ALBERT

Description: Background: The group of rare hereditary anemias includes a large variety of intrinsic defects of the red blood cell, as well as erythropoiesis. They include hemolytic anemias (e.g. enzyme deficiencies), hemoglobinopathies, hypoplastic anemias (e.g. Diamond-Blackfan Anemia, DBA), and dyserythropoietic anemias. As a result of the rapid developments in genetic testing and the subsequent increased knowledge of molecular defects underlying hereditary anemias, our understanding of the pathophysiology of rare anemias has increased during the last decade. However, in a substantial number of patients, the clinical phenotype does not fit classical criteria of a disease, response to therapy is less than expected, or a molecular defect cannot be found. In addition, in patients with well-described molecular defects, there is often no clear genotype-phenotype correlation. In order to better understand the underlying pathophysiological mechanisms driving ineffective erythropoiesis in patients and to improve their classification and clinical evaluation, novel functional tests are needed. Metabolomics is the large-scale, unbiased study of metabolites and their interactions within a biological system, directly reflecting the underlying biochemical activity and state of cells. Metabolomics can be used to identify novel disease biomarkers, study deregulated cellular pathways, and to determine the cellular responses to therapeutic interventions. In this study we demonstrate that dried blood spots (DBS) can be used as a minimal invasive and validated technical approach to perform large scale metabolomics in a variety of rare hereditary anemias. Methods: DBS samples from 〉100 patients suffering from a variety of rare anemiaswere collected during regular hospital visits. Quantification of metabolites was performed by direct infusion high resolution mass spectrometry (DI-HRMS) followed by an untargetedmetabolomics pipeline. For annotation, the Human Metabolome Database (HMDB) was used. Results were compared with DBS samples of 70 healthy adult controls and 35 pediatric patients negatively screened for metabolic diseases Results: For each patient sample, Z-scores were calculated for all mass peaks annotated with metabolites (HMDB, 3930). Mass peak, intensity and corresponding Z-scores were compared with two distinct groups of controls (∆Z-scores): pediatric patients who were screened for metabolic diseases but were found negative, and healthy adult controls. For data interpretation, two strategies were used. First, by untargeted statistical analysis in Metabo-analyst, we identified metabolites (and/or isomers) that showed either increased or decreased intensity. For the second strategy we specifically focused on red blood cell metabolic pathways, including glycolysis, the pentose phosphate pathway, ascorbate and glutathione metabolism, arginine and polyamine metabolism, and erythrocyte membrane turnover and transport. We corrected for a potential hematocrit effect and performed subgroup analyses correcting for reticulocyte counts. Our preliminary data indicate potential biomarkers for distinct disease entities, including altered polyamine metabolism (DBA, SCD), glycolysis (DBA, HS), and aberrant arginine metabolism (SCD) (Figure 1). Further in-depth pathway analyses, and targeted validation of biomarker profiles are currently being performed. Conclusion: Untargeted metabolomics using dried blood spots provides a novel functional tool to identify disease biomarkers and common and distinct deregulated cellular pathways. This will improve diagnostic evaluation and clinical management of patients with rare hereditary anemias, contribute to a better understanding of disease pathophysiology, and aid in the development of therapeutic strategies. Disclosures van Beers: Agios Pharmaceuticals, Inc.: Membership on an entity's Board of Directors or advisory committees, Research Funding; Novartis: Consultancy, Research Funding; Pfizer: Research Funding; RR Mechatronics: Research Funding. van Wijk:RR Mechatronics: Research Funding; Agios Pharmaceuticals: Consultancy, Research Funding.

Description: Chronic myeloid leukemia (CML) patients with the BCR-ABL T315I mutation do not benefit from therapy with currently approved tyrosine kinase inhibitors. Omacetaxine mepesuccinate is a protein synthesis inhibitor that has demonstrated activity in cells harboring the T315I mutation. This phase 2 trial assessed the efficacy of omacetaxine in CML patients with T315I and tyrosine kinase inhibitor failure. Patients received subcutaneous omacetaxine 1.25 mg/m2 twice daily, days 1-14, every 28 days until hematologic response or a maximum of 6 cycles, and then days 1-7 every 28 days as maintenance. Results for patients treated in chronic phase are reported here. Patients (n = 62) received a median of 7 (range, 1-41) cycles. Complete hematologic response was achieved in 48 patients (77%; 95% lower confidence limit, 65%); median response duration was 9.1 months. Fourteen patients (23%; 95% lower confidence limit, 13%) achieved major cytogenetic response, including complete cytogenetic response in 10 (16%). Median progression free-survival was 7.7 months. Grade 3/4 hematologic toxicity included thrombocytopenia (76%), neutropenia (44%), and anemia (39%) and was typically manageable by dose reduction. Nonhematologic adverse events were mostly grade 1/2 and included infection (42%), diarrhea (40%), and nausea (34%). Omacetaxine may provide a safe and effective treatment for CML patients with T315I mutation. This study is registered at www.clinicaltrials.gov as NCT00375219.

Description: Studies of the Syk −/− mouse have implicated spleen tyrosine kinase (Syk), a signaling protein with both kinase and scaffolding activities, in platelet signaling following engagement of GPVI and αIIbβ3 by collagen and fibrinogen, respectively. The present study was designed to determine whether specific inhibition of the kinase activity of Syk, without targeting the Syk scaffolding function, affected in vivo arterial thrombosis. In preliminary experiments, blood from wild-type and Syk−/− mice was perfused through collagen-coated capillaries under arterial shear rates to study ex vivo thrombosis. While blood from wild-type mice formed robust thrombi (37±4.7 μm3/μm2), none was observed in Syk−/− mice. Thrombi intermediate in size (16±3.9 μm3/μm2) developed in Syk+/− mice. To achieve specific pharmacological targeting of the kinase activity of Syk, P142-76, a potent (IC50 = 4 nM) and selective Syk kinase inhibitor was utilized. P142-76 was screened against a broad panel of 139 purified kinases at 50 nM. While Syk kinase was inhibited by 92%, all other kinases retained more than 70% of their activity. In washed human platelets, P142-76 inhibited convulxin (CVX)-induced phosphorylation of LAT (linker for activation of T-cells; IC50 = 111 nM) and intracellular calcium increases (IC50 = 31 nM). The GPVI/Syk-specificity of P142-76 activity was confirmed by its inability to inhibit intracellular calcium increases induced by the PAR1 thrombin receptor agonist TRAP. P142-76 also inhibited CVX-induced aggregation of both human washed platelets (IC50 = 87 nM) and platelet-rich plasma (IC50 = 2.5 μM). Considering the controversial data in respect to the participation of GPVI in arterial thrombosis in murine models, the dependence of arterial thrombosis on Syk function was studied in vivo in pigs. Cross-species activity of P142-76 was confirmed in vitro (CVX-induced PRP aggregation IC50= 350 nM; 5 μM P142-76 completely inhibited thrombosis triggered by collagen in the perfusion chamber assay). At a plasma concentration which abolished ex vivo CVX-induced but not ADP-induced pig platelet aggregation, P142-76 significantly inhibited the deposition of [111In]-labeled platelets in a carotid artery crush swine thrombosis model, without compromising primary hemostasis. % aggregation Swine (n=3) Platelet Deposition % inhibition Plasma Conc (ng/ml) Bleed Time (min) Activated Clotting Time (sec) ADP (20 μM) CVX (250 ng/ml) Control Artery 0 0 3±0.9 133±22 100 100 Treated Artery 76±6.5 1343±304 3.5±0.3 130±13 100 0 To clarify further the contribution of the kinase activity of Syk to arterial thrombosis, effects of P142-76 on human blood were evaluated in real time in the collagen-coated perfusion chamber. Low concentrations of P142-76 (0.3 μM) affected thrombus stability, while increasing concentrations (1–5 μM) delayed and then completely inhibited thrombus formation. Furthermore, P142-76 destabilized pre-formed thrombi, indicating a critical role for Syk in conferring strength to platelet-platelet interactions, i.e. αIIbβ3-mediated cohesion. Our data indicate that the kinase activity of Syk acts in arterial thrombosis through at least two distinct mechanisms. First, Syk kinase confers stability to platelet-platelet interactions downstream of αIIbβ3. Second, it initiates thrombus formation on collagen surfaces. This dual activity of the kinase activity of Syk makes it a preferred target for inhibition of arterial thrombosis, as it does not compromise primary hemostasis.

Description: Heparin-induced thrombocytopenia (HIT), in which patients develop antibodies to complexes formed by heparin and platelet factor 4 (PF4), is the most frequent drug-induced immune thrombocytopenia. Extensive studies in vitro and our previous studies in vivo using a transgenic mouse model of HIT have shown that antibodies reactive with heparin-PF4 complexes lead to FcgRIIa receptor-mediated platelet activation. In this study we investigated whether PRT060318 (PRT318), a novel Syk inhibitor, prevents HIT antibody-mediated platelet activation both in vitro and in vivo. PRT318 at concentrations of 0.3 to 3 μM completely inhibited HIT immune complex (IC)-induced aggregation in both human and transgenic mouse platelets. In the absence of the inhibitor, HIT IC-induced final aggregation was 50–60%. At concentrations of PRT318 less than 0.1 μM, or in the presence of vehicle only, there was no inhibition of aggregation. Aggregation was not inhibited by PRT318 at any concentration when platelets were stimulated by ADP (5–20 μM final concentration). We also show that PRT318 prevents HIT IC-induced thrombocytopenia in vivo using a transgenic mouse HIT model. All mice were treated with KKO, a mouse monoclonal HIT antibody. On days 1 to 4 following antibody injection, the experimental group (n = 13) received orally dosed PRT318 (30 mg/kg body weight) twice a day by gavage while the control group (n = 11) was similarly treated with vehicle only (water). Both experimental and control mice were injected with heparin (1600 U/kg body weight, SQ, once daily). Nadir platelet counts of PRT318-treated mice were significantly higher than control mice (89.8 ± 1.1% of baseline vs. 48.8 ± 6.7%; p = 0.00003). The PRT318 concentration, 2 hrs post dose, in mouse plasma from treated mice was measured as 7.1 μM, consistent with the concentration which blocked FcgRIIa-mediated platelet activation in vitro. These studies demonstrate that Syk inhibitor PRT318 is an active agent in HIT. Figure Figure

Description: Although current antiplatelet therapies provide potent antithrombotic effects, their efficacy is limited by a heightened risk of bleeding and failure to affect vascular remodeling after injury. New lines of research suggest that thrombosis and hemorrhage may be uncoupled at the interface of pathways controlling thrombosis and inflammation. Here, as one remarkable example, studies using a novel and highly selective pharmacologic inhibitor of the spleen tyrosine kinase Syk [PRT060318; 2-((1R,2S)-2-aminocyclohexylamino)-4-(m-tolylamino)pyrimidine-5-carboxamide] coupled with genetic experiments, demonstrate that Syk inhibition ameliorates both the acute and chronic responses to vascular injury without affecting hemostasis. Specifically, lack of Syk (murine radiation chimeras) attenuated shear-induced thrombus formation ex vivo, and PRT060318 strongly inhibited arterial thrombosis in vivo in multiple animal species while having minimal impact on bleeding. Furthermore, leukocyte-platelet–dependent responses to vascular injury, including inflammatory cell recruitment and neointima formation, were markedly inhibited by PRT060318. Thus, Syk controls acute and long-term responses to arterial vascular injury. The therapeutic potential of Syk may be exemplary of a new class of antiatherothrombotic agents that target the interface between thrombosis and inflammation.

Description: Introduction: Philadelphia chromosome-like acute lymphoblastic leukemia (Ph-like ALL) is a high-risk subtype characterized by kinase-activating alterations. One recurrent alteration is the ETV6-NTRK3 fusion, which results in constitutive activation of NTRK3, a member of the neurotrophic receptor kinase family. ETV6-NTRK3 has been identified in a range of malignancies, including breast cancer, pediatric glioma and infantile fibrosarcoma. The oncogenic role of ETV6-NTRK3 in B-cell ALL has not been investigated. The goals of this study were to assess the development of leukemia in genetically engineered models of ETV6-NTRK3, and to investigate efficacy of the specific TRK A, B and C inhibitor, LOXO-101, currently in clinical trials for the treatment of solid tumor patients who harbor NTRK fusions. Methods: For in vitro studies, kinase fusions were expressed in IL3 dependent Ba/F3 cells. To generate a genetically engineered mouse model, we used a previously reported conditional knockin model of Etv6-NTRK3 (Cancer Cell 2007;12:542-558), whereby the human portion of NTRK3 cDNA encoding the tyrosine kinase domain was inserted into exon 6 of the mouse Etv6 locus, downstream of a floxed transcriptional terminator sequence. Expression of the Etv6-NTRK3 protein was accomplished using Cre-recombinase driven by the B-lineage promoter CD19. A patient derived xenograft (PDX) model of ETV6-NTRK3 was established by engrafting primary human ALL cells expressing luciferase into NOD.Cg-Prkdcscid Il2rgtm1Wjl/SzJ (NSG) mice. Phosphoflow cytometry analysis and sensitivity to LOXO-101 was assessed in vitro and in vivo. Results: Etv6-NTRK3/+, CD19-Cre mice developed aggressive disease with 100% penetrance and a median latency of 38 days (n=27). The average body weight of Etv6-NTRK3/+, CD19-Cre mice was significantly reduced compared to age-matched Etv6-NTRK3/+ controls (13.9 vs 20.2g, p

Description: Abstract 5070 Title: A case of Hemophagocytic Lymphohistiocytosis with Acute Myelofibrosis Case report: A 60 year old previously healthy male was admitted for a four week history of non-productive cough, and intermittent fever which was getting worse since 1 week prior to presentation. He also had runny nose and nasal stuffiness 2 months prior to presentation. The patient also had significant weight loss of 30 lbs over the past month. Physical examination was remarkable for a temperature of 38°C. Patient was alert and oriented. No Lymphadenopathy or hepatosplenomegaly was found. Rest of the physical examination was otherwise unremarkable. Laboratory findings on admission: hemoglobin 10.1 g/dl, white blood cell count 1000 /ml (with 58.5% neutrophils, 36.9% lymphocytes 3.4% monocytes), platelet count 111,000 /ml, reticulocyte count 1.3 % and an erythrocyte sedimentation rate 4 mm, total bilirubin 0.8 mg/dL (with 0.4 mg/dL conjugated), alkaline phosphatase 62 U/L, AST 113 U/L, ALT 137 U/L, triglyceride 388 mg/dL, LDH 513 IU/L, ferritin was 〉 2000 ng/mL, PT 12.2 sec PTT 26.8 sec and fibrinogen 154 mg/dl No pathogens were isolated from throat, urine, feces, or blood. H1N1 testing; serological studies for hepatitis A, B, D, and E; Quantiferon test for tuberculosis; Serological studies for Human immunodeficiency virus (HIV), Cytomegalovirus (CMV), Epstein-Bar virus (EBV), parvovirus; antinuclear antibody, rheumatoid factor, lupus anticoagulant were all negative. Peripheral blood examination revealed normochromic-normocytic anemia. There was no evidence of micro-angiopathy or other marrow infiltration. A bone marrow biopsy was performed which showed a hypercellular marrow, with absence of myeloid precursors and decrease in erythroid cells. The predominant components were atypical megakaryocytes, plasma cells and eosinophils Reticulin stain showed marked increase in coarse reticulin. Occasional large histiocytes were visualized with engulfed lymphocytes, polymorphonuclear and red blood cells. Flow cytometry was negative for a myeloproliferative disorder. The platelet and WBC count nadirs were 73,000/mL and 800/mL (53% polymorphonuclear cells) at days 5–7 of admission. He continued to have cytopenias with intermittent febrile episodes despite being on broad spectrum antibiotics and antifungals. Based on pancytopenia, intermittent fever, elevated liver enzymes, very high ferritin level, high triglyceride level and evidence of hemophagocytosis on bone marrow exam a diagnosis of Hemophagocytic Lymphohistiocytosis was made. Patient received IV Ig for 2 days along with high dose steroids with prophylaxis with IV proton pump inhibitors, after which his fever resolved. LDH decreased from a peak of 900 to 300 and his leucopenia resolved with a WBC count of 3,000 /ml 5 days after 1st dose of IV Ig. Patient seemed to be responding very well to the treatment, but he had an episode of massive GI bleed on the fifteenth day of hospitalization with malena and he could not be resuscitated. No autopsy was performed. Discussion: We describe a case of possible secondary Hemophagocytic Lymphohistiocytosis (HLH) with Acute Myelofibrosis. A diagnosis of HLH was made based on the proposed diagnostic criteria, 2009 by Dr. Filipovich. Acute Myelofibrosis was evidenced by marked increase in reticulin stain with absence of splenomegaly or tear drop cells on peripheral blood smear. Viral infection could have been a trigger for HLH in this patient as he had runny nose 2 months before presentation. The patient responded very well to IVIg and high dose steroids as evidenced by an increase in WBC and platelet count, resolution of fever and decrease in LDH. HLH is a rare and potentially fatal condition with excessive activation of macrophages and T cells with an overwhelming systemic inflammatory reaction. Viruses are implicated as the most common triggers for secondary HLH. Our case adds to the literature on the rare disease and will help in understanding the disease better. Disclosures: No relevant conflicts of interest to declare.

Description: Heparin-induced thrombocytopenia (HIT) is a major cause of morbidity and mortality resulting from the associated thrombosis. Extensive studies using our transgenic mouse model of HIT have shown that antibodies reactive with heparin-platelet factor 4 complexes lead to FcγRIIA-mediated platelet activation in vitro as well as thrombocytopenia and thrombosis in vivo. We tested PRT-060318 (PRT318), a novel selective inhibitor of the tyrosine kinase Syk, as an approach to HIT treatment. PRT318 completely inhibited HIT immune complex-induced aggregation of both human and transgenic HIT mouse platelets. Transgenic HIT model mice were treated with KKO, a mouse monoclonal HIT-like antibody, and heparin. The experimental group received orally dosed PRT318, whereas the control group received vehicle. Nadir platelet counts of PRT318-treated mice were significantly higher than those of control mice. When examined with a novel thrombosis visualization technique, mice treated with PRT318 had significantly reduced thrombosis. The Syk inhibitor PRT318 thus prevented both HIT immune complex-induced thrombocytopenia and thrombosis in vivo, demonstrating its activity in HIT.

Description: Coagulation factor Xa (fXa) is a validated target for antithrombotic therapy and there are several on-going clinical trials testing direct fXa inhibitors. Current measures for monitoring coagulation status [activated partial thromboplastin time (aPTT), prothrombin time (PT), international normalized ratio (INR), activated clotting time (ACT), anti fXa units] have been developed for existing anticoagulants (i.e., heparins and warfarin). The available tests are not sensitive enough to evaluate therapeutic concentrations of direct fXa inhibitors. Since, the true target of fXa inhibitors is the membrane associated prothrombinase complex, we hypothesized that an assay measuring thrombin generation would be a superior measure of the level of anti-coagulation achieved in patients dosed with direct fXa inhibitors. PRT54021 (PRT021), an orally bioavailable fXa inhibitor in advanced stages of clinical development (Phase II), was used to validate this hypothesis. PRT021 is an active site directed, competitive inhibitor of human fXa (Ki=117pM ) and exhibits a 〉86,000 fold specificity against related proteases such as thrombin, factor VIIa, factor IXa, activated protein C, tissue plasminogen activator, plasmin and trypsin. PRT021 is a potent inhibitor of the purified prothrombinase complex (Ki=801pM). Evaluation in a whole blood prothrombinase assay was carried out to compare PRT021 to fondaparinux, an indirect inhibitor of fXa. Like fondaparinux, PRT021 dose-dependently inhibited platelet mediated prothrombinase activity in this test system. To measure prothrombinase inhibition, we developed a new bioassay which used a fluorogenic thrombin substrate to quantitate the amount of thrombin produced upon addition of tissue factor to human plasma. In order to determine if this new bioassay predicts in vivo antithrombotic activity, we tested PRT021 in a baboon model of arteriovenous shunt thrombosis. 111In labeled platelet and 125I fibrinogen deposition on the thrombogenic device were used as indicators of thrombotic activity. Dose-dependent inhibition of venous thrombosis was observed at four doses (0.05, 0.12, 0.21 and 0.49mg/kg) of PRT021. Ex vivo measurements of plasma thrombin generation, aPTT, PT, ACT, and anti fXa units were performed during the time course. In contrast to the observed antithrombotic activity (30 to 90% inhibition of platelet deposition, 0 to 87% inhibition of fibrin deposition), there were minimal extension of clotting parameters upon PRT021 treatment. Anti fXa units were below the limit of quantitation for the three lower doses and 0.31 Units/ml at the highest dose. Template bleeding times were not perturbed for any of the PRT021 treated animals. The only ex vivo parameter that correlated to the antithrombotic activity was the dose proportional inhibition (correlation coefficient R2=0.99) of plasma thrombin generation; which ranged from 13% inhibition at the lowest dose to 72% at the highest dose. Thus we have established that this new prothrombinase bioassay predicts in vivo antithrombotic activity. PRT021 is currently in clinical development for prevention and treatment of venous thromboembolic diseases. On-going work with plasma from PRT021 treated patients will verify if the bioassay correlates with clinical endpoints.

Description: Wnt/β-catenin signaling is central to bone development and homeostasis in adulthood and its deregulation is associated with bone pathologies. Dickkopf-1 (DKK1), a soluble inhibitor of Wnt/β-catenin signaling required for embryonic head development, regulates Wnt signaling by binding to the Wnt coreceptor lipoprotein-related protein-5 (LRP5)/Arrow. LRP5 mutations causing high bone mass syndromes disrupt DKK1-mediated regulation of LRP5. Forced overexpression of Dkk1 in osteoblasts causes osteopenia, disruption of the hematopoietic stem cell (HSC) niche, and defects in HSC function. Dkk1 also inhibits fracture repair. Studies suggest that DKK1 activation in osteoblasts is the underlying cause of glucocorticoid- and estrogen deficiency–mediated osteoporosis, and at least partially underlies the teratogenic effects of thalidomide on limb development. DKK1 induces proliferation of mesenchymal stem cells (MSC) in vitro and may play a role in the development of high-grade undifferentiated pleomorphic sarcomas derived from MSC and osteosarcomas. DKK1 has been implicated in causing erosive arthritis, the osteolytic phenotypes of multiple myeloma and metastatic breast cancer, and osteoblastic metastases of prostate cancer. Preclinical studies have shown that neutralizing DKK1/Dkk1 and/or enhancing Wnt/β-catenin signaling may prove effective in treating bone pathologies. Here, we review the rapidly growing body of literature defining a pivotal role for DKK1 in bone health and disease.