Proteomic profiling across breast cancer cell lines and models.

We performed quantitative proteomics on 60 human-derived breast cancer cell line models to a depth of ~13,000 proteins. The resulting high-throughput datasets were assessed for quality and reproducibility. We used the datasets to identify and characterize the subtypes of breast cancer and showed that they conform to known transcriptional subtypes, revealing that molecular subtypes are preserved even in under-sampled protein feature sets. All datasets are freely available as public resources on the LINCS portal. We anticipate that these datasets, either in isolation or in combination with complimentary measurements such as genomics, transcriptomics and phosphoproteomics, can be mined for the purpose of predicting drug response, informing cell line specific context in models of signalling pathways, and identifying markers of sensitivity or resistance to therapeutics.

Catalytic Degraders Effectively Address Kinase Site Mutations in EML4-ALK Oncogenic Fusions.

Heterobifunctional degraders, known as proteolysis targeting chimeras (PROTACs), theoretically possess a catalytic mode-of-action, yet few studies have either confirmed or exploited this potential advantage of event-driven pharmacology. Degraders of oncogenic EML4-ALK fusions were developed by conjugating ALK inhibitors to cereblon ligands. Simultaneous optimization of pharmacology and compound properties using ternary complex modeling and physicochemical considerations yielded multiple catalytic degraders that were more resilient to clinically relevant ATP-binding site mutations than kinase inhibitor drugs. Our strategy culminated in the design of the orally bioavailable derivative CPD-1224 that avoided hemolysis (a feature of detergent-like PROTACs), degraded the otherwise recalcitrant mutant L1196M/G1202R in vivo, and commensurately slowed tumor growth, while the third generation ALK inhibitor drug lorlatinib had no effect. These results validate our original therapeutic hypothesis by exemplifying opportunities for catalytic degraders to proactively address binding site resistant mutations in cancer.

Time-series transcriptomics and proteomics reveal alternative modes to decode p53 oscillations.

The cell stress-responsive transcription factor p53 influences the expression of its target genes and subsequent cellular responses based in part on its dynamics (changes in level over time). The mechanisms decoding p53 dynamics into subsequent target mRNA and protein dynamics remain unclear. We systematically quantified p53 target mRNA and protein expression over time under two p53 dynamical regimes, oscillatory and rising, using RNA-sequencing and TMT mass spectrometry. Oscillatory dynamics allowed for a greater variety of dynamical patterns for both mRNAs and proteins. Mathematical modeling of empirical data revealed three distinct mechanisms that decode p53 dynamics. Specific combinations of these mechanisms at the transcriptional and post-transcriptional levels enabled exclusive induction of proteins under particular dynamics. In addition, rising induction of p53 led to higher induction of proteins regardless of their functional class, including proteins promoting arrest of proliferation, the primary cellular outcome under rising p53. Our results highlight the diverse mechanisms cells employ to distinguish complex transcription factor dynamics to regulate gene expression.

Discovery and resistance mechanism of a selective CDK12 degrader.

Cyclin-dependent kinase 12 (CDK12) is an emerging therapeutic target due to its role in regulating transcription of DNA-damage response (DDR) genes. However, development of selective small molecules targeting CDK12 has been challenging due to the high degree of homology between kinase domains of CDK12 and other transcriptional CDKs, most notably CDK13. In the present study, we report the rational design and characterization of a CDK12-specific degrader, BSJ-4-116. BSJ-4-116 selectively degraded CDK12 as assessed through quantitative proteomics. Selective degradation of CDK12 resulted in premature cleavage and poly(adenylation) of DDR genes. Moreover, BSJ-4-116 exhibited potent antiproliferative effects, alone and in combination with the poly(ADP-ribose) polymerase inhibitor olaparib, as well as when used as a single agent against cell lines resistant to covalent CDK12 inhibitors. Two point mutations in CDK12 were identified that confer resistance to BSJ-4-116, demonstrating a potential mechanism that tumor cells can use to evade bivalent degrader molecules.

Targeting immunosuppressive macrophages overcomes PARP inhibitor resistance in BRCA1-associated triple-negative breast cancer.

Despite objective responses to PARP inhibition and improvements in progression-free survival compared to standard chemotherapy in patients with BRCA-associated triple-negative breast cancer (TNBC), benefits are transitory. Using high dimensional single-cell profiling of human TNBC, here we demonstrate that macrophages are the predominant infiltrating immune cell type in BRCA-associated TNBC. Through multi-omics profiling we show that PARP inhibitors enhance both anti- and pro-tumor features of macrophages through glucose and lipid metabolic reprogramming driven by the sterol regulatory element-binding protein 1 (SREBP-1) pathway. Combined PARP inhibitor therapy with CSF-1R blocking antibodies significantly enhanced innate and adaptive anti-tumor immunity and extends survival in BRCA-deficient tumors in vivo and is mediated by CD8+ T-cells. Collectively, our results uncover macrophage-mediated immune suppression as a liability of PARP inhibitor treatment and demonstrate combined PARP inhibition and macrophage targeting therapy induces a durable reprogramming of the tumor microenvironment, thus constituting a promising therapeutic strategy for TNBC.

Impact of Zika virus on the human type I interferon osteoimmune response.

BACKGROUND: The developing field of osteoimmunology supports importance of an interferon (IFN) response pathway in osteoblasts. Clarifying osteoblast-IFN interactions is important because IFN is used as salvage anti-tumor therapy but systemic toxicity is high with variable clinical results. In addition, osteoblast response to systemic bursts and disruptions of IFN pathways induced by viral infection may influence bone remodeling. ZIKA virus (ZIKV) infection impacts bone development in humans and IFN response in vitro. Consistently, initial evidence of permissivity to ZIKV has been reported in human osteoblasts. HYPOTHESIS: Osteoblast-like Saos-2 cells are permissive to ZIKV and responsive to IFN. METHODS: Multiple approaches were used to assess whether Saos-2 cells are permissive to ZIKV infection and exhibit IFN-mediated ZIKV suppression. Proteomic methods were used to evaluate impact of ZIKV and IFN on Saos-2 cells. RESULTS: Evidence is presented confirming Saos-2 cells are permissive to ZIKV and support IFN-mediated suppression of ZIKV. ZIKV and IFN differentially impact the Saos-2 proteome, exemplified by HELZ2 protein which is upregulated by IFN but non responsive to ZIKV. Both ZIKV and IFN suppress proteins associated with microcephaly/pseudo-TORCH syndrome (BI1, KI20A and UBP18), and ZIKV induces potential entry factor PLVAP. CONCLUSIONS: Transient ZIKV infection influences osteoimmune state, and IFN and ZIKV activate distinct proteomes in Saos-2 cells, which could inform therapeutic, engineered, disruptions.

The Activation State of CD4 T Cells Alters Cellular Peptidase Activities, HIV Antigen Processing, and MHC Class I Presentation in a Sequence-Dependent Manner.

CD4 T cell activation is critical to the initiation of adaptive immunity. CD4 T cells are also the main targets of HIV infection, and their activation status contributes to the maintenance and outcome of infection. Although the role of activation in the differentiation and proliferation of CD4 T cells is well studied, its impact on the processing and MHC class I (MHC-I) presentation of epitopes and immune recognition by CD8 T cells are not investigated. In this study, we show that the expression and hydrolytic activities of cellular peptidases are increased upon TCR-dependent and MHC-peptide activation of primary CD4 T cells from healthy or HIV-infected persons. Changes in peptidase activities altered the degradation patterns of HIV Ags analyzed by mass spectrometry, modifying the amount of MHC-I epitopes produced, the antigenicity of the degradation products, and the coverage of Ags by degradation peptides presentable by MHC-I. The computational analysis of 2237 degradation peptides generated during the degradation of various HIV-antigenic fragments in CD4 T cells identified cleavage sites that were predictably enhanced, reduced, or unchanged upon cellular activation. Epitope processing and presentation by CD4 T cells may be modulated by the activation state of cells in a sequence-dependent manner. Accordingly, cellular activation modified endogenous Ag processing and presentation and killing of HIV-infected CD4 T cells by CD8 T cells in a way that mirrored differences in in vitro epitope processing. The clearance of HIV-infected cells may rely on different immune responses according to activation state during HIV infection.

Discovery of Covalent CDK14 Inhibitors with Pan-TAIRE Family Specificity.

Cyclin-dependent kinase 14 (CDK14) and other TAIRE family kinases (CDKs 15-18) are proteins that lack functional annotation but are frequent off-targets of clinical kinase inhibitors. In this study we develop and characterize FMF-04-159-2, a tool compound that specifically targets CDK14 covalently and possesses a TAIRE kinase-biased selectivity profile. This tool compound and its reversible analog were used to characterize the cellular consequences of covalent CDK14 inhibition, including an unbiased investigation using phospho-proteomics. To reduce confounding off-target activity, washout conditions were used to deconvolute CDK14-specific effects. This investigation suggested that CDK14 plays a supporting role in cell-cycle regulation, particularly mitotic progression, and identified putative CDK14 substrates. Together, these results represent an important step forward in understanding the cellular consequences of inhibiting CDK14 kinase activity.

Leveraging Compound Promiscuity to Identify Targetable Cysteines within the Kinome.

Covalent kinase inhibitors, which typically target cysteine residues, represent an important class of clinically relevant compounds. Approximately 215 kinases are known to have potentially targetable cysteines distributed across 18 spatially distinct locations proximal to the ATP-binding pocket. However, only 40 kinases have been covalently targeted, with certain cysteine sites being the primary focus. To address this disparity, we have developed a strategy that combines the use of a multi-targeted acrylamide-modified inhibitor, SM1-71, with a suite of complementary chemoproteomic and cellular approaches to identify additional targetable cysteines. Using this single multi-targeted compound, we successfully identified 23 kinases that are amenable to covalent inhibition including MKNK2, MAP2K1/2/3/4/6/7, GAK, AAK1, BMP2K, MAP3K7, MAPKAPK5, GSK3A/B, MAPK1/3, SRC, YES1, FGFR1, ZAK (MLTK), MAP3K1, LIMK1, and RSK2. The identification of nine of these kinases previously not targeted by a covalent inhibitor increases the number of targetable kinases and highlights opportunities for covalent kinase inhibitor development.

A dynamic view of the proteomic landscape during differentiation of ReNcell VM cells, an immortalized human neural progenitor line.

The immortalized human ReNcell VM cell line represents a reproducible and easy-to-propagate cell culture system for studying the differentiation of neural progenitors. To better characterize the starting line and its subsequent differentiation, we assessed protein and phospho-protein levels and cell morphology over a 15-day period during which ReNcell progenitors differentiated into neurons, astrocytes and oligodendrocytes. Five of the resulting datasets measured protein levels or states of phosphorylation based on tandem-mass-tag (TMT) mass spectrometry and four datasets characterized cellular phenotypes using high-content microscopy. Proteomic analysis revealed reproducible changes in pathways responsible for cytoskeletal rearrangement, cell phase transitions, neuronal migration, glial differentiation, neurotrophic signalling and extracellular matrix regulation. Proteomic and imaging data revealed accelerated differentiation in cells treated with the poly-selective CDK and GSK3 inhibitor kenpaullone or the HMG-CoA reductase inhibitor mevastatin, both of which have previously been reported to promote neural differentiation. These data provide in-depth information on the ReNcell progenitor state and on neural differentiation in the presence and absence of drugs, setting the stage for functional studies.

MS3-IDQ: Utilizing MS3 Spectra beyond Quantification Yields Increased Coverage of the Phosphoproteome in Isobaric Tag Experiments.

Protein phosphorylation is critically important for many cellular processes, including progression through the cell cycle, cellular metabolism, and differentiation. Isobaric labeling, for example, tandem mass tags (TMT), in phosphoproteomics workflows enables both relative and absolute quantitation of these phosphorylation events. Traditional TMT workflows identify peptides using fragment ions at the MS2 level and quantify reporter ions at the MS3 level. However, in addition to the TMT reporter ions, MS3 spectra also include fragment ions that can be used to identify peptides. Here we describe using MS3 spectra for both phosphopeptide identification and quantification, a process that we term MS3-IDQ. To maximize quantified phosphopeptides, we optimize several instrument parameters, including the modality of mass analyzer (i.e., ion trap or Orbitrap), MS2 automatic gain control (AGC), and MS3 normalized collision energy (NCE), to achieve the best balance of identified and quantified peptides. Our optimized MS3-IDQ method included the following parameters for the MS3 scan: NCE = 37.5 and AGC target = 1.5 × 105, and scan range = 100-2000. Data from the MS3 scan were complementary to those of the MS2 scan, and the combination of these scans can increase phosphoproteome coverage by >50%, thereby yielding a greater number of quantified and accurately localized phosphopeptides.

Analysis of Major Histocompatibility Complex-Bound HIV Peptides Identified from Various Cell Types Reveals Common Nested Peptides and Novel T Cell Responses.

UNLABELLED: Despite the critical role of epitope presentation for immune recognition, we still lack a comprehensive definition of HIV peptides presented by HIV-infected cells. Here we identified 107 major histocompatibility complex (MHC)-bound HIV peptides directly from the surface of live HIV-transfected 293T cells, HIV-infected B cells, and primary CD4 T cells expressing a variety of HLAs. The majority of peptides were 8 to 12 amino acids (aa) long and mostly derived from Gag and Pol. The analysis of the total MHC-peptidome and of HLA-A02-bound peptides identified new noncanonical HIV peptides of up to 16 aa that could not be predicted by HLA anchor scanning and revealed an heterogeneous surface peptidome. Nested sets of surface HIV peptides included optimal and extended HIV epitopes and peptides partly overlapping or distinct from known epitopes, revealing new immune responses in HIV-infected persons. Surprisingly, in all three cell types, a majority of Gag peptides derived from p15 rather than from the most immunogenic p24. The cytosolic degradation of peptide precursors in corresponding cells confirmed the generation of identified surface-nested peptides. Cytosolic degradation revealed peptides commonly produced in all cell types and displayed by various HLAs, peptides commonly produced in all cell types and selectively displayed by specific HLAs, and peptides produced in only one cell type. Importantly, we identified areas of proteins leading to common presentations of noncanonical peptides by several cell types with distinct HLAs. These peptides may benefit the design of immunogens, focusing T cell responses on relevant markers of HIV infection in the context of HLA diversity. IMPORTANCE: The recognition of HIV-infected cells by immune T cells relies on the presentation of HIV-derived peptides by diverse HLA molecules at the surface of cells. The landscape of HIV peptides displayed by HIV-infected cells is not well defined. Considering the diversity of HLA molecules in the human population, it is critical for vaccine design to identify HIV peptides that may be displayed despite the HLA diversity. We identified 107 HIV peptides directly from the surface of three cell types infected with HIV. They corresponded to nested sets of HIV peptides of canonical and novel noncanonical lengths not predictable by the presence of HLA anchors. Importantly, we identified areas of HIV proteins leading to presentation of noncanonical peptides by several cell types with distinct HLAs. Including such peptides in vaccine immunogen may help to focus immune responses on common markers of HIV infection in the context of HLA diversity.

HIV Protease Inhibitor-Induced Cathepsin Modulation Alters Antigen Processing and Cross-Presentation.

Immune recognition by T cells relies on the presentation of pathogen-derived peptides by infected cells, but the persistence of chronic infections calls for new approaches to modulate immune recognition. Ag cross-presentation, the process by which pathogen Ags are internalized, degraded, and presented by MHC class I, is crucial to prime CD8 T cell responses. The original degradation of Ags is performed by pH-dependent endolysosomal cathepsins. In this article, we show that HIV protease inhibitors (PIs) prescribed to HIV-infected persons variably modulate cathepsin activities in human APCs, dendritic cells and macrophages, and CD4 T cells, three cell subsets infected by HIV. Two HIV PIs acted in two complementary ways on cathepsin hydrolytic activities: directly on cathepsins and indirectly on their regulators by inhibiting Akt kinase activities, reducing NADPH oxidase 2 activation, and lowering phagolysosomal reactive oxygen species production and pH, which led to enhanced cathepsin activities. HIV PIs modified endolysosomal degradation and epitope production of proteins from HIV and other pathogens in a sequence-dependent manner. They altered cross-presentation of Ags by dendritic cells to epitope-specific T cells and T cell-mediated killing. HIV PI-induced modulation of Ag processing partly changed the MHC self-peptidome displayed by primary human cells. This first identification, to our knowledge, of prescription drugs modifying the regulation of cathepsin activities and the MHC-peptidome may provide an alternate therapeutic approach to modulate immune recognition in immune disease beyond HIV.

In vitro evaluation of digestive and endolysosomal enzymes to cleave CML-modified Ara h 1 peptides.

Ara h 1 is a major peanut allergen. Processing-induced modifications may modulate the allergenic potency of Ara h 1. Carboxymethyl lysine (CML) modifications are a commonly described nonenzymatic modification on food proteins. In the current study, we tested the ability of digestive and endolysosomal proteases to cleave CML-modified and unmodified Ara h 1 peptides. Mass spectrometric analyses of the digested peptides demonstrate that carboxymethylation of lysine residues renders these peptides refractory to trypsin digestion. We did not detect observable differences in the simulated gastric fluid or endolysosomal digestion between the parental and CML-modified peptides. One of the tested peptides contains a lysine residue previously shown to be CML modified laying in a previously mapped linear IgE epitope, but we did not observe a difference in IgE binding between the modified and parental peptides. Our findings suggest a molecular mechanism for the increased resistance of peanut allergens modified by thermal processing, such as Ara h 1, to digestion in intestinal fluid after heating and could help explain how food processing-induced modifications may lead to more potent food allergens by acting to protect intact IgE epitopes from digestion by proteases targeting lysine residues.

Variable processing and cross-presentation of HIV by dendritic cells and macrophages shapes CTL immunodominance and immune escape.

Dendritic cells (DCs) and macrophages (Møs) internalize and process exogenous HIV-derived antigens for cross-presentation by MHC-I to cytotoxic CD8⁺ T cells (CTL). However, how degradation patterns of HIV antigens in the cross-presentation pathways affect immunodominance and immune escape is poorly defined. Here, we studied the processing and cross-presentation of dominant and subdominant HIV-1 Gag-derived epitopes and HLA-restricted mutants by monocyte-derived DCs and Møs. The cross-presentation of HIV proteins by both DCs and Møs led to higher CTL responses specific for immunodominant epitopes. The low CTL responses to subdominant epitopes were increased by pretreatment of target cells with peptidase inhibitors, suggestive of higher intracellular degradation of the corresponding peptides. Using DC and Mø cell extracts as a source of cytosolic, endosomal or lysosomal proteases to degrade long HIV peptides, we identified by mass spectrometry cell-specific and compartment-specific degradation patterns, which favored the production of peptides containing immunodominant epitopes in all compartments. The intracellular stability of optimal HIV-1 epitopes prior to loading onto MHC was highly variable and sequence-dependent in all compartments, and followed CTL hierarchy with immunodominant epitopes presenting higher stability rates. Common HLA-associated mutations in a dominant epitope appearing during acute HIV infection modified the degradation patterns of long HIV peptides, reduced intracellular stability and epitope production in cross-presentation-competent cell compartments, showing that impaired epitope production in the cross-presentation pathway contributes to immune escape. These findings highlight the contribution of degradation patterns in the cross-presentation pathway to HIV immunodominance and provide the first demonstration of immune escape affecting epitope cross-presentation.