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Purpose: Inhibitors of Bruton's tyrosine kinase (BTKi) and PI3K (PI3Ki) have significantly improved therapy of chronic lymphocytic leukemia (CLL). However, the emergence of resistance to BTKi has introduced an unmet therapeutic need. Hence, we sought evidence for essential roles of PI3K-δi and PI3K-γi in treatment-naïve and BTKi-refractory CLL. Experimental Design: Responses to PI3K-δi, PI3K-γi, and the dual-inhibitor duvelisib in each B, T, and myeloid cell compartments of CLL were studied in vitro, and in a xenograft mouse model using primary cells from treatment-naïve and ibrutinib-resistant patients, and finally, in a patient with ibrutinib-resistant CLL treated with duvelisib. Results: We demonstrate the essential roles of PI3K-δ for CLL B-cell survival and migration, of PI3K-γ for T-cell migration and macrophage polarization, and of dual inhibition of PI3K-δ,γ for efficacious reduction of leukemia burden. We also show that samples from patients whose disease progressed on ibrutinib were responsive to duvelisib therapy in a xenograft model, irrespective of BTK mutations. In support of this, we report a patient with ibrutinib-resistant CLL, bearing a clone with BTK and PLCγ2 mutations, who responded immediately to single-agent duvelisib with redistribution lymphocytosis followed by a partial clinical remission associated with modulation of T and myeloid cells. Conclusions: Our data define the mechanism of action whereby dual inhibition of PI3K-δ,γ affects CLL B-cell numbers and T and myeloid cell pro-leukemia functions and support the use of duvelisib as a valuable approach for therapeutic interventions, including for patients refractory to BTKi.

This article covers exciting developments of recent microfluidics-based single-cell proteomics methods and their utilizations to tackle important biological questions for both basic and translational research.

The fundamental pursuit to complete the human proteome atlas and the unmet clinical needs in lung adenocarcinoma have prompted us to study the functional role of uncharacterized proteins and explore their implications in cancer biology. In this study, we characterized SEL1L3, a previously uncharacterized protein encoded from chromosome 4 as a dysregulated protein in lung adenocarcinoma from the large-scale tissue proteogenomics data set established using the cohort of Taiwan Cancer Moonshot. SEL1L3 was expressed in abundance in the tumor parts compared with paired adjacent normal tissues in 90% of the lung adenocarcinoma patients in our cohorts. Moreover, survival analysis revealed the association of SEL1L3 with better clinical outcomes. Intriguingly, silencing of SEL1L3 imposed a reduction in cell viability and activation of ER stress response pathways, indicating a role of SEL1L3 in the regulation of cell stress. Furthermore, the immune profiles of patients with higher SEL1L3 expression were corroborated with its active role in immunophenotype and favorable clinical outcomes in lung adenocarcinoma. Taken together, our study revealed that SEL1L3 might play a vital role in the regulation of cell stress, interaction with cancer cells and the immune microenvironment. Our research findings provide promising insights for further investigation of its molecular signaling network and also suggest SEL1L3 as a potential emerging adjuvant for immunotherapy in lung adenocarcinoma.

International audience; The 2022 Metrics of the Human Proteome from the HUPO Human Proteome Project (HPP) illustrates that protein expression has now been credibly detected (neXtProt PE1 level) for 18,407 (93.2%) of the 19,750 predicted proteins coded in the human genome, a net gain of 50 since 2021 from datasets generated around the world and reanalyzed by the HPP. Conversely, the number of neXtProt PE2, PE3, and PE4 missing proteins has been reduced by 78 from 1421 to 1343. This represents continuing experimental progress on the proteome parts list across all the chromosomes, as well as significant reclassifications. Meanwhile, applying proteomics in a vast array of biological and clinical studies continues to yield significant findings and growing integration with other omics platforms. We present highlights from the Chromosome-Centric HPP, Biology and Disease-driven HPP, and HPP Resource Pillars, compare features of mass spectrometry and Olink and Somalogic platforms, note the emergence of translation products from ribosome profiling of small open reading frames, and discuss the launch of the initial HPP Grand Challenge Project, "A Function for Each Protein".

Effects of inhibition of PI3K isoforms on CLL-cell proliferation, survival, and migration.

Purpose:Inhibitors of Bruton's tyrosine kinase (BTKi) and PI3K (PI3Ki) have significantly improved therapy of chronic lymphocytic leukemia (CLL). However, the emergence of resistance to BTKi has introduced an unmet therapeutic need. Hence, we sought evidence for essential roles of PI3K-δi and PI3K-γi in treatment-naïve and BTKi-refractory CLL.Experimental Design:Responses to PI3K-δi, PI3K-γi, and the dual-inhibitor duvelisib in each B, T, and myeloid cell compartments of CLL were studied in vitro, and in a xenograft mouse model using primary cells from treatment-naïve and ibrutinib-resistant patients, and finally, in a patient with ibrutinib-resistant CLL treated with duvelisib.Results:We demonstrate the essential roles of PI3K-δ for CLL B-cell survival and migration, of PI3K-γ for T-cell migration and macrophage polarization, and of dual inhibition of PI3K-δ,γ for efficacious reduction of leukemia burden. We also show that samples from patients whose disease progressed on ibrutinib were responsive to duvelisib therapy in a xenograft model, irrespective of BTK mutations. In support of this, we report a patient with ibrutinib-resistant CLL, bearing a clone with BTK and PLCγ2 mutations, who responded immediately to single-agent duvelisib with redistribution lymphocytosis followed by a partial clinical remission associated with modulation of T and myeloid cells.Conclusions:Our data define the mechanism of action whereby dual inhibition of PI3K-δ,γ affects CLL B-cell numbers and T and myeloid cell pro-leukemia functions and support the use of duvelisib as a valuable approach for therapeutic interventions, including for patients refractory to BTKi.

IC50 values of PI3K isoform inhibitors used. Duvelisib5, IPI-3063 (PI3K-d inhibitor)5, IPI-549 (PI3K-g inhibitor used in vitro)50, IPI-5243 (PI3K-g inhibitor used in vivo).

Application of B-cell receptor (BCR) pathway inhibitor ibrutinib for chronic lymphocytic leukemia (CLL) is a major breakthrough, yet the downstream effects following inhibition of BCR signaling and during relapse await further clarification. By comparative phosphoproteomic profiling of B cells from patients with CLL and healthy donors, as well as CLL B cells collected at multiple time points during the course of ibrutinib treatment, we provided the landscape of dysregulated phosphoproteome in CLL and its dynamic alterations associated with ibrutinib treatment. Particularly, differential phosphorylation events associated with several signaling pathways, including BCR pathway, were enriched in patient CLL cells. A constitutively elevated phosphorylation level of KAP1 at serine 473 (S473) was found in the majority of CLL samples prior to treatment. Further verification showed that BCR activation promoted KAP1 S473 phosphorylation, whereas ibrutinib treatment abolished it. Depletion of KAP1 in primary CLL cells decelerated cell-cycle progression and ectopic expression of a KAP1 S473 phospho-mimicking mutant accelerated G2–M cell-cycle transition of CLL cells. Moreover, temporal phosphoproteomic profiles using a series of CLL cells isolated from one patient during the ibrutinib treatment revealed the dynamic changes of several molecules associated with BCR signaling in the ibrutinib responsive and recurrent stages.Implications:This phosphoproteomic analysis and functional validation illuminated the phosphorylation of KAP1 at S473 as an important downstream BCR signaling event and a potential indicator for the success of ibrutinib treatment in CLL.

Supplementary Data from Phosphoproteomics Reveals the Role of Constitutive KAP1 Phosphorylation by B-cell Receptor Signaling in Chronic Lymphocytic Leukemia

Application of B-cell receptor (BCR) pathway inhibitor ibrutinib for chronic lymphocytic leukemia (CLL) is a major breakthrough, yet the downstream effects following inhibition of BCR signaling and during relapse await further clarification. By comparative phosphoproteomic profiling of B cells from patients with CLL and healthy donors, as well as CLL B cells collected at multiple time points during the course of ibrutinib treatment, we provided the landscape of dysregulated phosphoproteome in CLL and its dynamic alterations associated with ibrutinib treatment. Particularly, differential phosphorylation events associated with several signaling pathways, including BCR pathway, were enriched in patient CLL cells. A constitutively elevated phosphorylation level of KAP1 at serine 473 (S473) was found in the majority of CLL samples prior to treatment. Further verification showed that BCR activation promoted KAP1 S473 phosphorylation, whereas ibrutinib treatment abolished it. Depletion of KAP1 in primary CLL cells decelerated cell-cycle progression and ectopic expression of a KAP1 S473 phospho-mimicking mutant accelerated G2–M cell-cycle transition of CLL cells. Moreover, temporal phosphoproteomic profiles using a series of CLL cells isolated from one patient during the ibrutinib treatment revealed the dynamic changes of several molecules associated with BCR signaling in the ibrutinib responsive and recurrent stages. Implications: This phosphoproteomic analysis and functional validation illuminated the phosphorylation of KAP1 at S473 as an important downstream BCR signaling event and a potential indicator for the success of ibrutinib treatment in CLL.

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Supplementary Table S5. is a List of Intact glycopeptides of purified FAM198B identified by Orbitrap Velos MS analysis and Byonic processing.

Purpose: The comprehensive understanding of mechanisms involved in the tumor metastasis is urgently needed for discovering novel metastasis-related genes for developing effective diagnoses and treatments for lung cancer.Experimental Design: FAM198B was identified from an isogenic lung cancer metastasis cell model by microarray analysis. To investigate the clinical relevance of FAM198B, the FAM198B expression of 95 Taiwan lung adenocarcinoma patients was analyzed by quantitative real-time PCR and correlated to patients' survivals. The impact of FAM198B on cell invasion, metastasis, and tumor growth was examined by in vitro cellular assays and in vivo mouse models. In addition, the N-glycosylation–defective FAM198B mutants generated by site-directed mutagenesis were used to study protein stability and subcellular localization of FAM198B. Finally, the microarray and pathway analyses were used to elucidate the underlying mechanisms of FAM198B-mediated tumor suppression.Results: We found that the high expression of FAM198B was associated with favorable survival in Taiwan lung adenocarcinoma patients and in a lung cancer public database. Enforced expression of FAM198B inhibited cell invasion, migration, mobility, proliferation, and anchorage-independent growth, and FAM198B silencing exhibited opposite activities in vitro. FAM198B also attenuated tumor growth and metastasis in vivo. We further identified MMP-1 as a critical downstream target of FAM198B. The FAM198B-mediated MMP-1 downregulation was via inhibition of the phosphorylation of ERK. Interestingly deglycosylation nearly eliminated the metastasis suppression activity of FAM198B due to a decrease of protein stability.Conclusions: Our results implicate FAM198B as a potential tumor suppressor and to be a prognostic marker in lung adenocarcinoma. Clin Cancer Res; 24(4); 916–26. ©2017 AACR.

Supplementary Methods. Description of additional methods and procedures used in the study.

Supplementary Figures S1-S11. The co-expression profile among ALDH activity, memGrp78 and Glut3 in SAS Sphere cells and cisplatin-resistant (cisPtR) SAS cells was examined by FACS (S1); Single-cell suspension from parental SAS or sphere SAS cells was stained with CellROX Deep Red reagent and DCF (S2); Enrichment of the drug-resistant population after in vitro drug treatment (S3); ROSLow cells were sorted using DCFDA staining from SAS sphere cells (S4); Differentially expressed genes of reactive oxygen species scavenging in Parental cells and Sphere cells under 2, 3, 5, or 9 weeks of cultivation with defined serum-free selection medium were collected and analyzed (S5); Treatment of sphere cells with catalase inhibitor 3AT reduced catalase activity (S6); Combinatorial treatment of ROS scavenger inhibitor and cisplatin induces apoptosis cell death (S7); Knockdown of CAT or SOD2 gene expression diminished spheres-forming capability, stemness marker expression of HN-CICs (S8); Overexpression of CAT in HNCICs promotes stemness properties (S9); Overexpression of CAT in HNSCC under defined serum-free selection medium promotes stemness properties (S10); Cell viability of drugs treated hematopoietic stem cells (S11).

Supplementary Table S1. Antibodies and reagents used.

Alterations of protein glycosylation are closely related with pathophysiological regulation. Due to the structural macro- and microheterogeneity, low stoichiometry, and low ionization efficiency of glycopeptides, high-performance tools to enrich glycopeptides, especially the negatively charged and labile sialoglycopeptides, are essential to enhance the identification of the underexplored glycoproteome. Here, we present the first implementation of zwitterionic hydrophilic interaction chromatography with the exposed choline group (ZIC-cHILIC) in StageTip for simultaneous enrichment and fractionation of intact glycopeptides. In a model study using lung cancer cells, early elution by a high percentage of acetonitrile prominently prefilters nonglycopeptides, facilitating high enrichment specificity for glycopeptides (92-96%) and sialoglycopeptides (77-89%) in the subsequent hydrophilic fractions. The stepwise elution shows a high glycopeptide fractionation efficiency by a 80% in triplicate analysis) as well as superior coverage of 4.6- to 12.0-fold and 2.1- to 35.6-fold more glycopeptides and sialoglycopeptides compared to conventional TiO2 and ZIC-HILIC, respectively. To the best of our knowledge, the result with 2742 sialoglycopeptides among 7367 unique glycopeptides and 166 glycans from 2434 N-glycosites of 1118 glycoproteins (Byonic score > 100) provides one of the deepest glycoproteomic profiles in single-cell type. Without the immunoprecipitation step, the large-scale glycoproteomic atlas also reveals site-specific glycosylation of many druggable receptor proteins, such as EGFR, MET, ERBB2, ERBB3, AXL, and IGF1R. The demonstrated high enrichment specificity and identification depth show that stepwise ZIC-cHILIC is an efficient method to explore the under-represented sialoglycoproteome.

Achieving sufficient coverage of regulatory phosphorylation sites by mass spectrometry (MS)-based phosphoproteomics for signaling pathway reconstitution is challenging when analyzing tiny sample amounts. We present a novel hybrid data-independent acquisition (DIA) strategy (hybrid-DIA) that combines targeted and discovery proteomics through an Application Programming Interface (API) to dynamically intercalate DIA scans with accurate triggering of multiplexed tandem MS scans of predefined (phospho)peptide targets. By spiking-in heavy stable isotope labeled phosphopeptide standards covering seven major signaling pathways, we benchmarked hybrid-DIA against state-of-the-art targeted MS methods (i.e. SureQuant) using EGF-stimulated HeLa cells and found the quantitative accuracy and sensitivity to be comparable while hybrid-DIA also profiled the global phosphoproteome. To demonstrate the robustness, sensitivity and potential of hybrid-DIA, we profiled chemotherapeutic agents in single colon carcinoma multicellular spheroids and evaluated the difference of cancer cells in 2D vs 3D culture. Altogether, we showed that hybrid-DIA is the way-to-go method in highly sensitive phospho-proteomics experiments.

In 2021, the Asia-Oceania Human Proteome Organization (AOHUPO) initiated a new endeavor named the AOHUPO Online Education Series with the aim to promote scientific education and collaboration, exchange of ideas and culture among the young scientists in the AO region. Following the warm participation, the AOHUPO organized the second series in 2022, with the theme "The Renaissance of Clinical Proteomics: Biomarkers, Imaging and Therapeutics". This time, the second AOHUPO Online Education Series was hosted by the UKM Medical Molecular Biology Institute (UMBI) affiliated to the National University of Malaysia (UKM) in Kuala Lumpur, Malaysia on three consecutive Fridays (11th, 18th and 25th of March). More than 300 participants coming from 29 countries/regions registered for this 3-days event. This event provided an amalgamation of six prominent speakers and all participants whose interests lay mainly in applying MS-based and non-MS-based proteomics for clinical investigation.

Kinases are responsible for regulating cellular and physiological processes, and abnormal kinase activity is associated with various diseases. Therefore, kinases are being used as biomarkers for disease and developing methods for their sensing is highly important. Usually more than one kinase is involved in phosphorylating a target protein. However, kinase detection methods usually detect the activity of only one specific kinase. Here we describe an electrochemical kinase sensing tool for the selective detection of two kinases using the same target peptide. We demonstrate the sensing of kinases ERK2 and PKCδ. This is based on a single sensing element, a peptide that contains two distinct phosphorylation sites of these two kinases. Reversibility experiments with alkaline phosphatase and reaction with the electrochemically active ferrocene-labeled ATP showed that the mechanism of sensing is by detecting the enzymatic phosphorylation. Our approach can be further utilized to develop devices for the detection of multiple kinases and can be expanded to other types of enzymes involved in disease.

T315I mutation of Bcr-Abl in chronic myeloid leukemia (CML) leads to therapeutic resistance. It is known that Bcr-Abl transformation causes ROS-induced DNA damage, which can be exploited for anti-nucleotide treatment. We developed a small compound, JMF4073, which inhibits pyrimidylate kinases. Intriguingly, treatment of JMF4073 selectively eliminated WT-, but not T315I-, Bcr-Abl-transformed cells. We found that the higher level of cellular glutathione(GSH) in T315I-Bcr-Abl cells confers replication fork progression and sustains dTTP pool, leading to JMF4073 insusceptibility. Blocking mitochondrial pyruvate carrier (MPC) by UK-5099 reduced GSH and induced DNA replication stress in T315I-Bcr-Abl cells, thus increasing JMF4073 sensitivity in vitro and in vivo. We also observed the increases in glutamine flux to GSH production in T315I-Bcr-Abl cells, and reductive carboxylation of glutamate metabolism, together with mitochondrial dysfunction. The proteomic assay revealed the deficiencies in respiration complex I, II, and IV proteins in T315I-Bcr-Abl cells. Most interestingly, calcineurin inhibition remarkably elevated mitochondrial oxidative stress to cause cytotoxicity specifically in T315I-Bcr-Abl cells. Our results demonstrate the metabolic shift by T315I mutation of Bcr-Abl CML and provide the therapeutic options by co-targeting mitochondria and pyrimidylate kinases. Citation Format: Chang-Yu Huang, Yin-Hsuan Chung, Sheng-Yang Wu, Hsin-Yang Wang, Chih-Yu Lin, Tsung-Jung Yang, Jim-Ming Fang, Yu-Ju Chen, Chun-Mei Hu, Zee-Fen Chang. Co-targeting mitochondria and nucleotide metabolism in T315I-BCR-ABL myeloid leukemia for therapy [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 1602.

Phosphoproteomics can provide insights into cellular signaling dynamics. To achieve deep and robust quantitative phosphoproteomics profiling for minute amounts of sample, we here develop a global phosphoproteomics strategy based on data-independent acquisition (DIA) mass spectrometry and hybrid spectral libraries derived from data-dependent acquisition (DDA) and DIA data. Benchmarking the method using 166 synthetic phosphopeptides shows high sensitivity (, Phosphoproteomics can provide systematic insights into disease-associated cell signaling changes. Here, the authors present a sensitive workflow integrating library-based and direct data-independent acquisition approaches, and a hybrid spectral library resource for in-depth phosphoproteomic profiling.

The data-independent acquisition mass spectrometry (DIA-MS) has rapidly evolved as a powerful alternative for highly reproducible proteome profiling with a unique strength of generating permanent digital maps for retrospective analysis of biological systems. Recent advancements in data analysis software tools for the complex DIA-MS/MS spectra coupled to fast MS scanning speed and high mass accuracy have greatly expanded the sensitivity and coverage of DIA-based proteomics profiling. Here, we review the evolution of the DIA-MS techniques, from earlier proof-of-principle of parallel fragmentation of all-ions or ions in selected m/z range, the sequential window acquisition of all theoretical mass spectra (SWATH-MS) to latest innovations, recent development in computation algorithms for data informatics, and auxiliary tools and advanced instrumentation to enhance the performance of DIA-MS. We further summarize recent applications of DIA-MS and experimentally-derived as well as in silico spectra library resources for large-scale profiling to facilitate biomarker discovery and drug development in human diseases with emphasis on the proteomic profiling coverage. Toward next-generation DIA-MS for clinical proteomics, we outline the challenges in processing multi-dimensional DIA data set and large-scale clinical proteomics, and continuing need in higher profiling coverage and sensitivity.

Tripeptidyl peptidase II (TPPII or TPP2) degrades N-terminal tripeptides from proteins and peptides. Studies in both humans and mice have shown that TPPII deficiency is linked to cellular immune-senescence, lifespan regulation and the aging process. However, the mechanism of how TPPII participates in these processes is less clear. In this study, we established a chemical probe-based assay and found that although the mRNA and protein levels of TPPII were not altered during senescence, its enzymatic activity was reduced in senescent human fibroblasts. We also showed that elevation of the levels of the serine protease inhibitor serpinB2 reduced TPPII activity in senescent cells. Moreover, suppression of TPPII led to elevation in the amount of lysosomal contents as in well as TPPI (TPP1) and β-galactosidase activities, suggesting that lysosome biogenesis is induced to compensate for the reduction of TPPII activity in senescent cells. Together, this study discloses a critical role of the serpinB2-TPPII signaling pathway in proteostasis during senescence. Since serpinB2 levels can be increased by a variety of cellular stresses, reduction of TPPII activity through activation of serpinB2 might represent a common pathway for cells to respond to different stress conditions. This article has an associated First Person interview with the first author of the paper.

Meleagrin B is a terpene-alkaloid hybrid natural product that contains both the conidiogenone and meleagrin scaffold. Their derivatives show diverse biological activities. We characterized the biosynthesis of (-)-conidiogenone B (1), which involves a diterpene synthase and a P450 monooxygenase. In addition, an α,β-hydrolase (Con-ABH) was shown to catalyze an aza-Michael addition between 1 and imidazole to give 3S-imidazolyl conidiogenone B (6). Compound 6 was more potent than 1 against Staphylococcus aureus strains.