Enriched Plasma Membrane Research Articles

Abstract 1090: GPC2 directed CAR T cells are efficacious against fusion-positive and fusion-negative rhabdomyosarcomas

Abstract Background: Rhabdomyosarcoma (RMS) is an aggressive pediatric solid tumor with poor survival and new targeted therapies are needed. Aims: Using an integrative proteogenomic quantitative approach, we sought to identify candidate immunotherapeutic targets then validate top targets in RMS preclinical models. Methods: We first performed plasma membrane enrichment followed by mass spectrometry (MS) to define the surfaceome of 7 fusion-positive (FP) and 14 fusion-negative (FN) RMS patient-derived xenograft models. Surface proteins were scored by IMMUNOTAR, our custom pipeline which extracts and integrates quantitative features from diverse proteogenomic and annotation datasets to provide a score for each protein, using mean-average-precision for score optimization. Cell surface expression of IMMUNOTAR-prioritized proteins was validated using flow cytometry, focusing on targets with immunotherapies in development. CAR T cells targeting the lead protein were engineered and studied in RMS preclinical models. Results: A total of 847 and 882 high confidence cell surface proteins were detected in FP and FN RMS surfaceome, respectively, with 94% of proteins overlapping the phenotypes. IMMUNOTAR ranked anaplastic lymphoma kinase (ALK), a putative immunotherapeutic target in FP RMS, as the top FP target. Glypican 2 (GPC2), a differentially expressed cell surface proteoglycan identified in neuroblastoma and other neural-derived tumors, targetable with CAR T cells and antibody-drug conjugates [Bosse et al. Cancer Cell 2017], ranked as the top protein in FN and 5th protein in FP RMS. Overall, GPC2 was abundantly expressed in 16 of 21 RMS models profiled. Flow cytometry revealed expression of GPC2 comparable to neuroblastoma in 5 of 8 RMS cells lines. Additionally, both FP and FN GPC2+ RMS cell lines (FN: RH36, SMS-CTR; FP: RH41, RH30) activated GPC2 CAR T cells with either a CD28 or a 4-1BB costimulatory domain, inducing release of IFN-gamma and IL-2 upon co-incubation across different E:T ratios as quantified by ELISA. Further, GPC2 CAR T cells induced potent RMS cell cytotoxicity compared to control CD19 CAR T cells with near 100% specific cytotoxicity at 1:1 E:T ratio. In a pilot study, GPC2 CAR T cells with 4-1BB costimulatory domains induced significant tumor regression (p=0.03) and prolonged progression-free survival (p=0.01) compared to CD19 CAR T cells in an established murine RH41 flank xenograft model. Conclusion: In summary, we probed the RMS surfaceome using MS and identified candidate proteins amenable for immunotherapeutic targeting using our IMMUNOTAR algorithm. We validated GPC2 as a candidate target in FP and FN RMS and are corroborating our findings in additional pre-clinical RMS models. Demonstration of GPC2 CAR efficacy in these models could lead to inclusion of RMS in our ongoing phase 1 clinical trial (NCT05650749) in a planned expansion phase. Citation Format: Rawan Shraim, Patrick M. Schuerch, Anna Maria Giudice, Karina L. Conkrite, Amber K. Weiner, Alexander B. Radaoui, Brian Mooney, Raushan T. Kurmasheva, Peter J. Houghton, Gregg B. Morin, Yael B. Mosse, Poul H. Sorensen, Benjamin A. Garcia, Ahmet Sacan, John M. Maris, Kristopher R. Bosse, Sharon J. Diskin. GPC2 directed CAR T cells are efficacious against fusion-positive and fusion-negative rhabdomyosarcomas [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 1090.

Actin-binding protein profilin1 is an important determinant of cellular phosphoinositide control

Membrane polyphosphoinositides (PPIs) are lipid-signaling molecules that undergo metabolic turnover and influence a diverse range of cellular functions. PPIs regulate the activity and/or spatial localization of a number of actin-binding proteins (ABPs) through direct interactions; however, it is much less clear whether ABPs could also be an integral part in regulating PPI signaling. In this study, we show that ABP profilin1 (Pfn1) is an important molecular determinant of the cellular content of PI(4,5)P2 (the most abundant PPI in cells). In growth factor (EGF) stimulation setting, Pfn1 depletion does not impact PI(4,5)P2 hydrolysis but enhances plasma membrane (PM) enrichment of PPIs that are produced downstream of activated PI3-kinase, including PI(3,4,5)P3 and PI(3,4)P2, the latter consistent with increased PM recruitment of SH2-containing inositol 5' phosphatase (SHIP2) (a key enzyme for PI(3,4)P2 biosynthesis). Although Pfn1 binds to PPIs invitro, our data suggest that Pfn1's affinity to PPIs and PM presence in actual cells, if at all, is negligible, suggesting that Pfn1 is unlikely to directly compete with SHIP2 for binding to PM PPIs. Additionally, we provide evidence for Pfn1's interaction with SHIP2 in cells and modulation of this interaction upon EGF stimulation, raising an alternative possibility of Pfn1 binding as a potential restrictive mechanism for PM recruitment of SHIP2. In conclusion, our findings challenge the dogma of Pfn1's binding to PM by PPI interaction, uncover a previously unrecognized role of Pfn1 in PI(4,5)P2 homeostasis and provide a new mechanistic avenue of how an ABP could potentially impact PI3K signaling byproducts in cells through lipid phosphatase control.

The EXO70 inhibitor Endosidin2 alters plasma membrane protein composition in Arabidopsis roots.

To sustain normal growth and allow rapid responses to environmental cues, plants alter the plasma membrane protein composition under different conditions presumably by regulation of delivery, stability, and internalization. Exocytosis is a conserved cellular process that delivers proteins and lipids to the plasma membrane or extracellular space in eukaryotes. The octameric exocyst complex contributes to exocytosis by tethering secretory vesicles to the correct site for membrane fusion; however, whether the exocyst complex acts universally for all secretory vesicle cargo or just for specialized subsets used during polarized growth and trafficking is currently unknown. In addition to its role in exocytosis, the exocyst complex is also known to participate in membrane recycling and autophagy. Using a previously identified small molecule inhibitor of the plant exocyst complex subunit EXO70A1, Endosidin2 (ES2), combined with a plasma membrane enrichment method and quantitative proteomic analysis, we examined the composition of plasma membrane proteins in the root of Arabidopsis seedlings, after inhibition of the ES2-targetted exocyst complex, and verified our findings by live imaging of GFP-tagged plasma membrane proteins in root epidermal cells. The abundance of 145 plasma membrane proteins was significantly reduced following short-term ES2 treatments and these likely represent candidate cargo proteins of exocyst-mediated trafficking. Gene Ontology analysis showed that these proteins play diverse functions in cell growth, cell wall biosynthesis, hormone signaling, stress response, membrane transport, and nutrient uptake. Additionally, we quantified the effect of ES2 on the spatial distribution of EXO70A1 with live-cell imaging. Our results indicate that the plant exocyst complex mediates constitutive dynamic transport of subsets of plasma membrane proteins during normal root growth.

Abstract A009: Proteogenomic prioritization of immunotherapeutic targets in rhabdomyosarcoma nominate MEGF10 for preclinical development

Abstract Background: Rhabdomyosarcoma (RMS) has a high unmet need in terms of precision therapy development as there are currently no approved immunotherapies or targeted therapies, and few in the developmental pipeline. Here we sought to identify cell surface oncoproteins as a target for novel RMS-directed immunotherapies. Methods: We first performed plasma membrane enrichment followed by mass spectrometry to define the cell surface landscape of 7 fusion(+) and 14 fusion(-) RMS patient-derived xenograft (PDX) models. The surfaceome data was filtered to only “high confidence” surface proteins by querying protein localization databases, Compartments (https://compartments.jensenlab.org/) and CIRFESS (https://gundrylab.shinyapps.io/cirfess/). We then developed a prioritization algorithm that uses a rank-product approach to score surface proteins. The input to the algorithm is a matrix that integrates multiple datasets to score the surface proteins based on their suitability to be an optimal immunotherapeutic target. In addition to the surfaceome data generated here, we also integrated matched RNA-sequencing data from eleven of the RMS PDX models, RNA-sequencing data from GTEx (n=15,253) and a recently developed normal tissue proteomics dataset (n=201) [Jiang. Cell. 2020], a list from Gene Ontology that included genes involved in muscle development pathways, and the gene dependency list for RMS in DepMap (https://depmap.org/portal/). Results: A total of 913 and 937 high confidence surface proteins were annotated from the mass spectrometry data for fusion(+) and fusion(−) samples, respectively. A dendrogram separated the surface protein profiles into two clusters based on fusion(+) and fusion(−) RMS subtypes, thus the algorithm was run separately on each subtype. Within the top 50% of prioritized targets, 88% and 86% of the targets overlapped and 12% and 14% were identified exclusively in fusion(+) and fusion(−) subtypes, respectively. ALK, a previously putative protein marker in fusion(+) RMS, scored in the top 10% of the fusion(+) targets based on the algorithm, and surprisingly we saw abundant ALK expression in 6/14 fusion(−) PDX samples. MEGF10, a novel target, was ranked as the top target for both fusion(+) and fusion(−) RMS. MEGF10 plays a role in cell adhesion, motility, and proliferation. It scored as a significant dependency in DepMap for RMS (p-value=0.0002). Based on RNA-sequencing and proteomics, MEGF10 shows no expression in most healthy tissues surveyed, with several orders of magnitude lower expression detected in RNASeq in muscle and brain tissue, but not in the proteomic datasets. Conclusion: Here, we defined the surfaceome of RMS, and found substantial overlap in surface proteins between fusion(+) and fusion(−) RMS subtypes. We validated previous observations that ALK is expressed in RMS, here verifying that the protein is expressed on the plasma membrane. MEGF10 appears to be a strong novel candidate target for RMS immunotherapies, and ongoing work to validate our proteogenomic findings will be reported. Citation Format: Rawan Shraim, Amber K. Weiner, Karina L. Conkrite, Alexander B. Radaoui, John M. Maris, Yael P. Mosse, Sharon J. Diskin, Ahmet Sacan, Benjamin A. Garcia, Peter J. Houghton, Raushan T. Kurmasheva, Poul Sorensen, Gregg B. Morin, Brian Mooney. Proteogenomic prioritization of immunotherapeutic targets in rhabdomyosarcoma nominate MEGF10 for preclinical development [abstract]. In: Proceedings of the AACR Special Conference: Sarcomas; 2022 May 9-12; Montreal, QC, Canada. Philadelphia (PA): AACR; Clin Cancer Res 2022;28(18_Suppl):Abstract nr A009.

Influence of sphingomyelin metabolism during epithelial-mesenchymal transition associated with aging in the renal papilla.

The renal collecting ducts (CD) are formed by a fully differentiated epithelium, and their tissue organization and function require the presence of mature cell adhesion structures. In certain circumstances, the cells can undergo de-differentiation by a process called epithelial-mesenchymal transition (EMT), in which the cells lose their epithelial phenotype and acquire the characteristics of the mesenchymal cells, which includes loss of cell-cell adhesion. We have previously shown that in renal papillary CD cells, cell adhesion structures are located in sphingomyelin (SM)-enriched plasma membrane microdomains and the inhibition of SM synthase 1 activity induced CD cells to undergo an EMT process. In the present study, we evaluated the influence of SM metabolism during the EMT of the cells that form the CD of the renal papilla during aging. To this end, primary cultures of renal papillary CD cells from young, middle-, and aged-rats were performed. By combining biochemical and immunofluorescence studies, we found experimental evidence that CD cells undergo an increase in spontaneous and reversible EMT during aging and that at least one of the reasons for this phenomenon is the decrease in SM content due to the combination of decreased SM synthase activity and an increase in SM degradation mediated by neutral sphingomyelinase. Age is a risk factor for many diseases, among which renal fibrosis is included. Our findings highlight the importance of sphingolipids and particularly SM as a modulator of the fate of CD cells and probably contribute to the development of treatments to avoid or reverse renal fibrosis during aging.

Circulating linoleic acid at the time of myocardial infarction and risk of primary ventricular fibrillation

Primary ventricular fibrillation (PVF) is a major driver of cardiac arrest in the acute phase of ST-segment elevation myocardial infarction (STEMI). Enrichment of cardiomyocyte plasma membranes with dietary polyunsaturated fatty acids (PUFA) reduces vulnerability to PVF experimentally, but clinical data are scarce. PUFA status in serum phospholipids is a valid surrogate biomarker of PUFA status in cardiomyocytes within a wide range of dietary PUFA. In this nested case–control study (n = 58 cases of STEMI-driven PVF, n = 116 control non-PVF STEMI patients matched for age, sex, smoking status, dyslipidemia, diabetes mellitus and hypertension) we determined fatty acids in serum phospholipids by gas-chromatography, and assessed differences between cases and controls, applying the Benjamini–Hochberg procedure on nominal P-values to control the false discovery rate (FDR). Significant differences between cases and controls were restricted to linoleic acid (LA), with PVF patients showing a lower level (nominal P = 0.002; FDR-corrected P = 0.027). In a conditional logistic regression model, each one standard deviation increase in the proportion of LA was related to a 42% lower prevalence of PVF (odds ratio = 0.58; 95% confidence interval, 0.37, 0.90; P = 0.02). The association lasted after the inclusion of confounders. Thus, regular consumption of LA-rich foods (nuts, oils from seeds) may protect against ischemia-driven malignant arrhythmias.

Plant "helper" immune receptors are Ca2+-permeable nonselective cation channels.

Plant nucleotide-binding leucine-rich repeat receptors (NLRs) regulate immunity and cell death. In Arabidopsis, a subfamily of "helper" NLRs is required by many "sensor" NLRs. Active NRG1.1 oligomerized, was enriched in plasma membrane puncta, and conferred cytoplasmic calcium ion (Ca2+) influx in plant and human cells. NRG1.1-dependent Ca2+ influx and cell death were sensitive to Ca2+ channel blockers and were suppressed by mutations affecting oligomerization or plasma membrane enrichment. Ca2+ influx and cell death mediated by NRG1.1 and ACTIVATED DISEASE RESISTANCE 1 (ADR1), another helper NLR, required conserved negatively charged N-terminal residues. Whole-cell voltage-clamp recordings demonstrated that Arabidopsis helper NLRs form Ca2+-permeable cation channels to directly regulate cytoplasmic Ca2+ levels and consequent cell death. Thus, helper NLRs transduce cell death signals directly.

Histone Deacetylase Activity and the Renin-Angiotensin-Aldosterone System: Key Elements in Cardiorenal Alterations Provoked by Chronic Malnutrition in Male Adult Rats.

Chronic malnutrition (M) affects >1 billion people worldwide. Epidemiological data point to long-term renal and cardiovascular outcomes (e.g. arterial hypertension, cardiorenal syndromes). The renin-angiotensin-aldosterone system (RAAS) has been implicated in the physiopathology of these disturbances, but M-induced alterations in RAAS-modulated renal Na+ handling and their cardiovascular repercussions are not known. Moreover, altered tissue-specific histone deacetylases (HDAC) results in arterial hypertension and the use of sodium Valproate (Val; a HDAC inhibitor) reduces blood pressure. However, there are no reports regarding the renal and cardiovascular effects of HDAC inhibition in M, or on the signaling pathways involved. The central aim of our study has been to investigate whether alterations in the HDAC/RAAS axis underpin alterations in active Na+ transport in the kidney and heart, and affects blood pressure. Male rats aged 28 days were given either a control (C) or a multideficient diet (Regional Basic Diet, RBD), which mimics alimentary habits from developing countries. Subgroups received Losartan (Los), a blocker of type 1 Angiotensin II receptors. When the rats reached 70 days, new subgroups received Val until they were 90 days of age. Homogenates and enriched plasma membrane fractions from renal cortex corticis and cardiomyocytes were obtained by differential centrifugation of the tissues. The activity of renal and cardiac deacetylases was assayed by measuring - after incubation with the membranes - the amount of deacetylated lysines in a substrate containing an acetylated lysine side chain. Protein kinases activities were measured following the incorporation of the γ-phosphoryl group of [γ-32P]ATP into Ser/Thr residues of histone type III-S. The activity of Na+-transporting ATPases (kidney and heart) was quantified by measuring the release of Pi from ATP that was sensitive to ouabain ((Na++K+)ATPase), or sensitive to furosemide (Na+-ATPase). Tail-cuff plethysmography was used to measure systolic blood pressure and heart rate. M provoked HDAC downregulation, which was reversed by Los and Val, either alone or in combination, with selective upregulation of protein kinases C and A (PKC, PKA) in renal cortex corticis, but not in left ventricle cardiomyocytes. The 2 kinases were strongly inhibited by Los and Val in both organs. Malnourished rats developed elevated systolic arterial pressure (SAP) and heart rate (HR) at 70 days of age; Los and Val restored the control SAP, but not HR. Functional and the above biochemical alterations were associated with the deregulation of renal and cardiac Na+-transporting ATPases. (Na++K+)ATPase activities were downregulated in M rats in both organs, and were further inhibited by the pharmacological treatments in the renal cortex corticis (C and M groups) and the left ventricle (only in C rats). No additional effect was found in cardiac (Na++K+)ATPase from M rats. Ouabain-resistant Na+-ATPase was upregulated in renal cortex corticis and downregulated in cardiomyocytes, returning to C values after administration of Los and Val. The HDAC/RAAS axis appears to be a key regulator of Na+-transporting ATPases in renal cortex corticis and cardiomyocytes via an appropriate balance of PKC and PKA activities. Modifications within the HDAC/RAAS axis provoked by chronic M - with repercussions in renal and cardiac Na+ transport - underpin alterations in bodily Na+ homeostasis that culminate with the onset of arterial hypertension and potential cardiorenal syndrome.

Multi-Omic Based Antigen Discovery for the Immunotherapy of Pediatric Acute T Cell Lymphoblastic Leukemia

Pediatric T-cell acute lymphoblastic leukemia (T-ALL) is a high-risk disease due to treatment related complications and poor prognosis of patients with relapsed disease. Immunotherapy with monoclonal antibodies (MAbs) and/or chimeric antigen receptor (CAR) T-cells for T-ALL is limited by identification of tumor specific target antigens. Differential expression is necessary to prevent on-target/off-tumor toxicities and fratricide of activated T-cells. Targeting multiple antigens can bypass immune escape and result in improved T-cell effector function, since antigen density correlates with T-cell activation. Here we designed a pipeline (Figure 1) to identify unique surface antigens expressed in T-ALL using proteomic and transcriptomic analyses followed by flow cytometry validation, and functional studies with CAR T cells targeting the identified antigens. We generated an Illumina total stranded RNAseq library from healthy donor myeloid and lymphoid cells of bone marrow, peripheral blood and cord blood (N= 116). We compared data to 265 St. Jude pediatric T-ALL samples and against 53 normal tissue expression data from the GTEx (Genotype-Tissue Expression) project. To analyze the T-cell surface proteome, we isolated plasma membrane fractions from 11 samples including healthy T-cells and T-ALL cell lines using a differential centrifugation-based method. The purity of the plasma membrane fraction was confirmed by western blot. Na+/K+ ATPase and GAPDH were used as controls for the plasma membrane and cytosolic fractions respectively. Following plasma membrane enrichment, the membrane proteins were applied for proteomic analysis using an advanced TMT-L/LC-MS/MS pipeline, and the acquired proteomic data were further processed via the JUMP software suite. 997 unique proteins were quantified from the membrane fractions. Integrated analysis the transcriptomic and proteomic datasets showed significant correlation and yielded a list of candidate genes, which were validated by flow cytometry on a panel of T-ALL cell lines (CCRF, RPMI8402, and MOLT3) and resting and activated T-cells from healthy donors. We identified GRP78 as one of the differentially expressed cell surface antigens and further confirmed its expression on additional T-ALL cell lines (KE37, PF382, PEER, CEMC7) and 3 PDX samples. Finally, we generated GRP78-CAR T cells and demonstrate that GRP78-CAR T cells recognize and kill GRP78+ T-ALL cells and have potent antitumor activity in xenograft and PDX models. We have established an unbiased pipeline to identify differentially expressed antigens on the cell surface of T-ALL blasts and created a healthy tissue RNAseq library. The results from our analyses are encouraging and interrogation of our pipeline has yielded differentially expressed immunotherapy targets for the treatment of relapsed refractory T-ALL. Our results highlight the importance of integrated surface proteomics and transcriptomics analysis. Figure 1: Outline of strategy for target selection: Figure Disclosures Hebbar: St. Jude: Patents & Royalties. Epperly:St. Jude: Patents & Royalties. Gottschalk:Inmatics and Tidal: Membership on an entity's Board of Directors or advisory committees; TESSA Therapeutics: Other: research collaboration; Patents and patent applications in the fields of T-cell & Gene therapy for cancer: Patents & Royalties; Merck and ViraCyte: Consultancy. Mullighan:AbbVie, Inc.: Research Funding; Illumina: Consultancy, Honoraria, Speakers Bureau; Pfizer: Honoraria, Research Funding, Speakers Bureau. Velasquez:Rally! Foundation: Membership on an entity's Board of Directors or advisory committees; St. Jude: Patents & Royalties.

Selective Enrichment Coupled with Proteomics to Identify S-Acylated Plasma Membrane Proteins in Arabidopsis.

Protein S-acylation, predominately in the form of palmitoylation, is a reversible lipid post-translational modification on cysteines that plays important roles in protein localization, trafficking, activity, and complex assembly. The functions and regulatory mechanisms of S-acylation have been extensively studied in mammals owing to remarkable development of high-resolution proteomics and the discovery of the S-acylation-related enzymes. However, the advancement of S-acylation studies in plants lags behind that in mammals, mainly due to the lack of knowledge about proteins responsible for this process, such as protein acyltransferases and their substrates. In this article, a set of systematic protocols to study global S-acylation in Arabidopsis seedlings is described. The procedures are presented in detail, including preparation of Arabidopsis seedlings, enrichment of plasma membrane (PM) proteins, ensuing enrichment of S-acylated proteins/peptides based on the acyl-biotin exchange method, and large-scale identification of S-acylated proteins/peptides via mass spectrometry. This approach enables researchers to study S-acylation of PM proteins in plants in a systematic and straightforward way. © 2020 Wiley Periodicals LLC. Basic Protocol 1: Preparation of Arabidopsis seedling materials Basic Protocol 2: Isolation and enrichment of plasma membrane proteins Support Protocol 1: Determination of protein concentration using BCA assay Basic Protocol 3: Enrichment of S-acylated proteins by acyl-biotin exchange method Support Protocol 2: Protein precipitation by methanol/chloroform method Basic Protocol 4: Trypsin digestion and proteomic analysis Alternate Protocol: Pre-resin digestion and peptide-level enrichment.

Critical roles of phosphoinositides and NF2 in Hippo pathway regulation.

The Hippo pathway is a master regulator of tissue homeostasis and organ size. NF2 is a well-established tumor suppressor, and loss of NF2 severely compromises Hippo pathway activity. However, the precise mechanism of how NF2 mediates upstream signals to regulate the Hippo pathway is not clear. Here we report that, in mammalian cells, NF2's lipid-binding ability is critical for its function in activating the Hippo pathway in response to osmotic stress. Mechanistically, osmotic stress induces PI(4,5)P2 plasma membrane enrichment by activating the PIP5K family, allowing for NF2 plasma membrane recruitment and subsequent downstream Hippo pathway activation. An NF2 mutant deficient in lipid binding is unable to activate the Hippo pathway in response to osmotic stress, as measured by LATS and YAP phosphorylation. Our findings identify the PIP5K family as novel regulators upstream of Hippo signaling, and uncover the importance of phosphoinositide dynamics, specifically PI(4,5)P2, in Hippo pathway regulation.

TM4SF5-mediated CD44v8-10 splicing variant promotes survival of type II alveolar epithelial cells during idiopathic pulmonary fibrosis

Reactive oxygen species (ROS) regulate cell fate, although signaling molecules that regulate ROS hormesis remain unclear. Here we show that transmembrane 4 L six family member 5 (TM4SF5) in lung epithelial cells induced the alternatively spliced CD44v8-10 variant via an inverse ZEB2/epithelial splicing regulatory proteins (ESRPs) linkage. TM4SF5 formed complexes with the cystine/glutamate antiporter system via TM4SF5- and CD44v8-10-dependent CD98hc plasma-membrane enrichment. Dynamic TM4SF5 binding to CD98hc required CD44v8-10 under ROS-generating inflammatory conditions. TM4SF5 and CD44v8-10 upregulated cystine/glutamate antiporter activity and intracellular glutathione levels, leading to ROS modulation for cell survival. Tm4sf5-null mice exhibited attenuated bleomycin-induced pulmonary fibrosis with lower CD44v8-10 and ESRPs levels than wild-type mice. Primary mouse alveolar epithelial cells (AECs) revealed type II AECs (AECII), but not type I, to adapt the TM4SF5-mediated characteristics, suggesting TM4SF5-mediated AECII survival following AECI injury during idiopathic pulmonary fibrosis (IPF). Thus, the TM4SF5-mediated CD44v8-10 splice variant could be targeted against IPF.

Abstract 2938: Transmembrane chloride intracellular channel 1 (tmCLIC1) correlates with metastatic potential of colorectal cancer cells

Abstract Colorectal cancer (CRC) is the third most common and lethal tumor worldwide. Although CRC can be efficiently eradicated when diagnosed in the early stage, no effective therapies are currently available for advanced metastatic disease. Therefore, targeting metastasis represents a critical challenge for the successful treatment of CRC. To achieve this purpose, our laboratory has found a promising candidate inthe transmembrane localized Chloride Intracellular Channel 1 (tmCLIC1) protein. Growing scientific evidence have suggested an involvement of tmCLIC1 in different human malignancies, including CRC, with an unknown mechanism. CLIC1 is a metamorphic protein able to shuttle between a soluble cytoplasmic and a membrane integrated form, the latter characterized by ionic channel function. In normal cells, it is mostly cytoplasmic and cooperates to the maintenance of several physiological processes. Otherwise, in cancer cells CLIC1 undergoes to chronic membrane insertion, where contributes to the abnormal cellular hyperproliferative rate.In the present investigation, using CRC human cell lines at different stage of tumor development, we demonstrate that tmCLIC1 functional expression correlates with CRC cells increasing aggressiveness, while it is almost absent in normal colon cells. We also show that CLIC1 enrichment in plasma membrane is involved in promotion of CRC metastasis. CLIC1 inhibition using different pharmacological treatments led to drastic reduction of cell proliferation, migration and invasiveness.tmCLIC1 function permits to discriminate between normal and malignant cells and its downregulation is able to arrest metastatic cells. This is not the case of other upregulated proteins not only involved in tumorigenesis but also in physiological cell functions. Since metastasis are considered the leading cause of treatment failure and tumor relapse, CLIC1 inhibition coupled to conventional therapies can represent a novel strategy to successfully treat CRC with reduced side effects. (This work is supported by a GRANT from AIRC to MM) Citation Format: Valentina Carlini, Francesca Cianci, Ivan Verduci, Michele Mazzanti. Transmembrane chloride intracellular channel 1 (tmCLIC1) correlates with metastatic potential of colorectal cancer cells [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 2938.

Lapatinib-induced annexin A6 upregulation as an adaptive response of triple-negative breast cancer cells to EGFR tyrosine kinase inhibitors.

The epidermal growth factor receptor (EGFR) is a major oncogene in triple-negative breast cancer (TNBC), but the use of EGFR-targeted tyrosine kinase inhibitors (TKI) and therapeutic monoclonal antibodies is associated with poor response and acquired resistance. Understanding the basis for the acquired resistance to these drugs and identifying biomarkers to monitor the ensuing resistance remain a major challenge. We previously showed that reduced expression of annexin A6 (AnxA6), a calcium-dependent membrane-binding tumor suppressor, not only promoted the internalization and degradation of activated EGFR but also sensitized TNBC cells to EGFR-TKIs. Here, we demonstrate that prolong (>3 days) treatment of AnxA6-low TNBC cells with lapatinib led to AnxA6 upregulation and accumulation of cholesterol in late endosomes. Basal extracellular signal-regulated kinase 1 and 2 (ERK1/2) activation was EGFR independent and significantly higher in lapatinib-resistant MDA-MB-468 (LAP-R) cells. These cells were more sensitive to cholesterol depletion than untreated control cells. Inhibition of lapatinib-induced upregulation of AnxA6 by RNA interference (A6sh) or withdrawal lapatinib from LAP-R cells not only reversed the accumulation of cholesterol in late endosomes but also led to enrichment of plasma membranes with cholesterol, restored EGFR-dependent activation of ERK1/2 and sensitized the cells to lapatinib. These data suggest that lapatinib-induced AnxA6 expression and accumulation of cholesterol in late endosomes constitute an adaptive mechanism for EGFR-expressing TNBC cells to overcome prolong treatment with EGFR-targeted TKIs and can be exploited as an option to inhibit and/or monitor the frequently observed acquired resistance to these drugs.

Intratumoral Heterogeneity: Tools to Understand and Exploit Clone Wars in AML

In this issue of Cancer Cell, de Boer etal. refine a set of acute myeloid leukemia (AML)-enriched plasma membrane markers that can be used to identify, prospectively isolate, and longitudinally track leukemic subclones within individual AML patients, correlating immunophenotypic profiles with specific mutational signatures, transcription, functional behavior, and therapeutic outcomes.

Isx9 Regulates Calbindin D28K Expression in Pancreatic β Cells and Promotes β Cell Survival and Function.

Pancreatic β-cell dysfunction and death contribute to the onset of diabetes, and novel strategies of β-cell function and survival under diabetogenic conditions need to be explored. We previously demonstrated that Isx9, a small molecule based on the isoxazole scaffold, drives neuroendocrine phenotypes by increasing the expression of genes required for β-cell function and improves glycemia in a model of β cell regeneration. We further investigated the role of Isx9 in β-cell survival. We find that Isx9 drives the expression of Calbindin-D28K (D28K), a key regulator of calcium homeostasis, and plays a cytoprotective role through its calcium buffering capacity in β cells. Isx9 increased the activity of the calcineurin (CN)/cytoplasmic nuclear factor of the activated T-cells (NFAT) transcription factor, a key regulator of D28K, and improved the recruitment of NFATc1, cAMP response element-binding protein (CREB), and p300 to the D28K promoter. We found that nutrient stimulation increased D28K plasma membrane enrichment and modulated calcium channel activity in order to regulate glucose-induced insulin secretion. Isx9-mediated expression of D28K protected β cells against chronic stress induced by serum withdrawal or chronic inflammation by reducing caspase 3 activity. Consequently, Isx9 improved human islet function after transplantation in NOD-SCID mice in a streptozotocin-induced diabetes model. In summary, Isx9 significantly regulates expression of genes relevant to β cell survival and function, and may be an attractive therapy to treat diabetes and improve islet function post-transplantation.

A bidirectional crosstalk between glioblastoma and brain endothelial cells potentiates the angiogenic and proliferative signaling of sphingosine-1-phosphate in the glioblastoma microenvironment

Glioblastoma is one of the most malignant, angiogenic, and incurable tumors in humans. The aberrant communication between glioblastoma cells and tumor microenvironment represents one of the major factors regulating glioblastoma malignancy and angiogenic properties. Emerging evidence implicates sphingosine-1-phosphate signaling in the pathobiology of glioblastoma and angiogenesis, but its role in glioblastoma-endothelial crosstalk remains largely unknown. In this study, we sought to determine whether the crosstalk between glioblastoma cells and brain endothelial cells regulates sphingosine-1-phosphate signaling in the tumor microenvironment. Using human glioblastoma and brain endothelial cell lines, as well as primary brain endothelial cells derived from human glioblastoma, we report that glioblastoma-co-culture promotes the expression, activity, and plasma membrane enrichment of sphingosine kinase 2 in brain endothelial cells, leading to increased cellular level of sphingosine-1-phosphate, and significant potentiation of its secretion. In turn, extracellular sphingosine-1-phosphate stimulates glioblastoma cell proliferation, and brain endothelial cells migration and angiogenesis. We also show that, after co-culture, glioblastoma cells exhibit enhanced expression of S1P1 and S1P3, the sphingosine-1-phosphate receptors that are of paramount importance for cell growth and invasivity. Collectively, our results envision glioblastoma-endothelial crosstalk as a multi-compartmental strategy to enforce pro-tumoral sphingosine-1-phosphate signaling in the glioblastoma microenvironment.

Mapping of Plasma Membrane Proteins Interacting With Arabidopsis thaliana Flotillin 2.

Arabidopsis flotillin 2 (At5g25260) belongs to the group of plant flotillins, which are not well characterized. In contrast, metazoan flotillins are well known as plasma membrane proteins associated with membrane microdomains that act as a signaling hub. The similarity of plant and metazoan flotillins, whose functions most likely consist of affecting other proteins via protein–protein interactions, determines the necessity of detecting their interacting partners in plants. Nevertheless, identifying the proteins that form complexes on the plasma membrane is a challenging task due to their low abundance and hydrophobic character. Here we present an approach for mapping Arabidopsis thaliana flotillin 2 plasma membrane interactors, based on the immunoaffinity purification of crosslinked and enriched plasma membrane proteins with mass spectrometry detection. Using this approach, 61 proteins were enriched in the AtFlot-GFP plasma membrane fraction, and 19 of them were proposed to be flotillin 2 interaction partners. Among our proposed partners of Flot2, proteins playing a role in the plant response to various biotic and abiotic stresses were detected. Additionally, the use of the split-ubiquitin yeast system helped us to confirm that plasma-membrane ATPase 1, early-responsive to dehydration stress protein 4, syntaxin-71, harpin-induced protein-like 3, hypersensitive-induced response protein 2 and two aquaporin isoforms interact with flotillin 2 directly. Based on the results of our study and the reported properties of Flot2 interactors, we propose that Flot2 complexes may be involved in plant–pathogen interactions, water transport and intracellular trafficking.

The inhibition of sphingomyelin synthase 1 activity induces collecting duct cells to lose their epithelial phenotype

Epithelial tissue requires that cells attach to each other and to the extracellular matrix by the assembly of adherens junctions (AJ) and focal adhesions (FA) respectively. We have previously shown that, in renal papillary collecting duct (CD) cells, both AJ and FA are located in sphingomyelin (SM)-enriched plasma membrane microdomains. In the present work, we investigated the involvement of SM metabolism in the preservation of the epithelial cell phenotype and tissue organization. To this end, primary cultures of renal papillary CD cells were performed. Cultured cells preserved the fully differentiated epithelial phenotype as reflected by the presence of primary cilia. Cells were then incubated for 24h with increasing concentrations of D609, a SM synthase (SMS) inhibitor. Knock-down experiments silencing SMS 1 and 2 were also performed. By combining biochemical and immunofluorescence studies, we found experimental evidences suggesting that, in CD cells, SMS 1 activity is essential for the preservation of cell-cell adhesion structures and therefore for the maintenance of CD tissue/tubular organization. The inhibition of SMS 1 activity induced CD cells to lose their epithelial phenotype and to undergo an epithelial-mesenchymal transition (EMT) process.

Isolation and Purity Assessment of Membranes from Norway Spruce.

Gaining membrane vesicles from different plant species and tissue types is crucial for membrane studies. Membrane vesicles can be used for further purification of individual membrane types, and, for example, in studies of membrane enzyme activities, transport assays, and in proteomic analysis. Membrane isolation from some species, such as conifers, has proved to be more difficult than that of angiosperm species. In this paper, we describe steps for isolating cellular membranes from developing xylem, phloem, and lignin-forming tissue-cultured cells of Norway spruce, followed by partial enrichment of plasma membranes by aqueous polymer two-phase partitioning and purity analyses. The methods used are partially similar to the ones used for mono- and dicotyledonous plants, but some steps require discreet optimization, probably due to a high content of phenolic compounds present in the tissues and cultured cells of Norway spruce.