Recent improvement in patient outcomes with the use of novel cellular immunotherapies for multiple myeloma (MM) has raised the prospect for the emergence of a curative treatment. While BCMA-targeted chimeric antigen receptor (CAR)-T cell therapies have been successful in treating MM, CAR-T cell manufacturing challenges preventing broad patient access and treatment relapse drive the need for additional targeted therapies with emphasis on multi-antigen targeting and off-the-shelf availability. GPRC5D, a tumor-associated orphan G-protein-coupled receptor found to be highly expressed in MM, is a potentially attractive target that has demonstrated promising clinical benefit when targeted via immunotherapy modalities. Here, we describe the development of FT555, an induced pluripotent stem cell (iPSC)-derived CAR-NK (CAR-iNK) cell product with the unique and effective ability to simultaneously co-target GPRC5D and CD38 (an additional tumor-associated antigen of MM) via combination with daratumumab, and which can be mass produced and is available off-the-shelf to support broad patient access. FT555 is a CAR-NK cell derived from an iPSC master cell line that has been multiplexed-engineered at the clonal level to contain four unique modalities; a novel GPRC5D-specific CAR fine-tuned for NK cell biology; a high-affinity, non-cleavable CD16 (hnCD16) to maximize antibody-dependent cellular cytotoxicity (ADCC) when combined with a monoclonal antibody (mAb); a unique IL-15/IL-15 receptor fusion protein (IL15RF) to promote cytokine-independent function; and CD38 knockout to promote NK cell fitness and uniquely prevent anti-CD38 mAb-mediated fratricide. Sourced from a renewable engineered iPSC master cell line, FT555 is a pure population of engineered NK cells (>95% CD56+) exhibiting uniform expression of CAR-GPRC5D (>90%), hnCD16 (>90%) and IL15RF (>90%), with complete elimination of CD38 expression (not detected). In cytotoxicity assays, FT555 shows antigen specificity, dose-dependent potency and importantly, when combined with the anti-CD38 mAb daratumumab, exhibits resistance to fratricide. In a serial restimulation killing assay, FT555 demonstrates persistent tumor-specific activity against GPRC5D-positive MM.1S WT target cells when compared to isogenic GPRC5D knock-out (KO) targets (70.9% control of tumor by AUC against WT targets vs. 3.5% control against GPRC5D KO targets in the third round of stimulation). When used in combination with daratumumab, FT555 targets CD38 and eliminates CAR-resistant GPRC5D KO target cells (57.1% target killing when combined with daratumumab vs. 3.5% target killing in the absence of mAb in the third round of stimulation), demonstrating the capacity for FT555 to target GPRC5D+ and CD38+ cells through CAR and hnCD16 engagement, respectively. In a disseminated xenograft in vivo model of MM where the MM.1S cell line shows comprehensive tumor engraftment, FT555 demonstrates robust killing kinetics and tumor clearance as a single dose, resulting in control of MM progression for up to 42 days and increased survival to 80 days vs. 37 days for the untreated control arm (Figure 1A, 1B, FT555; 99.9% tumor growth inhibition (TGI) at D37, 151% increased lifespan (ILS)). Durability of FT555 is further enhanced with the addition of daratumumab, where tumor growth inhibition deepened and survival improved, with 2 of 5 mice showing complete clearance of tumor cells at Day 80, demonstrating the synergy of anti-tumor activity between CAR and hnCD16 (Figure 1A, 1B, FT555+Dara; 100% TGI at D37, >207% ILS). Furthermore, in a distinct xenograft model of MM, treatment with FT555 resulted in significantly improved TGI against OPM2 tumor targets (FT555; 100% TGI at D51, p < 0.05). Together these studies demonstrate that FT555 is a multiplexed-engineered CAR-NK cell derived from a clonal master iPSC line, which utilizes the intrinsic versatility of NK cells to enable a highly effective combination therapy with daratumumab to simultaneously target both GPRC5D and CD38 in a single, standardized, and scalable off-the-shelf platform. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

PLAUR encodes the urokinase receptor (uPAR), which promotes cell survival, migration, and resistance to targeted cancer therapeutics in glioblastoma cells in culture and in mouse model systems. Herein, we show that patient survival correlates inversely with PLAUR mRNA expression in gliomas of all grades, in glioblastomas, and in the subset of glioblastomas that demonstrate the mesenchymal gene expression signature. PLAUR clusters with genes that define the more aggressive mesenchymal subtype in transcriptome profiles of glioblastoma tissue and glioblastoma cells in neurospheres, which are enriched for multipotent cells with stem cell-like qualities. When PLAUR was over-expressed or silenced in glioblastoma cells, neurosphere growth and expression of mesenchymal subtype biomarkers correlated with uPAR abundance. uPAR also promoted glioblastoma cell survival in neurospheres. Constitutively-active EGF Receptor (EGFRvIII) promoted neurosphere growth; however, unlike uPAR, EGFRvIII did not induce the mesenchymal gene expression signature. Immunohistochemical analysis of human glioblastomas showed that uPAR is typically expressed by a small sub-population of the cancer cells; it is thus reasonable to conclude that this subpopulation of cells is responsible for the effects of PLAUR on patient survival. We propose that uPAR-expressing glioblastoma cells demonstrate a mesenchymal gene signature, an increased capacity for cell survival, and stem cell-like properties.

The fibrinolysis proteinase tissue-type plasminogen activator (tPA, also known as PLAT) triggers cell signaling and regulates cell physiology. In PC12 cells, Schwann cells and macrophages, the N-methyl-D-aspartate receptor (NMDA-R) mediates tPA signaling. Plasminogen activator inhibitor-1 (PAI1, also known as SERPINE1) is a rapidly acting inhibitor of tPA enzyme activity. Although tPA-initiated cell signaling is not dependent on its enzyme active site, we show that tPA signaling is neutralized by PAI1. In PC12 cells, PAI1 blocked the ERK1/2 activation mediated by tPA as well as neurite outgrowth. In Schwann cells, PAI1 blocked tPA-mediated ERK1/2 activation and cell migration. In macrophages, PAI1 blocked the ability of tPA to inhibit IκBα phosphorylation and cytokine expression. The cell signaling activity of tPA-PAI1 complex was rescued when the complex was formed with PAI1R76E, which binds to LRP1 with decreased affinity, by pre-treating cells with the LRP1 antagonist receptor-associated protein and upon LRP1 gene silencing. The inhibitory role of LRP1 in tPA-PAI1 complex-initiated cell signaling was unanticipated given the reported role of LRP1 as an NMDA-R co-receptor in signaling responses elicited by free tPA or α2-macroglobulin. We conclude that PAI1 functions as an in-hibitor not only of the enzyme activity of tPA but also of tPA receptor-mediated activities.

LDL receptor-related proteins (LRPs) are transmembrane receptors involved in endocytosis, cell-signaling, and trafficking of other cellular proteins. Considerable work has focused on LRPs in the fields of vascular biology and neurobiology. How these receptors affect cancer progression in humans remains largely unknown. Herein, we mined provisional databases in The Cancer Genome Atlas (TCGA) to compare expression of thirteen LRPs in ten common solid malignancies in patients. Our first goal was to determine the abundance of LRP mRNAs in each type of cancer. Our second goal was to determine whether expression of LRPs is associated with improved or worsened patient survival. In total, data from 4,629 patients were mined. In nine of ten cancers studied, the most abundantly expressed LRP was LRP1; however, a correlation between LRP1 mRNA expression and patient survival was observed only in bladder urothelial carcinoma. In this malignancy, high levels of LRP1 mRNA were associated with worsened patient survival. High levels of LDL receptor (LDLR) mRNA were associated with decreased patient survival in pancreatic adenocarcinoma. High levels of LRP10 mRNA were associated with decreased patient survival in hepatocellular carcinoma, lung adenocarcinoma, and pancreatic adenocarcinoma. LRP2 was the only LRP for which high levels of mRNA expression correlated with improved patient survival. This correlation was observed in renal clear cell carcinoma. Insights into LRP gene expression in human cancers and their effects on patient survival should guide future research.

In the CNS, microglia are activated in response to injury or infection and in neurodegenerative diseases. The endocytic and cell signaling receptor, LDL receptor-related protein-1 (LRP1), is reported to suppress innate immunity in macrophages and oppose microglial activation. The goal of this study was to identify novel mechanisms by which LRP1 may regulate microglial activation. Using primary cultures of microglia isolated from mouse brains, we demonstrated that LRP1 gene silencing increases expression of proinflammatory mediators; however, the observed response was modest. By contrast, the LRP1 ligand, receptor-associated protein (RAP), robustly activated microglia, and its activity was attenuated in LRP1-deficient cells. An important element of the mechanism by which RAP activated microglia was its ability to cause LRP1 shedding from the plasma membrane. This process eliminated cellular LRP1, which is anti-inflammatory, and generated a soluble product, shed LRP1 (sLRP1), which is potently proinflammatory. Purified sLRP1 induced expression of multiple proinflammatory cytokines and the mRNA encoding inducible nitric-oxide synthase in both LRP1-expressing and -deficient microglia. LPS also stimulated LRP1 shedding, as did the heat-shock protein and LRP1 ligand, calreticulin. Other LRP1 ligands, including (2)-macroglobulin and tissue-type plasminogen activator, failed to cause LRP1 shedding. Treatment of microglia with a metalloproteinase inhibitor inhibited LRP1 shedding and significantly attenuated RAP-induced cytokine expression. RAP and sLRP1 both caused neuroinflammation in vivo when administered by stereotaxic injection into mouse spinal cords. Collectively, these results suggest that LRP1 shedding from microglia may amplify and sustain neuroinflammation in response to proinflammatory stimuli.

Tissue-type plasminogen activator (tPA) is the major intravascular activator of fibrinolysis and a ligand for receptors involved in cell signaling. In cultured macrophages, tPA inhibits the response to lipopolysaccharide (LPS) by a pathway that apparently requires low-density lipoprotein receptor-related protein-1 (LRP1). Herein, we show that the mechanism by which tPA neutralizes LPS involves rapid reversal of IκBα phosphorylation. tPA independently induced transient IκBα phosphorylation and extracellular signal-regulated kinase 1/2 (ERK1/2) activation in macrophages; however, these events did not trigger inflammatory mediator expression. The tPA signaling response was distinguished from the signature of signaling events elicited by proinflammatory LRP1 ligands, such as receptor-associated protein (RAP), which included sustained IκBα phosphorylation and activation of all 3 MAP kinases (ERK1/2, c-Jun kinase, and p38 MAP kinase). Enzymatically active and inactive tPA demonstrated similar immune modulatory activity. Intravascular administration of enzymatically inactive tPA in mice blocked the toxicity of LPS. In mice not treated with exogenous tPA, the plasma concentration of endogenous tPA increased 3-fold in response to LPS, to 116 ± 15 pM, but remained below the approximate threshold for eliciting anti-inflammatory cell signaling in macrophages (∼2.0 nM). This threshold is readily achieved in patients when tPA is administered therapeutically for stroke. In addition to LRP1, we demonstrate that the N-methyl-D-aspartic acid receptor (NMDA-R) is expressed by macrophages and essential for anti-inflammatory cell signaling and regulation of cytokine expression by tPA. The NMDA-R and Toll-like receptor-4 were not required for proinflammatory RAP signaling. By mediating the tPA response in macrophages, the NMDA-R provides a pathway by which the fibrinolysis system may regulate innate immunity.

In high grade glioma (HGG), extensive tumor cell infiltration of normal brain typically precludes identifying effective margins for surgical resection or irradiation. Pertussis toxin (PT) is a multimeric complex that inactivates diverse Gi/o G-protein coupled receptors (GPCRs). Despite the broad continuum of regulatory events controlled by GPCRs, PT may be applicable as a therapeutic. We have shown that the urokinase receptor (uPAR) is a major driver of HGG cell migration. uPAR-initiated cell-signaling requires a Gi/o GPCR, N-formyl Peptide Receptor 2 (FPR2), as an essential co-receptor and is thus, PT-sensitive. Herein, we show that PT robustly inhibits migration of three separate HGG-like cell lines that express a mutated form of the EGF Receptor (EGFR), EGFRvIII, which is constitutively active. PT also almost completely blocked the ability of HGG cells to invade Matrigel. In the equivalent concentration range (0.01-1.0 μg/mL), PT had no effect on cell survival and only affected proliferation of one cell line. Neutralization of EGFRvIII expression in HGG cells, which is known to activate uPAR-initiated cell-signaling, promoted HGG cell migration. The increase in HGG cell migration, induced by EGFRvIII neutralization, was entirely blocked by silencing FPR2 gene expression or by treating the cells with PT. When U87MG HGG cells were cultured as suspended neurospheres in serum-free, growth factor-supplemented medium, uPAR expression was increased. HGG cells isolated from neurospheres migrated through Transwell membranes without loss of cell contacts; this process was inhibited by PT by >90%. PT also inhibited expression of vimentin by HGG cells; vimentin is associated with epithelial-mesenchymal transition and worsened prognosis. We conclude that PT may function as a selective inhibitor of HGG cell migration and invasion.

You have accessJournal of UrologyBladder Cancer: Basic Research & Pathophysiology IV1 Apr 2016MP83-19 UPAR AND MTORC2: COUPLED TARGETS FOR THERAPEUTICS DEVELOPMENT IN BLADDER CANCER Andrew Hau, Andrew Gilder, Jingjing Hu, Steven Gonias, and Donna Hansel Andrew HauAndrew Hau More articles by this author , Andrew GilderAndrew Gilder More articles by this author , Jingjing HuJingjing Hu More articles by this author , Steven GoniasSteven Gonias More articles by this author , and Donna HanselDonna Hansel More articles by this author View All Author Informationhttps://doi.org/10.1016/j.juro.2016.02.2201AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookTwitterLinked InEmail INTRODUCTION AND OBJECTIVES Bladder cancer currently ranks as the fifth most common and the single most expensive cancer to manage in the United States. Although it is established that invasive behavior is a major predictor of diminished outcomes for patients with bladder cancer, the molecular mechanisms governing bladder cancer cell invasion are not well understood. The urokinase receptor (uPAR) and mammalian target of rapamycin complex 2 (mTORC2) represent two powerful pro-invasion candidates that have increased expression in high-grade, invasive bladder cancer, though the former has not been characterized in detail in bladder cancer. Therefore, the aims of this study are to characterize the uPAR signaling network and delineate the signaling interplay between mTORC2 and uPAR in bladder cancer. METHODS Using immunoblot and RT-qPCR analyses, we evaluated uPAR expression in a panel of immortalized bladder cancer cell lines: UROtsa, RT4, UMUC3, T24 and J82. uPAR influence on mTORC1 and mTORC2 signaling was determined by immunoblot analysis following targeted gene-silencing of uPAR using siRNA. Additionally, the effects of uPAR knockdown on cell migration and invasion were investigated using modified scratch-wound migration and transwell invasion assays. Lastly, signaling interplay between uPAR and mTORC2 was investigated by evaluating the effects of uPAR and mTORC2 silencing on Rac1 activity RESULTS uPAR knockdown in a subset (T24 and J82) of invasive bladder cancer cell lines inhibited mTORC2, but not mTORC1, activity as measured by P-AKT S473 and P-S6 levels. We found that uPAR silencing in T24 and J82 cells resulted in significant reductions in cell migration and invasion through Matrigel, which is likely attributed to inhibition of Rac1 and decreased lamellipodia formation. CONCLUSIONS Collectively, our results identify uPAR and mTORC2 as major regulators of bladder cancer cell invasion and that these two systems are linked through Rac1. Further investigation of uPAR and mTORC2 inhibition using uPAR-targeting antibodies and mTOR inhibitors in an in vivo mouse model of bladder cancer will determine if these signaling pathways are therapeutically beneficial for the treatment of bladder cancer. © 2016FiguresReferencesRelatedDetails Volume 195Issue 4SApril 2016Page: e1088 Advertisement Copyright & Permissions© 2016MetricsAuthor Information Andrew Hau More articles by this author Andrew Gilder More articles by this author Jingjing Hu More articles by this author Steven Gonias More articles by this author Donna Hansel More articles by this author Expand All Advertisement Advertisement PDF downloadLoading ...

LDL receptor‐related protein‐1 (LRP1) is an endocytic and phagocytic receptor. In neurons and neuron‐like cells, binding and endocytosis of specific ligands is coupled to robust cell‐signaling responses. The majority of the LRP1, present at the cell‐surface in most cells, localizes to clathrin‐coated pits. However, in some cells, LRP1 also local‐izes to lipid rafts. We previously demonstrated that, in PC12 and N2A neuron‐like cells, tissue‐type plasminogen activator (tPA) and activated α 2 ‐macroglobulin (α 2 M*) phos‐phorylate TrkA and activate ERK1/2 in a response that requires LRP1 and the NMDA Receptor. We have now shown that in PC12 and N2A cells, LRP1 is partially localized to lipid rafts. In these cells, both enzymatically‐inactive tPA and α 2 M* acti‐vated ERK1/2, confirming our earlier result. When PC12 or N2A cells were treated with methyl‐β‐cyclodextrin (MβCD), to disrupt lipid rafts, cell‐signaling in response to tPA and α 2 M* was blocked. MβCD had no effect on the total cellular abundance of LRP1, as deter‐mined by immunoblot analysis or on the cell‐surface abundance of LRP1, as deter‐mined by measuring the binding of radioiodinated α 2 M* at 4º C. MβCD also failed to af‐fect endocytosis of radioiodinated α 2 M* at 37º C. The selective effects of MβCD on LRP1‐initiated cell‐signaling was apparent in neuritogenesis experiments in PC12 cells; when lipid rafts were disrupted, tPA and α 2 M* failed to induce neurite outgrowth, which was observed in control cultures. Our studies suggest that LRP1 signaling is initiated in lipid rafts. The fact that LRP1 localizes to lipid rafts only in some cell types may partially explain why its activity in cell‐signaling is cell type‐specific.

Mammalian target of rapamycin complex 2 (mTORC2) has been identified as a major regulator of bladder cancer cell migration and invasion. Upstream pathways that mediate mTORC2 activation remain poorly defined. Urokinase-type plasminogen activator receptor (uPAR) is a GPI-anchored membrane protein and known activator of cell-signaling. We identified increased uPAR expression in 94% of invasive human bladder cancers and in 54–71% of non-invasive bladder cancers, depending on grade. Normal urothelium was uPAR-immunonegative. Analysis of publicly available datasets identified uPAR gene amplification or mRNA upregulation in a subset of bladder cancer patients with reduced overall survival. Using biochemical approaches, we showed that uPAR activates mTORC2 in bladder cancer cells. Highly invasive bladder cancer cell lines, including T24, J82 and UM-UC-3 cells, showed increased uPAR mRNA expression and protein levels compared with the less aggressive cell lines, UROtsa and RT4. uPAR gene-silencing significantly reduced phosphorylation of Serine-473 in Akt, an mTORC2 target. uPAR gene-silencing also reduced bladder cancer cell migration and Matrigel invasion. S473 phosphorylation was observed by immunohistochemistry in human bladder cancers only when the tumors expressed high levels of uPAR. S473 phosphorylation was not controlled by uPAR in bladder cancer cell lines that are PTEN-negative; however, this result probably did not reflect altered mTORC2 regulation. Instead, PTEN deficiency de-repressed alternative kinases that phosphorylate S473. Our results suggest that uPAR and mTORC2 are components of a single cell-signaling pathway. Targeting uPAR or mTORC2 may be beneficial in patients with bladder cancer.

447 Background: Bladder cancer currently ranks as the fifth most common and the single most expensive cancer to manage in the United States. Although it is established that invasive behavior is a major predictor of diminished outcomes for patients with bladder cancer, the molecular mechanisms governing bladder cancer cell invasion are not well understood. The urokinase receptor (uPAR) and mammalian target of rapamycin complex 2 (mTORC2) represent two powerful pro-invasion candidates that have increased expression in high-grade, invasive bladder cancer, though the former has not been characterized in detail in bladder cancer. Therefore, the aims of this study are to characterize the uPAR signaling network and delineate the signaling interplay between mTORC2 and uPAR in bladder cancer. Methods: Using immunoblot and RT-qPCR analyses, we evaluated uPAR expression in a panel of immortalized bladder cancer cell lines: UROtsa, RT4, UMUC3, T24 and J82. uPAR influence on mTORC1 and mTORC2 signaling was determined by immunoblot analysis following targeted gene-silencing of uPAR using siRNA. Additionally, the effects of uPAR knockdown on cell migration and invasion were investigated using modified scratch-wound migration and transwell invasion assays. Lastly, signaling interplay between uPAR and mTORC2 was investigated by evaluating the effects of uPAR and mTORC2 silencing on Rac1 activity. Results: uPAR knockdown in a subset (T24 and J82) of invasive bladder cancer cell lines inhibited mTORC2, but not mTORC1, activity as measured by P-AKT S473 and P-S6 levels. We found that uPAR silencing in T24 and J82 cells resulted in significant reductions in cell migration and invasion through Matrigel. This is likely attributed to inhibition of Rac1 and decreased lamellipodia formation. Conclusions: Collectively, our results identify uPAR and mTORC2 as major regulators of bladder cancer cell invasion and that these two systems are linked through Rac1. Further investigation of uPAR and mTORC2 inhibition using uPAR-targeting antibodies and mTOR inhibitors in an in vivo mouse model of bladder cancer will determine if these signaling pathways are therapeutically beneficial for the treatment of bladder cancer.

LDL Receptor-related Protein-1 (LRP1) is an endocytic receptor for diverse ligands. In neurons and neuron-like cells, ligand-binding to LRP1 initiates cell-signaling. Herein, we show that in PC12 and N2a neuron-like cells, LRP1 distributes into lipid rafts and non-raft plasma membrane fractions. When lipid rafts were disrupted, using methyl-β-cyclodextrin or fumonisin B1, activation of Src family kinases and ERK1/2 by the LRP1 ligands, tissue-type plasminogen activator and activated α2-macroglobulin, was blocked. Biological consequences of activated LRP1 signaling, including neurite outgrowth and cell growth, also were blocked. The effects of lipid raft disruption on ERK1/2 activation and neurite outgrowth, in response to LRP1 ligands, were reproduced in experiments with cerebellar granule neurons in primary culture. Because the reagents used to disrupt lipid rafts may have effects on the composition of the plasma membrane outside lipid rafts, we studied the effects of these reagents on LRP1 activities unrelated to cell-signaling. Lipid raft disruption did not affect the total ligand binding capacity of LRP1, the affinity of LRP1 for its ligands, or its endocytic activity. These results demonstrate that well described activities of LRP1 require localization of this receptor to distinct plasma membrane microdomains.

LDL receptor-related protein-1 (LRP1) is an endocytic and cell-signaling receptor. In mice in which LRP1 is deleted in myeloid cells, the response to lipopolysaccharide (LPS) was greatly exacerbated. LRP1 deletion in macrophages in vitro, under the control of tamoxifen-activated Cre-ER(T) fusion protein, robustly increased expression of proinflammatory cytokines and chemokines. In LRP1-expressing macrophages, proinflammatory mediator expression was regulated by LRP1 ligands in a ligand-specific manner. The LRP1 agonists, α2-macroglobulin and tissue-type plasminogen activator, attenuated expression of inflammatory mediators, even in the presence of LPS. The antagonists, receptor-associated protein (RAP) and lactoferrin (LF), and LRP1-specific antibody had the entirely opposite effect, promoting inflammatory mediator expression and mimicking LRP1 deletion. NFκB was rapidly activated in response to RAP and LF and responsible for the initial increase in expression of proinflammatory mediators. RAP and LF also significantly increased expression of microRNA-155 (miR-155) after a lag phase of about 4 h. miR-155 expression reflected, at least in part, activation of secondary cell-signaling pathways downstream of TNFα. Although miR-155 was not involved in the initial induction of cytokine expression in response to LRP1 antagonists, miR-155 was essential for sustaining the proinflammatory response. We conclude that LRP1, NFκB, and miR-155 function as members of a previously unidentified system that has the potential to inhibit or sustain inflammation, depending on the continuum of LRP1 ligands present in the macrophage microenvironment.