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CD39 (ENTPD1) is a key enzyme responsible for degradation of extracellular ATP and is upregulated in the tumor microenvironment (TME). Extracellular ATP accumulates in the TME from tissue damage and immunogenic cell death, potentially initiating proinflammatory responses that are reduced by the enzymatic activity of CD39. Degradation of ATP by CD39 and other ectonucleotidases (e.g., CD73) results in extracellular adenosine accumulation, constituting an important mechanism for tumor immune escape, angiogenesis induction, and metastasis. Thus, inhibiting CD39 enzymatic activity can inhibit tumor growth by converting a suppressive TME to a proinflammatory environment. SRF617 is an investigational, anti-CD39, fully human IgG4 Ab that binds to human CD39 with nanomolar affinity and potently inhibits its ATPase activity. In vitro functional assays using primary human immune cells demonstrate that inhibiting CD39 enhances T-cell proliferation, dendritic cell maturation/activation, and release of IL-1β and IL-18 from macrophages. In vivo, SRF617 has significant single-agent antitumor activity in human cell line–derived xenograft models that express CD39. Pharmacodynamic studies demonstrate that target engagement of CD39 by SRF617 in the TME inhibits ATPase activity, inducing proinflammatory mechanistic changes in tumor-infiltrating leukocytes. Syngeneic tumor studies using human CD39 knock-in mice show that SRF617 can modulate CD39 levels on immune cells in vivo and can penetrate the TME of an orthotopic tumor, leading to increased CD8+ T-cell infiltration. Targeting CD39 is an attractive approach for treating cancer, and, as such, the properties of SRF617 make it an excellent drug development candidate.

supplementary Table S1 to S3; Supplementary Figure S1 to S9

The ectonucleotidases CD39 and CD73 catalyze extracellular ATP to immunosuppressive adenosine, and as such, represent potential cancer targets. We investigated biological impacts of CD39 and CD73 in pancreatic ductal adenocarcinoma (PDAC) by studying clinical samples and experimental mouse tumors. Stromal CD39 and tumoral CD73 expression significantly associated with worse survival in human PDAC samples and abolished the favorable prognostic impact associated with the presence of tumor-infiltrating CD8+ T cells. In mouse transplanted KPC tumors, both CD39 and CD73 on myeloid cells, as well as CD73 on tumor cells, promoted polarization of infiltrating myeloid cells towards an M2-like phenotype, which enhanced tumor growth. CD39 on tumor-specific CD8+ T cells and pancreatic stellate cells also suppressed IFNγ production by T cells. Although therapeutic inhibition of CD39 or CD73 alone significantly delayed tumor growth in vivo, targeting of both ectonucleotidases exhibited markedly superior antitumor activity. CD73 expression on human and mouse PDAC tumor cells also protected against DNA damage induced by gemcitabine and irradiation. Accordingly, large-scale pharmacogenomic analyses of human PDAC cell lines revealed significant associations between CD73 expression and gemcitabine chemoresistance. Strikingly, increased DNA damage in CD73-deficient tumor cells associated with activation of the cGAS–STING pathway. Moreover, cGAS expression in mouse KPC tumor cells was required for antitumor activity of the CD73 inhibitor AB680 in vivo. Our study, thus, illuminates molecular mechanisms whereby CD73 and CD39 seemingly cooperate to promote PDAC progression.

Guanylyl cyclase C (GCC) is a unique therapeutic target with expression restricted to the apical side of epithelial cell tight junctions thought to be only accessible by intravenously administered agents on malignant tissues where GCC expression is aberrant. In this study, we sought to evaluate the therapeutic potential of a second-generation investigational antibody–dug conjugate (ADC), TAK-164, comprised of a human anti-GCC mAb conjugated via a peptide linker to the highly cytotoxic DNA alkylator, DGN549. The in vitro binding, payload release, and in vitro activity of TAK-164 was characterized motivating in vivo evaluation. The efficacy of TAK-164 and the relationship to exposure, pharmacodynamic marker activation, and biodistribution was evaluated in xenograft models and primary human tumor xenograft (PHTX) models. We demonstrate TAK-164 selectively binds to, is internalized by, and has potent cytotoxic effects against GCC-expressing cells in vitro. A single intravenous administration of TAK-164 (0.76 mg/kg) resulted in significant growth rate inhibition in PHTX models of metastatic colorectal cancer. Furthermore, imaging studies characterized TAK-164 uptake and activity and showed positive relationships between GCC expression and tumor uptake which correlated with antitumor activity. Collectively, our data suggest that TAK-164 is highly active in multiple GCC-positive tumors including those refractory to TAK-264, a GCC-targeted auristatin ADC. A strong relationship between uptake of 89Zr-labeled TAK-164, levels of GCC expression and, most notably, response to TAK-164 therapy in GCC-expressing xenografts and PHTX models. These data supported the clinical development of TAK-164 as part of a first-in-human clinical trial (NCT03449030).

Supplemental Figure 1: heavy and light chain sequences for 5F9, the human IgG1 monoclonal antibody (mAb) targeting the extracellular domain of GCC as described previously Supplemental Figure 2: Characterization of TAK-164 and chKTI-DGN549 by A) SDS PAGE gel and B) by Liquid chromatography-mass spectrometry (LC-MS) Supplemental Figure 3: In vivo activity of TAK-164 in PHTXM-46C Supplemental Table 1: Growth rate inhibition following treatment with TAK-164 and B) chKTI-ADC in PHTX and cell derived xenograft models Supplemental Table 2: Growth Rate Inhibition and associated statistics for HEK293 GCC#2 Supplemental Table 3: Growth Rate Inhibition and associated statistics for PHTX-09C Supplemental Table 4: Growth Rate Inhibition and associated statistics for PHTX-17C Supplemental Table 5: Growth Rate Inhibition and associated statistics for PHTX-11C

The ectonucleotidases CD39 and CD73 catalyze extracellular ATP to immunosuppressive adenosine, and as such, represent potential cancer targets. We investigated biological impacts of CD39 and CD73 in pancreatic ductal adenocarcinoma (PDAC) by studying clinical samples and experimental mouse tumors. Stromal CD39 and tumoral CD73 expression significantly associated with worse survival in human PDAC samples and abolished the favorable prognostic impact associated with the presence of tumor-infiltrating CD8+ T cells. In mouse transplanted KPC tumors, both CD39 and CD73 on myeloid cells, as well as CD73 on tumor cells, promoted polarization of infiltrating myeloid cells towards an M2-like phenotype, which enhanced tumor growth. CD39 on tumor-specific CD8+ T cells and pancreatic stellate cells also suppressed IFNγ production by T cells. Although therapeutic inhibition of CD39 or CD73 alone significantly delayed tumor growth in vivo, targeting of both ectonucleotidases exhibited markedly superior antitumor activity. CD73 expression on human and mouse PDAC tumor cells also protected against DNA damage induced by gemcitabine and irradiation. Accordingly, large-scale pharmacogenomic analyses of human PDAC cell lines revealed significant associations between CD73 expression and gemcitabine chemoresistance. Strikingly, increased DNA damage in CD73-deficient tumor cells associated with activation of the cGAS–STING pathway. Moreover, cGAS expression in mouse KPC tumor cells was required for antitumor activity of the CD73 inhibitor AB680 in vivo. Our study, thus, illuminates molecular mechanisms whereby CD73 and CD39 seemingly cooperate to promote PDAC progression.

Guanylyl cyclase C (GCC) is a unique therapeutic target with expression restricted to the apical side of epithelial cell tight junctions thought to be only accessible by intravenously administered agents on malignant tissues where GCC expression is aberrant. In this study, we sought to evaluate the therapeutic potential of a second-generation investigational antibody–dug conjugate (ADC), TAK-164, comprised of a human anti-GCC mAb conjugated via a peptide linker to the highly cytotoxic DNA alkylator, DGN549. The in vitro binding, payload release, and in vitro activity of TAK-164 was characterized motivating in vivo evaluation. The efficacy of TAK-164 and the relationship to exposure, pharmacodynamic marker activation, and biodistribution was evaluated in xenograft models and primary human tumor xenograft (PHTX) models. We demonstrate TAK-164 selectively binds to, is internalized by, and has potent cytotoxic effects against GCC-expressing cells in vitro. A single intravenous administration of TAK-164 (0.76 mg/kg) resulted in significant growth rate inhibition in PHTX models of metastatic colorectal cancer. Furthermore, imaging studies characterized TAK-164 uptake and activity and showed positive relationships between GCC expression and tumor uptake which correlated with antitumor activity. Collectively, our data suggest that TAK-164 is highly active in multiple GCC-positive tumors including those refractory to TAK-264, a GCC-targeted auristatin ADC. A strong relationship between uptake of 89Zr-labeled TAK-164, levels of GCC expression and, most notably, response to TAK-164 therapy in GCC-expressing xenografts and PHTX models. These data supported the clinical development of TAK-164 as part of a first-in-human clinical trial (NCT03449030).

Background IL-27 is a heterodimeric cytokine consisting of IL 27p28 and Epstein-Barr virus-induced gene 3 (EBI3) that binds the IL-27 receptor subunit alpha and glycoprotein 130. IL-27 is produced by activated macrophages and dendritic cells and limits the intensity and duration of immune responses in the tumor microenvironment by inducing the expression of immunoregulatory receptors (PD-L1, TIM3, LAG-3, TIGIT) and inhibiting production of proinflammatory cytokines (IFNγ, IL-17, TNFα). The IL-27 subunit EBI3 is elevated in plasma from patients with certain cancers including renal cell carcinoma, where it correlates with poor outcome. Based on high expression of IL-27 transcript in tumors from patients with hepatocellular carcinoma (HCC), the role of IL-27 was further explored in patient samples and a mouse model of HCC. Methods Gene expression profiles from the Cancer Genome Atlas (TCGA) were analyzed to identify tumors with elevated IL-27 transcripts. Serum from patients with HCC was analyzed for levels of the IL-27 subunit EBI3. The ability of SRF388, a first-in-class IL-27-blocking antibody that binds to IL-27p28, to reverse IL-27-induced inhibition of cytokine production in human immune cell cultures from patients with HCC was assessed in vitro. Finally, the anti-tumor activity of SRF388 was assessed in an orthotopic murine model of HCC. Results TCGA expression data revealed that IL-27p28 transcripts were elevated in tumors from patients with HCC relative to other indications. Serum levels of EBI3 were: 1) elevated in a subset of HCC patients; 2) inversely correlated with survival; 3) independent of serum alpha-fetoprotein levels; and 4) elevated in both hepatitis B/C virus positive and negative patients. Treatment with SRF388 stimulated increased cytokine production in activated peripheral blood mononuclear cells from patients with HCC that was further enhanced when combined with PD-1 blockade. Furthermore, SRF388 inhibited the growth of orthotopic Hepa1-6 liver tumors. mRNA transcriptional profiling of treated tumors revealed that SRF388 profoundly altered the transcriptional landscape in this model. In particular, treatment with SRF388 inhibited expression of immunoregulatory receptors PD-L1 and TIGIT, repressed transcripts associated with TGF-β signaling, and altered myeloid and natural killer cell transcripts. Conclusions These data indicate that elevated IL-27 subunit EBI3 is a hallmark of HCC and is associated with poor outcomes in these patients. Blockade of IL-27 with SRF388, currently being evaluated in a Phase 1 clinical trial in patients with advanced solid tumors (NCT04374877), may represent a promising therapy for patients with HCC where it can potentiate anti-tumor immune responses.

BackgroundThe purine nucleotide adenosine plays an important role in dampening both the innate and adaptive arms of the immune system. In contrast, extracellular adenosine triphosphate (ATP), which can be generated at high levels due to cell stress, initiates proinflammatory responses. Extracellular ATP can be produced in cancerous tumors, and its conversion to adenosine monophosphate by CD39 (ENTPD1) and subsequent degradation to free adenosine by CD73 (NT5E) in the tumor microenvironment (TME) is a major contributor to tumor immune evasion. Accordingly, CD39 serves as an important immune regulator. CD39 is highly expressed in the TME of various tumor types, including lung, prostate, colon, ovarian, and pancreatic cancers, making CD39 an attractive target for immune-based anticancer therapy.SRF617 is a fully human anti-CD39 antibody designed to target human CD39 and inhibit its enzymatic activity. SRF617 provides a dual mechanism of immune activation by increasing inflammatory responses through accumulation of free ATP and alleviating immune suppression by reducing adenosine production. In this way, SRF617 shifts the TME to a proinflammatory state leading to reactivation of the immune system and antitumor function.MethodsA pharmacokinetic/pharmacodynamic profile was determined in CT26 tumor-bearing human CD39 knock-in (hCD39 KI) mice by measuring plasma SRF617 concentration, peripheral blood target occupancy (TO), and ATP hydrolysis activity in frozen tissue sections. Wild-type mice bearing subcutaneous (s.c.) KPC1245 tumors were treated with anti-murine CD39 antibody, alone or in combination with gemcitabine.ResultsCirculating SRF617 was observed in the plasma after intravenous injection of CT26 tumor-bearing hCD39 KI mice, which correlated with CD39-bound SRF617 on B cells. The presence of SRF617 correlated with a decrease in ATPase activity and CD39 expression in tissues. In the KPC tumor model, a combination effect was observed for anti-CD39 treatment with gemcitabine. Treatment with anti-CD39 antibody resulted in an increase in CD8+ T cells in the TME as measured by flow cytometry.ConclusionsSRF617 treatment of hCD39 KI mice led to reduction of ATPase activity in the TME that correlated with peripheral TO and plasma concentration. Anti-CD39 treatment alone or in combination with gemcitabine led to tumor growth inhibition and increased tumor-infiltrating lymphocytes as measured by flow cytometry in s.c. KPC tumors. These studies demonstrate the therapeutic potential of targeting CD39 for the treatment of cancer. SRF617 is currently being evaluated in a Phase 1 clinical trial in patients with advanced solid tumors (NCT04336098) and in combination with chemotherapy in patients with pancreatic cancer.

BackgroundInterleukin (IL)-27 is a heterodimeric immunoregulatory cytokine that signals through the JAK/STAT pathway to increase the expression of coinhibitory receptors on immune cells (e.g. PD-L1, TIM-3, LAG-3) and dampen inflammatory cytokine production. Blockade of IL-27 leads to antitumor activity in preclinical mouse models of lung metastases. A Phase 1 trial of SRF388 (NCT04374877), a first-in-class anti–IL-27 antibody, has demonstrated monotherapy antitumor activity in a patient with non-small cell lung cancer (NSCLC).1 The current study aimed to characterize the immunoregulatory impact of IL-27 signaling by gene expression profiling.MethodsGene expression changes induced by IL-27 were examined in activated human CD4+ T cells, human peripheral blood mononuclear cells (PBMCs), and the IL-27RA–expressing lung cancer cell line NCI-H2228 by microarray or single cell RNA-sequencing. The resulting IL-27 signature genes were interrogated by Gene Set Enrichment Analysis (GSEA) using publicly available datasets, including single cell RNA-seq analysis of the tumor microenvironment, from patients with NSCLC.2ResultsIL-27 induced a robust gene expression program in human immune cells that included several inhibitory receptors and canonical interferon regulated genes such as guanylate-binding proteins and interferon regulatory factors. GSEA and interferon signature analysis showed a striking overlap with those genes regulated by interferon-beta, a cytokine known to drive immune suppression associated with chronic viral infection and that is used therapeutically for controlling inflammation associated with the autoimmune disease multiple sclerosis. Moreover, interferon regulated pathways have recently emerged as a mechanism of resistance to immune checkpoint blockade in cancer. Exploration of the IL-27 gene signature in published datasets showed enrichment in macrophage populations associated with progressive disease in patients with NSCLC. While many of the properties of IL-27–mediated immune regulation have focused on hematopoietic cells, IL-27RA is also expressed on tumor cells from NSCLC patients with progressive disease as well as lung cancer cell lines in which IL-27 can upregulate PD-L1, IDO1, and other canonical interferon regulated genes.ConclusionsThese studies elucidate the transcriptional networks that are engaged after IL-27 signaling in immune and cancer cells and highlight the parallels with interferon-associated immune regulation. Blockade of IL-27 provides a novel therapeutic strategy to alleviate a gene transcriptional program implicated in immune suppression and checkpoint resistance.ReferencesPatnaik A, Morgensztern D, Mantia C, et al. Results of a phase 1 study of SRF388, a first-in-human, first-in-class, high-affinity anti-IL-27 antibody in advanced solid tumors. J Clin Oncol 2021;39:2551–2551.Maynard A, McCoach CE, Rotow JK, et al. Therapy-induced evolution of human lung cancer revealed by single-cell RNA sequencing. Cell 2020;182:1232–1251.

CD39 (ENTPD1) is a key enzyme responsible for the degradation of extracellular adenosine triphosphate (ATP) and is upregulated in the tumor microenvironment (TME). High levels of extracellular ATP, often generated in the TME as a result of tissue damage and immunogenic cell death, can initiate proinflammatory responses that are blocked by the enzymatic activity of CD39. In addition, extracellular adenosine accumulates in cancerous tissues through the degradation of ATP by CD39 and other ectonucleotidases (eg, CD73), and constitutes an important mechanism of tumor immune escape, induction of angiogenesis, and metastasis. Thus, inhibition of CD39 can convert a suppressive TME to a proinflammatory environment. SRF617 is an investigational fully human anti-CD39 antibody that binds to human CD39 with nanomolar affinity and potently inhibits its enzymatic function. Results of the current studies show that CD39 is predominantly expressed in tumor stroma and on tumor-infiltrating immune cells in samples from patients with cancer. Similar expression patterns are observed in various murine tumor models. In vivo, SRF617 has significant single-agent anti-tumor activity in a variety of cell line-derived xenograft models that express CD39. Cancer therapies, such as immunogenic cell death agents, can increase inflammation and ATP levels in the TME; combination studies of SRF617 with these agents showed improved preclinical efficacy and led to significant improvement in survival. In addition, a mouse-specific anti-CD39 surrogate antibody demonstrated potent binding and enzymatic inhibition of murine CD39 in vitro and significantly decreased tumor growth in a syngeneic murine tumor model. Pharmacodynamic studies demonstrated mechanistic changes in the tumor-infiltrating leukocytes and plasma chemokine levels. Combination treatment of a murine anti-CD39 surrogate and an anti-mouse PD-1 displayed improved activity and an increase in overall survival in the CT-26 mouse model. In addition, administration of SRF617 in combination with other immunotherapies also demonstrated a similar improvement in activity and survival. In summary, these studies demonstrate that SRF617 is a potent inhibitor of CD39 enzymatic activity both in vitro and in vivo. Inhibition of CD39 potentiates the activity of chemotherapy and immunotherapy agents to improve tumor growth inhibition and survival in mice. These findings support future clinical studies of SRF617 as monotherapy and in combination with other therapeutic agents in treating patients with cancer. Citation Format: Sonia G. Das, Austin Dulak, Gege Tan, Marc Johnson, Tauqeer H. Zaidi, Michael C. Warren, Secil Koseoglu, Erik Devereaux, Marisa O. Peluso, Alison M. Paterson, Benjamin H. Lee, Vito J. Palombella, Pamela M. Holland, Andrew C. Lake. SRF617, a potent enzymatic inhibitor of CD39, demonstrates single-agent activity and cooperates with various cancer therapies in both solid tumor and hematologic malignancies [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 6639.

Platelet exocytosis is regulated partially by the granular/cellular membrane lipids and proteins. Some platelets contain a membrane-bound tube, called an open canalicular system (OCS), which assists in granular release events and increases the membrane surface area for greater spreading. The OCS is not found in all species, and variations in membrane composition can cause changes in platelet secretion. Since platelet studies use various animal models, it is important to understand how platelets differ in both their composition and granular release to draw conclusions among various models. The relative phospholipid composition of the platelets with (mouse, rabbit) and without (cow) an OCS was quantified using UPLC-MS/MS. Cholesterol and protein composition was measured using an Amplex Red Assay and BCA Assay. TEM and dark field platelet images were gathered and analyzed with Image J. Granular release was monitored with single cell carbon fiber microelectrode amperometry. Cow platelets contained greater amounts of cholesterol and sphingomyelin. In addition, they yield greater serotonin release and longer δ granule secretion times. Finally, they showed greater spreading area with a greater range of spread. Platelets containing an OCS had more similarities in their membrane composition and secretion kinetics compared to cow platelets. However, cow platelets showed greater fusion pore stability which could be due to extra sphingomyelin and cholesterol, the primary components of lipid rafts. In addition, their greater stability may lead to many granules assisting in spreading. This study highlights fundamental membrane differences and their effects on platelet secretion.

The cellular phospholipid membrane plays an important role in cell function and cell-cell communication, but its biocomplexity and dynamic nature presents a challenge for examining cellular uptake of phospholipids and the resultant effects on cell function. Platelets, small anuclear circulating cell bodies that influence a wide variety of physiological functions through their dynamic secretory and adhesion behavior, present an ideal platform for exploring the effects of exogenous phospholipids on membrane phospholipid content and cell function. In this work, a broad range of platelet functions are quantitatively assessed by leveraging a variety of analytical chemistry techniques, including ultraperformance liquid chromatography-tandem electrospray ionization mass spectrometry (UPLC-MS/MS), vasculature-mimicking microfluidic analysis, and single cell carbon-fiber microelectrode amperometry (CFMA). The relative enrichments of phosphatidylserine (PS) and phosphatidylethanolamine (PE) were characterized with UPLC-MS/MS, and the effects of the enrichment of these two phospholipids on both platelet secretory behavior and adhesion were examined. Results show that, in fact, both PS and PE influence platelet adhesion and secretion. PS was enriched dramatically and decreased platelet adhesion as well as secretion from δ-, α-, and lysosomal granules. PE enrichment was moderate and increased secretion from platelet lysosomes. These insights illuminate the critical connection between membrane phospholipid character and platelet behavior, and both the methods and results presented herein are likely translatable to other mammalian cell systems.

Objective— Platelet granule exocytosis serves a central role in hemostasis and thrombosis. Recently, single-cell amperometry has shown that platelet membrane fusion during granule exocytosis results in the formation of a fusion pore that subsequently expands to enable the extrusion of granule contents. However, the molecular mechanisms that control platelet fusion pore expansion and collapse are not known. Methods and Results— We identified dynamin-related protein-1 (Drp1) in platelets and found that an inhibitor of Drp1, mdivi-1, blocked exocytosis of both platelet dense and α-granules. We used single-cell amperometry to monitor serotonin release from individual dense granules and, thereby, measured the effect of Drp1 inhibition on fusion pore dynamics. Inhibition of Drp1 increased spike width and decreased prespike foot events, indicating that Drp1 influences fusion pore formation and expansion. Platelet-mediated thrombus formation in vivo after laser-induced injury of mouse cremaster arterioles was impaired after infusion of mdivi-1. Conclusion— These results demonstrate that inhibition of Drp1 disrupts platelet fusion pore dynamics and indicate that Drp1 can be targeted to control thrombus formation in vivo.

TAK-164 is an antibody-drug conjugate comprising a full-length, fully human IgG1 monoclonal antibody (mAb) directed towards the extracellular domain of guanylyl cyclase C (GCC). The mAb is conjugated using the peptide-linked indolino-benzodiazepine DNA alkylator DGN549 (also known as IGN-P1). TAK-164 demonstrates efficient binding to antigen-expressing cells in vitro, and GCC-dependent uptake that results in cytotoxicity. Following a single administration of TAK-164 in female SCID mice bearing GCC-positive xenografts, the pharmacokinetics of the conjugated antibody component of TAK-164 displayed low plasma clearance. Exposure to the ADC resulted in a dose- and time-dependent increase in DNA damage as measured by the pharmacodynamic marker pH2A.X in GCC-positive tumor-bearing xenografts. To further these observations, antitumor activity of TAK-164 was evaluated in multiple GCC-positive patient-derived xenografts. In these studies, a single intravenous administration of TAK-164 resulted in durable antitumor activity. Furthermore, the use of preclinical imaging demonstrated that TAK-164 preferentially accumulates in GCC-positive tumors, and that treatment response can be monitored using FDG-PET. These promising preclinical data warrant advancement of this ADC to clinical evaluation. Citation Format: Adnan O. Abu-Yousif, Bret M. Bannerman, Donna Cvet, Melissa Gallery, Michelle L. Ganno, Michael D. Smith, Katharine C. Lai, Thomas A. Keating, Jayaprakasam Bolleddula, Bradley Stringer, Mark G. Qian, Afrand Kamali, Kurt Eng, Secil Koseoglu, Cindy Q. Xia, O. Petter Veiby. TAK-164, a GCC-targeted antibody-drug conjugate (ADC) for the treatment of colorectal cancers and other GI malignancies [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2017 Oct 26-30; Philadelphia, PA. Philadelphia (PA): AACR; Mol Cancer Ther 2018;17(1 Suppl):Abstract nr B120.

While ion-selective electrodes (ISEs) based on anion ionophores with guanidine groups were reported in the past, their response mechanism was poorly understood. In this study, an ionophore with one guanidine group was prepared as a model compound, and the chloride and sulfate responses of ISE membranes containing this ionophore were investigated. The electrodes exhibit Nernstian responses resulting from a neutral or charged ionophore mode when added cationic or anionic sites are used, respectively. Based on this observation, it might be expected that membranes without added ionic sites exhibit Nernstian responses according to a charged carrier mechanism. However, experimental results show an apparently ‘two–thirds Nernstian’ equilibrium response, which can be explained on the basis of the phase boundary model.

International audience; Platelet activation results in profound morphologic changes accompanied by release of granule contents. Recent evidence indicates that fusion of granules with the plasma membrane during activation provides auxiliary membrane to cover growing actin structures. Yet little is known about how membrane fusion is coupled with actin reorganization. Vesicle-associated membrane protein (VAMP)-7 is found on platelet vesicles and possesses an N-terminal longin domain capable of linking exocytosis to cytoskeletal remodeling. We have evaluated platelets from VAMP-7(-/-) mice to determine whether this VAMP isoform contributes to granule release and platelet spreading. VAMP-7(-/-) platelets demonstrated a partial defect in dense granule exocytosis and impaired aggregation. α Granule exocytosis from VAMP-7(-/-) platelets was diminished both in vitro and in vivo during thrombus formation. Consistent with a role of VAMP-7 in cytoskeletal remodeling, spreading on matrices was decreased in VAMP-7(-/-) platelets compared to wild-type controls. Immunoprecipitation of VAMP-7 revealed an association with VPS9-domain ankyrin repeat protein (VARP), an adaptor protein that interacts with both membrane-bound and cytoskeleton proteins and with Arp2/3. VAMP-7, VARP, and Arp2/3 localized to the platelet periphery during spreading. These studies demonstrate that VAMP-7 participates in both platelet granule secretion and spreading and suggest a mechanism whereby VAMP-7 links granule exocytosis with actin reorganization.

To inhibit pathological thrombus formation without impairing hemostasis is the holy grail of anticoagulant therapy. Recent data from animal models have indicated that factor XII (FXII) may be a promising new antithrombotic target that is particularly intriguing due to the longstanding clinical observation that severe congenital FXII deficiency is not associated with a bleeding diathesis in humans. FXII is thought to participate in thrombus formation after being activated in high shear arterial environments. FXIIa then initiates downstream activation of the contact pathway, culminating in thrombin generation. However, the relevant cell surface for FXII activation remains unclear. Here we compare the role of platelets versus endothelial cells in FXII activation and study the function of surface phospholipids in this process. To explore the effect of FXII inhibition on thrombus formation in vivo, we used antibody X210-C01, a novel human IgG1 developed using phage-display technology that blocks both mouse and human FXIIa. Using a mouse laser injury model of arterial thrombosis, we showed that X210-C01 inhibited both fibrin formation and platelet accumulation at sites of vascular injury. Plasma removed from animals after completion of these experiments was used to quantify the concentrations of X210-C01 achieved in vivo at a given dose. FXII inhibition was somewhat more potent in preventing platelet accumulation (IC50 dose = 27 mg/kg, R2=0.93) than fibrin formation (IC50 dose = 43 mg/kg, R2=0.95). Importantly, treatment with X210-C01 at 100 mg/kg did not prolong bleeding times or increase total blood loss in a tail bleeding assay. To evaluate the mechanism underlying our in vivo observations, we studied the differential role of FXII in thrombin generation by stimulated platelets and endothelium. X210-C01 did not globally inhibit SFLLRN-induced platelet aggregation or granule release. We next performed a fluorogenic thrombin generation assay (TGA) using human platelets treated with the peptide agonist SFLLRN. X210-C01 inhibited platelet-based thrombin generation in a dose-dependent fashion, whereas anti-tissue factor (TF) and anti-factor VIIa (FVIIa) antibodies did not. By contrast, in a similar TGA using SFLLRN-stimulated endothelial cells, X210-C01 had no effect, while anti-TF antibodies abrogated thrombin generation. These results indicate that stimulated endothelium generates thrombin by a mechanism distinct from that of platelets. FXII is known to be activated in vitro by anionic surfaces. Because phosphatidylserine (PS) is a negatively-charged phospholipid expressed on the surface of stimulated platelets, we reasoned that PS may serve as the platelet-based activator of FXII. To test this hypothesis, we used lactadherin, a potent and specific inhibitor of PS, in the platelet-based TGA and showed that PS blockade inhibited platelet-based thrombin generation at concentrations as low as 10 nM. We then used a chromogenic FXIIa activity assay to test the ability of PS-containing liposomes to activate FXII. Liposomes containing 80% phosphatidylcholine (PC) and 20% PS (PC-PS 80/20) failed to activate FXII at concentrations as high as 100 µM. In this assay, SFLLRN-stimulated platelets led to significantly greater FXII activation than either resting platelets or the PC-PS 80/20 liposomes tested. In summary, we have made the unexpected finding that thrombin generation on the surface of stimulated platelets proceeds by a FXIIa-dependent pathway and does not require FVIIa or TF. By contrast, thrombin generation on endothelium requires TF but not FXIIa. Additionally, surface PS is necessary but not sufficient for platelet-based FXII activation and thrombin generation, pointing to the involvement of a second platelet component. Further studies will be directed towards investigating the in vivo role of platelet-based FXII activation in arterial thrombus formation. Disclosures Mason: Shire Pharmaceuticals: Employment. Kenniston:Shire Pharmaceuticals: Employment.

Secreted bioactive lipids play critical roles in cell-to-cell communication and have been implicated in inflammatory immune responses such as anaphylaxis, vasodilation, and bronchoconstriction. Analysis of secreted bioactive lipids can be challenging due to their relatively short lifetimes and structural diversity. Herein, a method has been developed using ultraperformance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) to quantify five cell-secreted, structurally and functionally diverse bioactive lipids (PGD2, LTC4, LTD4, LTE4, PAF) that play roles in inflammation. Sample analysis time is 5 min, and isotopically labeled internal standards are used for quantification. This method was applied to an immortal secretory cell line (RBL-2H3), a heterogeneous primary cell culture containing peritoneal mast cells, and murine platelets. In RBL cell supernatant samples, intrasample precisions ranged from 7.32-21.6%, averaging 17.0%, and spike recoveries in cell supernatant matrices ranged from 88.0-107%, averaging 97.0%. Calibration curves were linear from 10 ng mL(-1) to 250 ng mL(-1), and limits of detection ranged from 0.0348 ng mL(-1) to 0.803 ng mL(-1). This method was applied to the determination of lipid secretion from mast cells and platelets, demonstrating broad applicability for lipid measurement in primary culture biological systems.

This review will provide an overview of several recent advances in the field of platelet granule biology.The past few years have witnessed a substantial evolution in our knowledge of platelet granules based on a number of discoveries and new experimental approaches. This article will cover recent studies in five areas. First, the vesicle trafficking pathways responsible for α-granule formation are beginning to be assembled as a result of the characterization of patients with α-granule deficiencies. Second, a revision of our understanding of which SNARE isoforms mediate platelet granule exocytosis has occurred following evaluation of patients with defects in platelet granule exocytosis and the generation of mice lacking specific SNAREs. Third, investigators have begun to establish how cargos are segregated among α-granules and determine whether or not different α-granule subpopulations exist in platelets. Fourth, an unanticipated role for α-granules in platelet spreading has been identified. Fifth, single-cell amperometry has revealed secretion kinetics with submillisecond temporal resolution enabling evaluation of the molecular control of the platelet fusion pore.These new observations reveal a previously unappreciated complexity to platelet granule formation and exocytosis and challenge our earlier notions of how these granules are organized within platelets and contribute to the multitude of physiological activities in which platelets function.

The use of carbon as an electrode material was first popularized by Ralph Adams in the 1950s.1 Since its inception, carbon has been a popular choice for electrochemical applications, as carbon electrodes demonstrate a wide working potential window, stable background currents, low noise, and high sensitivity. The development of carbon electrodes with active surfaces on the scale of several microns introduces several additional advantages. For voltammetry experiments, microelectrodes offer the ability to observe diffusion-limited currents at high scan rates and thus provide very fast time resolution.2 The small surface area of microelectrodes enables the use of high scan rates with relatively low background currents. Because of the small number of molecules that must be detected at the single cell level, however, most electrochemical measurements made with carbon microelectrodes produce currents in the nanoampere or picoampere range, depending on the method used.2 These current levels require electrodes that are both sensitive and exhibit low levels of noise, and carbon microelectrodes meet both of these criteria. Conveniently, using microelectrodes requires only a two-electrode system, as such low currents render the use of an auxiliary electrode unnecessary. Perhaps most importantly, microelectrodes provide much greater spatial resolution, and thus permit electrochemical measurements to be made from single cells.3 Although other forms of carbon can also be used to fabricate microelectrodes, carbon-fiber microelectrodes are often favored because they can be fabricated in large quantities and are relatively inexpensive. R. Mark Wightman and coworkers first demonstrated the utility of carbon-fiber microelectrodes for the detection of exocytosis from single cells in 1990.4 Carbon-fiber microelectrodes of different geometries are used depending on the intended application; for single-cell electrochemical techniques, beveled and polished disc microelectrodes are most widely used. While Wightman's initial demonstration explored exocytosis from chromaffin cells, the cells responsible for the release of electroactive adrenaline, exocytosis is highly conserved across several cell types including neurons, chromaffin cells, platelets, and a variety of immune cells such as mast cells and macrophages. Accordingly, the technique has the capacity to reveal critical mechanistic details about exocytosis in these cell types, providing new insight into the fundamental behavior of normal and dysfunctional secretory cells.

Platelets are traditionally recognized as critical cells in hemostasis and thrombosis but have also recently been identified to play a significant role in many diseases, including bacterial infections, cancer, and allergic asthma. Platelets store and release important messenger molecules via exocytosis from two populations of granules (α- granules with adhesive protein species and δ- granules with small molecule/ion species). However, very little known about how chemical messengers are stored in these granules and the driving forces for secretion into the blood stream, thus limiting the development of new therapeutic approaches to manage the role of platelets in many physiological events. Carbon-fiber microelectrode amperometry (CFMA) enables both quantal and kinetic analysis of the exocytotic event of single cell with sub-ms time resolution but is limited to measuring electroactive species from δ-granules. Localized surface plasmon resonance (LSPR) spectroscopy is a complementary tool that offers an outstanding sensing ability by measuring the extremely small changes in the sensing media. This work aims to overcome the gap in fundamental knowledge about platelet granular storage and secretion by detecting serotonin secretion from δ- granules using CFMA and Platelet factor 4 (PF4) secretion from α- granules using LSPR. These measurements are performed while exposing platelets to different stimulants, including ionomycin, adenosine diphosphate (ADP), and thrombin. Significant differences are seen in both α- and δ-granule secretion with varied stimulant exposure, and these combined measurements yield biophysical insight into the mechanism behind these secretory behaviors.

Cell–cell communication is often achieved via granular exocytosis, as in neurons during synaptic transmission or neuroendocrine cells during blood hormone control. Owing to its critical role in membrane properties and SNARE function, cholesterol is expected to play an important role in the highly conserved process of exocytosis. In this work, membrane cholesterol concentration is systematically varied in primary culture mouse chromaffin cells, and the change in secretion behavior of distinct vesicle pools as well as pool recovery following stimulation is measured using carbon-fiber microelectrode amperometry. Amperometric traces obtained from activation of the younger readily releasable and slowly releasable pool (RRP/SRP) vesicles at depleted cholesterol levels showed fewer sustained fusion pore features (6.1 ± 1.1% of spikes compared with 11.2 ± 1.0% for control), revealing that cholesterol content influences fusion pore formation and stability during exocytosis. Moreover, subsequent stimulation of RRP/SRP vesicles showed that cellular cholesterol level influences both the quantal recovery and kinetics of the later release events. Finally, diverging effects of cholesterol on RRP and the older reserve pool vesicle release suggest two different mechanisms for the release of these two vesicular pools.

Fluorous media are the least polar and polarizable condensed phases known. Their use as membrane materials considerably increases the selectivity and robustness of ion-selective electrodes (ISEs). In this research, a fluorous amorphous perfluoropolymer was used for the first time as a matrix for an ISE membrane. Electrodes for pH measurements with membranes composed of poly[4,5-difluoro-2,2-bis(trifluoromethyl)-1,3-dioxole]-co-poly(tetrafluoroethylene) (87% dioxole monomer content; known as Teflon AF2400) as polymer matrix, a linear perfluorooligoether as plasticizer, sodium tetrakis[3,5-bis(perfluorohexyl)phenyl]borate providing for ionic sites, and bis[(perfluorooctyl)propyl]-2,2,2-trifluoroethylamine as H+ ionophore were investigated. All electrodes had excellent potentiometric selectivities, showed Nernstian responses to H+ over a wide pH range, exhibited enhanced mechanical stability, and maintained their selectivity over at least 4 weeks. For membranes of low ionophore concentration, the polymer affected the sensor selectivity noticeably at polymer concentrations exceeding 15%. Also, the membrane resistance increased quite strongly at high polymer concentrations, which cannot be explained by the Mackie-Meares obstruction model. The selectivities and resistances depend on the polymer concentration because of a functional group associated with Teflon AF2400, with a concentration of one functional group per 854 monomer units of the polymer. In the fluorous environment of these membranes, this functional group binds to Na+, K+, Ca2+, and the unprotonated ionophore with binding constants of 10(3.5), 10(1.8), 10(6.8), and 10(4.4) M(-1), respectively. Potentiometric and spectroscopic evidence indicates that these functional groups are COOH groups formed by the hydrolysis of carboxylic acid fluoride (COF) groups originally present in Teflon AF2400. The use of higher ionophore concentrations removes the undesirable effect of these COOH groups almost completely. Alternatively, the C(=O)F groups can be eliminated chemically, or they can be used to readily introduce new functionalities.

Platelet granule secretion is important not only for hemostasis and thrombosis, but also for a variety of physiological processes including inflammation, angiogenesis and malignancy. Vesicle Associated Membrane Proteins (VAMPs) are a group of v-SNARE proteins resident on the platelet granule surface that participate in granule secretion. Platelets contain several VAMP isoforms including VAMP-2, VAMP-3, VAMP-7, and VAMP-8. VAMP-7 is unique in that it contains an N-terminal profilin-like longin domain. Previous work by our group demonstrated spatial segregation of granules expressing different VAMPs during platelet spreading. Granules expressing VAMP-3 and VAMP-8 localized to the granulomere of spreading platelets, while those expressing VAMP-7 moved towards the periphery. Based on this observation, we proposed that VAMP-7+ granules move to the periphery of the spreading platelet to add membrane to growing actin structures. To assess this hypothesis, platelets from VAMP-7 null mice were used to analyze the role of VAMP-7 in platelet spreading, aggregation and secretion. VAMP-7 null platelets were normal in size, shape, and number. When compared to wild-type platelets, VAMP-7 null platelets did not show any defects in aggregation upon exposure to increasing doses of the PAR4 agonist peptide, AYPGKF, or collagen. In contrast, the surface area of VAMP-7 null platelets following 15 min of spreading on poly-L-lysine was only 51% that of wild-type of platelets (P < 0.05). To assess mechanisms of the movement of VAMP-7 to the platelet periphery, the association of VAMP-7 to the Triton X-100-insoluble platelet cytoskeleton was evaluated and results showed that VAMP-7 associated with the actin cytoskeleton. Moreover, VAMP-7 null platelets showed impaired P-selectin surface expression and PF4 secretion at low concentrations of AYPGKF. TIMP-2 and VEGF localize to VAMP-7 expressing granules in the periphery of spread platelets. We therefore evaluated the secretion of TIMP-2 and VEGF from VAMP-7 null platelets. Secretion of TIMP-2 and VEGF was reduced even at saturating doses of agonist (300 mM AYPGKF). To examine the role of VAMP-7 in a-granule exocytosis during platelet activation in vivo, PF4 release was monitored following laser-induced injury of cremaster arterioles. Platelet accumulation at sites of laser injury was identical in wild-type and VAMP-7 null mice. In wild-type mice, PF4 was secreted by activated platelets and bound back to activated endothelium and platelets producing a localized concentration of PF4 that accumulated over 15 min following injury. PF4 release from platelets lacking VAMP-7 was decreased to 47% of that of control. These results demonstrate that VAMP-7 interacts with the actin cytoskeleton and functions selectively in a-granule exocytosis. VAMP-7 associates with the actin cytoskeleton and functions during platelet spreading, adding further support to the premise that membrane fusion occurring during granule secretion is an essential component of normal platelet spreading. This VAMP-7 mediated, actin-dependent mechanism of secretion is not important for platelet thrombus formation, but rather functions in the release of particular granular contents, such as PF4, at sites of vascular injury. Disclosures: No relevant conflicts of interest to declare.

Abstract 361 Platelet granule secretion serves a central role in hemostasis and thrombosis. During platelet secretion, fusion of granule membranes with those of the plasma membrane results in the release of granule contents. Recently electrochemical techniques using single-cell amperometry have shown that platelet membrane fusion results in the formation of a fusion pore. The fusion pore subsequently expands to enable the complete extrusion of granule contents. However, the molecular mechanisms that control platelet fusion pore formation and expansion are not known. To discover novel components of the platelet secretory machinery, we tested >300,000 compounds in a forward chemical genetic screen designed to identify inhibitors of dense granule secretion. A compound, ML160, was found that blocked dense granule release with an IC50 of approximately 0.5 μM. ML160 was also identified in an unrelated high throughput screen designed to detect inhibitors of dynamin-related protein-1 (Drp-1). Although best known as mediators of membrane fission, dynamins also contribute to granule exocytosis by controlling fusion pore expansion. Immunoblot analysis of platelet pellets and supernatants confirmed the presence of Drp-1 in platelets and demonstrated nearly equal distribution between platelet membranes and cytosol. mDivi-1, a well-characterized small molecule inhibitor of Drp-1 that acts outside of the GTP binding site, blocked PAR1-mediated platelet dense granule and α-granule release with an IC50 of approximately 20 μM. mDivi-1 also inhibited granule release induced by the thromboxane receptor agonist U46619, PMA, or Ca2+ ionophore, indicating that Drp-1 acts distally in the secretory pathway. To assess whether Drp-1 functions in platelet fusion pore dynamics, we tested the effect of mDivi-1 on the release of dense granules from rabbit platelets using single-cell amperometry. This technique monitors the release of serotonin from single granules in real-time with sub-millisecond temporal resolution. mDivi-1 exposure (10 μM) retarded each release event, resulting in a prolonged spike width of 23.00 ± 1.702 msec compared to the control value of 14.71 ± 1.194 msec. Although this concentration of mDivi-1 did not change the overall percentage of the fusion pore events or the amount of serotonin released through the fusion pore, it showed a distinct effect on the transition from stable fusion pore to maximal fusion pore dilation (% foot= 17.46 ± 1.809%, 9.464 ± 2.014% for control and mDivi-1 conditions, respectively). Evaluation of fluorescein-dextran incorporation into activated platelets by fluorescence microscopy enabled visualization of fusion pore dynamics and confirmed the effect of mDivi-1 on fusion pore expansion. To assess whether Drp-1 participates in platelet function in vivo, we determined the effect of mDivi-1 on thrombus formation following laser-induced injury of mouse cremaster arterioles. mDivi-1 inhibited platelet accumulation at the site of vascular injury by 74%. In contrast, mDivi-1 had no significant effect on fibrin formation under the same conditions. These results identify Drp-1 in platelets, demonstrate a role for Drp-1 in fusion pore dynamics, and indicate that pharmacological regulation of platelet fusion pore expansion can be used to control thrombus formation in vivo. Disclosures: No relevant conflicts of interest to declare.