Juneteenth in STEMM and the barriers to equitable science

We are 52 Black scientists. Here, we establish the context of Juneteenth in STEMM and discuss the barriers Black scientists face, the struggles they endure, and the lack of recognition they receive. We review racism's history in science and provide institutional-level solutions to reduce the burdens on Black scientists

Transcriptomic Analysis Reveals Erythroferrone Modulates BMP6 Signaling Pathways Involved in Iron Homeostasis and Metabolism

Abstract Introduction: Erythroferrone (ERFE), the recently identified erythroid regulator of iron absorption, is a member of the C1q/TNF-related protein (CTRP) family. It is produced in erythroblasts in response to an increased erythropoietic drive, downregulates hepcidin expression and thereby promotes intestinal iron absorption and mobilization. The levels of Erfe are inappropriately high under conditions of ineffective erythropoiesis in inherited anemias such as thalassemia and congenital dyserythropoietic anemias. However, the mechanism of Erfe mediated Hepcidin inhibition remains unknown. Here, we use transcriptomic analysis of the human hepatoma cell line, Huh7 to identify genes and pathways involved in hepatocyte response to Erfe. Methods: Huh7 cells were seeded in a 6-well cell culture plate. Twenty four hours later, the cells were washed with PBS, and treated with a recombinant murine ERFE monomeric Fc (mEFRE-FC, 10µg/ml) protein or a control IgG (10 ug/ml) for 1h, 6h or 24h in growth media. RNA was isolated, followed by RNA quantification and quality assessment using a 2100 Agilent Bioanalyzer. ERFE is known to regulate Hepcidin antimicrobial peptide (HAMP) transcription and a treatment effect on HAMP expression was demonstrated by qPCR prior to sequencing. A total of 27 mRNA sequencing libraries were constructed from 1ug of human total RNA with the Illumina TruSeq Stranded mRNA Sample Prep protocol and single-end 75 bp reads were generated on an Illumina NextSeq 500. DESeq2 statistical package was used for differential expression testing. Multiple comparisons were adjusted for using a false discovery rate of 5%. Additionally, results were filtered to consider only those genes that demonstrated a fold-change point estimate &gt;2 in either direction. Pathway analysis was performed using the Biological Process gene sets from the Gene Ontology annotation. Results: Compared to control treated Huh7 cells, 3 transcripts were differentially regulated following a one hour treatment with mERFE-Fc, ID1 (Log2 fold-change -1.55, q-value 4.99E-09), BPIFB2 (Log2 fold-change -1.88, q-value 0.036) and ANO1 (Log2 fold-change -1.43, q-value 0.019). A larger number of genes were differentially regulated with longer treatments, 32 genes at 6h and 828 genes at 24h. Among selected genes differentially expressed at 24h, between control treated and Erfe treated cells, we observed a significant reduction in expression of genes known to be directly upregulated by bone morphogenetic proteins (BMPs), including DNA binding protein inhibitor ID1(ID1), DNA binding protein inhibitor ID2 (ID2), DNA binding protein inhibitor ID3 (ID3) and HAMP. BMP6 has been demonstrated to regulate several biological processes such as iron metabolism in the liver, adipogenesis, and insulin sensitization. Interestingly, we also observed upregulation of SLC27A1 (or FATP1, fatty acid transporter, Log2 fold-change 2.27, q-value 0.003), GDNF (Log2 fold-change 2.57, q-value 2.4E-05), GIPR (Log2 fold-change 2.53, q-value 7.9E-07) upon treatment with Erfe at 24h. Previous work has demonstrated that all of these genes are downregulated by BMP6 treatment 1-3. Pathway analysis indicated a number of genes differentially regulated in the GOBP (gene ontology biological processes) iron ion homeostasis pathway, BMP signaling or TGF-β receptor signaling pathway. This assessment further indicated downregulation of cellular hormone levels and metabolic processes and ion homeostasis as biological processes impacted by erythroferrone. Conclusions: Significant differences in gene expression occur in hepatocytes upon interaction with erythroferrone. Many genes in the iron homeostasis, BMP6 signaling metabolic processes were also differentially regulated. This further supports the mechanism of ERFE on iron homeostasis. Disclosures Jasuja: Pfizer: Employment. Sawant:Pfizer: Employment. Pittman:Pfizer: Employment. Quan:Pfizer: Employment. </jats:sec

An Antibody to Tissue Factor Pathway Inhibitor (TFPI) Restores Hemostasis after the Onset of Bleeding in Hemophilic a Mouse Injury Models

Abstract Hemophilia is a family of rare bleeding disorders characterized by inadequate levels of intrinsic coagulation factors, Factor VIII (FVIII) in hemophilia A and Factor IX (FIX) in hemophilia B. This leads to insufficient thrombin generation for the conversion of fibrinogen to fibrin for the development of a stable clot. Replacement factor therapies are provided as a prophylactic treatment to prevent bleeds or for on-demand treatment to treat an active bleed. Some patients develop inhibitory antibodies making them refractory to treatment. Although hemophilia patients have defects in the intrinsic pathway, the extrinsic pathway remains intact. An alternative approach to therapy would be to augment the extrinsic tissue factor pathway. TFPI is a Kunitz domain type inhibitor that negatively regulates thrombin generation within the extrinsic pathway of coagulation by rapidly inactivating the protease functions of Factor Xa and the Factor VIIa/Tissue Factor complex. PF-06741086 is a fully human monoclonal antibody directed against TFPI. Here, we investigated the activity of PF-06741086 in controlling bleeding in hemophilic mouse injury models when administered after a bleeding injury. PF-06741086 restores hemostasis prior to the onset of an injury. As a model for on demand treatment of bleeds, the effect of PF-06741086 on restoring hemostasis in hemophilia A mice following the induction of a severe bleed was monitored. PF-06741086 (1, 3, and 6 mg/kg), recombinant FVIII (200 U/mg) or vehicle were administered to mice, at 2 minutes into an active bleed after the 3 mm tail transection. A dose-dependent decrease in blood loss was observed with the infusion of PF-06741086, 51 % (3 mg/kg), and 76 % (6 mg/kg) compared to vehicle treated mice. A decrease in blood loss (81 %) was also observed for mice receiving recombinant FVIII, compared to vehicle treated mice. The magnitude of the effect in mice that received PF-06741086 (6 mg/kg) at 2 minutes post injury was similar to hemostatic effects of recombinant FVIII administered 2 minutes after the onset of bleeding. In a second model, the cremaster microvessel laser injury model was used to investigate the hemostatic effect of PF-06741086 in hemophilia A mice after the induction of a vessel injury. In each individual mouse, an injury was made without administration of PF-06741086 and data was recorded for platelet accumulation and fibrin generation. A second laser injury was made in the same mouse and a single intravenous dose of PF-06741086 was infused at 6 mg/kg immediately following the laser injury. When compared to untreated mice, enhanced platelet accumulation and fibrin deposition at the site of injury was observed when PF-06741086 was administered in hemophilia A mice during an ongoing bleed induced with laser injury. In summary, PF-06741086, an inhibitor of TFPI, restores hemostasis in on demand hemophilia mouse injury models when administered after the onset of a bleeding injury. PF 06741086 is being developed for the treatment of hemophilia A and hemophilia B, with and without inhibitors. All experiments were within guidelines and were reviewed and approved by Pfizer Institutional Animal Care and use Committee. Disclosures Jasuja: Pfizer: Employment. Barakat:Pfizer: Employment. Murphy:Pfizer: Employment. Pittman:Pfizer: Employment. </jats:sec

Efficacy of Anti-TFPI Antibody PF-06741086 Alone and in Combination with Activated Prothrombin Complex Concentrates in Mouse Hemophilia a Bleeding Model

Abstract BACKGROUND: Tissue Factor Pathway inhibitor (TFPI) is a plasma serine protease inhibitor that modulates the initiation of coagulation by directly binding and inhibiting the Tissue Factor (TF)/Factor VIIa/Factor Xa complex. TFPI is a multi-Kunitz domain protein that directly binds to and inhibits both activated Factor Xa (FXa) and FVIIa. Blocking TFPI can act as a bypass therapy by facilitating hemostasis initiated by tissue factor/FVIIa, thereby, compensating for loss of Factor VIII or Factor IX (in hemophilia A or B). PF-06741086, a fully human antibody engineered to inhibit TFPI, exhibits broad cross reactivity to TFPI from numerous species, including mouse. PF-06741086 is being developed as a potential treatment for bleeding disorders including hemophilia A and hemophilia B with and without inhibitors. aPCC (activated Prothrombin complex concentrates or FEIBA, Factor Eight Inhibitor Bypass Agent) is a bypass agent for to control bleed in Hemophilia patients with inhibitors. Since it is a plasma-derived concentrate containing various prothrombin complex coagulation factors in their enzymatic or zymogen form, it is possible that FEIBA could potentially impact the activity of PF-06741086. AIMS: Here, we directly compare the hemostatic effect of PF-06741086 alone, and in combination with aPCC in Hemophilia A mouse model using severe tail vein transection. METHODS: Male hemophilia A mice were dosed with a single intravenous dose of PF-06741086 (0.5, 1, 2 or 6 mg/kg) 30 minutes prior to a 3mm tail clip, or aPCC (50, 100 or 200 U/kg) was administered 5 minutes before the tail clip. Mice were also treated with a combined dose of 0.5 mg/kg anti-TFPI PF-06741086 and aPCC at 50, 100 or 200 U/kg. Blood was collected for 10 minutes and quantified against a standard curve of hemoglobin as volume blood loss. RESULTS: PF-06741086 demonstrated a dose dependent response in improving hemostasis in Hemophilia A mice after tail clip. PF-06741086 was able to restore hemostasis at 1 mg/kg (49%), and higher doses further improved hemostasis at 2 mg/kg (63%), and 6 mg/kg (78%). aPCC also demonstrated a dose dependent reduction in blood loss and improved hemostasis with all tested doses of 50 U/kg (25%), 100 U/kg (23%) and 200 U/kg (66%). At a dose of 0.5 mg/kg, PF-06741086 did not show any improvement in hemostasis over vehicle control. We used this dose for all combination studies with aPCC. Combined use of low dose PF-06741086 (0.5 mg/kg) and 100 U/kg aPCC shows a trend towards improvement in hemostasis compared to either drug alone. A higher dose of aPCC (200 U/kg) combined with low dose PF-06741086 (0.5 mg/kg) significantly reduces blood loss (86%) in Hemophilia A mice in tail clip model compared to saline, TFPI or aPCC alone used at the same dose of 0.5 mg/kg or 200 U/kg respectively. CONCLUSIONS: Prophylactic administration of PF-06741086 exhibits a dose response and improves hemostasis in an injury model in Hemophilia A mice. The addition of aPCC alone restores hemostasis at 200 U/kg and this effect was enhanced in combination with PF-06741086 in this mouse model. Disclosures Barakat: Pfizer: Employment. Jasuja:Pfizer: Employment. Murphy:Pfizer: Employment. Pittman:Pfizer: Employment. </jats:sec

A Neutralizing Monoclonal Antibody (PF-06741086) Against Tissue Factor Pathway Inhibitor in Combination with Activated Prothrombin Complex Concentrate Increases Hemostasis in Inhibitor Hemophilia Plasmas without Excessive Thrombin Generation

Abstract Hemophilia A and B are hereditary bleeding disorders caused by intrinsic coagulation pathway deficiencies of Factor VIII or Factor IX, respectively. Tissue factor pathway inhibitor (TFPI) is a Kunitz-type serine protease inhibitor that negatively regulates thrombin generation within the extrinsic pathway of coagulation. PF-06741086 is a fully human monoclonal antibody which binds the Kunitz-2 domain and neutralizes the inhibitory activity of human tissue factor pathway inhibitor and is currently under development as a potential prophylactic treatment to prevent bleeding episodes in hemophilia A and hemophilia B patients with and without inhibitors. Activated prothrombin complex concentrate (aPCC) is used as bypass treatment for the resolution of bleeding in some hemophilia patients with inhibitors. Hemophilia inhibitor patients receiving PF-06741086 have a possibility to also receive treatment with aPCC. The aim of the current study was to assess the potential additive effect of PF-06741086 with aPCC added in vitro to Hemophilia A and B inhibitor plasmas using a thrombin generation assay (TGA). Thrombin generation in the presence of 1 pM tissue factor and 4 µM phospholipid, was measured using the calibrated automated thrombogram (CAT) system in citrated platelet poor hemophilia A inhibitor (88-160 Bethesda Units) donor plasma or hemophilia B inhibitor (FIX immune-depleted and spiked with FIX neutralizing antibody, 14 Bethesda Units) plasma following the addition of PF-06741086 or aPCC (FEIBA) either alone or in combination. All donors had less than 1% coagulation factor activity. Non-hemophilic plasma from healthy donors alone or spiked in vitro with 16 µg/mL of PF-06741086 was also included in the analysis. Non-hemophilic plasma would have the full complement of coagulation factors. Dose-dependent increases in peak thrombin were observed with the addition of aPCC alone or PF-06741086 alone to the hemophilia plasmas. For combination studies, the aPCC concentration of 1 Unit/mL was selected to correspond to plasma levels that could be achieved clinically post-dosing. The concentration of PF-06741086 at 16µg/mL in these studies was chosen to approximate the Cmax concentration following a single 300 mg subcutaneous dose. Both PF-06741086 (16 µg/mL) and aPCC (1 Unit/mL) decreased the lag time in hemophilia plasma, however, there was not an additive decrease in the lag time with the combination of PF-06741086 and aPCC. The addition of PF-06741086 in combination with aPCC to hemophilia plasma resulted in an increase in thrombin generation including a higher peak thrombin concentration compared to the addition of either alone, but was within the range reported in studies for non-hemophilic normal plasma. To summarize, the addition of aPCC (1 Unit/mL) in combination with PF-06741086 (16µg/mL) in vitro resulted in increased thrombin generation in hemophilia A and hemophilia B inhibitor plasmas without inducing excessive coagulation. Disclosures Rakhe: Pfizer: Employment. Bowley:Pfizer: Employment. Murphy:Pfizer: Employment. Pittman:Pfizer: Employment. </jats:sec