Tellurophene-Tagged Carfilzomib Enables Single-Cell Mass Cytometric Mapping of Proteasome Activity.

Effective targeting of primitive AML CD34+ cells by the second-generation proteasome inhibitor carfilzomib.

Metastasis is the major cause of cancer-related deaths due to insufficient therapeutic effects of conventional treatment. Individual tumor cells exhibit heterogeneous features and growth potentials within the same tumor. This intra-tumor heterogeneity influences the ability of the tumor to form metastasis. The underlying mechanisms of why some tumor cells can give rise to metastases, whereas others cannot are remarkably poorly understood. Tumor cells acquire complex properties in order to invade into surrounding tissue, intravasate into the circulation, survive the journey to the distant site, where they extravasate into the new tissue and give rise to a secondary tumor. During this multistep process, tumor cells adapt their phenotype to various microenvironments that are distinct from their original site and are shaped by different immune and stromal cell populations. Metastatic tumor cells are heterogeneous and different from the primary tumor cells and may depend on the microenvironment at the distant site. Our study aims to better understand how the tumor heterogeneity influences the tumor-immune cell axis and how, in turn, immune cells support tumor cell plasticity and metastasis formation. We established patient-derived xenograft models of breast cancer with different metastatic potential and preserved tumor heterogeneity. We analyzed the gene expression of individual tumor and metastatic cells and their associated immune cell signature using a novel multiplexing approach for high-throughput single-cell RNA sequencing (MULTI-seq). In our study, we characterize the heterogeneity of different myeloid cells and their involvement in metastatic progression. Our data show that primary tumors from different individuals share a similar immune cell signature. However, metastasis-associated immune cells show distinct expression patterns related to the metastatic potential of the primary tumor and metastatic progression. In addition, prior to metastatic seeding, we identify an altered immune cell signature in the tissue distant from the primary breast tumor. Additionally, our data reveal that the plasticity of tumor cells is beneficial for the formation of metastasis. Tumors with higher levels of plastic tumor cells that undergo epithelial-mesenchymal-transition (EMT), have a greater ability to form metastasis. Our data indicate that subsets of immune cells of the myeloid lineage could promote this tumor plasticity and are enriched in tumors with higher metastatic potential. Thus, these innate immune cells provide a potential target for immune cell therapy, which may inhibit tumor cell plasticity and thereby the development of metastasis. Citation Format: Juliane Winkler, Weilun Tan, Christopher McGinnis, Zev Gartner, Spyros Darmanis, Zena Werb. Alterations in immune cell signatures during breast cancer metastasis at single cell resolution [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 351.

Background Understanding the genetic heterogeneity of circulating immune and tumors cells at single-cell resolution will enable one to decipher the interplay between cancer and immune cells, and ultimately lead to better design of immunotherapy assays. Marker-free methods for isolation of cells are attractive because they provide an opportunity to analyze a larger set of these cells that may otherwise be missed due to variable or no expression of protein (marker) markers. Integration of the Biolidics ClearCell® FX System and the Fluidigm® Polaris™ system enabled us not only to develop a marker-free workflow to isolate immune and circulating tumor cells (CTCs) but also to seamlessly prepare amplified cDNA from immune and CTCs for full-length mRNA-seq analysis. Method and Results The ClearCell FX System processes blood samples and isolates CTCs in a marker-free manner. During the CTC isolation process the immune cells are separated from CTCs on the microfluidic device. To differentiate larger blood cells from CTCs, we stained the enriched cells with Alexa Fluor® 647-conjugated CD45 and CD31 to identify leukocytes and endothelial cells, respectively. Calcein AM (live cell marker) and CellTracker™ Orange (universal cell marker) were added to identify live cells. Different enrichment strategies on Polaris allowed us to either specifically select for CTCs (live+, universal cell marker+, CD45-, CD31-) or immune cells (live+, universal cell marker+, CD45+). The enriched cells were then lysed, mRNA were reverse-transcribed, and cDNA were preamplified on a Polaris integrated fluidic circuit (IFC). Sequencing libraries were generated (off-IFC) using the Nextera® XT DNA library prep protocol and sequenced on Illumina® MiSeq™ systems. We successfully profiled full-length mRNA of 81 CTCs from stage III and IV subjects. Based on hormone receptor status, these six subjects were categorized as three types: (1) ER-/PR-/HER2- (triple-negative breast cancer; n=1), (2) ER+/PR+/HER2- (n=3), and (3) ER+/PR+/HER2- (n=2). For immune cell comparative analysis, we included CD45+ (FACS-sorted) from healthy donor. Unsupervised hierarchal clustering of gene expression data showed clustering by subject with a unique set of genes expressed by triple-negative breast cancer only. Heterogeneity was noted in immune cells from subjects at baseline (pretreatment), under treatment with drugs and a healthy subject without cancer. Using TraCeR computational method, we successfully reconstructed full-length, paired T cell receptor (TCR) sequences from CD45+ single-cell RNA sequence data from breast cancer subjects. Conclusion We present the feasibility of integrating two microfluidics platforms to isolate circulating immune and tumor cells for transcriptome and functional study. Our data suggests that the heterogeneity of immune and cancer cells can be elucidated from single-cell mRNA sequencing data and full-length, paired T cell receptor sequences can be reconstructed from immune cells of breast cancer subjects. Citation Format: Naveen Ramalingam, Yifang Lee, Lukasz Szpankowski, Anne Leyrat, Brian Fowler, Ninez Delos Angeles, Chad Sanada, Andrew Wu, Yoon Sim Yap, Jay West, Ali Asgar Bhagat, Kyle Hukari, Mark Lynch, David King. Full-Length mRNA transcriptome analysis of matched circulating tumor and immune cells from breast cancer subjects [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr LB-326.

The accumulation of the intermediate filament protein, glial fibrillary acidic protein (GFAP), in astrocytes of Alexander disease (AxD) impairs proteasome function in astrocytes. We have explored the molecular mechanism that underlies the proteasome inhibition. We find that both assembled and unassembled wild type (wt) and R239C mutant GFAP protein interacts with the 20 S proteasome complex and that the R239C AxD mutation does not interfere with this interaction. However, the R239C GFAP accumulates to higher levels and forms more protein aggregates than wt protein. These aggregates bind components of the ubiquitin-proteasome system and, thus, may deplete the cytosolic stores of these proteins. We also find that the R239C GFAP has a greater inhibitory effect on proteasome system than wt GFAP. Using a ubiquitin-independent degradation assay in vitro, we observed that the proteasome cannot efficiently degrade unassembled R239C GFAP, and the interaction of R239C GFAP with proteasomes actually inhibits proteasomal protease activity. The small heat shock protein, alphaB-crystallin, which accumulates massively in AxD astrocytes, reverses the inhibitory effects of R239C GFAP on proteasome activity and promotes degradation of the mutant GFAP, apparently by shifting the size of the mutant protein from larger oligomers to smaller oligomers and monomers. These observations suggest that oligomeric forms of GFAP are particularly effective at inhibiting proteasome activity.

Quantitation of protein abundance is a vital component in the proteomic analysis of biological systems, which can be achieved by differential stable isotopic labeling. To analyze tissue-derived samples, the isotopic labeling can be performed using chemical labeling of the peptides post-digestion. Standard chemical labeling procedures often require many manual sample handling steps, reducing the accuracy of measurements. Here, we describe a fully automated, online (in nanoLC columns), labeling procedure, which allows protein quantitation using differential isotopic dimethyl labeling of peptide N termini and lysine residues. We show that the method allows reliable quantitation over a wide dynamic range and can be used to quantify differential protein abundances in lysates and, more targeted, differences in composition between purified protein complexes. We apply the method to determine the differences in composition between bovine liver and spleen 20 S core proteasome complexes. We find that although all catalytically active immunoproteasome subunits were up-regulated in spleen (compared with liver), only one of the normal catalytic subunits was down-regulated, suggesting that the tissue-specific immunoproteasome assembly is more diverse than previously assumed.

PKA rapidly enhances proteasome assembly and activity in in vivo canine hearts

Huntington's disease (HD) is one of eight established triplet repeat neurodegenerative disorders, which are collectively caused by the genetic expansion of polyglutamine repeats. While the mechanism(s) by which polyglutamine expansion causes neurodegeneration in each of these disorders is being intensely investigated, the underlying cause of polyglutamine toxicity has not been fully elucidated. A number of studies have focused on the potential role of protein aggregation and disruption of the proteasome proteolytic pathway in polyglutamine-mediated neurodegeneration. However, at present it is not clear whether polyglutamine-mediated protein aggregation is sufficient to induce cell death, nor has it been clearly determined whether proteasome inhibition precedes, coincides, or occurs as the result of the formation of polyglutamine-associated protein aggregation. To address these important components of polyglutamine toxicity, in the present study we utilized neural SH-SY5Y cells stably transfected with polyglutamine-green fluorescent protein constructs to examine the effects of polyglutamine expansion on protein aggregation, proteasome activity, and neural cell survival. Data from the present study demonstrate that polyglutamine expansion does not dramatically impair proteasome activity or elevate protein aggregate formation under basal conditions, but does significantly impair the ability of the proteasome to respond to stress, and increases stress-induced protein aggregation following stress, all in the absence of neural cell death.

Lithium affects several enzymatic activities, however, the molecular mechanisms of lithium actions are not fully understood. We previously showed that LiCl interacts synergistically with all-trans-retinoic acid to promote terminal differentiation of WEHI-3B D(+) cells, a phenomenon accompanied by the recovery of the retinoid-induced loss of retinoic acid receptor alpha protein pools. Here, we demonstrate the effects of LiCl on proteasome-dependent degradation of retinoic acid receptor alpha proteins. LiCl alone, or in combination with all-trans-retinoic acid, increased cellular levels of ubiquitinated retinoic acid receptor alpha and markedly reduced chymotryptic-like activity of WEHI-3B D(+) 20 S and 26 S proteasome enzymes. Neither KCl nor all-trans-retinoic acid affected enzyme activity, whereas NaCl produced a modest reduction at relatively high concentrations. In addition, LiCl inhibited 20 S proteasome chymotryptic-like activity from rabbits but had no effect on tryptic-like activity of the 26 S proteasome. This effect has significant consequences in stabilizing the retinoic acid receptor alpha protein levels that are necessary to promote continued differentiation of leukemia cells in response to all-trans-retinoic acid. In support of this concept, combination of proteasome inhibitors beta-clastolactacystin or benzyloxycarbonyl-Leu-Leu-Phe with all-trans-retinoic acid increased differentiation of WEHI-3B D(+) cells in a manner that was analogous to the combination of LiCl and all-trans-retinoic acid.

BackgroundParadigm shifting next-generation immuno-oncology therapeutics such as CAR-T cells and bispecific engagers are rapidly gaining interest as investigational therapies. However, the discovery and preclinical development of these therapies involve 2-D...

S4-3 - Proteasome activation as a novel anti-aging strategy

Single-Cell Analysis of the Classical Hodgkin Lymphoma Immune Environment Reveals a Clonally-Expanded CD8+ T Cell Population with a Cytotoxic Phenotype

Interactions between immune cells within the tumor immune microenvironment (TIME) dictate immune cell function and their ability to respond to immunotherapy. Here, we have developed and applied a targeted imaging mass cytometry (IMC) approach to spatially interrogate immune cell interactions at a sufficient scale to associate them with patient outcomes. IMC uses heavy metal-conjugated antibodies and time of flight mass-spectrometry to visualize multiplexed protein targets at single-cell resolution on formaldehyde fixed paraffin embedded tissues. Application of this method to a RCC patient cohort requires the development of a tailored approach. To identify clinically relevant RCC immune cell states, fresh surgical renal cell carcinoma (RCC) samples were procured at the University Health Network through the REnal cancer MicroEnvironment DiscoverY (REMEDY) project and have undergone single cell RNA sequencing (scRNA-seq) and single cell suspension mass cytometry (SMC). Immune cell populations identified through scRNA-seq informed the selection of 43 protein targets sufficient to capture the protein profile and spatial relationships of select cell populations. Heavy metal-conjugated antibodies for each of these protein targets were tested and developed into a novel multiplexed IMC panel to quantify immune cell populations in RCC and applied to the REMEDY cohort. An established analytic pipeline was applied to map clusters of IMC-defined immune cell populations, using both supervised and unsupervised methods of cell identification, and benchmarked to those identified by scRNA-seq and SMC. Going forward, this approach will be repeated on a larger cohort of over 500 RCC patients with known clinical outcomes to identify immune cell networks as signatures associated with disease progression. RCC is amongst the most immune infiltrated solid tumors having varied response to immune checkpoint inhibitors, highlighting the potential for immune cell interactions to effect patient overall response to immunotherapy. With this tailored application of IMC to RCC we aim to characterize cell-cell interactions within the TIME to elucidate mechanisms that enable these interactions to dictate cell function and clinical outcomes. Using this approach, we have characterized over 30 cell populations, including clinically relevant RCC immune cell states, and quantified their spatial relationships within the TIME. We have identified preliminary relationships of immune cell networks within and between patients and determined their association to clinical features. In this preliminary data we observed heterogeneity of immune cell populations between patients as well as intratumoral regional spatial heterogeneity, highlighting the importance of utilizing spatial technologies to characterize immune cell interactions more comprehensively. ​​In summary, we have developed scalable IMC measurements which capture spatially resolved immune cell networks at single-cell resolution within the RCC TIME that will enable the first association of immune cell networks to clinical outcomes. Citation Format: Jennifer Pfeil, Daniel Stueckmann, Shirley Hui, Lisa Martin, Sally Zhang, Maria Komisarenko, Keith Lawson, Gary Bader, Antonio Finelli, Hartland Jackson. Spatially resolved immune cell networks in renal cell carcinoma [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Tumor Immunology and Immunotherapy; 2023 Oct 1-4; Toronto, Ontario, Canada. Philadelphia (PA): AACR; Cancer Immunol Res 2023;11(12 Suppl):Abstract nr A040.

The accumulation of aggregated alpha-synuclein is thought to contribute to the pathogenesis of Parkinson's disease. Recent studies indicate that aggregated alpha-synuclein binds to S6', a component of the 19 S subunit in the 26 S proteasome and inhibits 26 S proteasomal degradation, both ubiquitin-independent and ubiquitin-dependent. The IC(50) of aggregated alpha-synuclein for inhibition of the 26 S ubiquitin-independent proteasomal activity is approximately 1 nm. alpha-Synuclein has two close homologues, termed beta-synuclein and gamma-synuclein. In the present study we compared the effects of the three synuclein homologues on proteasomal activity. The proteasome exists as a 26 S and a 20 S species, with the 26 S proteasome containing the 20 S core and 19 S cap. Monomeric alpha- and beta-synucleins inhibited the 20 S and 26 S proteasomal activities only weakly, but monomeric gamma-synuclein strongly inhibited ubiquitin-independent proteolysis. The IC(50) of monomeric gamma-synuclein for the 20 S proteolysis was 400 nm. In monomeric form, none of the three synuclein proteins inhibited 26 S ubiquitin-dependent proteasomal activity. Although beta-synuclein had no direct effect on proteasomal activity, co-incubating monomeric beta-synuclein with aggregated alpha-synuclein antagonized the inhibition of the 26 S ubiquitin-independent proteasome by aggregated alpha-synuclein when added before the aggregated alpha-synuclein. Co-incubating beta-synuclein with gamma-synuclein had no effect on the inhibition of the 20 S proteasome by monomeric gamma-synuclein. Immunoprecipitation and pull-down experiments suggested that antagonism by beta-synuclein resulted from binding to alpha-synuclein rather than binding to S6'. Pull-down experiments demonstrated that recombinant monomeric beta-synuclein does not interact with the proteasomal subunit S6', unlike alpha-synuclein, but beta-synuclein does bind alpha-synuclein and competes with S6' for binding to alpha-synuclein. Based on these data, we hypothesize that the alpha- and gamma-synucleins regulate proteasomal function and that beta-synuclein acts as a negative regulator of alpha-synuclein.

Dynamics of leukemic blast and immune cell populations in Acute Myeloid Leukemia

The 26S proteasome is a multi-catalytic protein complex responsible for the degradation of almost all short-lived and most long-lived proteins. Aberrations of the ubiquitin/proteasome system contribute to many human diseases, including cancer. Many aspects of its structural organization and regulation are still unknown. Among many other functions, the 26S proteasome as the major proteolytic machinery in the cell plays a central role in all phases of the cell cycle. At present, the activity of the 26S proteasome is considered static throughout the cell cycle with protein degradation only depending on their ubiquitination status. We hypothesized that regulation of the activity of the proteasome complex itself could be an additional layer of regulation of cell cycle progression. To test our hypothesis we synchronized two human cell lines, PC-3 prostate cancer cells and MDA-MB-231 breast cancer cells, by treatment with Mimosine and measured 26S proteasome activity in these cells in the presence of the drug and 3, 6, 12 and 24h after restarting the cell cycle. Cells cycle distribution was assessed by Propidium Iodide staining and analyzed by flow cytometry. In parallel, using a proteomics approach we investigated changes of the expression levels and posttranslational modifications of proteasome subunits. Therefore, intact 26S proteasomes from PC3 prostate cancer cell stably expressing a 19S subunit, Rpn11, tagged with 6xHis-TEV-biotin-6xHis, were pulled down from synchronized and untreated (normally cycling) cells. Proteasome subunits and proteasome interacting proteins were separated by 2-dimensional gel electrophoresis (2DE). Differences between synchronized and un-treated cells were analyzed by specific staining for total protein and posttranslational modifications. Differentially expressed or post-translationally modified spots were identified by mass spectrometry. Using this approach, we observed that all three activities of the proteasome were regulated in a similar fashion with down regulation of 26S proteasome activity in cells arrested in the G1-phase of the cell cycle. Treatment of isolated proteasome with Mimosine did not affect its function, excluding that the drug could serve as proteasome inhibitor. After restarting the cell cycle, all three activities sharply increased with maximum activities in cells in the G2/M phase of the cell cycle, which exceeded the 26S proteasome activity of non-synchronized cells about 1.5fold. 2DE and mass spectrometry revealed that proteasome subunit expression was not affected. However, cell cycle synchronization caused specific changes in the phosphorylation pattern of 20S alpha proteasome subunits. In conclusion, we demonstrated that protein degradation and accumulation throughout the cell cycle is not only controlled at the level of proteins involved in cell cycle progression but also by fine-tuning of the activity of the 26S proteasome. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 1063.