Determinant factors leading from stem cells to megakaryocytes (MKs) and subsequently platelets have yet to be identified. We now report that a combination of nuclear factor erythroid-derived 2 p45 unit (p45NF-E2), Maf G, and Maf K can convert mouse fibroblast 3T3 cells and adult human dermal fibroblasts into MKs. To screen MK-inducing factors, gene expressions were compared between 3T3 cells that do not differentiate into MKs and 3T3-L1 cells known to differentiate into MKs. 3T3 cells transfected with candidate factors were cultured in a defined MK lineage induction medium. Among the tested factors, transfection with p45NF-E2/MafG/MafK lead to the highest frequency of CD41-positive cells. Adult human dermal fibroblasts transfected with these genes were cultured in MK lineage induction medium. Cultured cells had megakaryocytic features, including surface markers, ploidy, and morphology. More than 90% of MK-sized cells expressed CD41, designated induced MK (iMK). Infusion of these iMK cells into immunodeficient mice led to a time-dependent appearance of CD41-positive, platelet-sized particles. Blood samples from iMK-infused into thrombocytopenic immunodeficient mice were perfused on a collagen-coated chip, and human CD41-positive platelets were incorporated into thrombi on the chip, demonstrating their functionality. These findings demonstrate that a combination of p45NF-E2, Maf G, and Maf K is a key determinant of both megakaryopoiesis and thrombopoiesis.
This phase 3 pivotal study evaluated the safety, efficacy, and pharmacokinetics of a recombinant FVIII Fc fusion protein (rFVIIIFc) for prophylaxis, treatment of acute bleeding, and perioperative hemostatic control in 165 previously treated males aged ≥12 years with severe hemophilia A. The study had three treatment arms: arm 1, individualized prophylaxis (25-65 IU/kg every 3-5 days, n = 118); arm 2, weekly prophylaxis (65 IU/kg, n = 24); and arm 3, episodic treatment (10-50 IU/kg, n = 23). A subgroup compared rFVIII and rFVIIIFc pharmacokinetics. Endpoints included annualized bleeding rate (ABR), inhibitor development, and adverse events. The terminal half-life of rFVIIIFc (19.0 h) was extended 1.5-fold versus rFVIII (12.4 h; P<.001). Median ABRs observed in arms 1, 2, and 3 were 1.6, 3.6, and 33.6, respectively. In arm 1, the median weekly dose was 77.9 IU/kg; approximately 30% of subjects achieved a 5-day dosing interval (last 3 months on study). Across arms, 87.3% of bleeding episodes resolved with one injection. Adverse events were consistent with those expected in this population; no subjects developed inhibitors. rFVIIIFc was well-tolerated, had a prolonged half-life compared with rFVIII, and resulted in low ABRs when dosed prophylactically 1-2 times per week. This study is registered at www.clinicaltrials.gov, ID: NCT01181128.
Treatment options for patients with heavily pretreated relapsed and/or refractory multiple myeloma remain limited. We evaluated a novel therapeutic regimen consisting of carfilzomib, pomalidomide, and dexamethasone in an open-label, multicenter, phase 1, dose-escalation study. Patients that relapsed after prior therapy or were refractory to the most recently received therapy were eligible. All patients were refractory to prior lenalidomide. Patients received carfilzomib intravenously on days 1, 2, 8, 9, 15, and 16 (starting dose of 20/27 mg/m(2)), pomalidomide once daily on days 1-21 (4 mg as the initial dose level) and dexamethasone (40 mg oral or IV) on days 1, 8, 15, and 22 of 28-day cycles. The primary objective was to evaluate the safety and determine the maximum tolerated dose (MTD) of the regimen. A total of 32 patients were enrolled. The MTD of the regimen was dose level 1 (carfilzomib 20/27 mg/m(2), pomalidomide 4 mg, dexamethasone 40 mg). Hematological adverse events occurred in ≥60% of all patients, including eleven patients with grade ≥3 anemia. Dyspnea was limited to grade ½ in ten patients. Peripheral neuropathy was uncommon and limited to grade ½. Eight patients had dose reductions during therapy, and seven patients discontinued treatment due to adverse events. Two deaths were noted on study due to pneumonia and pulmonary embolism (n=1 each). The combination of carfilzomib, pomalidomide, and dexamethasone is well-tolerated and highly active in patients with RRMM. This study was funded by Onyx Pharmaceuticals, Inc., an Amgen subsidiary, and Celgene Corporation. The trial was registered with www.Clinicaltrials.gov: identifier NCT01464034.
Neonatal bone marrow transplantation (nBMT) could offer a novel therapeutic opportunity for genetic disorders by providing sustainable levels of the missing protein at birth thus preventing tissue damage. We tested this concept in mucopolysaccharidosis type I (MPS IH; Hurler syndrome), a lysosomal storage disorder caused by deficiency of α-L-iduronidase (IDUA). MPS IH is characterized by a broad spectrum of clinical manifestations including severe progressive skeletal abnormalities. Although BMT increases the life span of MPS IH patients, musculoskeletal manifestations are only minimally responsive if the timing of BMT delays, suggesting already irreversible bone damage. In this study, we tested the hypothesis that transplanting normal bone marrow into newborn MPS I mice, soon after birth, can prevent skeletal dysplasia. We observed that nBMT was effective at restoring IDUA activity and clearing elevated glycosaminoglycans in blood and multiple organs. At 37 weeks of age, we observed an almost complete normalization of all bone tissue parameters using radiographic, micro-CT, biochemical, and histological analyses. Overall, the magnitude of improvements correlated with the extent of hematopoietic engraftment. We conclude that BMT at a very early stage in life markedly reduces signs and symptoms of MPS I before they appear.
ACE910 is a recombinant humanized bispecific antibody that binds to activated factor IX and factor X and mimics the cofactor function of factor VIII (FVIII). This first-in-human study examined the safety, tolerability, pharmacokinetics (PK), and pharmacodynamics (PD) of ACE910 in healthy male adults. A total of 40 Japanese and 24 Caucasian subjects were randomized to receive a single subcutaneous injection of ACE910 (Japanese: 0.001, 0.01, 0.1, 0.3, or 1 mg/kg; Caucasian: 0.1, 0.3, or 1 mg/kg; n = 6 per dose group) or placebo (n = 2 per dose group). ACE910 exhibited a linear PK profile and had a half-life of approximately 4 to 5 weeks. In FVIII-neutralized plasma, ACE910 shortened activated partial thromboplastin time and increased peak height of thrombin generation in a dose-dependent manner. All adverse events were non-serious and did not lead to any subject’s withdrawal. Neither clinical findings nor laboratory abnormalities indicating hypercoagulability were observed. Two of 48 subjects receiving ACE910 (1 Japanese and 1 Caucasian) were positive for anti-ACE910 antibodies (anti-drug antibodies; ADA). One subject tested positive for ADA both before and after ACE910 administration, whereas the other became ADA-positive after receiving ACE910. The PK and PD profiles of ACE910 were similar in healthy Japanese and Caucasian subjects, and suggest that ACE910 will be an effective and convenient prophylactic treatment for hemophilia A. This trial was registered at http://www.clinicaltrials.jp (JapicCTI-121934).
Duvelisib (also known as IPI-145) is an oral, dual inhibitor of phosphoinositide 3-kinase (PI3K) δ and -γ being developed for treatment of hematologic malignancies. PI3K-δ,γ signaling can promote B cell proliferation and survival in clonal B cell malignancies, such as chronic lymphocytic leukemia (CLL)/small lymphocytic lymphoma (SLL). In a Phase 1 study, duvelisib showed clinically meaningful activity and acceptable safety in CLL/SLL patients. We report here the results of DUO™, a global Phase 3 randomized study of duvelisib versus ofatumumab monotherapy for patients with relapsed or refractory (RR) CLL/SLL. Patients were randomized 1:1 to oral duvelisib 25 mg twice daily (BID) (n = 160) or ofatumumab intravenous (IV) (n = 159). The study met the primary study endpoint by significantly improving progression-free survival (PFS) per Independent Review Committee (IRC) assessment compared to ofatumumab for all patients (median 13.3 months versus 9.9 months; hazard ratio [HR] = 0.52; p < 0.0001), including those with high-risk chromosome 17p13.1 deletions [del(17p)] and/or TP53 mutations (HR = 0.40; p = 0.0002). The overall response rate (ORR) was significantly higher with duvelisib (74% versus 45%; p < 0.0001) regardless of del(17p) status. The most common adverse events (AEs) were diarrhea, neutropenia, pyrexia, nausea, anemia, and cough on the duvelisib arm, and neutropenia and infusion reactions on the ofatumumab arm. The DUO trial data support duvelisib as a potentially effective treatment option for patients with RR CLL/SLL. This trial is registered at ClinicalTrials.gov as NCT02004522.
Older patients with acute myeloid leukemia (AML) respond poorly to standard induction therapy. BCL-2 overexpression is implicated in survival of AML cells and treatment resistance. We report safety and efficacy of venetoclax with decitabine or azacitidine from a large, multicenter, phase 1b dose-escalation and expansion study. Patients (N=145) were ≥65 years with treatment-naive AML ineligible for intensive chemotherapy. During dose escalation, oral venetoclax was administered at 400, 800, or 1200 mg daily in combination with either decitabine (20 mg/m2, days 1-5; intravenously [IV]) or azacitidine (75 mg/m2, days 1-7; IV or subcutaneously). In the expansion, 400 mg or 800 mg venetoclax with either hypomethylating agent (HMA) was given. Median age was 74 years, with poor-risk cytogenetics in 49% of patients. Common adverse events (>30%) included nausea, diarrhea, constipation, febrile neutropenia, fatigue, hypokalemia, decreased appetite, and decreased white blood cell count. No tumor lysis syndrome was observed. With a median time on study of 8.9 months, 67% of patients (all doses) achieved complete remission (CR) + CR with incomplete count recovery (CRi), with a CR+CRi rate of 73% in the venetoclax 400-mg + HMA cohort. Patients with poor-risk cytogenetics and those ≥75 years had CR+CRi rates of 60% and 65%, respectively. The median duration of CR+CRi (all patients) was 11.3 months and median overall survival (mOS) was 17.5 months; mOS has not been reached for the 400 mg venetoclax cohort. The novel combination of venetoclax with decitabine or azacitidine was effective and well tolerated in elderly patients with AML (https://clinicaltrials.gov/, NCT02203773).
The clinical needs for platelet transfusions are increasing. Donor-dependent platelet transfusions are, however, associated with practical problems, such as the limited supply and the risk of infection. Thus, we developed a manufacturing system for platelets from donor-independent cell source, human adipose-derived mesenchymal stromal/stem cell line (ASCL). ASCL was obtained by upside-down culture flask method. ASCL satisfied the minimal criteria for defining mesenchymal stem cell (MSC) by The International Society for Cellular Therapy. ASCL showed their proliferation capacity for at least 2 months without any abnormal karyotypes. ASCL was cultured in megakaryocyte induction media. ASCL-derived megakaryocyte (ASCL-MK) were obtained with a peak at Day 8 of culture. ASCL-derived platelets (ASCL-PLT) were obtained with a peak at Day 12 of culture. We observed that CD42b-positive cells expressed an MSC marker CD90 in relation to cell adhesion. Compared with peripheral platelets, ASCL-PLT exhibits higher levels of PAC1 binding, P-selectin surface exposure, ristocetin-induced platelet aggregation, and ADP-induced platelet aggregation; similar levels of fibrinogen binding, and collagen-induced platelet aggregation. ASCL-PLT has lower epinephrine-induced platelet aggregation. The pattern of in vivo kinetics after being infused into irradiated immunodeficient NSG mice was similar to that of platelet concentrates. ASCL-PLT has characterization observed in other platelet populations and might have an additional function as MSC. Neither the establishment of ASCL nor its differentiation into ASCL-PLT requires gene transfer, and endogenous thrombopoietin is utilized for differentiation. The present protocol is a simple method that requires no feeder cells, further enhancing the clinical application of our approach.
COVID-19 affects millions of patients worldwide with clinical presentation ranging from isolated thrombosis to acute respiratory distress syndrome (ARDS) requiring ventilator support. Neutrophil extracellular traps (NETs) originate from decondensed chromatin released to immobilize pathogens and can trigger immunothrombosis. We studied the connection between NETs and COVID-19 severity and progression. We conducted a prospective cohort study of COVID-19 patients (n=33) with age- and sex-matched controls (n=17). We measured plasma myeloperoxidase (MPO)-DNA complexes (NETs), Platelet Factor 4, RANTES, and selected cytokines. Three COVID-19 lung autopsies were examined for NETs and platelet involvement. We assessed NET formation ex vivo in COVID-19 neutrophils and in healthy neutrophils incubated with COVID-19 plasma. We also tested the ability of neonatal NET-Inhibitory Factor (nNIF) to block NET formation induced by COVID-19 plasma. Plasma MPO-DNA complexes increased in COVID-19 with intubation (P<0.0001) and death as outcome (P<0.0005). Illness severity correlated directly with plasma MPO-DNA complexes (P=0.0360), while PaO2/FiO2 correlated inversely(P=0.0340). Soluble and cellular factors triggering NETs were significantly increased in COVID-19 and pulmonary autopsies confirmed NET-containing microthrombi with neutrophil-platelet infiltration. Finally, COVID-19 neutrophils ex vivo displayed excessive NETs at baseline and COVID-19 plasma triggered NET formation which was blocked by nNIF. Thus, NETs triggering immunothrombosis may, in part, explain the prothrombotic clinical presentations in COVID-19 and NETs may represent targets for therapeutic intervention.
Effective treatment options are limited for patients with acute myeloid leukemia (AML) who cannot tolerate intensive chemotherapy.