Cellular Growth Control Research Articles

CRISPR perfect adaptation for robust control of cellular immune and apoptotic responses.

A central challenge in the quest for precise gene regulation within mammalian cells is the development of regulatory networks that can achieve perfect adaptation-where outputs consistently return to a set baseline post-stimulus. Here, we present such a system that leverages the CRISPR activation (CRISPRa) and anti-CRISPR proteins as two antithetic elements to establish perfect adaptation in mammalian cells and dynamically regulate gene expression. We demonstrate that this system can maintain stable expression levels of target genes in the face of external perturbations, thus providing a robust platform for biological applications. The versatility of our system is further showcased through its integration with endogenous regulatory mechanisms in T cells, such as the NF-κB-mediated immune response, and its ability to program apoptosis responses for precise spatial and temporal control of cellular growth and death. This study not only advances our understanding of gene regulation in mammalian cells but also opens new avenues for therapeutic intervention, particularly in diseases characterized by dysregulated gene expression.

Chronic Kidney Disease Interplay with Comorbidities and Carbohydrate Metabolism: A Review.

Chronic kidney disease (CKD) poses a global health challenge, engendering various physiological and metabolic shifts that significantly impact health and escalate the susceptibility to severe illnesses. This comprehensive review delves into the intricate complexities of CKD, scrutinizing its influence on cellular growth homeostasis, hormonal equilibrium, wasting, malnutrition, and its interconnectedness with inflammation, oxidative stress, and cardiovascular diseases. Exploring the genetic, birth-related, and comorbidity factors associated with CKD, alongside considerations of metabolic disturbances, anemia, and malnutrition, the review elucidates how CKD orchestrates cellular growth control. A pivotal focus lies on the nexus between CKD and insulin resistance, where debates persist regarding its chronological relationship with impaired kidney function. The prevalence of insulin abnormalities in CKD is emphasized, contributing to glucose intolerance and raising questions about its role as a precursor or consequence. Moreover, the review sheds light on disruptions in the growth hormone and insulin-like growth factor axis in CKD, underscoring the heightened vulnerability to illness and mortality in cases of severe growth retardation. Wasting, a prevalent concern affecting up to 75% of end-stage renal disease (ESRD) patients, is analyzed, elucidating the manifestations of cachexia and its impact on appetite, energy expenditure, and protein reserves. Taste disturbances in CKD, affecting sour, umami, and salty tastes, are explored for their implications on food palatability and nutritional status. Independent of age and gender, these taste alterations have the potential to sway dietary choices, further complicating the management of CKD. The intricate interplay between CKD, inflammation, oxidative stress, and cardiovascular diseases is unraveled, emphasizing the profound repercussions on overall health. Additionally, the review extends its analysis to CKD's broader impact on cognitive function, emotional well-being, taste perception, and endothelial dysfunction. Concluding with an emphasis on dietary interventions as crucial components in CKD management, this comprehensive review navigates the multifaceted dimensions of CKD, providing a nuanced understanding essential for developing targeted therapeutic strategies.

SLIT2/ROBO1 signaling suppresses mTORC1 for organelle control and bacterial killing.

SLIT/ROBO signaling impacts many aspects of tissue development and homeostasis, in part, through the regulation of cell growth and proliferation. Recent studies have also linked SLIT/ROBO signaling to the regulation of diverse phagocyte functions. However, the mechanisms by which SLIT/ROBO signaling acts at the nexus of cellular growth control and innate immunity remain enigmatic. Here, we show that SLIT2-mediated activation of ROBO1 leads to inhibition of mTORC1 kinase activity in macrophages, leading to dephosphorylation of its downstream targets, including transcription factor EB and ULK1. Consequently, SLIT2 augments lysosome biogenesis, potently induces autophagy, and robustly promotes the killing of bacteria within phagosomes. Concordant with these results, we demonstrate decreased lysosomal content and accumulated peroxisomes in the spinal cords of embryos from Robo1 -/- , Robo2 -/- double knockout mice. We also show that impediment of auto/paracrine SLIT-ROBO signaling axis in cancer cells leads to hyperactivation of mTORC1 and inhibition of autophagy. Together, these findings elucidate a central role of chemorepellent SLIT2 in the regulation of mTORC1 activity with important implications for innate immunity and cancer cell survival.

The Cdc48 N-terminal domain has a molecular switch that mediates the Npl4-Ufd1-Cdc48 complex formation

Cdc48 (VCP/p97) is a major AAA-ATPase involved in protein quality control, along with its canonical cofactors Ufd1 and Npl4 (UN). Here, we present novel structural insights into the interactions within the Cdc48-Npl4-Ufd1 ternary complex. Using integrative modeling, we combine subunit structures with crosslinking mass spectrometry (XL-MS) to map the interaction between Npl4 and Ufd1, alone and in complex with Cdc48. We describe the stabilization of the UN assembly upon binding with the N-terminal-domain (NTD) of Cdc48 and identify a highly conserved cysteine, C115, at the Cdc48-Npl4-binding interface which is central to the stability of the Cdc48-Npl4-Ufd1 complex. Mutation of Cys115 to serine disrupts the interaction between Cdc48-NTD and Npl4-Ufd1 and leads to a moderate decrease in cellular growth and protein quality control in yeast. Our results provide structural insight into the architecture of the Cdc48-Npl4-Ufd1 complex as well as its invivo implications.

Genomic landscape and gene expression profiles of feline oral squamous cell carcinoma.

Feline oral squamous cell carcinoma (FOSCC) is a cancer of the squamous cell lining in the oral cavity and represents up to 80% of all oral cancers in cats, with a poor prognosis. We have used whole exome sequencing (WES) and RNA sequencing of the tumor to discover somatic mutations and gene expression changes that may be associated with FOSCC occurrence. FOSCC offers a potential comparative model to study human head and neck squamous cell carcinoma (HNSCC) due to its similar spontaneous formation, and morphological and histological features. In this first study using WES to identify somatic mutations in feline cancer, we have identified tumor-associated gene mutations in six cats with FOSCC and found some overlap with identified recurrently mutated genes observed in HNSCC. Four samples each had mutations in TP53, a common mutation in all cancers, but each was unique. Mutations in other cellular growth control genes were also found such as KAT2B and ARID1A. Enrichment analysis of FOSCC gene expression profiles suggests a molecular similarity to human OSCC as well, including alterations in epithelial to mesenchymal transition and IL6/JAK/STAT pathways. In this preliminary study, we present exome and transcriptome results that further our understanding of FOSCC.

Mechanisms of hedgehog, calcium and retinoic acid signalling pathway inhibitors: Plausible modes of action along the MLL–EZH2-p53 axis in cellular growth control

Understanding the molecular mechanism(s) of small compounds in cellular growth control are essential for using those against the disease(s). Oral cancers exhibit a very high mortality rate due to higher metastatic potential. Aberrant EGFR, RAR, HH signalling, enhanced [Ca2+] and oxidative stress are some of the important characteristics of oral cancer. So, we target these for our study. Herein, we tested the effect of fendiline hydrochloride (FH) as an LTCC Ca2+-channel inhibitor, erismodegib (a SMO inhibitor of HH-signalling) and all-trans retinoic acid (RA) inducer of RAR signalling that causes cellular differentiation. OCT4 activating compound (OAC1) counters differentiation and induces stemness properties. Cytosine β-D arabinofuranoside (Cyto-BDA), a DNA replication inhibitor was used to reduce high proliferative capacity. Treatment of FaDu cells with OAC1, Cyto-BDA and FH increase G0/G1 population by 3%, 20% and 7% respectively, and lead to reduction of cyclin D1, CDK4/6 levels. Erismodegib arrests the cells in S-phase with reduced cyclin-E1&A1 levels, whereas RA-treatment causes G2/M phase arrest with reduced cyclin-B1. There was a decrease in the expression of EGFR and mesenchymal markers, Snail/Slug/Vim/Zeb/Twist, and increased E-cadherin expression in all the drug treatments, indicating a reduction in proliferative signal and EMT. Enhanced MLL2 (Mll4) and reduced EZH2 expression associated overexpression of p53 and p21 were traced out. We conclude that these drugs impact expression of epigenetic modifiers by modulating signalling pathways and the epigenetic modifiers then controls the expression of cell cycle control genes, including p53 and p21.

Regulatory architecture of housekeeping genes is driven by promoter assemblies

Genes that are key to cell identity are generally regulated by cell-type-specific enhancer elements bound by transcription factors, some of which facilitate looping to distant gene promoters. In contrast, genes that encode housekeeping functions, whose regulation is essential for normal cell metabolism and growth, generally lack interactions with distal enhancers. We find that Ronin (Thap11) assembles multiple promoters of housekeeping and metabolic genes to regulate gene expression. This behavior is analogous to how enhancers are brought together with promoters to regulate cell identity genes. Thus, Ronin-dependent promoter assemblies provide a mechanism to explain why housekeeping genes can forgo distal enhancer elements and why Ronin is important for cellular metabolism and growth control. We propose that clustering ofregulatory elements is a mechanism common to cell identity and housekeeping genes but is accomplished by different factors binding distinct control elements to establish enhancer-promoter or promoter-promoter interactions, respectively.

Relationship between 4-Hydroxynonenal (4-HNE) as Systemic Biomarker of Lipid Peroxidation and Metabolomic Profiling of Patients with Prostate Cancer.

An oxidative degradation product of the polyunsaturated fatty acids, 4-hydroxynonenal (4-HNE), is of particular interest in cancer research due to its concentration-dependent pleiotropic activities affecting cellular antioxidants, metabolism, and growth control. Although an increase in oxidative stress and lipid peroxidation was already associated with prostate cancer progression a few decades ago, the knowledge of the involvement of 4-HNE in prostate cancer tumorigenesis is limited. This study investigated the appearance of 4-HNE-protein adducts in prostate cancer tissue by immunohistochemistry using a genuine 4-HNE monoclonal antibody. Plasma samples of the same patients and samples of the healthy controls were also analyzed for the presence of 4-HNE-protein adducts, followed by metabolic profiling using LC-ESI-QTOF-MS and GC-EI-Q-MS. Finally, the analysis of the metabolic pathways affected by 4-HNE was performed. The obtained results revealed the absence of 4-HNE-protein adducts in prostate carcinoma tissue but increased 4-HNE-protein levels in the plasma of these patients. Metabolomics revealed a positive association of different long-chain and medium-chain fatty acids with the presence of prostate cancer. Furthermore, while linoleic acid positively correlated with the levels of 4-HNE-protein adducts in the blood of healthy men, no correlation was obtained for cancer patients indicating altered lipid metabolism in this case. The metabolic pathway of unsaturated fatty acids biosynthesis emerged as significantly affected by 4-HNE. Overall, this is the first study linking 4-HNE adduction to plasma proteins with specific alterations in the plasma metabolome of prostate cancer patients. This study revealed that increased 4-HNE plasma protein adducts could modulate the unsaturated fatty acids biosynthesis pathway. It is yet to be determined if this is a direct result of 4-HNE or whether they are produced by the same underlying mechanisms. Further mechanistic studies are needed to grasp the biological significance of the observed changes in prostate cancer tumorigenesis.

Sensors and Techniques for On-Line Determination of Cell Viability in Bioprocess Monitoring.

In recent years, the bioprocessing industry has experienced significant growth and is increasingly emerging as an important economic sector. Here, efficient process management and constant control of cellular growth are essential. Good product quality and yield can only be guaranteed with high cell density and high viability. Whereas the on-line measurement of physical and chemical process parameters has been common practice for many years, the on-line determination of viability remains a challenge and few commercial on-line measurement methods have been developed to date for determining viability in industrial bioprocesses. Thus, numerous studies have recently been conducted to develop sensors for on-line viability estimation, especially in the field of optical spectroscopic sensors, which will be the focus of this review. Spectroscopic sensors are versatile, on-line and mostly non-invasive. Especially in combination with bioinformatic data analysis, they offer great potential for industrial application. Known as soft sensors, they usually enable simultaneous estimation of multiple biological variables besides viability to be obtained from the same set of measurement data. However, the majority of the presented sensors are still in the research stage, and only a few are already commercially available.

Directional control of neurite outgrowth: emerging technologies for Parkinson's disease using magnetic nanoparticles and magnetic field gradients.

A challenge in current stem cell therapies for Parkinson's disease (PD) is controlling neuronal outgrowth from the substantia nigra towards the targeted area where connectivity is required in the striatum. Here we present progress towards controlling directional neurite extensions through the application of iron-oxide magnetic nanoparticles (MNPs) labelled neuronal cells combined with a magnetic array generating large spatially variant field gradients (greater than 20 T m-1). We investigated the viability of this approach in both two-dimensional and organotypic brain slice models and validated the observed changes in neurite directionality using mathematical models. Results showed that MNP-labelled cells exhibited a shift in directional neurite outgrowth when cultured in a magnetic field gradient, which broadly agreed with mathematical modelling of the magnetic force gradients and predicted MNP force direction. We translated our approach to an ex vivo rat brain slice where we observed directional neurite outgrowth of transplanted MNP-labelled cells from the substantia nigra towards the striatum. The improved directionality highlights the viability of this approach as a remote-control methodology for the control and manipulation of cellular growth for regenerative medicine applications. This study presents a new tool to overcome challenges faced in the development of new therapies for PD.

Insulin signaling couples growth and early maturation to cholesterol intake in Drosophila

Nutrition is one of the most important influences on growth and the timing of maturational transitions including mammalian puberty and insect metamorphosis. Childhood obesity is associated with precocious puberty, but the assessment mechanism that links body fat to early maturation is unknown. During development, the intake of nutrients promotes signaling through insulin-like systems that govern the growth of cells and tissues and also regulates the timely production of the steroid hormones that initiate the juvenile-adult transition. We show here that the dietary lipid cholesterol, which is required as a component of cell membranes and as a substrate for steroid biosynthesis, also governs body growth and maturation in Drosophila via promoting the expression and release of insulin-like peptides. This nutritional input acts via the nutrient sensor TOR, which is regulated by the Niemann-Pick-type-C 1 (Npc1) cholesterol transporter, in the glia of the blood-brain barrier and cells of the adipose tissue to remotely drive systemic insulin signaling and body growth. Furthermore, increasing intracellular cholesterol levels in the steroid-producing prothoracic gland strongly promotes endoreduplication, leading to an accelerated attainment of a nutritional checkpoint that normally ensures that animals do not initiate maturation prematurely. These findings, therefore, show that a Npc1-TOR signaling system couples the sensing of the lipid cholesterol with cellular and systemic growth control and maturational timing, which may help explain both the link between cholesterol and cancer as well as the connection between body fat (obesity) and early puberty.

Conserved heterodimeric GTPase Rbg1/Tma46 promotes efficient translation in eukaryotic cells.

Conserved developmentally regulated guanosine triphosphate (GTP)-binding proteins (Drgs) and their binding partner Drg family regulatory proteins (Dfrps) are important for embryonic development, cellular growth control, differentiation, and proliferation. Here, we report that the yeast Drg1/Dfrp1 ortholog Rbg1/Tma46 facilitates translational initiation, elongation, and termination by suppressing prolonged ribosome pausing. Consistent with the genome-wide observations, deletion of Rbg1 exacerbates the growth defect resulting from translation stalling, and Rbg1 stabilizes mRNAs against no-go decay. Furthermore, we provide a cryoelectron microscopy (cryo-EM) structure of the 80S ribosome bound with Rbg1/Tma46 that reveals the molecular interactions responsible for Rbg1/Tma46 function. The Rbg1 subunit binds to the GTPase association center of the ribosome and the A-tRNA, and the N-terminal zinc finger domain of the Tma46 subunit binds to the 40S, establishing an interaction critical for the ribosomal association. Our results answer the fundamental question of how a paused ribosome resumes translation and show that Drg1/Dfrp1 play a critical role in ensuring orderly translation.

Abstract LB220: Cellular Differentiation and Growth Control in the Human Colonic epithelium: Comparing Clinical Trial Outcomes to Responses in Human Colonoid Culture

Abstract Introduction: As part of our effort to understand the role of trace elements in colon polyp chemoprevention, we have conducted both colonoid culture (ex vivo) and human subject (in vivo) studies using Aquamin®— a calcium-, magnesium-, trace elements-rich, multi-mineral product derived from red marine algae as an intervention. Calcium alone was used as a comparator. Methods: Thirty subjects at-risk for colorectal cancer were enrolled and randomized to receive Aquamin® or calcium alone (each providing 800-mg calcium per day) or placebo. Colon biopsies were obtained before and after 90 days of intervention. We employed histologically normal colonic tissue from the same subjects (at baseline), to propagate in colonoid culture. Once established and expanded, we used the same interventions for a period of two weeks and compared the response (at 1.5mM) to the outcome of the studies conducted in human colonic biopsies after 90 days of intervention. Specifically, colonoid tissue and biopsies were subjected to analysis for markers of growth and differentiation by quantitative immunohistochemistry and tandem mass tag mass spectrometry-based proteomics. Results: The normal tissue colonoids demonstrated a high degree of differentiation as indicated by gross and microscopic appearance, and cytokeratin 20 (CK20) expression without either intervention. Only modest increases were seen in the CK20 expression with either calcium alone or Aquamin®. Similarly, CK20 expression was higher in baseline biopsies and there was no increase in the expression with calcium but a 5% increase seen with Aquamin® after 90 days of intervention. On proteomic screen, CK20 expression was increased by 40% in colonoid tissue in response to both interventions at 1.5mM, while CK20 expression increased by 10% and 21% with Calcium and Aquamin® in colon biopsies. When the colonoids were assessed for Ki67 (growth marker) expression, it was decreased by 17.2% and 18.4% with calcium alone and Aquamin® respectively as compared to the control. Analyzing colon biopsies after 90 days of intervention revealed that Aquamin® reduced the level of Ki67 expression by 20%, while no change was seen with calcium alone. Differential proteomic expression of proliferating cell nuclear antigen was decreased by 32% and 39% by calcium and Aquamin® at 1.5mM in colonoid tissue, whereas Ki67 protein was decreased by 4% and 22% with calcium and Aquamin®, respectively in colon biopsies. Conclusion: Colon tissue maintained in colonoid culture, thus, provides a good surrogate for human tissue collected in a clinical trial. It can be used to assess personalized responses or can provide a quick snapshot of the response in a relatively short time. Differential proteomic expression appears to be more sensitive than quantitative immunohistochemistry. A combination of calcium and additional trace elements proved to be more effective than calcium alone in altering differentiation and growth markers in either setting. Citation Format: Muhammad N. Aslam, Shannon McClintock, Mohamed Ali H Jawad-Makki, Karsten Knuver, Daniyal M. Nadeem, Haris Ahmad, Durga Attili, Danielle (Kim) Turgeon, James Varani. Cellular Differentiation and Growth Control in the Human Colonic epithelium: Comparing Clinical Trial Outcomes to Responses in Human Colonoid Culture [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 LB220.

Overexpression of BRD4 Is an Adverse Prognostic Factor in Acute Myeloid Leukemia

Background:Bromodomain containing protein 4 (BRD4), a member of the bromodomain and extra terminal domain (BET) protein family, has been implicated in the pathogenesis of a number of hematological and solid tumors. BRD4 is an epigenetic reader that plays a central role in transcriptional regulation, cellular growth control, and cell cycle progression. As a consequence, BET inhibitors elicit anticancer activity in numerous malignant contexts. While deregulation of BRD4 activity has been shown to be essential for the maintenance of AML, its expression profile and prognostic value in AML have not been investigated. Additionally, how BRD4 is regulated in cancer cells remains largely unknown.Methodology: We therefore evaluated the level of BRD4 expression using Reverse Phase Protein Array (RPPA) technology in marrow and blood samples from 511 newly diagnosed AML patients, and determined a correlation between BRD4 expression and clinical characteristics and overall survival in AML patients. We also correlated expression of BRD4 with 232 other proteins (encompassing signal transduction, metabolism, apoptosis and cellular proliferation pathways) that were included in RPPA. Potential role of molecular chaperone heat shock protein 90 (Hsp90) in regulating BRD4 protein stability was also investigated.Results: BRD4 expression was similar in peripheral blood and bone marrow and expression was found to be higher at diagnosis than at relapse (P = .035). Compared with normal bone marrow derived CD34+ cells, BRD4 expression in AML was higher in 28% of patients and that higher BRD4 levels significantly predicted shorter overall survival (P = .002). There was no significant difference in BRD4 expression on the basis of age, sex, or performance status, antecedent malignancies, and previous chemotherapy and/or radiotherapy. BRD4 expression levels were not strongly correlated with French-American-British classification (FAB), although levels were slightly higher in M5 subtype. BRD4 levels were found to be significantly higher in patients with NPM1 mutation (P = .003) or FLT3-ITD (P = .057) or between those with both FLT3-D835F and NPM1 mutation (P = .01). BRD4 levels were not associated with the attainment of complete remission (CR), although rates were somewhat lower for those with high. However, patients with high levels of BRD4 had significantly inferior remission duration (25.9 vs 50 weeks; P = 0.03) compared with those with normal levels of BRD4. The overall survival (OS) of patients with higher levels of BRD4 was significantly inferior compared with those with normal levels of BRD4 (28.3 vs 55.3 weeks; P = .002). Patients with unfavorable cytogenetics and above-normal levels of BRD4 had inferior survival compared with those with normal levels of BRD4 (22.4 vs 38.9 weeks; P = .0043; Figure 3C). BRD4 expression was found to be strongly correlated (R > 0.2 and P < 0.0001 or 0.00001) either positively (n = 48) or negatively (n = 14) with proteins known to be important in AML. BRD4 expression was positively correlated with various cell proliferation-related proteins (Hsp90, pSTAT5, nucleolin and mTOR), anti-apoptotic protein (Bcl-2), oncoproteins (FLI1, CREB and ELK1), proteins important for stem cell functions (Ash2L and LSD1), hematopoietic transcription factors (ERG and PU.1/Spi1), and Smad proteins, signal transducers for the members of the transforming growth factor-β (TGF-β) superfamily. The stability, activity and function of several mutated, chimeric, and overexpressed signaling proteins crucial for oncogenesis are often maintained by Hsp90. Our study also demonstrates that BRD4 stability is dependent upon Hsp90 activity and pharmacologic inhibition of Hsp90 promoted ubiquitination followed by proteasome-dependent degradation of BRD4 protein.Conclusions: These findings suggest BRD4 as a prognostic biomarker for unfavorable outcomes and supports the clinical rational for using BRD4 inhibitors in AML. Future studies could identify mechanistic insights into BRD4-associated proteins as biomarkers for response to BET inhibition. DisclosuresKarnad:ADC Therapeutics: Research Funding. Weitman:Tolero Pharmaceuticals, Inc.: Employment. Iyer:Takeda: Research Funding; Genentech: Research Funding.

Interpretations on the Interaction between Protein Tyrosine Phosphatase and E7 Oncoproteins of High and Low-Risk HPV: A Computational Perception.

The most prevalent and common sexually transmitted infection is caused by human papillomavirus (HPV) among sexually active women. Numerous genotypes of HPV are available, among which the major oncoproteins E6 and E7 lead to the progression of cervical cancer. The E7 oncoprotein interacts with cytoplasmic tumor suppressor protein PTPN14, which is the key regulator of cellular growth control pathways effecting the reduction of steady-state level. Disrupting the interaction between the tumor suppressor and the oncoprotein is vital to cease the development of cancer. Hence, the mechanism of interaction between E7 and tumor suppressor is explored through protein–protein and protein–ligand binding along with the conformational stability studies. The obtained results state that the LXCXE domain of HPV E7 of high and low risks binds with the tumor suppressor protein. Also, the small molecules bind in the interface of E7–PTPN14 that disrupts the interaction between the tumor suppressor and oncoprotein. These results were further supported by the dynamics simulation stating the stability over the bounded complex and the energy maintained during postdocking as well as postdynamics calculations. These observations possess an avenue in the drug discovery that leads to further validation and also proposes a potent drug candidate to treat cervical cancer caused by HPV.

Curcumin arrests G-quadruplex in the nuclear hyper-sensitive III1 element of c-MYC oncogene leading to apoptosis in metastatic breast cancer cells

c-MYC is deregulated in triple negative breast cancer (TNBC) pointing to be a promising biomarker for breast cancer treatment. Precise level of MYC expression is important in the control of cellular growth and proliferation. Designing of c-MYC-targeted antidotes to restore its basal level of cellular expression holds an optimistic approach towards anti-cancer treatment. MYC transcription is dominantly controlled by Nuclear Hypersensitive Element III-1 (NHEIII1) upstream of the promoter region possessing G-Quadruplex silencer element (Pu-27). We have investigated the selective binding-interaction profile of a natural phytophenolic compound Curcumin with native MYC G-quadruplex by conducting an array of biophysical experiments and in silico based Molecular Docking and Molecular Dynamic (MDs) simulation studies. Curcumin possesses immense anti-cancerous properties. We have observed significantly increased stability of MYC-G Quadruplex and thermodynamic spontaneity of Curcumin-MYC GQ binding with negative ΔG value. Transcription of MYC is tightly regulated by a complex mechanism involving promoters, enhancers and multiple transcription factors. We have used Curcumin as a model drug to understand the innate mechanism of controlling deregulated MYC back to its basal expression level. We have checked MYC-expression at transcriptional and translational level and proceeded for Chromatin Immuno-Precipitation assay (ChIP) to study the occupancy level of SP1, Heterogeneous nuclear ribonucleoprotein K (hnRNPK), Nucleoside Diphosphate Kinase 2 (NM23-H2) and Nucleolin at NHEIII1 upon Curcumin treatment of MDA-MB-231 cells. We have concluded that Curcumin binding tends to drive the equilibrium towards stable G-quadruplex formation repressing MYC back to its threshold-level. On retrospection of the synergistic effect of upregulated c-MYC and BCL-2 in cancer, we have also reported a new pathway [MYC-E2F-1-BCL-2-axis] through which Curcumin trigger apoptosis in cancer cells. Communicated by Ramaswamy H. Sarma

Conserved Heterodimeric GTPase Rbg1/Tma46 Promotes Efficient Translation in Eukaryotic Cells

Conserved developmentally-regulated GTP-binding (Drg) proteins and their binding partner Dfrp proteins are known to be important for embryonic development, cellular growth control, differentiation and proliferation. Here, we report that the yeast Drg1/Dfrp1 ortholog, Rbg1/Tma46, facilitates translational initiation, elongation and termination via suppressing prolonged ribosome pausing. Consistent with the genome-wide observation, Rbg1 reverses the growth defect resulting from translation stalling and stabilizes mRNAs against no-go decay. Furthermore, we provide a cryoEM structure of the 80S ribosome bound with Rbg1/Tma46 that reveals the molecular interactions responsible for Rbg1/Tma46 function. The Rbg1 subunit binds to the GTPase association center of the ribosome and the A-tRNA, and the N-terminal zinc finger domain of Tma46 subunit binds to the 40S, establishing an interaction critical for the complex ribosomal association. Our results answer the fundamental question on how a paused ribosome resumes translation and show that Drg1/Dfrp1 proteins play a critical role in ensuring orderly translation.

Less Can Be More: The Hormesis Theory of Stress Adaptation in the Global Biosphere and Its Implications.

A dose-response relationship to stressors, according to the hormesis theory, is characterized by low-dose stimulation and high-dose inhibition. It is non-linear with a low-dose optimum. Stress responses by cells lead to adapted vitality and fitness. Physical stress can be exerted through heat, radiation, or physical exercise. Chemical stressors include reactive species from oxygen (ROS), nitrogen (RNS), and carbon (RCS), carcinogens, elements, such as lithium (Li) and silicon (Si), and metals, such as silver (Ag), cadmium (Cd), and lead (Pb). Anthropogenic chemicals are agrochemicals (phytotoxins, herbicides), industrial chemicals, and pharmaceuticals. Biochemical stress can be exerted through toxins, medical drugs (e.g., cytostatics, psychopharmaceuticals, non-steroidal inhibitors of inflammation), and through fasting (dietary restriction). Key-lock interactions between enzymes and substrates, antigens and antibodies, antigen-presenting cells, and cognate T cells are the basics of biology, biochemistry, and immunology. Their rules do not obey linear dose-response relationships. The review provides examples of biologic stressors: oncolytic viruses (e.g., immuno-virotherapy of cancer) and hormones (e.g., melatonin, stress hormones). Molecular mechanisms of cellular stress adaptation involve the protein quality control system (PQS) and homeostasis of proteasome, endoplasmic reticulum, and mitochondria. Important components are transcription factors (e.g., Nrf2), micro-RNAs, heat shock proteins, ionic calcium, and enzymes (e.g., glutathion redox enzymes, DNA methyltransferases, and DNA repair enzymes). Cellular growth control, intercellular communication, and resistance to stress from microbial infections involve growth factors, cytokines, chemokines, interferons, and their respective receptors. The effects of hormesis during evolution are multifarious: cell protection and survival, evolutionary flexibility, and epigenetic memory. According to the hormesis theory, this is true for the entire biosphere, e.g., archaia, bacteria, fungi, plants, and the animal kingdoms.

Targeting NF-κB-Inducing Kinase (NIK) in Immunity, Inflammation, and Cancer.

NF-κB-inducing kinase (NIK), the essential upstream kinase, which regulates activation of the noncanonical NF-κB pathway, has important roles in regulating immunity and inflammation. In addition, NIK is vital for maintaining cellular health through its control of fundamental cellular processes, including differentiation, growth, and cell survival. As such aberrant expression or regulation of NIK is associated with several disease states. For example, loss of NIK leads to severe immune defects, while the overexpression of NIK is observed in inflammatory diseases, metabolic disorders, and the development and progression of cancer. This review discusses recent studies investigating the therapeutic potential of NIK inhibitors in various diseases.

Abstract IA31: Modeling and targeting double-hit lymphoma

Abstract High-grade B-cell lymphoma with concurrent activation of MYC and BCL2, also known as double-hit lymphoma (DHL), shows dismal prognosis with current front-line therapies (e.g., R-CHOP), calling for the development of new therapeutic regimens. The synergy between MYC and BCL2 in lymphomagenesis is explained by the ability of BCL2 to block the proapoptotic activity of MYC, while leaving intact its proliferative potential; on this basis, we reasoned that compounds that exacerbate MYC-induced apoptosis might cooperate with BCL2 inhibitors in killing DHL cells. We previously observed that genes encoding components of the mitochondrial ribosome were coordinately activated in MYC-driven lymphoma (1) and were critical for tumor maintenance in transgenic mice (2). In line with the genetic data, inhibition of mitochondrial translation with the antibiotic tigecycline was synthetic-lethal with MYC activation and extended lifespan in lymphoma-bearing mice (2). Combining tigecycline with the BCL2 inhibitor venetoclax revealed synergy in the induction of apoptosis in human DHL cell lines, as well as marked antitumoral activity in xenografted mice (3). These preclinical data warrant the repurposing of venetoclax and tigecycline for the treatment of refractory and/or relapsed DHL. By blocking the synthesis of mitochondrially encoded proteins, tigecycline impairs the assembly of the electron-transport chain (ETC) complexes and oxidative phosphorylation. We thus hypothesized that direct ETC inhibitors might also preferentially kill MYC-overexpressing cells and synergize with venetoclax against DHL. We will present data that verify these predictions for IACS-010759, a small-molecule inhibitor of mitochondrial complex I. A current limitation for further preclinical development and mechanistic analysis lies in the lack of a bona fide mouse model of DHL. While the cooperation between MYC and BCL2 in lymphomagenesis was amply documented over almost three decades, the models described so far led to tumor onset at early stages of B-cell ontogeny; as a consequence, no model is available that reliably reproduces the development of DHL from germinal center (GC) B-cells, as observed in the clinic. In order to overcome this limitation, we combined CRE-activated alleles of MYC and BCL2 with Cgamma1-CRE, a CRE-expressing transgene that is specifically expressed in GC B cells. Concerted activation of both oncogenes led to increased expansion of GC B cells upon immunization, followed within a few months by the onset of aggressive, invasive B-cell lymphomas. Our progress in characterizing this disease model will be presented at the meeting.