METTL3-Mediated DLGAP1 Antisense RNA 2 Promotes Gastric Cancer Tumorigenesis Through Facilitating C-Myc-Dependent Warburg Effect

Background The critical roles of N6-methyladenosine (m6A) modification have been demonstrated by more and more evidence. However, the cross-talking of m6A and long non-coding RNAs (lncRNAs) in gastric cancer (GC) tumorigenesis is still unclear. Here, our work focused on the functions and molecular mechanism of m6A-modified lncRNA DLGAP1 antisense RNA 2 (DLGAP1-AS2) in GC. Methods LncRNA expression profile data was derived from GEO. M6A profile was screened using Methylated RNA immunoprecipitation sequencing (MeRIP-Seq). The metabolism assays were conducted using quantitative analysis of glucose, lactate, ATP and extracellular acidification rate (ECAR). The m6A level of specific RNA was identified using MeRIP-qPCR. The molecular interaction was detected using RIP assay. Results Microarray analysis found that lncRNA DLGAP1-AS2 up-regulated in GC cells. Clinical data showed that DLGAP1-AS2 high-expression was correlated with advanced pathological stage and poor prognosis. Functionally, DLGAP1-AS2 promoted the Warburg effect (aerobic glycolysis) and knockdown of DLGAP1-AS2 suppressed the tumor growth of GC cells. Mechanistically, m6A methyltransferase METTL3 enhanced the stability of DLGAP1-AS2 via m6A site binding. Moreover, DLGAP1-AS2 interacted with YTHDF1 to enhance the stability of c-Myc mRNA through DLGAP1-AS2/m6A/YTHDF1/c-Myc mRNA. Conclusions In conclusion, our work indicates the functions of m6A-modified DLGAP1-AS2 in the GC aerobic glycolysis, disclosing a potential m6A-dependent manner for GC treatment.

1285. Identification of KPC Omega Loop Variant R163S Conferring Ceftazidime-Avibactam Resistance

Background We report on a 56 year-old male with prolonged COVID-19 pneumonia who initially improved with dexamethasone and intubation but quickly decompensated. Clinical and radiologic features were consistent with VAP. Tracheal aspirate cultures grew carbapenem-resistant Enterobacter cloacae; meropenem (MEM) MIC was >8 ug/ml (resistant) while ceftazidime-avibactam (CZA) MIC was 2/4 ug/ml (susceptible). Lateral flow antigen assay detected a KPC enzyme. The patient was treated with CZA with steady improvement in respiratory function over the next two weeks. He then experienced an episode of tachycardia, prompting repeat culture. At this point the patient had been extubated: sputum culture grew KPC+ E. cloacae that now showed CZA-resistance (MIC >8/4 ug/ml) and paradoxical decrease in MEM MIC (4 ug/ml); meropenem-vaborbactam (< 2/8 ug/ml) was susceptible. Methods The pre- & post-CZA therapy E. cloacae isolates underwent whole genome sequencing using the Illumina 150bp paired end protocol; sequences were quality trimmed and compared. Results A point mutation in the plasmid blaKPC3 gene was identified in the post-CZA therapy isolate, an R163S mutation in the omega loop of the enzyme. ompC and ompF porin genes were analyzed to rule-out decreased influx as a mechanism for CZA-resistance: the pre- and post-CZA isolates had identical porin sequences. Conclusion This case highlights emerging mutations within KPC carbapenemases that lead to resistance to ‘last-line’ antimicrobials like CZA. The presumptive mechanism is increased KPC active site promiscuity due to increased omega loop flexibility, allowing increased ceftazidime binding and hydrolysis, and decreased avibactam binding and beta lactamase inhibition. Paradoxically, MEM susceptibility improves after such omega loop mutations, likely due to decreased active site binding affinity, a ‘seesaw’ effect between MEM and CZA. While authors have reported MEM MICs falling into the ‘susceptible’ category after an omega loop variant, these bacteria invariably develop secondary mutations leading to MEM treatment failure. Fortunately, given our patient’s improved respiratory status, the post-CZA E. cloacae isolate was felt to reflect colonization and the patient was discharged home without antimicrobial therapy. Disclosures Romney Humphries, PhD D(ABMM), Accelerate Diagnostics (Individual(s) Involved: Self): Consultant, Shareholder; IHMA (Individual(s) Involved: Self): Consultant; Melinta (Individual(s) Involved: Self): Consultant; Momentum (Individual(s) Involved: Self): Grant/Research Support; Pattern (Individual(s) Involved: Self): Consultant; QPex (Individual(s) Involved: Self): Consultant; ThermoFisher (Individual(s) Involved: Self): Consultant; Torus (Individual(s) Involved: Self): Consultant

Design, Synthesis and Evaluation of WD-repeat containing protein 5 (WDR5) degraders

Histone H3K4 methylation serves as post-translational hallmark of actively transcribed genes and is introduced by histone methyltransferases (HMT) and its regulatory scaffolding proteins. One of these is the WD-repeat containing protein 5 (WDR5) that has also been associated with controlling long non-coding RNAs and transcription factors including MYC. The wide influence of dysfunctional HMTs complexes and the typically upregulated MYC levels in diverse tumor types suggested WDR5 as an attractive drug target. Indeed, protein-protein interface inhibitors for two protein interaction interfaces on WDR5 have been developed. While such compounds only inhibit a subset of WDR5 interactions, chemically induced proteasomal degradation of WDR5 might represent an elegant way to target all oncogenic function. This study presents the design, synthesis and evaluation of two diverse WDR5 degrader series based on two WIN site binding scaffolds and shows that linker nature and length strongly influence degradation efficacy.

Cryptic-site binding mechanism of medium-sized Bcl-xL inhibiting compounds elucidated by McMD-based dynamic docking simulations

We have performed multicanonical molecular dynamics (McMD) based dynamic docking simulations to study and compare the binding mechanism between two medium-sized inhibitors (ABT-737 and WEHI-539) that bind to the cryptic site of Bcl-xL, by exhaustively sampling the conformational and configurational space. Cryptic sites are binding pockets that are transiently formed in the apo state or are induced upon ligand binding. Bcl-xL, a pro-survival protein involved in cancer progression, is known to have a cryptic site, whereby the shape of the pocket depends on which ligand is bound to it. Starting from the apo-structure, we have performed two independent McMD-based dynamic docking simulations for each ligand, and were able to obtain near-native complex structures in both cases. In addition, we have also studied their interactions along their respective binding pathways by using path sampling simulations, which showed that the ligands form stable binding configurations via predominantly hydrophobic interactions. Although the protein started from the apo state, both ligands modulated the pocket in different ways, shifting the conformational preference of the sub-pockets of Bcl-xL. We demonstrate that McMD-based dynamic docking is a powerful tool that can be effectively used to study binding mechanisms involving a cryptic site, where ligand binding requires a large conformational change in the protein to occur.

Multifaceted Mechanism of Amicoumacin A Inhibition of Bacterial Translation

Amicoumacin A (Ami) halts bacterial growth by inhibiting the ribosome during translation. The Ami binding site locates in the vicinity of the E-site codon of mRNA. However, Ami does not clash with mRNA, rather stabilizes it, which is relatively unusual and implies a unique way of translation inhibition. In this work, we performed a kinetic and thermodynamic investigation of Ami influence on the main steps of polypeptide synthesis. We show that Ami reduces the rate of the functional canonical 70S initiation complex (IC) formation by 30-fold. Additionally, our results indicate that Ami promotes the formation of erroneous 30S ICs; however, IF3 prevents them from progressing towards translation initiation. During early elongation steps, Ami does not compromise EF-Tu-dependent A-site binding or peptide bond formation. On the other hand, Ami reduces the rate of peptidyl-tRNA movement from the A to the P site and significantly decreases the amount of the ribosomes capable of polypeptide synthesis. Our data indicate that Ami progressively decreases the activity of translating ribosomes that may appear to be the main inhibitory mechanism of Ami. Indeed, the use of EF-G mutants that confer resistance to Ami (G542V, G581A, or ins544V) leads to a complete restoration of the ribosome functionality. It is possible that the changes in translocation induced by EF-G mutants compensate for the activity loss caused by Ami.

A-769662 inhibits adipocyte glucose uptake in an AMPK-independent manner

Activation of AMP-activated protein kinase (AMPK) is considered a valid strategy for the treatment of type 2 diabetes. However, despite the importance of adipose tissue for whole-body energy homeostasis, the effect of AMPK activation in adipocytes has only been studied to a limited extent and mainly with the AMP-mimetic 5-aminoimidazole-4-carboxamide-1-β-d-ribofuranoside (AICAR), which has limited specificity. The aim of this study was to evaluate the effect of the allosteric AMPK activators A-769662 and 991 on glucose uptake in adipocytes. For this purpose, primary rat or human adipocytes, and cultured 3T3-L1 adipocytes, were treated with either of the allosteric activators, or AICAR, and basal and insulin-stimulated glucose uptake was assessed. Additionally, the effect of AMPK activators on insulin-stimulated phosphorylation of Akt and Akt substrate of 160 kDa was assessed. Furthermore, primary adipocytes from ADaM site binding drug-resistant AMPKβ1 S108A knock-in mice were employed to investigate the specificity of the drugs for the observed effects. Our results show that insulin-stimulated adipocyte glucose uptake was significantly reduced by A-769662 but not 991, yet neither activator had any clear effects on basal or insulin-stimulated Akt/AS160 signaling. The use of AMPKβ1 S108A mutant-expressing adipocytes revealed that the observed inhibition of glucose uptake by A-769662 is most likely AMPK-independent, a finding which is supported by the rapid inhibitory effect A-769662 exerts on glucose uptake in 3T3-L1 adipocytes. These data suggest that AMPK activation per se does not inhibit glucose uptake in adipocytes and that the effects of AICAR and A-769662 are AMPK-independent.