Cellular Growth Rate Research Articles

Grazer‐induced toxin production is energetically costly and significantly reduces growth of cylindrospermopsin‐producing cyanobacteria

AbstractToxins have been proposed as a defense mechanism for phytoplankton against zooplankton grazing. However, the associated costs are not understood. Here, we aim to reveal the costs in terms of both physiological and molecular responses in the cylindrospermopsin (CYN)‐producing cyanobacterium Raphidiopsis raciborskii. Exposure of R. raciborskii to Daphnia magna resulted in a significant increase in its CYN cell quota which was grazer cell density‐dependent, indicating an inducible effect on CYN production. A negative and significant relationship between cellular growth rate and CYN production suggested a growth‐defense trade‐off, highlighting the costly nature of toxin production. Despite strong transcriptional responses (121 differentially expressed genes) in R. raciborskii under grazing pressure, the CYN gene cluster remained largely unaffected, implying post‐transcriptional regulation of CYN biosynthesis. Significant variation was observed in transcripts for phosphorus metabolism and photosynthesis‐related genes, and evidenced that energy (ATP) metabolic processes were stimulated. This study is the first to demonstrate the high energy requirements for CYN production and provide insight into the molecular basis underlying the costs of toxin production.

The people behind the papers - Isabella Burda and Adrienne Roeder.

Morphogenesis in plants is often robust, resulting in a reproducible organ size and shape across organisms. In a new study, Adrienne Roeder and colleagues investigate this robustness in the developing Arabidopsis thaliana sepal. They find that the robustness is not affected by changes in the amount of cell division, but the variability of cellular growth rate must be uncorrelated or anti-correlated for robust development to occur. To find out more about the work, we caught up with first author Isabella Burda and corresponding author Adrienne Roeder, Associate Professor at Cornell University, USA.

EsxA, a type VII secretion system-dependent effector, reveals a novel function in the sporulation of Bacillus cereus ATCC14579

BackgroundBacillus cereus is a Gram-positive, spore-forming bacterium that produces a spectrum of effectors integral to bacterial niche adaptation and the development of various infections. Among those is EsxA, whose secretion depends on the EssC component of the type VII secretion system (T7SS). EsxA’s roles within the bacterial cell are poorly understood, although postulations indicate that it may be involved in sporulation. However, the T7SS repertoire in B. cereus has not been reported, and its functions are unestablished.MethodsWe used the type strain, B. cereus ATCC14579, to generate ΔessC mutant through homologous recombination using the homing endonuclease I-SceI mediated markerless gene replacement. Comparatively, we analyzed the culture supernatant of type strain and the ΔessC mutant through Liquid chromatography-tandem mass spectrometry (LC-MS/MS). We further generated T7SSb-specific gene mutations to explore the housekeeping roles of the T7SSb-dependent effectors. The sporulation process of B. cereus ATCC14579 and its mutants was observed microscopically through the classic Schaeffer-Fulton staining method. The spore viability of each strain in this study was established by enumerating the colony-forming units on LB agar.ResultsThrough LC-MS/MS, we identified a pair of nearly identical (94%) effector proteins named EsxA belonging to the sagEsxA-like subfamily of the WXG100 protein superfamily in the culture supernatant of the wild type and none in the ΔessC mutant. Homology analysis of the T7SSb gene cluster among B. cereus strains revealed diversity from the 3’ end of essC, encoding additional substrates. Deletions in esxA1 and esxA2 neither altered cellular morphology nor growth rate, but the ΔesxA1ΔesxA2 deletion resulted in significantly fewer viable spores and an overall slower sporulation process. Within 24 h culture, more than 80% of wild-type cells formed endospores compared to less than 5% in the ΔesxA1ΔesxA2 mutant. The maximum spore ratios for the wild type and ΔesxA1ΔesxA2 were 0.96 and 0.72, respectively. Altogether, these results indicated that EsxA1 and EsxA2 work cooperatively and are required for sporulation in B. cereus ATCC14567.ConclusionB. cereus ATCC14579 possesses two nearly identical T7SSb-dependent effectors belonging to the sagEsxA-like proteins. Simultaneous deletion of genes encoding these effectors significantly delayed and reduced sporulation, a novel finding for EsxA.

Suppression by RNA Polymerase I Inhibitors Varies Greatly Between Distinct RNA Polymerase I Transcribed Genes in Malaria Parasites.

Transcription of ribosomal RNA (rRNA) by RNA Polymerase I (Pol I) is the rate-limiting step in ribosome biogenesis and a major determinant of cellular growth rates. Unlike virtually every other eukaryote, which express identical rRNA from large tandem arrays of dozens to hundreds of identical rRNA genes in every cell, the genome of the human malaria parasite Plasmodium falciparum contains only a handful single-copy 47S rRNA loci that differ substantially from one another in length, sequence and expression in different cell-types. We found that growth of malaria parasite was acutely sensitive to the Pol I inhibitors 9-hydroxyellipticine and BMH-21 and demonstrate that they greatly reduce the transcription of 47S rRNAs as well as transcription of other non-coding RNA genes. Surprisingly, we found that the various types of Pol I-transcribed genes differed by more than two orders of magnitude in their susceptibility to these inhibitors and explore the implications of these findings for regulation of rRNA in P. falciparum.

Modulation of cell differentiation and growth underlies the shift from bud protection to light capture in cauline leaves.

Plant organs have evolved into diverse shapes for specialized functions despite emerging as simple protrusions at the shoot apex. Cauline leaves serve as photosynthetic organs and protective structures for emerging floral buds. However, the growth patterns underlying this dual function remain unknown. Here, we investigate the developmental dynamics shaping Arabidopsis (Arabidopsis thaliana) cauline leaves underlying their functional diversification from other laminar organs. We show that cauline leaves display a significant delay in overall elongation compared to rosette leaves. Using live imaging, we reveal that their functional divergence hinges on early modulation of the timing of cell differentiation and cellular growth rates. In contrast to rosette leaves and sepals, cell differentiation is delayed in cauline leaves, fostering extended proliferation, prolonged morphogenetic activity, and growth redistribution within the organ. Notably, cauline leaf growth is transiently suppressed during the early stages, keeping the leaf small and unfolded during the initiation of the first flowers. Our findings highlight the unique developmental timing of cauline leaves, underlying their shift from an early protective role to a later photosynthetic function.

How total mRNA influences cell growth

Experimental observations tracing back to the 1960s imply that ribosome quantities play a prominent role in determining a cell's growth. Nevertheless, in biologically relevant scenarios, growth can also be influenced by the levels of mRNA and RNA polymerase. Here, we construct a quantitative model of biosynthesis providing testable scenarios for these situations. The model explores a theoretically motivated regime where RNA polymerases compete for genes and ribosomes for transcripts and gives general expressions relating growth rate, mRNA concentrations, ribosome, and RNA polymerase levels. On general grounds, the model predicts how the fraction of ribosomes in the proteome depends on total mRNA concentration and inspects an underexplored regime in which the trade-off between transcript levels and ribosome abundances sets the cellular growth rate. In particular, we show that the model predicts and clarifies three important experimental observations, in budding yeast and Escherichia coli bacteria: i) that the growth-rate cost of unneeded protein expression can be affected by mRNA levels, ii) that resource optimization leads to decreasing trends in mRNA levels at slow growth, and iii) that ribosome allocation may increase, stay constant, or decrease, in response to transcription-inhibiting antibiotics. Since the data indicate that a regime of joint limitation may apply in physiological conditions and not only to perturbations, we speculate that this regime is likely self-imposed.

Polyhydroxyalkanoate Production from Eucalyptus Bark's Enzymatic Hydrolysate.

In recent years, polyhydroxyalkanoates (PHAs) have gained notoriety because of their desirable properties that include proven biodegradability, biocompatibility, and thermal stability, which make them suitable alternatives to fossil-based polymers. However, the widespread use of PHAs is still challenging because of their production costs, which are greatly associated with the cultivation medium used for bacterial cultivation. In Portugal, one-quarter of the forest area is covered by Eucalyptus globulus wood, making its residues a cheap, abundant, and sustainable potential carbon source for biotechnological uses. In this work, eucalyptus bark was used as the sole feedstock for PHA production in a circular bioeconomic approach. Eucalyptus bark hydrolysate was obtained after enzymatic saccharification using Cellic® CTec3, resulting in a sugar-rich solution containing glucose and xylose. Although with differing performances, several bacteria were able to grow and produce PHA with distinct compositions, using the enzymatic hydrolysate as the sole carbon source. Pseudomonas citronellolis NRRL B-2504 achieved a high cellular growth rate in bioreactor assays (24.4 ± 0.15 g/L) but presented a low accumulation of a medium-chain-length PHA (mcl-PHA) comprising the monomers hydroxydecanoate (HD, 65%), hydroxydodecanoate (HDd, 25%), and hydroxytetradecanoate (HTd, 14%). Burkholderia thailandensis E264, on the other hand, reached a lower cellular growth rate (8.87 ± 0.34 g/L) but showed a higher biopolymer accumulation, with a polyhydroxybutyrate (PHB) content in the cells of 12.3 wt.%. The new isolate, Pseudomonas sp., revealed that under nitrogen availability, it was able to reach a higher accumulation of the homopolymer PHB (31 wt.%). These results, although preliminary, demonstrate the suitability of eucalyptus bark's enzymatic hydrolysate as a feedstock for PHA production, thus offering an exciting avenue for achieving sustainable and environmentally responsible plastic products from an undervalued forestry waste.

Gene Expression Tradeoffs Determine Bacterial Survival and Adaptation to Antibiotic Stress.

To optimize their fitness, cells face the crucial task of efficiently responding to various stresses. This necessitates striking a balance between conserving resources for survival and allocating resources for growth and division. The fundamental principles governing these tradeoffs is an outstanding challenge in the physics of living systems. In this study, we introduce a coarse-grained theoretical framework for bacterial physiology that establishes a connection between the physiological state of cells and their survival outcomes in dynamic environments, particularly in the context of antibiotic exposure. Predicting bacterial survival responses to varying antibiotic doses proves challenging due to the profound influence of the physiological state on critical parameters, such as the minimum inhibitory concentration (MIC) and killing rates, even within an isogenic cell population. Our proposed theoretical model bridges the gap by linking extracellular antibiotic concentration and nutrient quality to intracellular damage accumulation and gene expression. This framework allows us to predict and explain the control of cellular growth rate, death rate, MIC, and survival fraction in a wide range of time-varying environments. Surprisingly, our model reveals that cell death is rarely due to antibiotic levels being above the maximum physiological limit, but instead survival is limited by the inability to alter gene expression sufficiently quickly to transition to a less susceptible physiological state. Moreover, bacteria tend to overexpress stress response genes at the expense of reduced growth, conferring greater protection against further antibiotic exposure. This strategy is in contrast to those employed in different nutrient environments, in which bacteria allocate resources to maximize growth rate. This highlights an important tradeoff between the cellular capacity for growth and the ability to survive antibiotic exposure.

A Response Surface Methodology Study for Chlorella vulgaris Mixotrophic Culture Optimization.

Glycerol is a carbon source that produces good biomass under mixotrophic conditions. Enhancing the composition of culture media in algae biomass production improves growth rates, biomass yield, nutrient utilization efficiency, and overall cost-effectiveness. Among the key nutrients in the medium, nitrogen plays a pivotal role. Urea can be effectively used as a nitrogen source and is considered a low-cost form of nitrogen compared to other sources. Urea metabolism releases some CO2 in photosynthesis, and magnesium plays a major role in urea uptake. Magnesium is another key nutrient that is key in photosynthesis and other metabolic reactions. To maximize glycerol consumption in the mixotrophic system and to obtain high biomass and lipid productions, the variations in MgSO4·7H2O and urea concentrations were evaluated in the growth medium of the microalgae. A response surface methodology (RSM) using a central composite design (CCD) was designed to maximize glycerol consumption at the initial cellular growth rates (up to four days). The magnesium and urea supply varied from 0.3 to 1.7 g L-1. Response surface methodology was utilized to analyze the results, and the highest glycerol consumption rate, 770.2 mg L-1 d-1, was observed when C. vulgaris was grown at 1.7 g L-1 urea, 1.0 g L-1 MgSO4·7H2O. Using the optimal urea and magnesium concentrations with acetate, glucose, and glycerol as carbon sources, the same lipid content (10% average) was achieved on day 4 of mixotrophic C. vulgaris culture. Overall, the results show that mixotrophic growth of C. vulgaris using urea with an optimum magnesium concentration yields large amounts of fatty acids and that the carbon source greatly influences the profile of the fatty acids.

Aging of the adrenal gland and its impact on the stress response.

This article discusses the physiological and anatomical changes of adrenal gland with age and the effects this has overall on how the organ responds to stress. Physiological changes entail a decrease in adrenocorticoid hormone secretion however cortisol levels remain intact leading to a disruptive stress response. Additionally, loss of zonation of the organ also occurs. Both characteristics in combination with chronic stress affect overall health. Complex interplay between adrenal aging and stress responsiveness is confounded further by the impact they expel on other systems, such as the thyroid hormone. The body undergoes age-related transformations modifying rate of cellular growth, differentiation, senescence, and hormone production. Given the multiplicity and complexity of hormones, their production must be considered to develop appropriate interventions to mitigate its effect on age related diseases in health.

Caveolin-1, a Determinant of the Fate of MCF-7 Breast Cancer Cells.

The scaffolding protein, caveolin-1 (Cav-1), participates in multiple cellular functions including promotion of sodium excretion from the kidney. Loss of expression of Cav-1 is associated with tumorigenesis of various types of cancer. We have shown the potential link between hypertension and breast cancer via abnormal function of the G protein-coupled receptor kinase type 4 (GRK4). The current studies tested the hypothesis that Cav-1 acts as a tumor-suppressive factor in breast cancer cells and enhances the sensitivity to the inhibitory effect of the type 1 dopaminergic receptor (D1R). Michigan Cancer Foundation (MCF) MCF-7 cells stably expressing a Cav-1/mCherry fusion protein or mCherry alone were used as models to examine the effect of Cav-1 on cell growth, apoptosis, and senescence. Cell proliferation was determined by cell counting, cell cycle analysis (flow cytometry), and BrdU incorporation. Apoptosis was determined using the Cell Death Detection ELISA kit from Roche Diagnosis. Senescence was determined using the senescence associated beta galactosidase (SA-β-gal) assay. Reactive oxygen species (ROS) was measured using 2',7'-dichlorodihydrofluorescein diacetate. Western blot analysis was used to measure activation of signaling pathway molecules. All statistical analyses were conducted with Microsoft Excel. Overexpression of Cav-1 in MCF-7 cells reduced cellular growth rate. Both inhibition of proliferation and induction of cell death are contributing factors. Multiple signaling pathways were activated in Cav-1-expressing MCF-7 cells. Activation of Akt was prominent. In MCF-7-expressing Cav-1 (MCF-7 Cav-1) cells, the levels of phosphorylated Akt at S473 and T308 were increased 28- and 8.7-fold, respectively. Instead of protecting cells from apoptosis, extremely high levels of activated Akt resulted in increased levels of ROS which led to apoptosis and senescence. The tumor-suppressive effect plus downregulation of GRK4 makes Cav-1-expressing MCF-7 cells significantly more sensitive to the inhibitory effect of the D1R agonist, SKF38393. Caveolin-1 acts as a tumor-suppressing factor via extreme activation of Akt and down regulation of survival factors such as GRK4, survivin, and cyclin D1.

Comparison of Transcription Elongation Rates of Three RNA Polymerases in Real Time.

RNA polymerases (RNAPs) across the bacterial kingdom have retained a conserved structure and function. In spite of the remarkable similarity of the enzyme in different bacteria, a wide variation is found in the promoter-polymerase interaction, transcription initiation, and termination. However, the transcription elongation was considered to be a monotonic process, although the rate of elongation could vary in different bacteria. Such variations in RNAP elongation rates could be important to fine-tune the transcription, which in turn would influence cellular metabolism and growth rates. Here, we describe a quantitative study to measure the transcription rates for the RNAPs from three bacteria, namely, Mycobacterium tuberculosis, Mycobacterium smegmatis, and Escherichia coli, which exhibit different growth kinetics. The RNA synthesis rates of the RNAPs were calculated from the real-time elongation kinetic profile using surface plasmon resonance through a computational flux flow model. The computational model revealed the modular process of elongation, with different rate profiles for the three RNAPs. Notably, the transcription elongation rates of these RNAPs followed the trend in the growth rates of these bacteria.

The Clinical Relevance and Functional Implications of Thymosin Beta-10 in Glioma.

Glioma is a highly aggressive form of brain cancer characterized by limited treatment options and poor patient prognosis. In this study, we aimed to elucidate the oncogenic role of thymosin beta-10 (TMSB10) in glioma through comprehensive analyses of patient data from the TCGA and GTEx databases. Our investigation encompassed several key aspects, including the analysis of patients' clinical characteristics, survival analysis, in vitro and in vivo functional experiments, and the exploration of correlations between TMSB10 expression and immune cell infiltration. Our findings revealed a significant upregulation of TMSB10 expression in glioma tissues compared to normal brain tissues, with higher expression levels observed in tumors of advanced histological grades. Moreover, we observed positive correlations between TMSB10 expression and patient age, while no significant association with gender was detected. Additionally, TMSB10 exhibited marked elevation in gliomas with wild-type IDH and noncodeletion of 1p/19q. Survival analysis indicated that high TMSB10 expression was significantly associated with worse overall survival, disease-specific survival, and progression-free survival in glioma patients. Functionally, knockdown of TMSB10 in glioma cells resulted in reduced cellular growth rates and impaired tumor growth in xenograft models. Furthermore, our study revealed intriguing correlations between TMSB10 expression and immune cell infiltration within the tumor microenvironment. Specifically, TMSB10 showed negative associations with plasmacytoid dendritic cells (pDC) and γδ T cells (Tgd), while displaying positive correlations with neutrophils and macrophages. These findings collectively provide valuable insights into the oncogenic properties of TMSB10 in glioma, suggesting its potential as a therapeutic target and a biomarker for patient stratification.

Trade-offs constrain adaptive pathways to type VI secretion system survival

The Type VI Secretion System (T6SS) is a nano-harpoon used by many bacteria to inject toxins into neighboring cells. While much is understood about mechanisms of T6SS-mediated toxicity, less is known about the ways that competitors can defend themselves against this attack, especially in the absence of their own T6SS. Here we subjected eight replicate populations of Escherichia coli to T6SS attack by Vibrio cholerae. Over ∼500 generations of competition, isolates of the E.coli populations evolved to survive T6SS attack an average of 27-fold better, through two convergently evolved pathways: apaH was mutated in six of the eight replicate populations, while the other two populations each had mutations in both yejM and yjeP. However, the mutations we identified are pleiotropic, reducing cellular growth rates, and increasing susceptibility to antibiotics and elevated pH. These trade-offs help us understand how the T6SS shapes the evolution of bacterial interactions.

A newly improved method of primary cell culture: Tissue block with continuous adhesion subculture in skin fibroblast

BackgroundFibroblasts (FBs) have been widely used as a typical in vitro cell model for investigating the biological processes and cell pathophysiological mechanisms. However, FBs are prone to senescence in cell culture process after several passages. Thus, a new approach to cell culture is quite required to enhance the viability of cells. ObjectiveTo explore a novel method of cell culture based on skin FBs. MethodsDermal tissue blocks were obtained from BALB/c neonatal mice and randomly divided into experimental group and control group. The experimental group received the newly improved culture method, namely, continuous adherence subculture of tissue block (CASTB) method; while the traditional subculture method was applied in the control group. Cells at 1st, 5th and 10th passages were collected and identified by using histological/immunohistochemical and western blot analysis. Cellular viability, proliferation, senescence and apoptosis were analyzed through application of cell growth curve, CCK-8 assay, Ki67 assay, PCNA protein analysis, β-galactosidase staining, flow cytometry and western blot analysis. ResultsCells under two culture patterns exhibited spindle/irregular shape and vimentin positive expression. With the increase of passage times, the cellular growth rate in the control group gradually decreased, but no alterations emerged from the experimental group. CASTB method remarkably promoted cell growth and proliferation. Moreover, a greatly lower apoptosis and senescence tendency appeared in the experimental group than the control group with passages increasing. ConclusionThe method of CASTB is superior to traditional subculture, offering a large number of primary FBs with higher efficiency and success rate and being worth of further popularization and application.

Mathematical properties of optimal fluxes in cellular reaction networks at balanced growth.

The physiology of biological cells evolved under physical and chemical constraints, such as mass conservation across the network of biochemical reactions, nonlinear reaction kinetics, and limits on cell density. For unicellular organisms, the fitness that governs this evolution is mainly determined by the balanced cellular growth rate. We previously introduced growth balance analysis (GBA) as a general framework to model and analyze such nonlinear systems, revealing important analytical properties of optimal balanced growth states. It has been shown that at optimality, only a minimal subset of reactions can have nonzero flux. However, no general principles have been established to determine if a specific reaction is active at optimality. Here, we extend the GBA framework to study the optimality of each biochemical reaction, and we identify the mathematical conditions determining whether a reaction is active or not at optimal growth in a given environment. We reformulate the mathematical problem in terms of a minimal number of dimensionless variables and use the Karush-Kuhn-Tucker (KKT) conditions to identify fundamental principles of optimal resource allocation in GBA models of any size and complexity. Our approach helps to identify from first principles the economic values of biochemical reactions, expressed as marginal changes in cellular growth rate; these economic values can be related to the costs and benefits of proteome allocation into the reactions' catalysts. Our formulation also generalizes the concepts of Metabolic Control Analysis to models of growing cells. We show how the extended GBA framework unifies and extends previous approaches of cellular modeling and analysis, putting forward a program to analyze cellular growth through the stationarity conditions of a Lagrangian function. GBA thereby provides a general theoretical toolbox for the study of fundamental mathematical properties of balanced cellular growth.

Response of two cyanobacterial strains to non-biodegradable glitter particles

Microplastic pollution is a growing concern mainly in aquatic environments due to its deleterious effects. Some types of microplastics, such as glitter, remain overlooked. Glitter particles are artificial reflective microplastics used by different consumers within arts and handcraft products. In nature, glitter can physically affect phytoplankton by causing shade or acting as a sunlight-reflective surface, influencing primary production. This study aimed to evaluate the effects of five concentrations of non-biodegradable glitter particles in two bloom-forming cyanobacterial strains, Microcystis aeruginosa CENA508 (unicellular) and Nodularia spumigena CENA596 (filamentous). Cellular growth rate, estimated by optical density (OD), demonstrated that the applied highest glitter dosage decreases cyanobacterial growth rate with a more evident effect on M. aeruginosa CENA508. The cellular biovolume of N. spumigena CENA596 increased following the application of high concentrations of glitter. Still, no significant difference was detected in chlorophyll-a and carotenoids’ contents for both strains. These results suggest that environmental concentrations of glitter, similar to the highest dosage tested (>200 mg glitter L−1), may negatively influence susceptible organisms of the aquatic ecosystems, as observed with M. aeruginosa CENA508 and N. spumigena CENA596.

Sodium oleate potentiates the concurrent overproduction of lipids and carotenoids in Chlorella vulgaris towards biofuel and nutraceutical applications

Microalgal metabolites have emerged as potential commodity components due to their notable beneficial nutritional and economic properties, and thus, their overproduction is of paramount importance to combat the existing ecological and dietary issues. However, the production titer of these metabolites by the algal systems at optimal cultivation conditions is inadequate to meet the burgeoning global demand for those metabolites. Therefore, a pressing demand remains to concurrently co-produce valuable metabolites without compromising cellular biomass production. Here, we report an effective strategy that simultaneously overproduces lipids, biomass and carotenoids in Chlorella vulgaris by providing sodium oleate (NaOL) in the cultivation medium. Supplement of sodium oleate at the optimal concentration (50 µg/mL) at increased light intensity enhances cellular growth rate in a dose-dependent manner. Total lipid content was quantified, which showed that lipid content was significantly increased in the NaOL-treated cells by 2.08-fold than that of the control cells. Interestingly, spectrophotometric determination of total carotenoids showed that carotenoid content was increased by 2.48-fold in the T5 (50 µg/mL) NaOL treated cells compared to the control. Interestingly antioxidant analysis showed that NaOL provision alleviated the antioxidant potential of the algal cells. Together, this report exemplifies the potential strategy to preclude the existing intricacies of co-producing metabolites in C. vulgaris by supplementing NaOL, thereby highlighting the viable chemical engineering approach for the biological production of commercially essential metabolites in microalgae.

Abstract 3456: Synergistic drug combinations promote the development of resistance in acute myeloid leukemia

Abstract Background: Combination therapies are frequently employed as part of cancer treatment, often with the goal of preventing or slowing the development of drug resistance. Empirically developed combination therapies have had remarkable impact on cancer survival, yet the task of using preclinical data to predict combinations that will yield clinical benefits remains a challenge. Current combination strategies often prioritize drug synergy, where short-term effect of a drug combination is more than additive. However, prior studies of antibiotic resistance have shown that synergistic combinations can accelerate the emergence of resistance in bacteria. This occurs because synergistic drugs rely on one another for activity, resistance to one drug in the combination thereby depletes the efficacy of both. Consistent with experimental data on antibiotics, computational models of tumor evolution have predicted that when comparing equally active synergistic and non-synergistic combinations, synergy will accelerate the development of resistance in cancer cells. Methods: In this study we aim to experimentally determine if synergistic drug combinations promote the development of resistance in acute myeloid leukemia cell lines, MOLM-13 and OCI-AML2. Using live-cell imaging we measured changes in growth rate over 21 days to observe the development of resistance to combination therapies ranging from additive to synergistic (Bliss score range - 1.6 to 19). We tested combinations of Venetoclax, Cytarabine, AZD5991, and AZD1775. We also measured the fitness benefit conferred from acquired resistance by comparing the difference between cellular growth rate during the first 72 hours of treatment and the maximum growth rate achieved by emerging resistant populations over the 21 day treatment. Results: When controlling for both activity and drug synergy using logistic regression, increased drug synergy is consistently predictive of increased likelihood of resistance development within 21 days (MOLM-13 p=0.001, OCI-AML2 p=0.007, n=150 per cell line). As previously predicted, there is also significant correlation between drug synergy and the fitness benefit of resistance (MOLM-13 R=0.71, p=1.6e-6). Furthermore, using a model of competing drug-sensitive and resistant subpopulations treated with combination therapy, we demonstrate that these observations are a mechanistic consequence of synergistic combination, whereby the fitness benefit acquired by gaining resistance to either drug in a synergistic combination is greater than the fitness benefit in a non-synergistic combination. Conclusion: Together these data have important implications for preclinical prioritization of combination therapies, and demonstrate that both short-term and long-term measures of drug efficacy can inform preclinical strategies aiming to develop novel combinations of cancer therapies with robust and durable efficacy. Citation Format: Emily Mason-Osann, Amy E. Pomeroy, Adam C. Palmer, Jerome T. Mettetal. Synergistic drug combinations promote the development of resistance in acute myeloid leukemia [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 3456.

Abstract 4254: The ratio of key metabolic transcripts is a predictive biomarker of breast cancer metastasis to the lung

Abstract Understanding the rewired metabolism of organ-specific metastasis in breast cancer is an under-appreciated problem, with implications for the treatment and prevention of metastatic disease. Here, we used a systems biology approach and the MDA-MB 231 model to compare metabolic fluxes used by parental breast cancer cells and their brain- and lung-homing derivatives. We combined metabolomic profiling, flux measurements, spatial tissue mimetic systems, and mathematical modeling to dissect the cells’ metabolic rewiring, and identified changes specific for lung metastatic selection. We validated our results in other cell lines as well as patient data from project GENIE and the MBCP. Our results produced five biological insights: First, levels of mRNA and metabolic intermediates may anticorrelate with flux. Second, different lineages evolved from the same line can have distinct heritable metabolic fluxes. Metastatic lineages in our model display higher glycolytic flux and bioenergetics than parental cells, with lung-homing cells exhibiting by far the greatest glucose uptake and lactate production. Importantly, this occurs despite low levels of glycolytic intermediates in lung-homing cells. Third, this apparent paradox can be reconciled if feedback inhibition of the glycolysis pathway is prevented, a finding we modeled with flux-balance analysis and confirmed by 13C glucose tracing and measuring glycolytic enzymatic activities directly. Fourth, this distinct metabolic behavior in lung-homing cells is maintained by a high ratio of lactate dehydrogenase (LDH) to pyruvate dehydrogenase (PDH) gene expression, which also correlates with lung metastases in patients with breast cancer. Feature classification models trained on clinical characteristics alone were unable to predict tropism; however, the LDH/PDH ratio was a significant predictor for lung but not brain metastases, independent of other transcriptomic signatures, suggesting that this feature is a potential biomarker for lung metastasis. Fifth, this metabolic effect did not increase cellular growth rate, suggesting that lactate secretion may itself be a trait under selection in breast cancer lung metastasis. Follow up experiments and game theory-based models in a spatially structured tissue-mimetic system showed that lung-homing cells grow more favorably than brain-homing cells in nutrient-deprived gradients, demonstrating how divergent metabolisms of the lineages could lead to selection in different environments. Notably, lung metastases in a mouse model of pancreatic cancer had higher lactate production than other metastases, suggesting that this metabolic trait could be important for other cancer types. Together, our in vitro, in silico, and clinical data analyses highlight that metabolism—currency of all physiological processes—plays an essential role in the connection from gene to phenotype in metastatic disease. Citation Format: Deepti Mathur, Chen Liao, Wendy Lin, Alessandro La Ferlita, Salvatore Alaimo, Alfredo Ferro, Yi Zhong, Christine Iacobuzio-Donahue, Joao Xavier. The ratio of key metabolic transcripts is a predictive biomarker of breast cancer metastasis to the lung. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 4254.