Substrate Inhibition Research Articles

Microbial Fermentation Processes of Lactic Acid: Challenges, Solutions, and Future Prospects.

The demand for lactic acid and lactic acid-derived products in the food, pharmaceutical, and cosmetic industries is increasing year by year. In recent decades, the synthesis of lactic acid by microbials has gained much attention from scientists due to the superior optical purity of the product, its low production costs, and its higher production efficiency compared to chemical synthesis. Microbial fermentation involves the selection of feedstock, strains, and fermentation modes. Each step can potentially affect the yield and purity of the final product. Therefore, there are still many critical challenges in lactic acid production. The costs of feedstocks and energy; the inhibition of substrates and end-product; the sensitivity to the inhibitory compounds released during pretreatment; and the lower optical purity are the main obstacles hindering the fermentation of lactic acid. This review highlights the limitations and challenges of applying microbial fermentation in lactic acid production. In addition, corresponding solutions to these difficulties are summarized in order to provide some guidance for the industrial production of lactic acid.

Contribution of Nudt12 enzyme to differentially methylated dinucleotides of 5’RNA cap structure

Recent findings have substantially broadened our knowledge about the diversity of modifications of the 5’end of RNAs, an issue generally attributed to mRNA cap structure (m7GpppN). Nudt12 is one of the recently described new enzymatic activities involved in cap metabolism. However, in contrast to its roles in metabolite-cap turnover (e.g., NAD-cap) and NADH/NAD metabolite hydrolysis, little is known regarding its hydrolytic activity towards dinucleotide cap structures. In order to gain further insight into this Nudt12 activity, comprehensive analysis with a spectrum of cap-like dinucleotides was performed with respect to different nucleotide types adjacent to the (m7)G moiety and its methylation status. Among the tested compounds, GpppA, GpppAm, and Gpppm6Am were identified as novel potent Nudt12 substrates, with KM values in the same range as that of NADH. Interestingly, substrate inhibition of Nudt12 catalytic activity was detected in the case of the GpppG dinucleotide, a phenomenon not reported to date. Finally, comparison of Nudt12 with DcpS and Nud16, two other enzymes with known activity on dinucleotide cap structures, revealed their overlapping and more specific substrates. Altogether, these findings provide a basis for clarifying the role of Nudt12 in cap-like dinucleotide turnover.

Human soluble CD39 displays substrate inhibition in a substrate-specific manner

CD39 (ectonucleoside triphosphate diphosphohydrolase-1; ENTPD1) metabolizes extracellular ATP and ADP to AMP. AMP is subsequently metabolized by CD79 to adenosine. CD39 activity is therefore a key regulator of purinergic signalling in cancer, thrombosis, and autoimmune diseases. In this study we demonstrate that soluble, recombinant CD39 shows substrate inhibition with ADP or ATP as the substrate. Although CD39 activity initially increased with increasing substrate concentration, at high concentrations of ATP or ADP, CD39 activity was markedly reduced. Although the reaction product, AMP, inhibits CD39 activity, insufficient AMP was generated under our conditions to account for the substrate inhibition seen. In contrast, inhibition was not seen with UDP or UTP as substrates. 2-methylthio-ADP also showed no substrate inhibition, indicating the nucleotide base is an important determinant of substrate inhibition. Molecular dynamics simulations revealed that ADP can undergo conformational rearrangements within the CD39 active site that were not seen with UDP or 2-methylthio-ADP. Appreciating the existence of substrate inhibition of CD39 will help the interpretation of studies of CD39 activity, including investigations into drugs that modulate CD39 activity.

Asymmetric Sulfoxidation by a Tyrosinase Biomimetic Dicopper Complex with a Benzimidazolyl Derivative of L-Phenylalanine.

A challenge in mimicking tyrosinase activity using model compounds is to reproduce its enantioselectivity. Good enantioselection requires rigidity and a chiral center close to the active site. In this study, the synthesis of a new chiral copper complex, [Cu2(mXPhI)]4+/2+, based on an m-xylyl-bis(imidazole)-bis(benzimidazole) ligand containing a stereocenter with a benzyl residue directly bound on the copper chelating ring, is reported. Binding experiments show that the cooperation between the two metal centers is weak, probably due to steric hindrance given by the benzyl group. The dicopper(II) complex [Cu2(mXPhI)]4+ has catalytic activity in the oxidations of enantiomeric couples of chiral catechols, with an excellent discrimination capability for Dopa-OMe enantiomers and a different substrate dependence, hyperbolic or with substrate inhibition, for the L- or D- enantiomers, respectively. [Cu2(mXPhI)]4+ is active in a tyrosinase-like sulfoxidation of organic sulfides. The monooxygenase reaction requires a reducing co-substrate (NH2OH) and yields sulfoxide with significant enantiomeric excess (e.e.). Experiments with 18O2 and thioanisole yielded sulfoxide with 77% incorporation of 18O, indicating a reaction occurring mostly through direct oxygen transfer from the copper active intermediate to the sulfide. This mechanism and the presence of the chiral center of the ligand in the immediate copper coordination sphere are responsible for the good enantioselectivity observed.

Acceptors and Effectors Alter Substrate Inhibition Kinetics of a Plant Glucosyltransferase NbUGT72AY1 and Its Mutants.

One of the main obstacles in biocatalysis is the substrate inhibition (SI) of enzymes that play important roles in biosynthesis and metabolic regulation in organisms. The promiscuous glycosyltransferase UGT72AY1 from Nicotiana benthamiana is strongly substrate-inhibited by hydroxycoumarins (inhibitory constant Ki < 20 µM), but only weakly inhibited when monolignols are glucosylated (Ki > 1000 µM). Apocarotenoid effectors reduce the inherent UDP-glucose glucohydrolase activity of the enzyme and attenuate the SI by scopoletin derivatives, which could also be achieved by mutations. Here, we studied the kinetic profiles of different phenols and used the substrate analog vanillin, which has shown atypical Michaelis-Menten kinetics in previous studies, to examine the effects of different ligands and mutations on the SI of NbUGT72AY1. Coumarins had no effect on enzymatic activity, whereas apocarotenoids and fatty acids strongly affected SI kinetics by increasing the inhibition constant Ki. Only the F87I mutant and a chimeric version of the enzyme showed weak SI with the substrate vanillin, but all mutants exhibited mild SI when sinapaldehyde was used as an acceptor. In contrast, stearic acid reduced the transferase activity of the mutants to varying degrees. The results not only confirm the multi-substrate functionality of NbUGT72AY1, but also reveal that the enzymatic activity of this protein can be fine-tuned by external metabolites such as apocarotenoids and fatty acids that affect SI. Since these signals are generated during plant cell destruction, NbUGT72AY1 likely plays an important role in plant defense by participating in the production of lignin in the cell wall and providing direct protection through the formation of toxic phytoalexins.

Application of Immersed Membrane Bioreactor for Semi-Continuous Production of Polyhydroxyalkanoates from Organic Waste-Based Volatile Fatty Acids.

Volatile fatty acids (VFAs) appear to be an economical carbon feedstock for the cost-effective production of polyhydroxyalkanoates (PHAs). The use of VFAs, however, could impose a drawback of substrate inhibition at high concentrations, resulting in low microbial PHA productivity in batch cultivations. In this regard, retaining high cell density using immersed membrane bioreactor (iMBR) in a (semi-) continuous process could enhance production yields. In this study, an iMBR with a flat-sheet membrane was applied for semi-continuous cultivation and recovery of Cupriavidus necator in a bench-scale bioreactor using VFAs as the sole carbon source. The cultivation was prolonged up to 128 h under an interval feed of 5 g/L VFAs at a dilution rate of 0.15 (d-1), yielding a maximum biomass and PHA production of 6.6 and 2.8 g/L, respectively. Potato liquor and apple pomace-based VFAs with a total concentration of 8.8 g/L were also successfully used in the iMBR, rendering the highest PHA content of 1.3 g/L after 128 h of cultivation. The PHAs obtained from both synthetic and real VFA effluents were affirmed to be poly(3-hydroxybutyrate-co-3-hydroxyvalerate) with a crystallinity degree of 23.8 and 9.6%, respectively. The application of iMBR could open an opportunity for semi-continuous production of PHA, increasing the feasibility of upscaling PHA production using waste-based VFAs.

Structural basis for substrate and inhibitor recognition of human multidrug transporter MRP4

Human multidrug resistance protein 4 (hMRP4, also known as ABCC4), with a representative topology of the MRP subfamily, translocates various substrates across the membrane and contributes to the development of multidrug resistance. However, the underlying transport mechanism of hMRP4 remains unclear due to a lack of high-resolution structures. Here, we use cryogenic electron microscopy (cryo-EM) to resolve its near-atomic structures in the apo inward-open and the ATP-bound outward-open states. We also capture the PGE1 substrate-bound structure and, importantly, the inhibitor-bound structure of hMRP4 in complex with sulindac, revealing that substrate and inhibitor compete for the same hydrophobic binding pocket although with different binding modes. Moreover, our cryo-EM structures, together with molecular dynamics simulations and biochemical assay, shed light on the structural basis of the substrate transport and inhibition mechanism, with implications for the development of hMRP4-targeted drugs.

The identification of a robust leucine dehydrogenase from a directed soil metagenome for efficient synthesis of L-2-aminobutyric acid.

L-2-aminobutyric acid (L-2-ABA) is a chiral precursor for the synthesis of anti-epileptic drug levetiracetam and anti-tuberculosis drug ethambutol. Asymmetric synthesis of L-2-ABA by leucine dehydrogenases has been widely developed. However, the limitations of natural enzymes, such as poor stability, low catalytic efficiency, and inhibition of high-concentration substrates, limit large-scale applications. Herein, by directed screening of a metagenomic library from unnatural amino acid-enriched environments, a robust leucine dehydrogenase, TvLeuDH, was identified, which exhibited high substrate tolerance and excellent enzymatic activity towards 2-oxobutyric acid. In addition, TvLeuDH has strong affinity for NADH. Subsequently, a three-enzyme co-expression system containing L-threonine deaminase, TvLeuDH, and glucose dehydrogenase was established. By optimizing reaction conditions, 1.5M L-threonine could be converted to L-2-ABA with a 99% molar conversion rate and a space-time yield of 51.5g·L-1 ·h-1 . In this process, no external coenzyme was added. The robustness of TvLeuDH allowed the reaction to be performed without the addition of extra salt as the buffer, demonstrating the simplest reaction system currently reported. These unique properties for the efficient and environmentally friendly production of chiral amino acids make TvLeuDH a particularly promising candidate for industrial applications, which reveals the great potential of directed metagenomics for industrial biotechnology.

ASC Transporters Mediate D-Serine Transport into Astrocytes Adjacent to Synapses in the Mouse Brain.

D-serine is an important signalling molecule, which activates N-methyl D-aspartate receptors (NMDARs) in conjunction with its fellow co-agonist, the neurotransmitter glutamate. Despite its involvement in plasticity and memory related to excitatory synapses, its cellular source and sink remain a question. We hypothesise that astrocytes, a type of glial cell that surrounds synapses, are likely candidates to control the extracellular concentration of D-Serine by removing it from the synaptic space. Using in situ patch clamp recordings and pharmacological manipulation of astrocytes in the CA1 region of the mouse hippocampal brain slices, we investigated the transport of D-serine across the plasma membrane. We observed the D-serine-induced transport-associated currents upon puff-application of 10 mM D-serine on astrocytes. Further, O-benzyl-L-serine and trans-4-hydroxy-proline, known substrate inhibitors of the alanine serine cysteine transporters (ASCT), reduced D-serine uptake. These results indicate that ASCT is a central mediator of astrocytic D-serine transport and plays a role in regulating its synaptic concentration by sequestration into astrocytes. Similar results were observed in astrocytes of the somatosensory cortex and Bergmann glia in the cerebellum, indicative of a general mechanism expressed across a range of brain areas. This removal of synaptic D-serine and its subsequent metabolic degradation are expected to reduce its extracellular availability, influencing NMDAR activation and NMDAR-dependent synaptic plasticity.

New findings on the action of hypericin in hypoxic cancer cells with a focus on the modulation of side population cells

The presence of key hypoxia regulators, namely, hypoxia-inducible factor (HIF)-1α or HIF-2α, in tumors is associated with poor patient prognosis. Hypoxia massively activates several genes, including the one encoding the BCRP transporter that proffers multidrug resistance to cancer cells through the xenobiotic efflux and is a determinant of the side population (SP) associated with cancer stem-like phenotypes. As natural medicine comes to the fore, it is instinctive to look for natural agents possessing powerful features against cancer resistance. Hypericin, a pleiotropic agent found in Hypericum plants, is a good example as it is a BCRP substrate and potential inhibitor, and an SP and HIF modulator. Here, we showed that hypericin efficiently accumulated in hypoxic cancer cells, degraded HIF-1/2α, and decreased BCRP efflux together with hypoxia, thus diminishing the SP population. On the contrary, this seemingly favorable result was accompanied by the stimulated migration of this minor population that preserved the SP phenotype. Because hypoxia unexpectedly decreased the BCRP level and SP fraction, we compared the SP and non-SP proteomes and their changes under hypoxia in the A549 cell line. We identified differences among protein groups connected to the epithelial–mesenchymal transition, although major changes were related to hypoxia, as the upregulation of many proteins, including serpin E1, PLOD2 and LOXL2, that ultimately contribute to the initiation of the metastatic cascade was detected. Altogether, this study helps in clarifying the innate and hypoxia-triggered resistance of cancer cells and highlights the ambivalent role of natural agents in the biology of these cells.

Structure of Rhizobium sp. 4-9 histamine dehydrogenase and analysis of the electron transfer pathway to an abiological electron acceptor

Histamine dehydrogenase from the gram-negative bacterium Rhizobium sp. 4-9 (HaDHR) is a member of a small family of dehydrogenases containing a covalently attached FMN, and the only member so far identified to date that does not exhibit substrate inhibition. In this study, we present the 2.1 Å resolution crystal structure of HaDHR. This new structure allowed for the identification of the internal electron transfer pathway to abiological ferrocene-based mediators. Alanine 437 was identified as the exit point of electrons from the Fe4S4 cluster. The enzyme was modified with a Ser436Cys mutation to facilitate covalent attachment of a ferrocene moiety. When modified with Fc-maleimide, this new construct demonstrated direct electron transfer from the enzyme to a gold electrode in a histamine concentration-dependent manner without the need for any additional electron mediators.

Mathematical modeling of bioethanol production from sweet sorghum juice under high gravity fermentation: Applicability of Monod-based, logistic, modified Gompertz and Weibull models

BackgroundMathematical modeling of a fermentation process is crucial in understanding and predicting dynamics of the process, which can be used in process improvement, design and control. The present study aimed to develop Monod-based kinetic models to describe cell growth, substrate consumption and ethanol production by Saccharomyces cerevisiae NP 01 under high gravity (HG) fermentation of sweet sorghum juice (SSJ). ResultsThe fermentation using an initial total sugar (TS) concentration of 240 g/L resulted in 113.3 g/L of ethanol production, with 90.9% TS consumption and a fermentation efficiency of 94.4%. Growth of the yeast in terms of specific growth rate was found to be inhibited at a threshold TS concentration of 65 g/L, and the maximum specific growth rate, Monod constant and inhibition constant were 0.45 1/h, 19.5 g/L and 0.002 L/(g·h), respectively. Monod-based models incorporating substrate and product inhibition terms showed high applicability to describe the changes of cell, TS and ethanol concentrations, based on the values of bias factor, accuracy factor, coefficient of determination and root mean square error. ConclusionsThe Monod-based models fitted the data equally well as compared with the logistic, modified Gompertz, and Weibull models, despite estimating the value of different kinetic parameters. These results demonstrated that all the models tested were applicable in modeling HG ethanol fermentation.How to cite: Salakkam A, Phukoetphim N, Laopaiboon P, et al. Mathematical modeling of bioethanol production from sweet sorghum juice under high gravity fermentation: Applicability of Monod-based, logistic, modified Gompertz and Weibull models. Electron J Biotechnol 2023;64. https://doi.org/10.1016/j.ejbt.2023.03.004.

Modulating Substrate Specificity of Rhizobium sp. Histamine Dehydrogenase through Protein Engineering for Food Quality Applications.

Histamine is a biogenic amine found in fish-derived and fermented food products with physiological relevance since its concentration is proportional to food spoilage and health risk for sensitive consumers. There are various analytical methods for histamine quantification from food samples; however, a simple and quick enzymatic detection and quantification method is highly desirable. Histamine dehydrogenase (HDH) is a candidate for enzymatic histamine detection; however, other biogenic amines can change its activity or produce false positive results with an observed substrate inhibition at higher concentrations. In this work, we studied the effect of site saturation mutagenesis in Rhizobium sp. Histamine Dehydrogenase (Rsp HDH) in nine amino acid positions selected through structural alignment analysis, substrate docking, and proximity to the proposed histamine-binding site. The resulting libraries were screened for histamine and agmatine activity. Variants from two libraries (positions 72 and 110) showed improved histamine/agmatine activity ratio, decreased substrate inhibition, and maintained thermal resistance. In addition, activity characterization of the identified Phe72Thr and Asn110Val HDH variants showed a clear substrate inhibition curve for histamine and modified kinetic parameters. The observed maximum velocity (Vmax) increased for variant Phe72Thr at the cost of an increased value for the Michaelis-Menten constant (Km) for histamine. The increased Km value, decreased substrate inhibition, and biogenic amine interference observed for variant Phe72Thr support a tradeoff between substrate affinity and substrate inhibition in the catalytic mechanism of HDHs. Considering this tradeoff for future enzyme engineering of HDH could lead to breakthroughs in performance increases and understanding of this enzyme class.

DEAD box RNA helicases act as nucleotide exchange factors for casein kinase 2.

DDX RNA helicases promote RNA processing, but DDX3X also activates casein kinase 1 (CK1ε). We show that other DDX proteins also stimulate the protein kinase activity of CK1ε and that this extends to casein kinase 2 (CK2). CK2 enzymatic activity was stimulated by various DDX proteins at high substrate concentrations. DDX1, DDX24, DDX41, and DDX54 were required for full kinase activity in vitro and in Xenopus embryos. Mutational analysis of DDX3X indicated that CK1 and CK2 kinase stimulation engages its RNA binding but not catalytic motifs. Mathematical modeling of enzyme kinetics and stopped-flow spectroscopy showed that DDX proteins function as nucleotide exchange factors toward CK2 and reduce unproductive reaction intermediates and substrate inhibition. Our study reveals protein kinase stimulation by nucleotide exchange as important for kinase regulation and as a generic function of DDX proteins.

Allosteric communication between ligand binding domains modulates substrate inhibition in adenylate kinase

Enzymes play a vital role in life processes; they control chemical reactions and allow functional cycles to be synchronized. Many enzymes harness large-scale motions of their domains to achieve tremendous catalytic prowess and high selectivity for specific substrates. One outstanding example is provided by the three-domain enzyme adenylate kinase (AK), which catalyzes phosphotransfer between ATP to AMP. Here we study the phenomenon of substrate inhibition by AMP and its correlation with domain motions. Using single-molecule FRET spectroscopy, we show that AMP does not block access to the ATP binding site, neither by competitive binding to the ATP cognate site nor by directly closing the LID domain. Instead, inhibitory concentrations of AMP lead to a faster and more cooperative domain closure by ATP, leading in turn to an increased population of the closed state. The effect of AMP binding can be modulated through mutations throughout the structure of the enzyme, as shown by the screening of an extensive AK mutant library. The mutation of multiple conserved residues reduces substrate inhibition, suggesting that substrate inhibition is an evolutionary well conserved feature in AK. Combining these insights, we developed a model that explains the complex activity of AK, particularly substrate inhibition, based on the experimentally observed opening and closing rates. Notably, the model indicates that the catalytic power is affected by the microsecond balance between the open and closed states of the enzyme. Our findings highlight the crucial role of protein motions in enzymatic activity.

Kinetic characterization and phylogenetic analysis of human ADP-dependent glucokinase reveal new insights into its regulatory properties

Although ADP-dependent sugar kinases were first described in archaea, at present, the presence of an ADP-dependent glucokinase (ADP-GK) in mammals is well documented. This enzyme is mainly expressed in hematopoietic lineages and tumor tissues, although its role has remained elusive. Here, we report a detailed kinetic characterization of the human ADP-dependent glucokinase (hADP-GK), addressing the influence of a putative signal peptide for endoplasmic reticulum (ER) destination by characterizing a truncated form. The truncated form revealed no significant impact on the kinetic parameters, showing only a slight increase in the Vmax value, higher metal promiscuity, and the same nucleotide specificity as the full-length enzyme. hADP-GK presents an ordered sequential kinetic mechanism in which MgADP is the first substrate to bind and AMP is the last product released, being the same mechanism described for archaeal ADP-dependent sugar kinases, in agreement with the protein topology. Substrate inhibition by glucose was observed due to sugar binding to nonproductive species.Although Mg2+ is an essential component for kinase activity, it also behaves as a partial mixed-type inhibitor for hADP-GK, mainly by decreasing the MgADP affinity. Regarding its distribution, phylogenetic analysis shows that ADP-GK's are present in a wide diversity of eukaryotic organisms although it is not ubiquitous. Eukaryotic ADP-GKs sequences cluster into two main groups, showing differences in the highly conserved sugar-binding motif reported for archaeal enzymes [NX(N)XD] where a cysteine residue is found instead of asparagine in a significant number of enzymes. Site directed mutagenesis of the cysteine residue by asparagine produces a 6-fold decrease in Vmax, suggesting a role for this residue in the catalytic process, probably by facilitating the proper orientation of the substrate to be phosphorylated.

Identification and characterization of extracellular GH3 β-glucosidase from the pink snow mold fungus, Microdochium nivale.

Glycoside hydrolase family 3 (GH3) β-glucosidase exists in many filamentous fungi. In phytopathogenic fungi, it is involved in fungal growth and pathogenicity. Microdochium nivale is a severe phytopathogenic fungus of grasses and cereals and is the causal agent of pink snow mold, but its β-glucosidase has not been identified. In this study, a GH3 β-glucosidase of M. nivale (MnBG3A) was identified and characterized. Among various p-nitrophenyl β-glycosides, MnBG3A showed activity on d-glucoside (pNP-Glc) and slight activity on d-xyloside. In the pNP-Glc hydrolysis, substrate inhibition occurred (Kis=1.6m m), and d-glucose caused competitive inhibition (Ki=0.5m m). MnBG3A acted on β-glucobioses with β1-3, -6, -4, and -2 linkages, in descending order of kcat/Km. In contrast, the regioselectivity for newly formed products was limited to β1-6 linkage. MnBG3A has similar features to those of β-glucosidases from Aspergillus spp., but higher sensitivity to inhibitory effects.

Limits of the thermal tolerance of two lentic crustaceans: The role of lactate dehydrogenase.

Limits of thermal tolerance in animal life is dependent on energy supply. Accordingly, the lowered ATP production capacity in ectotherms at high temperatures, which arises from a mismatch between oxygen supply and demand and the consequent switch from aerobic to anaerobic metabolism, affects the thermal resistance of these animals. The anaerobic ATP production capacity depends on the functional properties of the enzymes that reduce pyruvate. Thus, the present study focused on the role of the lactate dehydrogenase (LDH) of two daphnid species for anaerobic energy production at warm temperatures and the implications for their specific heat tolerances. Daphnia magna showed a higher thermal limit (indicated by immobilization time at 37°C) than Daphnia pulex, and in both species, this limit increased with rising acclimation temperature. In contrast to D. pulex, D. magna accumulated significant amounts of lactate at higher ambient temperatures. The intensity of anaerobic metabolism was also affected by acclimation temperature. Studying the functional enzyme properties revealed altered maximal reaction rates and substrate inhibitions of the LDH suites of the two daphnid species. D. magna LDH showed a significantly lower substrate inhibition than D. pulex LDH. The LDH isoform composition and the temperature-induced changes differed between both species. The detected qualitative modulations of the LDH suites may have resulted from differential isoform expression and different maturation processes. The species-specific LDH characteristics imply a higher anaerobic energy production at warm temperatures in D. magna, which likely contributes to the higher heat tolerance of this species.

Abstract 1143: Development of a CDK2-selective small molecule inhibitor INCB123667 for the treatment of CCNE1hi breast cancers

Abstract Cyclin-dependent kinases 4 and 6 (CDK4/6) inhibitors have been approved for treatment of metastatic ER+ HER2− breast cancer as monotherapy or in combination with endocrine therapy. However, many patients become resistant to CDK4/6 therapy due to various mechanisms such as loss of tumor suppressor Rb, hyperactivation of CDK4/6, and/or CDK2/Cyclin E1 expression and activity, resulting in a highly unmet clinical need. Triple-negative breast cancers (TNBC), owing to a lack of hormone receptor (HR) expression and extensive heterogeneity, remain notoriously aggressive and unresponsive to current therapies. CCNE1 overexpression or amplification has been shown to be an oncogenic driver associated with poor clinical outcomes in ovarian, gastric, endometrial, and HR+ breast cancers. CCNE1hi cancers are dependent on CDK2 for growth and survival. CDK2 in complex with CCNE1 phosphorylates Rb, resulting in E2F target gene expression and G1 to S-phase cell cycle progression. Using a potent and selective CDK2 inhibitor, INCB123667, we demonstrate the efficacy of CDK2 inhibition in CCNE1hi breast cancer models. Analysis of genome-wide CRISPR screen data identified a subset of CCNE1hi breast cancer models dependent on CDK2 comprised of basal and luminal breast cancer subtypes, including TNBC, ER+ HER2−, and HR− HER2+ models. Two CCNE1hi cell lines, HCC1569 and MDAMB157, were evaluated in cell assays monitoring pRb, S-phase, and growth inhibition, and found to be sensitive to treatment with INCB123667 and insensitive to multiple CDK4/6 inhibitors. Through extensive biochemical studies, INCB123667 was observed to be potently active against CDK2 at subnanomolar range and inactive against other CDKs (CDK1, CDK4, CDK6, CDK7, and CDK9). INCB123667 also selectively induced senescence in CCNE1hi HCC1569 cells. In contrast, CCNE1 nonamplified cell lines, MCF7 and T47D, were inactive in pRb, S-phase, and growth inhibition assays when treated with INCB123667. Significant tumor growth inhibition was observed in CCNE1hi PDX and xenograft models with minimal body weight loss. We also observed dose-dependent inhibition of CDK2 substrates, including pRb S780, both in vitro and in vivo. To further characterize downstream signaling and transcriptional changes with CDK2 inhibition, we identified a discrete gene signature associated with cell cycle regulation, which was notably distinct from CDK4/6 responsive target genes. In conclusion, we have demonstrated CDK2 activity in CCNE1hi breast cancers using INCB123667, a potent and selective small molecule inhibitor of CDK2 currently in clinical development. Treatment resulted in cell growth inhibition and antitumor activity in vitro and in vivo. Our data demonstrate the therapeutic potential of CDK2 inhibition in CCNE1hi HR+ HER2− and TNBC breast cancer models. Acknowledgments: R&amp;D services provided by Pharmaron, Crown Bioscience, and Champions Oncology Citation Format: Saswati Chand, Michael Hansbury, Yvonne Lo, Pat Feldman, Justine Carl, Cynthia Timmers, Joshua Hummel, Susan Wee, Sunkyu Kim. Development of a CDK2-selective small molecule inhibitor INCB123667 for the treatment of CCNE1hi breast cancers [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 1143.

Abstract 5073: NT219 induces tumor PD-L1 expression and potentiates anti-PD-1 efficacy

Abstract While the advent of immune checkpoint blockade (ICB) has dramatically improved the prognosis of many immune-infiltrated cancers, for others, unfortunately, these benefits have yet to be realized. The major challenge before the field, then, is to identify combination therapies that act both to combat evolved resistance. NT219 is a novel dual inhibitor of insulin receptor substrates 1 and 2 (IRS) and STAT3. NT219 demonstrated antitumor effects against both in situ and metastatic human melanoma models in mice as a stand-alone treatment and in combination with mutated BRAF and MEK inhibitors. The potential of NT219 to overcome resistance and increase efficacy was demonstrated in PDX models with multiple drug classes. Collectively, these findings provided preclinical proof-of-concept NT219 as a promising novel cancer therapy. Given this data, the goal of our study was to assess the efficacy of combining NT219 with anti-PD-1 and anti-CTLA-4 ICB, and test capacity of the combination to overcome immune resistance. To that end, we have examined PD-L1 expression levels in vitro following NT219 treatment in two different strains of melanoma cells, B16-tdTomato (TMT) and B16 3I-F4, ICB sensitive and ICB resistant derivative lines, respectively. In addition, to evaluate the combination effect of NT219 with ICB therapy in ICB-resistant PDX model, we used a humanized PDX model of pembrolizumab-resistant gastroesophageal tumor (GEJ). These mice were injected with PBMCs from the same patient. Using multiple syngeneic immunocompetent models of melanoma (TMT and 3I-F4 cells) and the humanized PDX model, we investigated the potential of each ICB with NT219. Our findings showed that NT219 increased PD-L1 expression levels on both melanoma cell lines in vitro, however, the PD-L1 expression levels were much more elevated 3I-F4 than TMT cells. Interestingly, the levels of both IRS and activated pSTAT3, the targets of NT219, which consist well known resistance mechanisms, were higher in the resistant cells as compared to the sensitive line. In accordance we found that NT219 was able to restore anti-PD-1 sensitivity in 3I-F4 model (TGI = 58%) in the syngeneic model, promoting tumor rejection and increasing survival rates. The combined approach between NT219 and anti-CTLA-4 displayed a moderate effect (TGI = 37%). In the TMT, NT219 combined with anti-PD-1 showed greater efficacy compared to anti-PD-1 alone (TGI = 63% and TGI = 47%, respectively). These results were confirmed using the humanized ICB-resistant PDX model, where impressive synergy between NT219 and the anti-PD-1 (TGI = 98%) was demonstrated. To summarize, we found a significant synergistic effect of NT219 combined with anti-PD-1 therapy, supported by a mechanism of PDL-1 induction making ICB resistant tumors amenable to ICB treatment. Collectively, these findings demonstrated that NT219 has the potential to reverse ICB resistance in both human PDX and murine syngeneic tumor model systems Citation Format: Ricardo Alexandre de Azevedo, Hadas Reuveni, Menashe Bar-Eli, Michael Curran. NT219 induces tumor PD-L1 expression and potentiates anti-PD-1 efficacy. [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 5073.