thousands of arginine methylation sites have been identified using high-resolution mass spectrometry-based proteomics technology. However, identification of arginine methylation using clinical samples at proteome level has not been reported yet. The objective of the present study was to identify, monomethyl-arginine (MMA) and asymmetric dimethyl-arginine (ADMA) sites in colorectal cancer (CRC) tissues at proteome level.MethodsPooled CRC tissue samples from 10 patients with stage II and III were digested by trypsin and these digests were further processed and lyophilized. Using monomethyl- or asymmetric dimethyl arginine (MMA or ADMA, respectively) motif kits, methylarginine-containing peptides were enriched and subsequently analyzed by high-resolution LC-MS/MS. DLD1 and HCT116 colon cancer cells were treated with type I PRMTs inhibitor (MS023) alone or combined with SN-38, and the effect of the drugs on CRC cell proliferation and apoptosis was measured by water-soluble tetrazolium salt (WST-1) assay and FACS analysis, respectively.ResultsIn the present study, 455 MMA sites of 272 proteins and 314 ADMA sites of 155 proteins were identified from CRC tissues acquired from patients. In addition, 216 methylation sites and 75 substrates for PRMTs were newly identified. These results reveal the significant presence of MMA and ADMA sites on nucleic acid binding proteins and protein complexes involved in transcription. To investigate the effect of protein arginine methylation in CRC proliferation and apoptosis, MS023 was treated to two CRC cell lines. After 48 h treatment with various concentrations of MS023, CRC cell proliferation was significantly suppressed, with concomitant apoptosis induction. Furthermore, MS023 treatment significantly enhanced the inhibitory effect of SN-38 on CRC cell proliferation.ConclusionThis work reports the first comprehensive analysis of arginine methylation with clinical sample and suggests that type I PRMTs are potential therapeutic targets for drug discovery in CRC.

Cardiomyocyte Specific Deletion of ADAR1 Causes Severe Cardiac Dysfunction and Increased Lethality.

Background: Adenosine deaminase acting on RNA 1 (ADAR1) is a double-stranded RNA-editing enzyme that is involved in several functions including the deamination of adenosine to inosine, RNA interference (RNAi) mechanisms and microRNA (miRNA) processing, rendering ADAR1 essential for life.Methods and Results: To investigate whether maintenance of ADAR1 expression is required for normal myocardial homeostasis, we bypassed the early embryonic lethality of ADAR1-null mice through the use of a tamoxifen-inducible Cre recombinase under the control of the cardiac-specific α-myosin heavy chain promoter (αMHC). Targeted ADAR1 deletion in adult mice caused a significant increase in lethality accompanied by severe ventricular remodeling and quick and spontaneous cardiac dysfunction, induction of stress markers and overall reduced expression of miRNAs. Administration of a selective inhibitor of the unfolded protein response (UPR) stress significantly blunted the deleterious effects and improved cardiac function thereby prolonging animal survival. In vitro restoring miR-199a-5p levels in cardiomyocytes lacking ADAR1 diminished UPR activation and concomitant apoptosis.Conclusions: Our findings demonstrate an essential role for ADAR1 in cardiomyocyte survival and maintenance of cardiac function through a mechanism that integrates ADAR1 dependent miRNA processing and the suppression of UPR stress.

Polyphenols-Induced Apoptosis by Murraya Koenigii against Lymphatic Filarial Parasite and Its Therapeutic Implication

ABSTRACTThe present study was designed to explore and validate the antifilarial activity of Murraya koenigii leaf extracts against Brugia malayi parasite. Anti-microfilarial activity of M. koenigii leaves extracted in methanol and ethanol (along with polyphenols depleted fractions) was determined. Ethanolic extract showed higher polyphenols content and anti-microfilarial activity compared to methanolic extract. Polyphenols depletion reduced such activity of ethanolic but not of methanolic extract. Ethanolic extract had lower alkaloids than methanolic extract. Ethanolic extract of M. koenigii leaves caused oxidative stress with concomitant apoptosis along with mitochondrial damage.Abbreviations: DEC: Diethyl carbamazine; DNPH: 2, 4 dinitro phenyl hydrazine reagent; DTNB: 5,5-dithio-bis-(2-nitrobenzoic acid); EB/AO: Ethidium bromide/Acridine orange; MDA: Mass Drug Administration; Mf: Microfilariae; PBS: Phosphate buffered saline; TCA: Trichloroacetic acid; WHO: World Health Organization

Genetic or Pharmacological Inhibition of the Polycomb Group Protein BMI1 Impairs Leukemic Transformation Mediated By the CALM-AF10 Fusion Oncoprotein

A subset of acute myeloid and lymphoid leukemia cases harbor a t(10;11)(p13;q14) translocation resulting in the CALM-AF10 fusion gene. Standard chemotherapeutic strategies are not very effective in treating patients with CALM-AF10 fusions. Hence, there is an urgent need to identify molecular pathways dysregulated in CALM-AF10 positive leukemias which may lay the foundation for novel targeted therapies. The polycomb repressive complex 1 gene BMI1 is consistently overexpressed in CALM-AF10 leukemias. Previous studies have shown that CALM-AF10 leukemias express high levels of BMI1, regardless of whether the leukemias are myeloid or lymphoid. Our analysis of TCGA acute myeloid leukemia (AML) data confirmed that AML cells with AF10-rearrangements displayed significantly higher expression of BMI1 transcripts compared to cells from non AF10-rearranged AML patients. These observations indicate that BMI1 may be directly activated by AF10-fusion oncogenes as suggested by our previous studies.We sought to investigate the role of BMI1 in CALM-AF10 mediated leukemogenesis using murine and human models of CALM-AF10-mediated AML. First, we tested whether BMI1 deficiency can affect CALM-AF10 mediated oncogenic transformation of hematopoietic stem and progenitor cells (HSPCs). Towards this end, we retrovirally transduced fetal liver cells from Bmi1 wild-type, heterozygous or homozygous null mice with the CALM-AF10 fusion oncogene. Upon plating these cells in colony forming unit (CFU) assays, we observed a significant decrease in the colony formation capacity of the CALM-AF10 fusion transduced cells on a Bmi1 deficient background. Next, we performed Cre-recombinase mediated excision of Bmi1 of already transformed CALM-AF10 myeloid leukemia cells (Bmi1 floxed background). Bmi1 deletion led to a significant reduction in the number of total CFUs compared to Bmi1 wild-type cells, with a particularly striking reduction in the number of blast-like colonies. These experiments, using Bmi1 constitutive or conditional knockout-mice, revealed that CALM-AF10 transformed AML cells are dependent on Bmi1.Recently, selective pharmacological BMI1 inhibitors have been developed. We tested the impact of pharmacologic BMI1 inhibition on a panel of CALM-AF10-driven mouse leukemias with the small molecule inhibitor PTC-209. PTC-209 treatment increased gene expression of the known BMI1-repressed targets Cdkn2a (p16) and Cdkn1a (p21) and led to a dose-dependent decrease in cell proliferation. We also observed a marked increase in Annexin V+ cells upon PTC-209 treatment. In addition, cell-cycle analysis using BrdU incorporation assays revealed a significant decrease in cells in the S-phase, demonstrating that PTC-209 treatment leads to growth arrest and apoptosis in CALM-AF10 AML cells.In order to confirm these findings in human AML with CALM-AF10 rearrangements, we treated human CALM-AF10 positive AML cell lines P31, U937 and KPMOTS with PTC-209. Consistent with our results in the murine AML model, we observed a time and dose-dependent decrease in proliferation of these human cell lines upon PTC-209 treatment. Drug treated human cells also showed concomitant cell-cycle arrest and apoptosis induction, coupled with an increase in expression of BMI1 repressed tumor suppressor genes such as CDKN2A and CDKN1A.In summary, our results demonstrate that BMI1 is a bonafide candidate for therapeutic targeting in AML with CALM-AF10 rearrangements and possibly other CALM-AF10 positive leukemias. We are now assessing clinical-grade BMI1 inhibitors for in vivo efficacy in mouse models of CALM-AF10-mediated AML. DisclosuresDeshpande:Salgomed Therapeutics: Membership on an entity's Board of Directors or advisory committees; A2A Pharma: Membership on an entity's Board of Directors or advisory committees.

ApoNETosis: discovery of a novel form of neutrophil death with concomitant apoptosis and NETosis

ApoNETosis: discovery of a novel form of neutrophil death with concomitant apoptosis and NETosis