EstroGene2.0: A multi-omic database of response to estrogens, ER-modulators, and resistance to endocrine therapies in breast cancer.

Endocrine therapies targeting the estrogen receptor (ER/ESR1) are the cornerstone to treat ER-positive breast cancers patients, but resistance often limits their effectiveness. Understanding the molecular mechanisms is thus key to optimize the existing drugs and to develop new ER-modulators. Notable progress has been made although the fragmented way data is reported has reduced their potential impact. Here, we introduce EstroGene2.0, an expanded database of its precursor 1.0 version. EstroGene2.0 focusses on response and resistance to endocrine therapies in breast cancer models. Incorporating multi-omic profiling of 361 experiments from 212 studies across 28 cell lines, a user-friendly browser offers comprehensive data visualization and metadata mining capabilities (https://estrogeneii.web.app/). Taking advantage of the harmonized data collection, our follow-up meta-analysis revealed substantial diversity in response to different classes of ER-modulators including SERMs, SERDs, SERCA and LDD/PROTAC. Notably, endocrine resistant models exhibit a spectrum of transcriptomic alterations including a contra-directional shift in ER and interferon signaling, which is recapitulated clinically. Furthermore, dissecting multiple ESR1-mutant cell models revealed the different clinical relevance of genome-edited versus ectopic overexpression model engineering and identified high-confidence mutant-ER targets, such as NPY1R. These examples demonstrate how EstroGene2.0 helps investigate breast cancer's response to endocrine therapies and explore resistance mechanisms.

Immunoproteasome function maintains oncogenic gene expression in KMT2A-complex driven leukemia.

Pharmacologic targeting of chromatin-associated protein complexes has shown significant responses in KMT2A-rearranged (KMT2A-r) acute myeloid leukemia (AML) but resistance frequently develops to single agents. This points to a need for therapeutic combinations that target multiple mechanisms. To enhance our understanding of functional dependencies in KMT2A-r AML, we have used a proteomic approach to identify the catalytic immunoproteasome subunit PSMB8 as a specific vulnerability. Genetic and pharmacologic inactivation of PSMB8 results in impaired proliferation of murine and human leukemic cells while normal hematopoietic cells remain unaffected. Disruption of immunoproteasome function drives an increase in transcription factor BASP1 which in turn represses KMT2A-fusion protein target genes. Pharmacologic targeting of PSMB8 improves efficacy of Menin-inhibitors, synergistically reduces leukemia in human xenografts and shows preserved activity against Menin-inhibitor resistance mutations. This identifies and validates a cell-intrinsic mechanism whereby selective disruption of proteostasis results in altered transcription factor abundance and repression of oncogene-specific transcriptional networks. These data demonstrate that the immunoproteasome is a relevant therapeutic target in AML and that targeting the immunoproteasome in combination with Menin-inhibition could be a novel approach for treatment of KMT2A-r AML.

PLCG1 is required for AML1-ETO leukemia stem cell self-renewal.

In an effort to identify novel drugs targeting fusion-oncogene-induced acute myeloid leukemia (AML), we performed high-resolution proteomic analysis. In AML1-ETO (AE)-driven AML, we uncovered a deregulation of phospholipase C (PLC) signaling. We identified PLCgamma 1 (PLCG1) as a specific target of the AE fusion protein that is induced after AE binding to intergenic regulatory DNA elements. Genetic inactivation of PLCG1 in murine and human AML inhibited AML1-ETO dependent self-renewal programs, leukemic proliferation, and leukemia maintenance in vivo. In contrast, PLCG1 was dispensable for normal hematopoietic stem and progenitor cell function. These findings are extended to and confirmed by pharmacologic perturbation of Ca++-signaling in AML1-ETO AML cells, indicating that the PLCG1 pathway poses an important therapeutic target for AML1-ETO+ leukemic stem cells.

Modulation of FLT3-ITD Localization and Targeting of Distinct Downstream Signaling Pathways as Potential Strategies to Overcome FLT3-Inhibitor Resistance.

OBJECTIVES: Internal tandem duplications (ITDs) of the Fms-like tyrosine kinase 3 (FLT3) represent the most frequent molecular aberrations in acute myeloid leukemia (AML) and are associated with an inferior prognosis. The pattern of downstream activation by this constitutively activated receptor tyrosine kinase is influenced by the localization of FLT3-ITD depending on its glycosylation status. Different pharmacological approaches can affect FLT3-ITD-driven oncogenic pathways by the modulation of FLT3-ITD localization. AIMS: The objective of this study was to investigate the effects of N-glycosylation inhibitors (tunicamycin or 2-deoxy-D-glucose) or the histone deacetylase inhibitor valproic acid (VPA) on FLT3-ITD localization and downstream activity. We sought to determine the potential differences between the distinct FLT3-ITD variants, particularly concerning their susceptibility towards combined treatment by addressing either N-glycosylation and the heat shock protein 90 (HSP90) by 17-AAG, or by targeting the PI3K/AKT/mTOR pathway by rapamycin after treatment with VPA. METHODS: Murine Ba/F3 leukemia cell lines were stably transfected with distinct FLT3-ITD variants resulting in IL3-independent growth. These Ba/F3 FLT3-ITD cell lines or FLT3-ITD-expressing human MOLM13 cells were exposed to tunicamycin, 2-deoxy-D-glucose or VPA, and 17-AAG or rapamycin, and characterized in terms of downstream signaling by immunoblotting. FLT3 surface expression, apoptosis, and metabolic activity were analyzed by flow cytometry or an MTS assay. Proteome analysis by liquid chromatography-tandem mass spectrometry was performed to assess differential protein expression. RESULTS: The susceptibility of FLT3-ITD-expressing cells to 17-AAG after pre-treatment with tunicamycin or 2-deoxy-D-glucose was demonstrated. Importantly, in Ba/F3 cells that were stably expressing distinct FLT3-ITD variants that were located either in the juxtamembrane domain (JMD) or in the tyrosine kinase 1 domain (TKD1), response to the sequential treatments with tunicamycin and 17-AAG varied between individual FLT3-ITD motifs without dependence on the localization of the ITD. In all of the FLT3-ITD cell lines that were investigated, incubation with tunicamycin was accompanied by intracellular retention of FLT3-ITD due to the inhibition of glycosylation. In contrast, treatment of Ba/F3-FLT3-ITD cells with VPA was associated with a significant increase of FLT3-ITD surface expression depending on FLT3 protein synthesis. The allocation of FLT3 to different cellular compartments that was induced by tunicamycin, 2-deoxy-D-glucose, or VPA resulted in the activation of distinct downstream signaling pathways. Whole proteome analyses of Ba/F3 FLT3-ITD cells revealed up-regulation of the relevant chaperone proteins (e.g., calreticulin, calnexin, HSP90beta1) that are directly involved in the stabilization of FLT3-ITD or in its retention in the ER compartment. CONCLUSION: The allocation of FLT3-ITD to different cellular compartments and targeting distinct downstream signaling pathways by combined treatment with N-glycosylation and HSP90 inhibitors or VPA and rapamycin might represent new therapeutic strategies to overcome resistance towards tyrosine kinase inhibitors in FLT3-ITD-positive AML. The treatment approaches addressing N-glycosylation of FLT3-ITD appear to depend on patient-specific FLT3-ITD sequences, potentially affecting the efficacy of such pharmacological strategies.

E3 ubiquitin ligase HECTD2 mediates melanoma progression and immune evasion.

The ubiquitin-proteasome system maintains protein homoeostasis, underpins the cell cycle, and is dysregulated in cancer. However, the role of individual E3 ubiquitin ligases, which mediate the final step in ubiquitin-mediated proteolysis, remains incompletely understood. Identified through screening for cancer-specific endogenous retroviral transcripts, we show that the little-studied E3 ubiquitin ligase HECTD2 exerts dominant control of tumour progression in melanoma. HECTD2 cell autonomously drives the proliferation of human and murine melanoma cells by accelerating the cell cycle. HECTD2 additionally regulates cancer cell production of immune mediators, initiating multiple immune suppressive pathways, which include the cyclooxygenase 2 (COX2) pathway. Accordingly, higher HECTD2 expression is associated with weaker anti-tumour immunity and unfavourable outcome of PD-1 blockade in human melanoma and counteracts immunity against a model tumour antigen in murine melanoma. This central, multifaceted role of HECTD2 in cancer cell-autonomous proliferation and in immune evasion may provide a single target for a multipronged therapy of melanoma.

Cooperative treatment effectiveness of ATR and HSP90 inhibition in Ewing's sarcoma cells.

INTRODUCTION: Ewing's sarcoma is an aggressive childhood malignancy whose outcome has not substantially improved over the last two decades. In this study, combination treatments of the HSP90 inhibitor AUY922 with either the ATR inhibitor VE821 or the ATM inhibitor KU55933 were investigated for their effectiveness in Ewing's sarcoma cells. METHODS: Effects were determined in p53 wild-type and p53 null Ewing's sarcoma cell lines by flow cytometric analyses of cell death, mitochondrial depolarization and cell-cycle distribution as well as fluorescence and transmission electron microscopy. They were molecularly characterized by gene and protein expression profiling, and by quantitative whole proteome analysis. RESULTS: AUY922 alone induced DNA damage, apoptosis and ER stress, while reducing the abundance of DNA repair proteins. The combination of AUY922 with VE821 led to strong apoptosis induction independent of the cellular p53 status, yet based on different molecular mechanisms. p53 wild-type cells activated pro-apoptotic gene transcription and underwent mitochondria-mediated apoptosis, while p53 null cells accumulated higher levels of DNA damage, ER stress and autophagy, eventually leading to apoptosis. Impaired PI3K/AKT/mTOR signaling further contributed to the antineoplastic combination effects of AUY922 and VE821. In contrast, the combination of AUY922 with KU55933 did not produce a cooperative effect. CONCLUSION: Our study reveals that HSP90 and ATR inhibitor combination treatment may be an effective therapeutic approach for Ewing's sarcoma irrespective of the p53 status.

Loss of metabolic plasticity underlies metformin toxicity in aged Caenorhabditis elegans.

Current clinical trials are testing the life-extending benefits of the diabetes drug metformin in healthy individuals without diabetes. However, the metabolic response of a non-diabetic cohort to metformin treatment has not been studied. Here, we show in C. elegans and human primary cells that metformin shortens lifespan when provided in late life, contrary to its positive effects in young organisms. We find that metformin exacerbates ageing-associated mitochondrial dysfunction, causing respiratory failure. Age-related failure to induce glycolysis and activate the dietary-restriction-like mobilization of lipid reserves in response to metformin result in lethal ATP exhaustion in metformin-treated aged worms and late-passage human cells, which can be rescued by ectopic stabilization of cellular ATP content. Metformin toxicity is alleviated in worms harbouring disruptions in insulin-receptor signalling, which show enhanced resilience to mitochondrial distortions at old age. Together, our data show that metformin induces deleterious changes of conserved metabolic pathways in late life, which could bring into question its benefits for older individuals without diabetes.

Spatially resolved analysis of FFPE tissue proteomes by quantitative mass spectrometry.

Bottom-up mass spectrometry-based proteomics relies on protein digestion and peptide purification. The application of such methods to broadly available clinical samples such as formalin-fixed and paraffin-embedded (FFPE) tissues requires reversal of chemical crosslinking and the removal of reagents that are incompatible with mass spectrometry. Here, we describe in detail a protocol that combines tissue disruption by ultrasonication, heat-induced antigen retrieval and two alternative methods for efficient detergent removal to enable quantitative proteomic analysis of limited amounts of FFPE material. To show the applicability of our approach, we used hepatocellular carcinoma (HCC) as a model system. By combining the described protocol with laser-capture microdissection, we were able to quantify the intra-tumor heterogeneity of a tumor specimen on the proteome level using a single slide with tissue of 10-µm thickness. We also demonstrate broader applicability to other tissues, including human gallbladder and heart. The procedure described in this protocol can be completed within 8 d.

Modulation of FLT3 signal transduction through cytoplasmic cysteine residues indicates the potential for redox regulation.

Oxidative modification of cysteine residues has been shown to regulate the activity of several protein-tyrosine kinases. We explored the possibility that Fms-like tyrosine kinase 3 (FLT3), a hematopoietic receptor-tyrosine kinase, is subject to this type of regulation. An underlying rationale was that the FLT3 gene is frequently mutated in Acute Myeloid Leukemia patients, and resulting oncogenic variants of FLT3 with 'internal tandem duplications (FLT3ITD)' drive production of reactive oxygen in leukemic cells. FLT3 was moderately activated by treatment of intact cells with hydrogen peroxide. Conversely, FLT3ITD signaling was attenuated by cell treatments with agents inhibiting formation of reactive oxygen species. FLT3 and FLT3ITD incorporated DCP-Bio1, a reagent specifically reacting with sulfenic acid residues. Mutation of FLT3ITD cysteines 695 and 790 reduced DCP-Bio1 incorporation, suggesting that these sites are subject to oxidative modification. Functional characterization of individual FLT3ITD cysteine-to-serine mutants of all 8 cytoplasmic cysteines revealed phenotypes in kinase activity, signal transduction and cell transformation. Replacement of cysteines 681, 694, 695, 807, 925, and 945 attenuated signaling and blocked FLT3ITD-mediated cell transformation, whereas mutation of cysteine 790 enhanced activity of both FLT3ITD and wild-type FLT3. These effects were not related to altered FLT3ITD dimerization, but likely caused by changed intramolecular interactions. The findings identify the functional relevance of all cytoplasmic FLT3ITD cysteines, and indicate the potential for redox regulation of this clinically important oncoprotein.

Author Correction: Metastatic-niche labelling reveals parenchymal cells with stem features.

An Amendment to this paper has been published and can be accessed via a link at the top of the paper.

Shedding light on the presymbiontic phase of C. arietinum.

A complex network of symbiotic events between plants and bacteria allows the biosphere to exploit the atmospheric reservoir of molecular nitrogen. In seeds, a series of presymbiotic steps are already identified during imbibition, while interactions between the host and its symbiont begin in the early stages of germination. In the present study, a detailed analysis of the substances' complex delivered by Cicer arietinum seeds during imbibition showed a relevant presence of proteins and amino acids, which, except for cysteine, occurred with the whole proteinogenic pool. The imbibing solution was found to provide essential probiotic properties able to sustain the growth of the specific chickpea symbiont Mesorhizobium ciceri. Moreover, the imbibing solution, behaving as a complete medium, was found to be critically important for the symbiont's attraction, a fact this that is strictly related to the presence of the amino acids glycine, serine, and threonine. Here, the presence of these amino acids is constantly supported by the presence of the enzymes serine hydroxymethyltransferase and formyltetrahydrofolate deformylase, which are both involved in their biosynthesis. The reported findings are discussed in the light of the pivotal role played by the imbibing solution in attracting and sustaining symbiosis between the host and its symbiont.

Metastatic-niche labelling reveals parenchymal cells with stem features.

Direct investigation of the early cellular changes induced by metastatic cells within the surrounding tissue remains a challenge. Here we present a system in which metastatic cancer cells release a cell-penetrating fluorescent protein, which is taken up by neighbouring cells and enables spatial identification of the local metastatic cellular environment. Using this system, tissue cells with low representation in the metastatic niche can be identified and characterized within the bulk tissue. To highlight its potential, we applied this strategy to study the cellular environment of metastatic breast cancer cells in the lung. We report the presence of cancer-associated parenchymal cells, which exhibit stem-cell-like features, expression of lung progenitor markers, multi-lineage differentiation potential and self-renewal activity. In ex vivo assays, lung epithelial cells acquire a cancer-associated parenchymal-cell-like phenotype when co-cultured with cancer cells and support their growth. These results highlight the potential of this method as a platform for new discoveries.

Preclinical assessment of MEK1/2 inhibitors for neurofibromatosis type 2-associated schwannomas reveals differences in efficacy and drug resistance development.

BACKGROUND: Neurofibromatosis type 2 (NF2) is a genetic tumor-predisposition disorder caused by NF2/merlin tumor suppressor gene inactivation. The hallmark of NF2 is formation of bilateral vestibular schwannomas (VS). Because merlin modulates activity of the Ras/Raf/mitogen-activated protein kinase kinase (MEK)/extracellular signal-regulated kinase (ERK) pathway, we investigated repurposing drugs targeting MEK1 and/or MEK2 as a treatment for NF2-associated schwannomas. METHODS: Mouse and human merlin-deficient Schwann cell lines (MD-MSC/HSC) were screened against 6 MEK1/2 inhibitors. Efficacious drugs were tested in orthotopic allograft and NF2 transgenic mouse models. Pathway and proteome analyses were conducted. Drug efficacy was examined in primary human VS cells with NF2 mutations and correlated with DNA methylation patterns. RESULTS: Trametinib, PD0325901, and cobimetinib were most effective in reducing MD-MSC/HSC viability. Each decreased phosphorylated pERK1/2 and cyclin D1, increased p27, and induced caspase-3 cleavage in MD-MSCs. Proteomic analysis confirmed cell cycle arrest and activation of pro-apoptotic pathways in trametinib-treated MD-MSCs. The 3 inhibitors slowed allograft growth; however, decreased pERK1/2, cyclin D1, and Ki-67 levels were observed only in PD0325901 and cobimetinib-treated grafts. Tumor burden and average tumor size were reduced in trametinib-treated NF2 transgenic mice; however, tumors did not exhibit reduced pERK1/2 levels. Trametinib and PD0325901 modestly reduced viability of several primary human VS cell cultures with NF2 mutations. DNA methylation analysis of PD0325901-resistant versus -susceptible VS identified genes that could contribute to drug resistance. CONCLUSION: MEK inhibitors exhibited differences in antitumor efficacy resistance in schwannoma models with possible emergence of trametinib resistance. The results support further investigation of MEK inhibitors in combination with other targeted drugs for NF2 schwannomas.

Cohesin-mediated NF-κB signaling limits hematopoietic stem cell self-renewal in aging and inflammation.

Organism aging is characterized by increased inflammation and decreased stem cell function, yet the relationship between these factors remains incompletely understood. This study shows that aged hematopoietic stem and progenitor cells (HSPCs) exhibit increased ground-stage NF-κB activity, which enhances their responsiveness to undergo differentiation and loss of self-renewal in response to inflammation. The study identifies Rad21/cohesin as a critical mediator of NF-κB signaling, which increases chromatin accessibility in the vicinity of NF-κB target genes in response to inflammation. Rad21 is required for normal differentiation, but limits self-renewal of hematopoietic stem cells (HSCs) during aging and inflammation in an NF-κB-dependent manner. HSCs from aged mice fail to down-regulate Rad21/cohesin and inflammation/differentiation signals in the resolution phase of inflammation. Inhibition of cohesin/NF-κB reverts hypersensitivity of aged HSPCs to inflammation-induced differentiation and myeloid-biased HSCs with disrupted/reduced expression of Rad21/cohesin are increasingly selected during aging. Together, Rad21/cohesin-mediated NF-κB signaling limits HSPC function during aging and selects for cohesin-deficient HSCs with myeloid-skewed differentiation.

Spatial Tissue Proteomics Quantifies Inter- and Intratumor Heterogeneity in Hepatocellular Carcinoma (HCC).

The interpatient variability of tumor proteomes has been investigated on a large scale but many tumors display also intratumoral heterogeneity regarding morphological and genetic features. It remains largely unknown to what extent the local proteome of tumors intrinsically differs. Here, we used hepatocellular carcinoma as a model system to quantify both inter- and intratumor heterogeneity across human patient specimens with spatial resolution. We defined proteomic features that distinguish neoplastic from the directly adjacent nonneoplastic tissue, such as decreased abundance of NADH dehydrogenase complex I. We then demonstrated the existence of intratumoral variations in protein abundance that re-occur across different patient samples, and affect clinically relevant proteins, even in the absence of obvious morphological differences or genetic alterations. Our work demonstrates the suitability and the benefits of using mass spectrometry-based proteomics to analyze diagnostic tumor specimens with spatial resolution. Data are available via ProteomeXchange with identifier PXD007052.

A new procedure for fast soft staining of BN-PAGEs on photosynthetic complexes.

We report a fast and sensitive procedure for blue native PAGE staining, in which the conventional staining step with CBB is avoided. After running, a short exposure to a mix of polar protic solvents (ethanol and acetic acid) leads to a fast and selective removal of the dye from the migration front and a specific binding to the protein bands, while the rest undergo a selective and complete background removal, leading to an intense contrast. This single-step staining-destaining technique is useful in protein samples that bind colored cofactors such as photosystems, which can be selectively discerned by their characteristic green color. After the staining of such samples, the green color persists, while the other unpigmented protein complexes and the molecular standard remain CBB stained, creating a useful reference system for the assignment of the bands. The advantages and chemical basis of this staining procedure are discussed.

An atomic model of HIV-1 capsid-SP1 reveals structures regulating assembly and maturation.

Immature HIV-1 assembles at and buds from the plasma membrane before proteolytic cleavage of the viral Gag polyprotein induces structural maturation. Maturation can be blocked by maturation inhibitors (MIs), thereby abolishing infectivity. The CA (capsid) and SP1 (spacer peptide 1) region of Gag is the key regulator of assembly and maturation and is the target of MIs. We applied optimized cryo-electron tomography and subtomogram averaging to resolve this region within assembled immature HIV-1 particles at 3.9 angstrom resolution and built an atomic model. The structure reveals a network of intra- and intermolecular interactions mediating immature HIV-1 assembly. The proteolytic cleavage site between CA and SP1 is inaccessible to protease. We suggest that MIs prevent CA-SP1 cleavage by stabilizing the structure, and MI resistance develops by destabilizing CA-SP1.

Isolation of Plant Photosystem II Complexes by Fractional Solubilization.

Photosystem II (PSII) occurs in different forms and supercomplexes in thylakoid membranes. Using a transplastomic strain of Nicotiana tabacum histidine tagged on the subunit PsbE, we have previously shown that a mild extraction protocol with ß-dodecylmaltoside enriches PSII characteristic of lamellae and grana margins. Here, we characterize residual granal PSII that is not extracted by this first solubilization step. Using affinity purification, we demonstrate that this PSII fraction consists of PSII-LHCII mega- and supercomplexes, PSII dimers, and PSII monomers, which were separated by gel filtration and functionally characterized. Our findings represent an alternative demonstration of different PSII populations in thylakoid membranes, and they make it possible to prepare PSII-LHCII supercomplexes in high yield.

New features of the cell wall of the radio-resistant bacterium Deinococcus radiodurans.

We have analyzed the cell wall of the radio-resistant bacterium Deinococcus radiodurans. Unexpectedly, the bacterial envelope appears to be organized in different complexes of high molecular weight. Each complex is composed of several proteins, most of which are coded by genes of unknown function and the majority are constituents of the inner/outer membrane system. One of the most abundant complexes is constituted by the gene DR_0774. This protein is a type of secretin which is a known subunit of the homo-oligomeric channel that represents the main bulk of the type IV piliation family. Finally, a minor component of the pink envelope consists of several inner-membrane proteins. The implications of these findings are discussed.

Src kinase modulates the apoptotic p53 pathway by altering HIPK2 localization.

Non-receptor tyrosine kinase Src is a master regulator of cell proliferation. Hyperactive Src is a potent oncogene and a driver of cellular transformation and carcinogenesis. Homeodomain-interacting protein kinase 2 (HIPK2) is a tumor suppressor mediating growth suppression and apoptosis upon genotoxic stress through phosphorylation of p53 at Ser46. Here we show that Src phosphorylates HIPK2 and changes its subcellular localization. Using mass spectrometry we identified 9 Src-mediated Tyr-phosphorylation sites within HIPK2, 5 of them positioned in the kinase domain. By means of a phosphorylation-specific antibody we confirm that Src mediates phosphorylation of HIPK2 at Tyr354. We demonstrate that ectopic expression of Src increases the half-life of HIPK2 by interfering with Siah-1-mediated HIPK2 degradation. Moreover, we find that hyperactive Src binds HIPK2 and redistributes HIPK2 from the cell nucleus to the cytoplasm, where both kinases partially colocalize. Accordingly, we find that hyperactive Src decreases chemotherapeutic drug-induced p53 Ser46 phosphorylation and apoptosis activation. Together, our results suggest that Src kinase suppresses the apoptotic p53 pathway by phosphorylating HIPK2 and relocalizing the kinase to the cytoplasm.