The small molecule inhibitor NAV-2729 has a complex target profile including multiple ADP-ribosylation factor regulatory proteins.

The ADP-ribosylation factor (Arf) GTPases and their regulatory proteins are implicated in cancer progression. NAV-2729 was previously identified as a specific inhibitor of Arf6 that reduced progression of uveal melanoma in an orthotopic xenograft. Here, our goal was to assess the inhibitory effects of NAV-2729 on the proliferation of additional cell types. We found NAV-2729 inhibited proliferation of multiple cell lines, but Arf6 expression did not correlate with NAV-2729 sensitivity, and knockdown of Arf6 affected neither cell viability nor sensitivity to NAV-2729. Furthermore, binding to native Arf6 was not detected; however, we determined that NAV-2729 inhibited both Arf exchange factors and Arf GTPase-activating proteins. ASAP1, a GTPase-activating protein linked to cancer progression, was further investigated. We demonstrated that NAV-2729 bound to the PH domain of ASAP1 and changed ASAP1 cellular distribution. However, ASAP1 knockdown did not fully recapitulate the cytoskeletal effects of NAV-2729 nor affect cell proliferation. Finally, our screens identified 48 other possible targets of NAV-2729. These results illustrate the complexities of defining targets of small molecules and identify NAV-2729 as a model PH domain-binding inhibitor.

Proteogenomic Analysis Unveils the HLA Class I-Presented Immunopeptidome in Melanoma and EGFR-Mutant Lung Adenocarcinoma.

Immune checkpoint inhibitors and adoptive lymphocyte transfer-based therapies have shown great therapeutic potential in cancers with high tumor mutational burden (TMB), such as melanoma, but not in cancers with low TMB, such as mutant epidermal growth factor receptor (EGFR)-driven lung adenocarcinoma. Precision immunotherapy is an unmet need for most cancers, particularly for cancers that respond inadequately to immune checkpoint inhibitors. Here, we employed large-scale MS-based proteogenomic profiling to identify potential immunogenic human leukocyte antigen (HLA) class I-presented peptides in melanoma and EGFR-mutant lung adenocarcinoma. Similar numbers of peptides were identified from both tumor types. Cell line and patient-specific databases (DBs) were constructed using variants identified from whole-exome sequencing. A de novo search algorithm was used to interrogate the HLA class I immunopeptidome MS data. We identified 12 variant peptides and several classes of tumor-associated antigen-derived peptides. We constructed a cancer germ line (CG) antigen DB with 285 antigens. This allowed us to identify 40 class I-presented CG antigen-derived peptides. The class I immunopeptidome comprised more than 1000 post-translationally modified (PTM) peptides representing 58 different PTMs, underscoring the critical role PTMs may play in HLA binding. Finally, leveraging de novo search algorithm and an annotated long noncoding RNA (lncRNA) DB, we developed a novel lncRNA-encoded peptide discovery pipeline to identify 44 lncRNA-derived peptides that are presented by class I. We validated tandem MS spectra of select variant, CG antigen, and lncRNA-derived peptides using synthetic peptides and performed HLA class I-binding assays to demonstrate binding to class I proteins. In summary, we provide direct evidence of HLA class I presentation of a large number of variant and tumor-associated peptides in both low and high TMB cancer. These results can potentially be useful for precision immunotherapies, such as vaccine or adoptive cell therapies in melanoma and EGFR-mutant lung cancers.

Synthetic and computational efforts towards the development of peptidomimetics and small-molecule SARS-CoV 3CLpro inhibitors.

Severe Acute Respiratory Syndrome (SARS) is a severe febrile respiratory disease caused by the beta genus of human coronavirus, known as SARS-CoV. Last year, 2019-n-CoV (COVID-19) was a global threat for everyone caused by the outbreak of SARS-CoV-2. 3CLpro, chymotrypsin-like protease, is a major cysteine protease that substantially contributes throughout the viral life cycle of SARS-CoV and SARS-CoV-2. It is a prospective target for the development of SARS-CoV inhibitors by applying a repurposing strategy. This review focuses on a detailed overview of the chemical synthesis and computational chemistry perspectives of peptidomimetic inhibitors (PIs) and small-molecule inhibitors (SMIs) targeting viral proteinase discovered from 2004 to 2020. The PIs and SMIs are one of the primary therapeutic inventions for SARS-CoV. The journey of different analogues towards the evolution of SARS-CoV 3CLpro inhibitors and complete synthetic preparation of nineteen derivatives of PIs and ten derivatives of SMIs and their computational chemistry perspectives were reviewed. From each class of derivatives, we have identified and highlighted the most compelling PIs and SMIs for SARS-CoV 3CLpro. The protein-ligand interaction of 29 inhibitors were also studied that involved with the 3CLpro inhibition, and the frequent amino acid residues of the protease were also analyzed that are responsible for the interactions with the inhibitors. This work will provide an initiative to encourage further research for the development of effective and drug-like 3CLpro inhibitors against coronaviruses in the near future.

Quantitative Mass Spectrometry to Interrogate Proteomic Heterogeneity in Metastatic Lung Adenocarcinoma and Validate a Novel Somatic Mutation CDK12-G879V.

Lung cancer is the leading cause of cancer death in both men and women. Tumor heterogeneity is an impediment to targeted treatment of all cancers, including lung cancer. Here, we sought to characterize tumor proteome and phosphoproteome changes by longitudinal, prospective collection of tumor tissue from an exceptional responder lung adenocarcinoma patient who survived with metastatic lung adenocarcinoma for over seven years while undergoing HER2-directed therapy in combination with chemotherapy. We employed "Super-SILAC" and TMT labeling strategies to quantify the proteome and phosphoproteome of a lung metastatic site and eight distinct metastatic progressive lymph nodes collected during these seven years, including five lymph nodes procured at autopsy. We identified specific signaling networks enriched in lung compared with the lymph node metastatic sites. We correlated the changes in protein abundance with changes in copy number alteration (CNA) and transcript expression. ERBB2/HER2 protein expression was higher in lung, consistent with a higher degree of ERBB2 amplification in lung compared with the lymph node metastatic sites. To further interrogate the mass spectrometry data, a patient-specific database was built by incorporating all the somatic and germline variants identified by whole genome sequencing (WGS) of genomic DNA from the lung, one lymph node metastatic site and blood. An extensive validation pipeline was built to confirm variant peptides. We validated 360 spectra corresponding to 55 germline and 6 somatic variant peptides. Targeted MRM assays revealed two novel variant somatic peptides, CDK12-G879V and FASN-R1439Q, expressed in lung and lymph node metastatic sites, respectively. The CDK12-G879V mutation likely results in a nonfunctional CDK12 kinase and chemotherapy susceptibility in lung metastatic sites. Knockdown of CDK12 in lung adenocarcinoma cells increased chemotherapy sensitivity which was rescued by wild type, but not CDK12-G879V expression, consistent with the complete resolution of the lung metastatic sites in this patient.

Osteoarthritis: Prognosis and emerging therapeutic approach for disease management.

Osteoarthritis (OA), a disorder of joints, is prevalent in older age. The contemporary cure for OA is aimed to confer symptomatic relief, consisting of temporary pain and swelling relief. In this paper, we discuss various modalities responsible for the onset of OA and associated with its severity. Inhibition of chondrocytes receptors such as DDR2, SDF-1, Asporin, and CXCR4 by specific pharmacological inhibitors attenuates OA, a critical step for finding potential disease modifying drugs. We critically analyzed recent OA studies with an emphasis on intermediate target molecules for OA intervention. We also explored some novel and safe treatments for OA by considering disease prognosis crosstalk with cellular signaling pathways.

Quantitative Tyrosine Phosphoproteomics of Epidermal Growth Factor Receptor (EGFR) Tyrosine Kinase Inhibitor-treated Lung Adenocarcinoma Cells Reveals Potential Novel Biomarkers of Therapeutic Response.

Mutations in the Epidermal growth factor receptor (EGFR) kinase domain, such as the L858R missense mutation and deletions spanning the conserved sequence 747LREA750, are sensitive to tyrosine kinase inhibitors (TKIs). The gatekeeper site residue mutation, T790M accounts for around 60% of acquired resistance to EGFR TKIs. The first generation EGFR TKIs, erlotinib and gefitinib, and the second generation inhibitor, afatinib are FDA approved for initial treatment of EGFR mutated lung adenocarcinoma. The predominant biomarker of EGFR TKI responsiveness is the presence of EGFR TKI-sensitizing mutations. However, 30-40% of patients with EGFR mutations exhibit primary resistance to these TKIs, underscoring the unmet need of identifying additional biomarkers of treatment response. Here, we sought to characterize the dynamics of tyrosine phosphorylation upon EGFR TKI treatment of mutant EGFR-driven human lung adenocarcinoma cell lines with varying sensitivity to EGFR TKIs, erlotinib and afatinib. We employed stable isotope labeling with amino acids in cell culture (SILAC)-based quantitative mass spectrometry to identify and quantify tyrosine phosphorylated peptides. The proportion of tyrosine phosphorylated sites that had reduced phosphorylation upon erlotinib or afatinib treatment correlated with the degree of TKI-sensitivity. Afatinib, an irreversible EGFR TKI, more effectively inhibited tyrosine phosphorylation of a majority of the substrates. The phosphosites with phosphorylation SILAC ratios that correlated with the TKI-sensitivity of the cell lines include sites on kinases, such as EGFR-Y1197 and MAPK7-Y221, and adaptor proteins, such as SHC1-Y349/350, ERRFI1-Y394, GAB1-Y689, STAT5A-Y694, DLG3-Y705, and DAPP1-Y139, suggesting these are potential biomarkers of TKI sensitivity. DAPP1, is a novel target of mutant EGFR signaling and Y-139 is the major site of DAPP1 tyrosine phosphorylation. We also uncovered several off-target effects of these TKIs, such as MST1R-Y1238/Y1239 and MET-Y1252/1253. This study provides unique insight into the TKI-mediated modulation of mutant EGFR signaling, which can be applied to the development of biomarkers of EGFR TKI response.

l-Cysteine-Conjugated Ruthenium Hydrous Oxide Nanomaterials with Anticancer Active Application.

Bioactive nanomaterials, namely: ruthenium hydrous oxide (or ruthenium oxy-hydroxide), RuOx(OH)y and also a surface-conjugated novel material of the same within the template of an amino acid molecule, l-cysteine, have been studied. These compounds have been prepared through a simple wet chemical route, under physiological conditions, such that they could be suitably used in anticancer applications. Several physical methods were used for the nanomaterial characterization, e.g.: thermal analysis of the as prepared ruthenium hydrous oxide by differential scanning calorimetry (DSC) followed by thermal gravimetric analysis (TGA). This confirms that the material is a precursor for anhydrous nanocrystalline ruthenium oxide (RuO2), as is affirmed by powder X-ray diffraction pattern. Also, optical spectroscopic absorption (UV-vis and FT-IR) study of these nanoparticles (NPs) to ascertain their surface conjugation with l-cysteine have been performed. Besides these, surface morphology of the NPs were studied by field emission scanning electron microscopy (FE-SEM) along with their elemental purity check through energy dispersive X-ray analysis (EDX). Their surface chemical microenvironments were examined by X-ray photo electron spectroscopy (XPS). The hydrodynamic size of the prepared NPs were measured through dynamic light scattering (DLS) studies. Further, biological consequences of these NPs on cancerous HeLa cells and their cytotoxicity effects have been reported with MTT assay, such an application has not been reported so far.

Microtubule-targeting agents augment the toxicity of DNA-damaging agents by disrupting intracellular trafficking of DNA repair proteins.

The paradigm that microtubule-targeting agents (MTAs) cause cell death via mitotic arrest applies to rapidly dividing cells but cannot explain MTA activity in slowly growing human cancers. Many preferred cancer regimens combine a MTA with a DNA-damaging agent (DDA). We hypothesized that MTAs synergize with DDAs by interfering with trafficking of DNA repair proteins on interphase microtubules. We investigated nine proteins involved in DNA repair: ATM, ATR, DNA-PK, Rad50, Mre11, p95/NBS1, p53, 53BP1, and p63. The proteins were sequestered in the cytoplasm by vincristine and paclitaxel but not by an aurora kinase inhibitor, colocalized with tubulin by confocal microscopy and coimmunoprecipitated with the microtubule motor dynein. Furthermore, adding MTAs to radiation, doxorubicin, or etoposide led to more sustained γ-H2AX levels. We conclude DNA damage-repair proteins traffic on microtubules and addition of MTAs sequesters them in the cytoplasm, explaining why MTA/DDA combinations are common anticancer regimens.

A Novel Tetraenoic Fatty Acid Isolated from Amaranthus spinosus Inhibits Proliferation and Induces Apoptosis of Human Liver Cancer Cells.

Amaranthus spinosus Linn. (Family: Amaranthaceae) has been shown to be useful in preventing and mitigating adverse pathophysiological conditions and complex diseases. However, only limited information is available on the anticancer potential of this plant. In this study, we examined the antiproliferative and pro-apoptotic effects of a novel fatty acid isolated from A. spinosus-(14E,18E,22E,26E)-methyl nonacosa-14,18,22,26 tetraenoate-against HepG2 human liver cancer cells. We used 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay to determine cell viability, flow cytometry assay for cell cycle analysis, and Western blot analysis to measure protein expression of Cdc2), cyclin B1, Bcl-2-associated X protein (Bax), and B-cell lymphoma 2 (Bcl-2). The MTT assay showed that the fatty acid markedly inhibited the proliferation of HepG2 cells in a dosage-dependent fashion, with a half maximal inhibitory concentration (IC50) value of 25.52 µmol/L. This antiproliferative result was superior to that of another known fatty acid, linoleic acid (IC50 38.65 µmol/L), but comparable to that of standard anticancer drug doxorubicin (IC50 24.68 µmol/L). The novel fatty acid also induced apoptosis mediated by downregulation of cyclin B1, upregulation of Bax, and downregulation of Bcl-2, resulting in the G2/M transition arrest. Our results provide the first experimental evidence that a novel fatty acid isolated from A. spinosus exhibits significant antiproliferative activity mediated through the induction of apoptosis in HepG2 cells. These encouraging results may facilitate the development of A. spinosus fatty acid for the prevention and intervention of hepatocellular carcinoma.

Antibacterial activity of a novel fatty acid (14E, 18E, 22E, 26E)-methyl nonacosa-14, 18, 22, 26 tetraenoate isolated from Amaranthus spinosus.

CONTEXT: Amaranthus spinosus Linn. (Amaranthaceae), commonly known as ''spiny pigweed'', is used in both Indian traditional system and folk medicine for treatment of infectious diseases for a long time in several traditional herbal medicinal preparations. A novel fatty acid [(14E, 18E, 22E, 26E)-methyl nonacosa-14, 18, 22, 26 tetraenoate] is the major metabolite present. OBJECTIVE: This study examines the antibacterial potential of the fatty acid isolated from the A. spinosus against some Gram-positive and Gram-negative bacteria. MATERIALS AND METHODS: Three Gram-positive and seven Gram-negative bacterial strains were used for antibacterial assay. The minimum inhibitory concentration (MIC) of the fatty acid was analysed by dilution method and the effects of the fatty acid on the bacterial membrane were studied in detail by flow cytometry analysis. RESULTS AND DISCUSSION: All the studied bacterial strains were found to be inhibited at a concentration of 100 µg/mL. Staphylococcus aureus ML-59, Bacillus lycheniformis 10341, Shigella boydii 8, Vibrio cholera 811, Vibrio cholera 854 and Vibrio alginolyteus were susceptible and sensitive to the tested fatty acid with a MIC value of 25 µg/mL. It proved a full spectrum of antibacterial activity associated with alterations in the permeability of bacterial membranes. CONCLUSION: The fatty acid from the A. spinosus possesses potent antibacterial action.

Evaluation of in vitro antioxidant, anticancer and in vivo antitumour activity of Termitomyces clypeatus MTCC 5091.

CONTEXT: Termitomyces clypeatus (Lyophyllaceae) is a filamentous edible mushroom, having ethnomedicinal uses. However, information about the antioxidant, anticancer and antitumour properties of this mushroom remains to be elucidated. OBJECTIVE: The study examines the in vitro antioxidant, anticancer and in vivo antitumour activity of T. clypeatus. MATERIALS AND METHODS: Antioxidant activity was evaluated with seven in vitro assays. Cytotoxicity of T. clypeatus was tested against a panel of cancer cells lines including U373MG, MDA-MB-468, HepG2, HL-60, A549, U937, OAW-42 and Y-79 using MTT assay. The antitumour activity of aqueous extract was evaluated against Ehrlich ascites carcinoma (EAC) tumour model in Swiss albino mice. RESULTS: HPLC analysis of aqueous extract revealed the presence of sugar entities. Termitomyces clypeatus showed excellent in vitro antioxidant activity. Termitomyces clypeatus was found cytotoxic against all cancer cells, among which it showed higher activity against U937 (IC50 25 ± 1.02 µg/mL). Treatment of EAC-bearing mice with varied doses of aqueous extract significantly (p < 0.01) reduced tumour volume, viable tumour cell count and improved haemoglobin content, RBC count, mean survival time, tumour inhibition and % increase life span. The enhanced antioxidant status in treated animals was evident from the decline in the levels of lipid peroxidation, increased levels of glutathione, catalase and superoxide dismutase. DISCUSSION: The analyzed data indicate that the aqueous extract of T. clypeatus exhibits significant antitumour activity, which might be due to the antioxidant effects on EAC bearing hosts. CONCLUSION: Termitomyces clypeatus possesses anticancer activity, valuable for application in food and drug products.

Identifying novel targets of oncogenic EGF receptor signaling in lung cancer through global phosphoproteomics.

Mutations in the epidermal growth factor receptor (EGFR) kinase domain occur in 10-30% of lung adenocarcinoma and are associated with tyrosine kinase inhibitor (TKI) sensitivity. We sought to identify the immediate direct and indirect phosphorylation targets of mutant EGFRs in lung adenocarcinoma. We undertook SILAC strategy, phosphopeptide enrichment, and quantitative MS to identify dynamic changes of phosphorylation downstream of mutant EGFRs in lung adenocarcinoma cells harboring EGFR(L858R) and EGFR(L858R/T790M) , the TKI-sensitive, and TKI-resistant mutations, respectively. Top canonical pathways that were inhibited upon erlotinib treatment in sensitive cells, but not in the resistant cells include EGFR, insulin receptor, hepatocyte growth factor, mitogen-activated protein kinase, mechanistic target of rapamycin, ribosomal protein S6 kinase beta 1, and Janus kinase/signal transducer and activator of transcription signaling. We identified phosphosites in proteins of the autophagy network, such as ULK1 (S623) that is constitutively phosphorylated in these lung adenocarcinoma cells; phosphorylation is inhibited upon erlotinib treatment in sensitive cells, but not in resistant cells. Finally, kinase-substrate prediction analysis from our data indicated that substrates of basophilic kinases from, AGC and Calcium and calmodulin-dependent kinase groups, as well as STE group kinases were significantly enriched and those of proline-directed kinases from, CMGC and Casein kinase groups were significantly depleted among substrates that exhibited increased phosphorylation upon EGF stimulation and reduced phosphorylation upon TKI inhibition. This is the first study to date to examine global phosphorylation changes upon erlotinib treatment of lung adenocarcinoma cells and results from this study provide new insights into signaling downstream of mutant EGFRs in lung adenocarcinoma. All MS data have been deposited in the ProteomeXchange with identifier PXD001101 (http://proteomecentral.proteomexchange.org/dataset/PXD001101).

A new ester of fatty acid from a methanol extract of the whole plant of Amaranthus spinosus and its α-glucosidase inhibitory activity.

CONTEXT: Amaranthus spinosus Linn. (Amaranthaceae), commonly known as "spiny pigweed", is used both in the Indian traditional system and in folk medicine to treat diabetes. OBJECTIVE: The present study evaluates the scientific basis of antidiabetic activity of chloroform fraction of methanol extract of A. spinosus and of an isolated constituent of A. spinosus. MATERIALS AND METHODS: HPLC analysis was performed to determine the purity and the amount of the constituent present in the plant extract. The yeast α-glucosidase inhibition technique was used to determine the antidiabetic activity of A. spinosus. Acarbose was used as a standard. An appropriate therapeutic approach for preventing diabetes mellitus and obesity is to retard the absorption of glucose by inhibition of α-glucosidase. RESULTS: One novel fatty acid with strong α-glucosidase inhibitory activity - (14E, 18E, 22E, 26E) - methyl nonacosa-14, 18, 22, 26 tetraenoate [1] (IC50 value 6.52 µM/mL) and ß-sitosterol [2] were purified. Compound 1 was found to be more potent than the methanol extract. HPLC quantitative analysis revealed that 0.15% of compound 1 and 0.06% of compound 2 were present in the plant extract. CONCLUSION: This novel fatty acid can potentially be developed as a novel natural nutraceutical for the management of diabetes.

Toll-like receptor agonists and febrile range hyperthermia synergize to induce heat shock protein 70 expression and extracellular release.

Heat shock protein (Hsp) 70 expression can be stimulated by febrile range temperature (FRT). Hsp70 has been shown to be elevated in serum of patients with sepsis, and when released from cells, extracellular Hsp70 exerts endotoxin-like effects through Toll-like receptor 4 (TLR4) receptors. Circulating TLR agonists and fever both persist for the first several days of sepsis, and each can activate Hsp70 expression; however, the effect of combined exposure to FRT and TLR agonists on Hsp70 expression is unknown. We found that concurrent exposure to FRT (39.5 °C) and agonists for TLR4 (LPS), TLR2 (Pam3Cys), or TLR3 (poly(IC)) synergized to increase Hsp70 expression and extracellular release in RAW264.7 macrophages. The increase in Hsp70 expression was associated with activation of p38 and ERK MAP kinases, phosphorylation of histone H3, and increased recruitment of HSF1 to the Hsp70 promoter. Pretreatment with the p38 MAPK inhibitor SB283580 but not the ERK pathway inhibitor UO126 significantly reduced Hsp70 gene modification and Hsp70 expression in RAW cells co-exposed to LPS and FRT. In mice challenged with intratracheal LPS and then exposed to febrile range hyperthermia (core temperature, ∼39.5 °C), Hsp70 levels in lung tissue and in cell-free lung lavage were increased compared with mice exposed to either hyperthermia or LPS alone. We propose a model of how enhanced Hsp70 expression and extracellular release in patients concurrently exposed to fever and TLR agonists may contribute to the pathogenesis of sepsis.

Synthetic GPR40/FFAR1 agonists: An exhaustive survey on the most recent chemical classes and their structure-activity relationships.

Free fatty acid receptor 1 (FFAR1 or GPR40) is a potential target for treating type 2 diabetes mellitus (T2DM) and related disorders that have been extensively researched for many years. GPR40/FFAR1 is a promising anti-diabetic target because it can activate insulin, promoting glucose metabolism. It controls T2DM by regulating glucose levels in the body through two separate mechanisms: glucose-stimulated insulin secretion and incretin production. In the last few years, various synthetic GPR40/FFAR1 agonists have been discovered that fall under several chemical classes, viz. phenylpropionic acid, phenoxyacetic acid, and dihydrobenzofuran acetic acid. However, only a few synthetic agonists have entered clinical trials due to various shortcomings like poor efficacy, low lipophilicity and toxicity issues. As a result, pharmaceutical firms and research institutions are interested in developing synthetic GPR40/FFAR1 agonists with superior effectiveness, lipophilicity, and safety profiles. This review encompasses the most recent research on synthetic GPR40/FFAR1 agonists, including their chemical classes, design strategies and structure-activity relationships. Additionally, we have emphasised the structural characteristics of the most potent GPR40/FFAR1 agonists from each chemical class of synthetic derivatives and analysed their chemico-biological interactions. This work will hopefully pave the way for developing more potent and selective synthetic GPR40/FFAR1 agonists for treating T2DM and related disorders.

Multiplexing the Identification of Microorganisms via Tandem Mass Tag Labeling Augmented by Interference Removal through a Novel Modification of the Expectation Maximization Algorithm.

Having fast, accurate, and broad spectrum methods for the identification of microorganisms is of paramount importance to public health, research, and safety. Bottom-up mass spectrometer-based proteomics has emerged as an effective tool for the accurate identification of microorganisms from microbial isolates. However, one major hurdle that limits the deployment of this tool for routine clinical diagnosis, and other areas of research such as culturomics, is the instrument time required for the mass spectrometer to analyze a single sample, which can take ∼1 h per sample, when using mass spectrometers that are presently used in most institutes. To address this issue, in this study, we employed, for the first time, tandem mass tags (TMTs) in multiplex identifications of microorganisms from multiple TMT-labeled samples in one MS/MS experiment. A difficulty encountered when using TMT labeling is the presence of interference in the measured intensities of TMT reporter ions. To correct for interference, we employed in the proposed method a modified version of the expectation maximization (EM) algorithm that redistributes the signal from ion interference back to the correct TMT-labeled samples. We have evaluated the sensitivity and specificity of the proposed method using 94 MS/MS experiments (covering a broad range of protein concentration ratios across TMT-labeled channels and experimental parameters), containing a total of 1931 true positive TMT-labeled channels and 317 true negative TMT-labeled channels. The results of the evaluation show that the proposed method has an identification sensitivity of 93-97% and a specificity of 100% at the species level. Furthermore, as a proof of concept, using an in-house-generated data set composed of some of the most common urinary tract pathogens, we demonstrated that by using the proposed method the mass spectrometer time required per sample, using a 1 h LC-MS/MS run, can be reduced to 10 and 6 min when samples are labeled with TMT-6 and TMT-10, respectively. The proposed method can also be used along with Orbitrap mass spectrometers that have faster MS/MS acquisition rates, like the recently released Orbitrap Astral mass spectrometer, to further reduce the mass spectrometer time required per sample.

The novel ATR inhibitor M1774 induces replication protein overexpression and broad synergy with DNA-targeted anticancer drugs.

Ataxia Telangiectasia and Rad3-related (ATR) checkpoint kinase inhibitors are in clinical trials. Here we explored the molecular pharmacology and therapeutic combination strategies of the oral ATR inhibitor M1774 (Tuvusertib) with DNA damaging agents (DDAs). As single agent, M1774 suppressed cancer cell viability at nanomolar concentrations, showing greater activity than ceralasertib and berzosertib, but less potency than gartisertib and elimusertib in the small-cell lung cancer H146, H82, and DMS114 cell lines. M1774 also efficiently blocked the activation of the ATR-CHK1 checkpoint pathway caused by replication stress induced by TOP1 inhibitors. Combination with non-toxic dose of M1774 enhanced TOP1 inhibitor-induced cancer cell death by enabling unscheduled replication upon replicative damage, thereby increasing genome instability. Tandem mass tag (TMT)-based quantitative proteomics uncovered that M1774, in the presence of DDA, forces the expression of proteins activating replication (CDC45) and G2/M-progression (PLK1 and CCNB1). In particular, the fork protection complex proteins (TIMELESS and TIPIN) were enriched. Low dose of M1774 was found highly synergistic with a broad spectrum of clinical DDAs including TOP1 inhibitors (SN-38/irinotecan, topotecan, exatecan, and exatecan), the TOP2 inhibitor etoposide, cisplatin, the RNA polymerase II inhibitor lurbinectedin, and the PARP inhibitor talazoparib in various models including cancer cell lines, patient-derived organoids, and mouse xenograft models. Furthermore, we demonstrate that M1774 reverses chemoresistance to anticancer DDAs in cancer cells lacking SLFN11 expression, suggesting that SLFN11 can be utilized for patient selection in upcoming clinical trials.

Functional Heterogeneity in MET Pathway Activation in PDX Models of Osimertinib-resistant EGFR-driven Lung Cancer.

MET pathway activation is one of the most common mechanisms of resistance to osimertinib in EGFR-mutant non-small cell lung cancer (NSCLC). We previously demonstrated spatial and temporal heterogeneity in MET pathway activation upon osimertinib resistance in EGFR-mutant NSCLC; however, the functional relevance of these findings is unclear. Here, we generated 19 patient-derived xenografts (PDX) from 9 patients with multi-region and temporal sampling of osimertinib-resistant tumor tissue from patients with EGFR-mutant NSCLC. MET pathway activation was a putative mechanism of osimertinib resistance in 66% (n = 6/9) patients from whom PDXs were generated. Significant spatial and temporal heterogeneity in MET pathway activation was evident. Osimertinib-resistant PDXs with MET amplification by FISH (defined as MET/CEP7 ratio ≥2.0 or mean MET ≥ 6.0 copies/cell) and high-level phospho-MET, but not c-MET expression, had better responses to osimertinib and savolitinib combination than to osimertinib alone. MET polysomy tumors by FISH from both PDXs and patients had evidence of subclonal phospho-MET expression. Select MET polysomy PDX tumors with phospho-MET expression responded better to osimertinib and savolitinib combination than MET polysomy PDX tumors without phospho-MET expression. Our results suggest osimertinib and savolitinib combination is most effective for osimertinib-resistant EGFR-mutant tumors with MET pathway activation as evidenced by phospho-MET. As subclonal MET amplification may be evident in MET polysomy tumor progression, MET polysomy warrants close clinical follow-up with phospho-MET IHC in parallel with FISH diagnostic. SIGNIFICANCE: Using a novel cohort of in vivo PDX models of MET pathway activation with acquired resistance to osimertinib in EGFR-mutant lung cancer, we demonstrate that phospho-MET may be a clinically relevant assay to guide treatment selection with osimertinib and savolitinib combination. In addition, our work shows that patients with MET polysomy tumors may have subclonal MET amplification and therefore require close follow up for the use of osimertinib and savolitinib combination.

Significance of TRAIL/Apo-2 ligand and its death receptors in apoptosis and necroptosis signalling: Implications for cancer-targeted therapeutics.

The human immune defensesystem routinely expresses the tumour necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL), which is the most prevalent element for antitumor immunity. TRAIL associates with its death receptors (DRs), DR4 (TRAIL-R1), and DR5 (TRAIL-R2), in cancer cells to initiate the intracellular apoptosis cascade. Accordingly, numerous academic institutions and pharmaceutical companies havetried to exploreTRAIL's capacity to kill tumourcells by producing recombinant versions of it (rhTRAIL) or TRAIL receptor agonists (TRAs) [monoclonal antibody (mAb), synthetic and natural compounds, etc.] and molecules that sensitize TRAIL signalling pathway for therapeutic applications. Recently, several microRNAs (miRs) have been found to activate or inhibit death receptor signalling. Therefore, pharmacological regulation of these miRs may activate or resensitize the TRAIL DRs signal, and this is a novel approach for developing anticancer therapeutics. In this article, we will discuss TRAIL and its receptors and molecular pathways by which it induces various cell death events. We will unravel potential innovative applications of TRAIL-based therapeutics, and other investigated therapeutics targeting TRAIL-DRs and summarize the current preclinical pharmacological studies and clinical trials. Moreover, we will also emphasizea few situations where future efforts may be addressed to modulate the TRAIL signalling pathway.

The homeobox transcription factor DUXBL controls exit from totipotency.

In mice, exit from the totipotent two-cell (2C) stage embryo requires silencing of the 2C-associated transcriptional program. However, the molecular mechanisms involved in this process remain poorly understood. Here we demonstrate that the 2C-specific transcription factor double homeobox protein (DUX) mediates an essential negative feedback loop by inducing the expression of DUXBL to promote this silencing. We show that DUXBL gains accessibility to DUX-bound regions specifically upon DUX expression. Furthermore, we determine that DUXBL interacts with TRIM24 and TRIM33, members of the TRIM superfamily involved in gene silencing, and colocalizes with them in nuclear foci upon DUX expression. Importantly, DUXBL overexpression impairs 2C-associated transcription, whereas Duxbl inactivation in mouse embryonic stem cells increases DUX-dependent induction of the 2C-transcriptional program. Consequently, DUXBL deficiency in embryos results in sustained expression of 2C-associated transcripts leading to early developmental arrest. Our study identifies DUXBL as an essential regulator of totipotency exit enabling the first divergence of cell fates.