Revertants Research Articles

Combination of Menin and IDH Mutant Inhibition in Acute Myeloid Leukemia

The interaction between menin and mixed lineage leukemia 1 (MLL1) protein contributes to abnormal proliferation in the MLL1-(KMT2A) rearranged and nucleophosmin 1 (NPM1)-mutated acute myeloid leukemia (AML). Indeed, small molecule inhibition of the menin/MLL1 interaction was shown to inhibit leukemia in both AML subtypes. Approximately 25% of NPM1 mutated AML also carry an IDH 1 or 2 mutation. Carrying both NPM1- and IDH mutations is associated with worse overall survival and relapse free survival, compared to having only the NPM1 mutation. MI-3454, a small molecule inhibitor of MLL1/menin interaction, has been shown to be effective in AML with KMT2A rearrangements and NPM1 mutations. A close analogue of this compound, KO-539 (Ziftomenib) is now being tested in clinical trials in refractory and relapsed AML patients (NCT 04067336). The aim of this study is to determine the efficacy of the combination of MI-3454 and IDH mutant inhibitors in AML carrying concurrent NPM1 and IDH1/2 mutations. Blood or bone marrow aspirate samples from patients with AML harboring both NPM1 and IDH1 and/or IDH2 mutations were obtained and used to assess the effect of combinatorial treatment of menin and IDH mutant inhibitors. Colony formation assays with these AML patient samples were performed in methylcellulose media with cells treated with a concentration range of MI-3454, ivosidenib (IDH1 inhibitor) or enasidenib (IDH2 inhibitor) as single agents and in combinations. At the end point of the experiments, colonies were counted. Cells were collected for 1) cytohistology to assess cell morphology 2) flow cytometry to evaluate markers of differentiation and apoptosis and 3) gene expression studies to assess the effect of the combinations on the expression level of genes implicated in leukemogenesis and differentiation. In the samples with concurrent NPM1 and IDH1 mutations, the combination of MI-3454 and ivosidenib caused synergistic reduction in colony number compared to the single agents, with combination indices (CI) ranging from 0.44 to 0.94. Furthermore, cells treated with this combination demonstrated more differentiating phenotypes than single agent treated cells. In the samples with concurrent NPM1 and IDH2 mutations, the combination of MI-3454 and enasidenib caused synergistic reduction in colony formation versus either single agent MI-3454 or enasidenib, with CI values 0.12-0.64. The combination of these agents also yielded stronger differentiation than single agents alone based on both morphologic assessment and enhanced expression of CD11B differentiation marker. Mechanistically, the combination of MI-3454 and IDH1/2 mutant inhibitors also more significantly decreased the expression of HOXA9, MEIS1, and HOXB2, which are oncogenes implicated in leukemogenesis, over single agents. The expression of MNDA was also significantly enhanced by these combinations, supporting differentiation. Overall, our study demonstrates that combination of MI-3454 and IDH mutant inhibitors outperforms the effect of single agents with respect to their anti-leukemic effects in clinically relevant pre-clinical models. These results support that combination of menin and IDH mutant inhibitors could be utilized in clinical trials to improve the ability of patients with AML to achieve remission.

Structure-based modification of ortho-amidophenylaminopyrimidines as a novel mutant EGFR inhibitor against resistant non-small cell lung cancer

The challenge of acquired drug resistance in patients with epidermal growth factor receptor (EGFR)-mediated non-small cell lung cancer (NSCLC) has not been overcome, especially the tertiary C797S mutation of EGFR. Herein, it was reported the structure-based design of ortho-amidophenylaminopyrimidines as new mutant EGFR (EGFRL858R/T790M/C797S and EGFRL858R/T790M) inhibitors (type I‒II). Among them, target Ⅱd exhibited the strongest activity against EGFRL858R/ T790M/C797S (IC50=5.51 nM) and EGFRL858R/T790M (IC50=33.35 nM) kinases, and suppressed Ba/F3 cells harboring EGFRL858R/ T790M/C797S and EGFR19Del/T790M/C797S mutants with IC50 values of 0.433 μM and 1.485 μM, respectively. In particular, compared with Ⅰb (IC50=3.656 μM) which was the best inhibitor of type I, Ⅱd (IC50=0.442 μM) displayed enhanced antiproliferative effect against gefitinib-resistant H1975 cells harboring the EGFRL858R/T790M mutation. Ⅱd also demonstrated promising EGFRL858R/T790M/C797S mutant selectivity, and was 70-fold less potent against the wild type EGFR. In addition, Ⅱd behaved low toxicity against the normal human liver cells L02, which was 28-fold less potent. Moreover, both DAPI staining and apoptosis assays demonstrated that Ⅱd induced apoptosis in the resistant H1975 cells. Cycle assays showed that Ⅱd arrested the cell cycle at the G2/M phase. Western blot analysis revealed that Ⅱd suppressed phosphorylation of the EGFR and the downstream signaling ERK1/2 level in NSCLC cells. Hence, these results suggested that Ⅱd might be useful as a potent mutant EGFR inhibitor and was effective in both third- and fourth-generation EGFR-mutant NSCLC, the activity would not be limited by the C797S mutation for further development in the treatment of resistant NSCLC.

PAK and PI3K pathway activation confers resistance to KRASG12C inhibitor sotorasib.

KRAS is a frequently mutated oncogene in human cancer. Clinical studies on the covalent inhibitors of the KRASG12C mutant have reported promising results. However, primary and acquired resistance may limit their clinical use. Sotorasib-resistant cell lines were established. We explored the signalling pathways activated in these resistant cell lines and their roles in sotorasib resistance. The resistant cells exhibited increased cell-matrix adhesion with increased levels of stress fibres and focal adherens. p21-activated kinases (PAKs) were activated in resistant cells, which phosphorylate MEK at serine 298 of MEK and serine 338 of c-Raf to activate the mitogen-activated protein kinase pathway. The PAK inhibitors FRAX597 and FRAX486 in synergy with sotorasib reduced the viability of KRASG12C mutant cancer cells. Furthermore, the PI3K/AKT pathway was constitutively active in sotorasib-resistant cells. The overexpression of constitutively activated PI3K or the knockdown of PTEN resulted in resistance to sotorasib. PI3K inhibitor alpelisib was synergistic with sotorasib in compromising the viability of KRASG12C mutant cancer cells. Moreover, PI3K and PAK pathways formed a mutual positive regulatory loop that mediated sotorasib resistance. Our results indicate that the cell-matrix interaction-dependent activation of PAK mediates resistance to sotorasib through the activation of MAPK and PI3K pathways.

Design, synthesis, and biological evaluation of novel quinoline derivatives as small molecule mutant EGFR inhibitors targeting resistance in NSCLC: In vitro screening and ADME predictions

Here in, we report the design, synthesis and in vitro anticancer activity of a novel series of 24 quinoline analogues of substituted amide and sulphonamide derivatives. The anticancer activity of the synthesised compounds was evaluated against the HCC827, H1975 (L858R/T790 M), A549 (WT EGFR), A-549 and BEAS-2B cell lines. The majority of quinoline compounds demonstrated a significant cytotoxic effect. Compound 21 was found to be the most potent, with IC50 values of 0.010 μM, 0.21 μM, 0.99 μM and 2.99 μM as compared to Osimertinib with IC50 values with of 0.0042 μM, 0.04 μM, 0.92 μM and 2.67 μM. Compound 21 exhibited promising inhibitory enzymatic activity against the EGFR L858R/T790 M with IC50 value of 138 nM, comparable to Osimertinib's 110 nM. Employing a Western blot assay on the phosphorylation of EGFR and the signalling pathways transmission in HCC827 cells, the anticancer activity of the synthesised compounds 18 and 21 was evaluated in terms of its mechanism of action. All the compounds were subjected to a comparative molecular docking study against various EGFR enzyme types, including the wild-type (PDB: 4I23) and T790 M mutant (PDB: 2JIV) enzymes. Furthermore, compounds were examined at the allosteric binding site of the EGFR enzyme with the L858R/T790 M/C797S mutation (PDB ID: 5D41). The MD simulation study was also performed for EGFR-compound 21 complex which indicates the stability compound 21 in both ATP and allosteric site of enzyme. Further, in silico ADME prediction studies of all derivatives were found promising, signifying the drug like properties.

Structure-Based Virtual Screening and De Novo Design to Identify Submicromolar Inhibitors of G2019S Mutant of Leucine-Rich Repeat Kinase 2.

Missense mutations of leucine-rich repeat kinase 2 (LRRK2), including the G2019S mutant, are responsible for the pathogenesis of Parkinson's disease. In this work, structure-based virtual screening of a large chemical library was carried out to identify a number of novel inhibitors of the G2019S mutant of LRRK2, the biochemical potencies of which ranged from the low micromolar to the submicromolar level. The discovery of these potent inhibitors was made possible due to the modification of the original protein-ligand binding energy function in order to include an accurate ligand dehydration energy term. The results of extensive molecular docking simulations indicated that the newly identified inhibitors were bound to the ATP-binding site of the G2019S mutant of LRRK2 through the multiple hydrogen bonds with backbone amide groups in the hinge region as well as the hydrophobic interactions with the nonpolar residues in the P-loop, hinge region, and interdomain region. Among 18 inhibitors derived from virtual screening, 4-(2-amino-5-phenylpyrimidin-4-yl)benzene-1,3-diol (Inhibitor 2) is most likely to serve as a new molecular scaffold to optimize the biochemical potency, because it revealed submicromolar inhibitory activity in spite of its low molecular weight (279.3 amu). Indeed, a highly potent inhibitor (Inhibitor 2n) of the G2019S mutant was derived via the structure-based de novo design using the structure of Inhibitor 2 as the molecular core. The biochemical potency of Inhibitor 2n surged to the nanomolar level due to the strengthening of hydrophobic interactions in the ATP-binding site, which were presumably caused by the substitutions of small nonpolar moieties. Due to the high biochemical potency against the G2019S mutant of LRRK2 and the putatively good physicochemical properties, Inhibitor 2n is anticipated to serve as a new lead compound for the discovery of antiparkinsonian medicines.

Effects of amikacin, polymyxin-B, and sulbactam combination on the pharmacodynamic indices of mutant selection against multi-drug resistant Acinetobacter baumannii.

Amikacin and polymyxins as monotherapies are ineffective against multidrug-resistant Acinetobacter baumannii at the clinical dose. When polymyxins, aminoglycosides, and sulbactam are co-administered, the combinations exhibit in vitro synergistic activities. The minimum inhibitory concentration (MIC) and mutant prevention concentration (MPC) were determined in 11 and 5 clinical resistant isolates of A. baumannii harboring OXA-23, respectively, in order to derive the fraction of time over the 24-h wherein the free drug concentration was within the mutant selection window (fTMSW) and the fraction of time that the free drug concentration was above the MPC (fT>MPC) from simulated pharmacokinetic profiles. The combination of these three antibiotics can confer susceptibility in multi-drug resistant A. baumannii and reduce the opportunity for bacteria to develop further resistance. Clinical intravenous dosing regimens of amikacin, polymyxin-B, and sulbactam were predicted to optimize fTMSW and fT>MPC from drug exposures in the blood. Mean fT>MPC were ≥ 60% and ≥ 80% for amikacin and polymyxin-B, whereas mean fTMSW was reduced to <30% and <15%, respectively, in the triple antibiotic combination. Due to the low free drug concentration of amikacin and polymyxin-B simulated in the epithelial lining fluid, the two predicted pharmacodynamic parameters in the lung after intravenous administration were not optimal even in the combination therapy setting.

PpGpp, the General Stress Response Alarmone, Is Required for the Expression of the α-Hemolysin Toxin in the Uropathogenic Escherichia coli Isolate, J96.

ppGpp is an intracellular sensor that, in response to different types of stress, coordinates the rearrangement of the gene expression pattern of bacteria to promote adaptation and survival to new environmental conditions. First described to modulate metabolic adaptive responses, ppGpp modulates the expression of genes belonging to very diverse functional categories. In Escherichia coli, ppGpp regulates the expression of cellular factors that are important during urinary tract infections. Here, we characterize the role of this alarmone in the regulation of the hlyCABDII operon of the UPEC isolate J96, encoding the toxin α-hemolysin that induces cytotoxicity during infection of bladder epithelial cells. ppGpp is required for the expression of the α-hemolysin encoded in hlyCABDII by stimulating its transcriptional expression. Prototrophy suppressor mutations in a ppGpp-deficient strain restore the α-hemolysin expression from this operon to wild-type levels, confirming the requirement of ppGpp for its expression. ppGpp stimulates hlyCABDII expression independently of RpoS, RfaH, Zur, and H-NS. The expression of hlyCABDII is promoted at 37 °C and at low osmolarity. ppGpp is required for the thermoregulation but not for the osmoregulation of the hlyCABDII operon. Studies in both commensal and UPEC isolates demonstrate that no UPEC specific factor is strictly required for the ppGpp-mediated regulation described. Our data further support the role of ppGpp participating in the coordinated regulation of the expression of bacterial factors required during infection.

In Silico Strategies for Designing of Peptide Inhibitors of Oncogenic K-Ras G12V Mutant: Inhibiting Cancer Growth and Proliferation.

Simple SummaryThe most well-known oncogene and one with the highest rates of mutation across all cancers is K-Ras, also known as the Kirsten rat sarcoma viral oncogene homologue. It is also linked to certain cancers with a high mortality rate, including pancreatic ductal adenocarcinoma (PDAC), non-small-cell lung cancer (NSCLC), and colorectal cancer (CRC). The aim of this study was to make peptides that inhibit K-Ras G12V by computer-aided drug design methods. Our results showed that the top four selected peptides interact with K-Ras more strongly than the reference-peptide and can stop K-Ras from activating. Our binding affinity analyses demonstrated that the developed peptides can inhibit K-Ras and slow cancer growth.Ras plays a pivotal function in cell proliferation and is an important protein in signal transduction pathways. Mutations in genes encoding the Ras protein drive the signaling cascades essential for malignant transformation, tumour angiogenesis, and metastasis and are responsible for above 30% of all human cancers. There is evidence that N-Ras, K-Ras, and H-Ras play significant roles in human cancer. The mutated K-Ras protein is typically observed in malignant growths. Mutant K-Ras is the most common in lung, colon, and pancreatic cancers. The purpose of this research was to create peptides that inhibit K-Ras G12V. The crystal structure of the mutant K-Ras G12V-H-REV107 complex was obtained from a protein data bank. Further, we used a residue scan approach to create unique peptides from the reference peptide (H-REV107). AMBER molecular dynamics simulations were used to test the stability of the top four proposed peptides (based on binding free energies). Our findings showed that the top four selected peptides had stronger interactions with K-Ras than the reference peptide and have the ability to block the activation function of K-Ras. Our extensive analyses of binding affinities showed that our designed peptide possesses the potential to inhibit K-Ras and to reduce the progression of cancer.

Screening of Natural Compounds Against SOD1 as a Therapeutic Target for Amyotrophic Lateral Sclerosis

Background: Amyotrophic lateral sclerosis (ALS) is an uncommon and progressive neurological illness that predominantly includes the neurons liable for voluntary muscular activities. Starting from weakness or stiffness in muscles, this gradually exploits the strength and ability to speak, eat, move, and even breathe. Its exact mechanism is still not clear, but mutations in the SOD1 gene have been reported to cause ALS, and some studies also found involvement of SOD1 overexpression in the pathogenesis of ALS. As of now, there is no remedy available for its cure. Objective: The objective of this study is to identify the potential inhibitors for wild type 1HL5, l113T mutant, and A4V mutant of SOD1 (Superoxide Dismutase 1) protein. Methods: In this study, in silico approaches like virtual screening, molecular docking, pharmacokinetic parameters study, and molecular dynamics simulation were used to identify the best potential inhibitors against wild type and mutant SOD1 protein. Results: On the basis of binding affinity and binding energy, the top three compounds ZINC000095486263, ZINC000095485989, and ZINC000028462577 were observed as the best compounds. In the case of 1HL5, ZINC000095486263 had the highest binding affinity with docking score - 10.62 Kcal/mol, 1UXM with ZINC000095485989 had the highest docking score -12.03 Kcal/mol, and 4A7V with ZINC000028462577 was found -11.72 Kcal/mol. Further, Molecular Dynamic Simulations (MDS) results showed that the ZINC000095486263, ZINC000095485989, and ZINC000095485956 compounds were formed a stable complex with 1HL5, 1UXM, and 4A7V respectively. Conclusions: After analyzing the results, we hereby conclude that natural compounds such as ZINC000095486263, ZINC000095485989, and ZINC000095485956 could be used as a potential inhibitor of 1HL5, 1UXM, and 4A7V respectively for ALS treatment and could be used as a drug. Further, In vivo/vitro study of these compounds could be a future direction in the field of drug discovery.

62 (PB052) - p53 mRNA rescue of tumor suppressor function prevents tumor growth and restores PARPi sensitivity in p53-deficient cancers in vitro and in vivo

Background: Loss of function in tumor suppressor genes is commonly associated with the onset/progression of cancer and the development of therapeutic resistance. The p53 tumor suppressor mutations resulting in loss of function are common across variety of tumors and control tumor cell proliferation in more than 50% of cancers. P53 null mutations are present in about 10% of p53 driven cancers. However, to date there is no effective treatment for rescuing p53 function in cancers. We have developed a new potent strategy combing p53 mRNA with a tumor selective nanocarrier, to rescue p53 tumor suppressor function as potential therapeutic approach in cancers. Methods: ADGN-technology is based on short amphipathic peptides that form stable neutral nanoparticles with mRNA. ADGN-531 nanoparticles containing full length p53-mRNA were evaluated on 20 different cancer cell lines (osteosarcoma, pancreas, colorectal, ovarian, lung, breast and prostate cancers) harboring different types of p53 mutations (null, deletion, nonsense, missense). Cell proliferation, cell cycle level and apoptosis activation were determined by flow cytometry, Elisa and Tunel assay. In-vivo efficacy of IV-administered ADGN-531 nanoparticles (0.5 mg/kg) was evaluated on colorectal SW403 (p53 deleted) and osteosarcoma SaOs2 (p53 null) mouse xenografts. Sensitivity to Veliparib (PARPi) following ADGN-531 treatment in vitro was evaluated on PARPI resistant SUM-149PT and OVCAR-8 cells and on PARPi sensitive MDA-MB436 cells. Results: ADGN-531 NPs markedly delay the growth of a large panel of cancer cells harboring p53-null or nonsense mutations, by inducing cell cycle arrest in G1 due to p21 upregulation and apoptosis following PUMA activation. We demonstrated that ADGN-531 mediated p53 function rescue is directly correlated to the type and level of p53 mutation in the cancer cells. ADGN-531 induces 60% to 80% cell proliferation inhibition in p53 null or nonsense mutated cells and 20 to 50% in p53 missense mutated cells. Intravenous-administration ADGN-531 containing wild type p53-mRNA (0.5 mg/kg) resulted in tumor growth inhibition of 90% and 70% in SaOs2 and SW403 xenografted mouse model, respectively. ADGN-531 NPs treatments are well tolerated, without inducing clinical toxicity or inflammatory response. We demonstrated that ADGN-531 mediated p53 rescue markedly improves (200 fold) and restores the sensitivity of ovarian and breast cancer cells to the PARPi veliparib. Conclusions: ADGN-531 nanoparticles are effective in rescuing P53 functions both in vitro and in vivo. Our study provides a proof-of-concept that restoration of tumor suppressor function by the ADGN-531 NP targeted delivery strategy could be combined together with other therapies for potent combinatorial cancer treatment. No conflict of interest.

TRNA methylation resolves codon usage bias at the limit of cell viability

Codon usage of each genome is closely correlated with the abundance of tRNA isoacceptors. How codon usage bias is resolved by tRNA post-transcriptional modifications is largely unknown. Here we demonstrate that the N1-methylation of guanosine at position 37 (m1G37) on the 3'-side of the anticodon, while not directly responsible for reading of codons, is a neutralizer that resolves differential decoding of proline codons. A genome-wide suppressor screen of a non-viable Escherichia coli strain, lacking m1G37, identifies proS suppressor mutations, indicating a coupling of methylation with tRNA prolyl-aminoacylation that sets the limit of cell viability. Using these suppressors, where prolyl-aminoacylation is decoupled from tRNA methylation, we show that m1G37 neutralizes differential translation of proline codons by the major isoacceptor. Lack of m1G37 inactivates this neutralization and exposes the need for a minor isoacceptor for cell viability. This work has medical implications for bacterial species that exclusively use the major isoacceptor for survival.

A pBBR1-based vector with IncP group plasmid compatibility for Methylorubrum extorquens.

Plasmids are one of the most important genetic tools for basic research and biotechnology, as they enable rapid genetic manipulation. Here we present a novel pBBR1‐based plasmid for Methylorubrum extorquens, a model methylotroph that is used for the development of C1‐based microbial cell factories. To develop a vector with compatibility to the so far mainly used pCM plasmid system, we transferred the pBBR1‐based plasmid pMiS1, which showed an extremely low transformation rate and caused a strong growth defect. Isolation of a suppressor mutant with improved growth led to the isolation of the variant pMis1_1B. Its higher transformation rate and less pronounced growth defect phenotype could be shown to be the result of a mutation in the promotor region of the rep gene. Moreover, cotransformation of pMis1_1B and pCM160 was possible, but the resulting transformants showed stronger growth defects in comparison with a single pMis1_1B transformant. Surprisingly, cotransformants carrying pCM160 and a pMis1_1B derivative containing a mCherry reporter construct showed higher fluorescence levels than strains containing only the pMis1_1B‐based reporter plasmids or a corresponding pCM160 derivative. Relative plasmid copy number determination experiments confirmed our hypothesis of an increased copy number of pMis1_1B in the strain carrying both plasmids. Despite the slight metabolic burden caused by pMis1_1B, the plasmid strongly expands the genetic toolbox for M. extorquens.

84 (PB074) - Identification of STX-721, an EGFR exon 20 mutant inhibitor with superior selectivity and a potential best-in-class profile

Background: EGFR mutations are well validated clinical targets in NSCLC. Osimertinib, a highly selective EGFR mutation-targeted drug, achieves an objective response rate (ORR) of >70% against cancers with L858R or exon 19 deletion. In contrast, patients with EGFR exon 20 insertion mutations have ORRs to currently approved or investigational therapies between 28 and 45%. This suggests suboptimal selectivity of existing EGFR inhibitors for exon 20 mutants versus wild-type EGFR.

The hydrophobic residue Leu73 is crucial for the high stability and low aggregation properties of murine transthyretin.

Destabilization of human transthyretin leads to its aggregation into amyloid fibrils, which causes a rare, progressive and fatal systemic disorder called ATTR amyloidosis. By contrast, murine transthyretin is known to be very stable and therefore does not aggregate into amyloid fibrils in vivo or in vitro. We examined the hydrophobic residues responsible for the high-stability and low-aggregation properties of murine transthyretin using site-directed mutagenesis. Urea-induced unfolding and thioflavin T fluorescence aggregation assay revealed that Leu73 of murine transthyretin largely contributes to its high stability and low aggregation properties: the I73L mutation stabilized human transthyretin, while the L73I mutation destabilized murine transthyretin. In addition, the I26V/I73L mutation stabilized the amyloidogenic V30M mutant of human transthyretin to the same degree as the suppressor mutation T119M, which protects transthyretin against amyloid fibril aggregation. The I73L mutation resulted in no significant differences in the overall structure of the transthyretin tetramer or the contacts of side-chains in the hydrophobic core of the monomer. We also found that Leu73 of murine transthyretin is conserved in many mammals, while Ile73 of human transthyretin is conserved in monkeys and cats. These studies will provide new insights into the stability and aggregation properties of transthyretin from various mammals.

Sustained Aurora Kinase B Expression Confers Resistance to PI3K Inhibition in Head and Neck Squamous Cell Carcinoma.

Aurora B signaling facilitates resistance to PI3K inhibition in head and neck squamous cell carcinoma, suggesting that combined inhibition of PI3K and Aurora kinase is a rational therapeutic strategy to overcome resistance.

Genomic landscape of pleural and peritoneal mesothelioma tumours

BackgroundMalignant pleural and peritoneal mesotheliomas are rare malignancies with unacceptable poor prognoses and limited treatment options. The genomic landscape is mainly characterised by the loss of tumour suppressor genes and mutations in DNA repair genes. Currently, data from next-generation sequencing (NGS) of mesothelioma tumours is restricted to a limited number of cases; moreover, data comparing molecular features of mesothelioma from the pleural and peritoneal origin with NGS are lacking.MethodsWe analysed 1113 pleural mesothelioma and 355 peritoneal mesothelioma samples. All tumours were sequenced with the FoundationOne® or FoundationOne®CDx assay for detection of substitutions, insertion–deletions, copy-number alterations and selected rearrangements in at least 324 cancer genes.ResultsThis analysis revealed alterations in 19 genes with an overall prevalence of at least 2%. Alterations in BAP1, CDKN2A, CDKN2B, NF2, MTAP, TP53 and SETD2 occurred with a prevalence of at least 10%. Peritoneal, compared to pleural mesothelioma, was characterised by a lower prevalence of alterations in CDKN2A, CDKN2B and MTAP. Moreover, we could define four distinct subgroups according to alterations in BAP1 and CDKN2A/B. Alterations in Hedgehog pathway-related genes (PTCH1/2 and SUFU) and Hippo pathway-related gene (NF2) as well as KRAS, EGFR, PDGFRA/B, ERBB2 and FGFR3 were detected in both cohorts.ConclusionHere, we report the molecular aberrations from the largest cohort of patients with mesothelioma. This analysis identified a proportion of patients with targetable alterations and suggests that molecular profiling can identify new treatment options for patients with mesothelioma.

Synthesis of BLU-945, an EGFR Mutant Inhibitor for Treatment-Resistant Non-Small-Cell Lung Cancer

Synthesis of BLU-945, an EGFR Mutant Inhibitor for Treatment-Resistant Non-Small-Cell Lung Cancer

Disruption of the tagF Orthologue in the epa Locus Variable Region of Enterococcus faecalis Causes Cell Surface Changes and Suppresses an eep-Dependent Lysozyme Resistance Phenotype.

The disease-producing capacity of the opportunistic pathogen Enterococcus faecalis is enhanced by the ability of the bacterium to evade killing by antimicrobial agents. Survival of E. faecalis in the presence of the human antimicrobial enzyme lysozyme is mediated in part by the site 2 metalloprotease Eep; however, a complete model of enterococcal lysozyme resistance has not been elucidated. To better understand the molecular basis for lysozyme resistance in E. faecalis, we analyzed Δeep suppressor mutants that acquire resistance to lysozyme through mutation of the gene OG1RF_11713, a predicted teichoic acid biosynthesis-encoding gene located within the variable region of the enterococcal polysaccharide antigen (epa) locus. Sequence comparisons revealed that OG1RF_11713 is most similar to the cytidine-5'-diphosphate (CDP)-glycerol:poly-(glycerolphosphate)glycerophosphotransferase TagF from Staphylococcus epidermidis. Inactivation of OG1RF_11713 in both the wild-type and Δeep genetic backgrounds was sufficient to increase the resistance of E. faecalis OG1RF to lysozyme. Minimal amounts of N-acetylgalactosamine were detectable in cell wall carbohydrate extracts of OG1RF_11713 deletion mutants, and this was associated with a reduction in negative cell surface charge. Targeted disruption of OG1RF_11713 was also associated with increased susceptibility to the antibiotic polymyxin B and membrane-targeting detergents and decreased susceptibility to the lantibiotic nisin. This work implicates OG1RF_11713 as a major determinant of cell envelope integrity and provides further validation that lysozyme resistance is intrinsically linked to the modification of enterococcal cell wall polysaccharides. IMPORTANCE Enterococcus faecalis is a leading cause of health-care-associated infections for which there are limited treatment options. E. faecalis is resistant to several antibiotics and to high concentrations of the human antimicrobial enzyme lysozyme. The molecular mechanisms that mediate lysozyme resistance in E. faecalis are complex and remain incompletely characterized. This work demonstrates that a gene located within the variable region of the enterococcal polysaccharide antigen locus of E. faecalis strain OG1RF (OG1RF_11713), which is predicted to encode a component of the teichoic acid biosynthesis machinery, is part of the lysozyme resistance circuitry and is important for enterococcal cell wall integrity. These findings suggest that OG1RF_11713 is a potential target for new therapeutic strategies to combat enterococcal infections.

Identification of the novel natural product inhibitors of SHP2 from the plant Toona sinensis: In vitro and in silico study

In this study, we tested the inhibitory activity of 45 natural products extracted from the plant Toona sinensis on SHP2 protein, and identified four natural product inhibitors. The natural product 1,2,3,6-Tetragalloylglucose (A-1) was first reported as a competitive inhibitor of SHP2, with an IC50 value of 0.20 ± 0.029 μM and the selectivity of 1.8-fold and 4.35-fold to high homologous proteins SHP1 and PTP1B, respectively. Compound A-1 also showed high inhibitory activity on SHP2-E76K and SHP2-E76A mutants, with IC50 values of 0.95 ± 0.21 μM and 0.29 ± 0.045 μM, respectively. Cell viability assay showed that compound A-1 could inhibit the proliferation of a variety of cancer cells. Apoptosis assay showed that compound A-1 could effectively induce apoptosis of KRASG12C-mut NCI-H23 and KRASG12S-mut A549 cells. Western blot assay showed that compound A-1 could down regulate the phosphorylation levels of Erk1/2 and Akt in NCI-H23 and A549 cells. Molecular docking showed that compound A-1 could effectively dock to the catalytic active region of SHP2. Molecular dynamics simulation explored the effect of compound A-1 on SHP2, revealing the deep-seated binding mechanism. This study would provide valuable clues for the development of SHP2 and its mutant inhibitors.

EP08.02-095 Successful Treatment of EGFR Exon 19 Deletion with TP53 and ERBB2 20ins Mutation in Stage IVB NSCLC Patient with Aumolertinib

Based on BENEFIT study, EGFR sensitive mutation NSCLC patients could benefit from first-line EGFR-TKI treatment regardless of combination with tumor suppressor or driver gene mutation. However, for patients combined with both tumor suppressor and driver gene mutations, it is still an unresolved challenge for clinical practice. Herein, we reported a case happened to be this intractable situation.